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Jean-Yves Sgro
Inst. for Mol.Virology
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1525 Linden Drive Madison, WI 53706

Table of Contents for this page:

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  • HIV-2 Depletes CD4 T Cells through Pyroptosis despite Vpx-Dependent Degradation of SAMHD1 [Virus-Cell Interactions]

  • Human immunodeficiency virus type 2 (HIV-2) infection results in a milder course of disease and slower progression to AIDS than does HIV-1. We hypothesized that this difference may be due to degradation of the sterile alpha motif and HD domain 1 (SAMHD1) host restriction factor by the HIV-2 Vpx gene product, thereby diminishing abortive infection and pyroptotic cell death within bystander CD4 T cells. We have compared CD4 T cell death in tonsil-derived human lymphoid aggregate cultures (HLACs) infected with wild-type HIV-2, HIV-2 Vpx, or HIV-1. In contrast to our hypothesis, HIV-2, HIV-2 Vpx, and HIV-1 induced similar levels of bystander CD4 T cell death. In all cases, cell death was blocked by AMD3100, a CXCR4 entry inhibitor, but not by raltegravir, an integrase, indicating that only early life cycle events were required. Cell death was also blocked by a caspase-1 inhibitor, a key enzyme promoting pyroptosis, but not by a caspase-3 inhibitor, an important enzyme in apoptosis. HIV-1-induced abortive infection and pyroptotic cell death were also not reduced by forced encapsidation of HIV-2 Vpx into HIV-1 virions. Together, these findings indicate that HIV-2 and HIV-1 support similar levels of CD4 T cell depletion in vitro despite HIV-2 Vpx-mediated degradation of the SAMHD1 transcription factor. The milder disease course observed with HIV-2 infection likely stems from factors other than abortive infection and caspase-1-dependent pyroptosis in bystander CD4 T cells.

    IMPORTANCE CD4 T cell depletion during HIV-1 infection involves the demise of bystander CD4 T cells due to abortive infection, viral DNA sensing, inflammasome assembly, and death by caspase-1-dependent pyroptosis. HIV-2 infection is associated with milder disease and lower rates of CD4 T cell loss. We hypothesized that HIV-2 infection produces lower levels of pyroptosis due to the action of its Vpx gene product. Vpx degrades the SAMHD1 restriction factor, potentially reducing abortive forms of infection. However, in tonsil cell cultures, HIV-2, HIV-2 Vpx, and HIV-1 induced indistinguishable levels of pyroptosis. Forced encapsidation of Vpx into HIV-1 virions also did not reduce pyroptosis. Thus, SAMHD1 does not appear to play a key role in the induction of bystander cell pyroptosis. Additionally, the milder clinical course of HIV-2-induced disease is apparently not explained by a decrease in this inflammatory form of programmed cell death.

  • Macromolecular Synthesis Shutoff Resistance by Myeloid Cells Is Critical to IRF7-Dependent Systemic Interferon Alpha/Beta Induction after Alphavirus Infection [Virus-Cell Interactions]

  • Alphavirus infection of fibroblastic cell types in vitro inhibits host cell translation and transcription, leading to suppression of interferon alpha/beta (IFN-aalpha;/bbeta;) production. However, the effect of infection upon myeloid cells, which are often the first cells encountered by alphaviruses in vivo, is unclear. Previous studies demonstrated an association of systemic IFN-aalpha;/bbeta; production with myeloid cell infection efficiency. Murine infection with wild-type Venezuelan equine encephalitis virus (VEEV), a highly myeloid-cell-tropic alphavirus, results in secretion of very high systemic levels of IFN-aalpha;/bbeta;, suggesting that stress responses in responding cells are active. Here, we infected myeloid cell cultures with VEEV to identify the cellular source of IFN-aalpha;/bbeta;, the timing and extent of translation and/or transcription inhibition in infected cells, and the transcription factors responsible for IFN-aalpha;/bbeta; induction. In contrast to fibroblast infection, myeloid cell cultures infected with VEEV secreted IFN-aalpha;/bbeta; that increased until cell death was observed. VEEV inhibited translation in most cells early after infection (llt;6 h postinfection [p.i.]), while transcription inhibition occurred later (ggt;6 h p.i.). Furthermore, the interferon regulatory factor 7 (IRF7), but not IRF3, transcription factor was critical for IFN-aalpha;/bbeta; induction in vitro and in sera of mice. We identified a subset of infected Raw 264.7 myeloid cells that resisted VEEV-induced translation inhibition and secreted IFN-aalpha;/bbeta; despite virus infection. However, in the absence of IFN receptor signaling, the size of this cell population was diminished. These results indicate that IFN-aalpha;/bbeta; induction in vivo is IRF7 dependent and arises in part from a subset of myeloid cells that are resistant, in an IFN-aalpha;/bbeta;-dependent manner, to VEEV-induced macromolecular synthesis inhibition.

    IMPORTANCE Most previous research exploring the interaction of alphaviruses with host cell antiviral responses has been conducted using fibroblast lineage cell lines. Previous studies have led to the discovery of virus-mediated activities that antagonize host cell antiviral defense pathways, such as host cell translation and transcription inhibition and suppression of STAT1 signaling. However, their relevance and impact upon myeloid lineage cell types, which are key responders during the initial stages of alphavirus infection in vivo, have not been well studied. Here, we demonstrate the different abilities of myeloid cells to resist VEEV infection compared to nonmyeloid cell types and begin to elucidate the mechanisms by which host antiviral responses are upregulated in myeloid cells despite the actions of virus-encoded antagonists.

  • Isolation and Characterization of Akhmeta Virus from Wild-Caught Rodents (Apodemus spp.) in Georgia [Genetic Diversity and Evolution]

  • In 2013, a novel orthopoxvirus was detected in skin lesions of two cattle herders from the Kakheti region of Georgia (country); this virus was named Akhmeta virus. Subsequent investigation of these cases revealed that small mammals in the area had serological evidence of orthopoxvirus infections, suggesting their involvement in the maintenance of these viruses in nature. In October 2015, we began a longitudinal study assessing the natural history of orthopoxviruses in Georgia. As part of this effort, we trapped small mammals near Akhmeta (n = 176) and Gudauri (n = 110). Here, we describe the isolation and molecular characterization of Akhmeta virus from lesion material and pooled heart and lung samples collected from five wood mice (Apodemus uralensis and Apodemus flavicollis) in these two locations. The genomes of Akhmeta virus obtained from rodents group into 2 clades: one clade represented by viruses isolated from A. uralensis samples, and one clade represented by viruses isolated from A. flavicollis samples. These genomes also display several presumptive recombination events for which gene truncation and identity have been examined.

    IMPORTANCE Akhmeta virus is a unique Orthopoxvirus that was described in 2013 from the country of Georgia. This paper presents the first isolation of this virus from small mammal (Rodentia; Apodemus spp.) samples and the molecular characterization of those isolates. The identification of the virus in small mammals is an essential component to understanding the natural history of this virus and its transmission to human populations and could guide public health interventions in Georgia. Akhmeta virus genomes harbor evidence suggestive of recombination with a variety of other orthopoxviruses; this has implications for the evolution of orthopoxviruses, their ability to infect mammalian hosts, and their ability to adapt to novel host species.

  • Differentiation into an Effector Memory Phenotype Potentiates HIV-1 Latency Reversal in CD4+ T Cells [Genome Replication and Regulation of Viral Gene Expression]

  • During antiretroviral therapy (ART), human immunodeficiency virus type 1 (HIV-1) persists as a latent reservoir in CD4+ T cell subsets in central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells. We have identified differences in mechanisms underlying latency and responses to latency-reversing agents (LRAs) in ex vivo CD4+ memory T cells from virally suppressed HIV-infected individuals and in an in vitro primary cell model of HIV-1 latency. Our ex vivo and in vitro results demonstrate the association of transcriptional pathways of T cell differentiation, acquisition of effector function, and cell cycle entry in response to LRAs. Analyses of memory cell subsets showed that effector memory pathways and cell surface markers of activation and proliferation in the TEM subset are predictive of higher frequencies of cells carrying an inducible reservoir. Transcriptional profiling also demonstrated that the epigenetic machinery (known to control latency and reactivation) in the TEM subset is associated with frequencies of cells with HIV-integrated DNA and inducible HIV multispliced RNA. TCM cells were triggered to differentiate into TEM cells when they were exposed to LRAs, and this increase of TEM subset frequencies upon LRA stimulation was positively associated with higher numbers of p24+ cells. Together, these data highlight differences in underlying biological latency control in different memory CD4+ T cell subsets which harbor latent HIV in vivo and support a role for differentiation into a TEM phenotype in facilitating latency reversal.

    IMPORTANCE By performing phenotypic analysis of latency reversal in CD4+ T cells from virally suppressed individuals, we identify the TEM subset as the largest contributor to the inducible HIV reservoir. Differential responses of memory CD4+ T cell subsets to latency-reversing agents (LRAs) demonstrate that HIV gene expression is associated with heightened expression of transcriptional pathways associated with differentiation, acquisition of effector function, and cell cycle entry. In vitro modeling of the latent HIV reservoir in memory CD4+ T cell subsets identify LRAs that reverse latency with ranges of efficiency and specificity. We found that therapeutic induction of latency reversal is associated with upregulation of identical sets of TEM-associated genes and cell surface markers shown to be associated with latency reversal in our ex vivo and in vitro models. Together, these data support the idea that the effector memory phenotype supports HIV latency reversal in CD4+ T cells.

  • Epstein-Barr Virus-Specific CD8 T Cells Selectively Infiltrate the Brain in Multiple Sclerosis and Interact Locally with Virus-Infected Cells: Clue for a Virus-Driven Immunopathological Mechanism [Pathogenesis and Immunity]

  • Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (lle;0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury.

    IMPORTANCE EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.

  • NF-{kappa}B Activation Promotes Alphavirus Replication in Mature Neurons [Virus-Cell Interactions]

  • Alphaviruses are enveloped, positive-sense RNA viruses that are important causes of viral encephalomyelitis. Sindbis virus (SINV) infects the neurons of rodents and is a model for studying factors that regulate infection of neuronal cells. The outcome of alphavirus infection of the central nervous system is dependent on neuronal maturation status. Differentiated mature neurons survive and control viral replication better than undifferentiated immature neurons. The cellular factors involved in age-dependent susceptibility include higher levels of antiapoptotic and innate immune factors in mature neurons. Because NF-B pathway activation is required for the initiation of both apoptosis and the host antiviral response, we analyzed the role of NF-B during SINV infection of differentiated and undifferentiated rat neuronal cells. SINV infection induced canonical NF-B activation, as evidenced by the degradation of IBaalpha; and the phosphorylation and nuclear translocation of p65. Inhibition or deletion of the upstream IB kinase substantially reduced SINV replication in differentiated but not in undifferentiated neuronal cells or mouse embryo fibroblasts. NF-B inhibition did not affect the establishment of infection, replication complex formation, the synthesis of nonstructural proteins, or viral RNA synthesis in differentiated neurons. However, the translation of structural proteins was impaired, phosphorylation of the aalpha; subunit of eukaryotic translation initiation factor 2 (eIF2aalpha;) was decreased, and host protein synthesis was maintained, suggesting that NF-B activation was involved in the regulation of translation during infection of mature neurons. Inhibition or deletion of double-stranded RNA-activated protein kinase (PKR) also decreased eIF2aalpha; phosphorylation, the translation of viral structural proteins, and virus production. Therefore, canonical NF-B activation synergizes with PKR to promote SINV replication in differentiated neurons by facilitating viral structural protein translation.

    IMPORTANCE Mosquito-borne alphaviruses are a significant and growing cause of viral encephalomyelitis worldwide. The outcome of alphaviral neuronal infections is host age dependent and greatly affected by neuronal maturation status, with differentiated, mature neurons being more resistant to infection than undifferentiated, immature neurons. The biological factors that change during neuronal maturation and that influence the outcome of viral infection are currently only partially defined. These studies investigated the role of NF-B in determining the outcome of alphaviral infection in mature and immature neurons. Inhibition of canonical NF-B activation decreased alphavirus replication in mature neurons by regulating protein synthesis and limiting the production of the viral structural proteins but had little effect on viral replication in immature neurons or fibroblasts. Therefore, NF-B is a signaling pathway that influences the maturation-dependent outcome of alphaviral infection in neurons and that highlights the importance of cellular context in determining the effects of signal pathway activation.

  • Activity-Based Protein Profiling Identifies ATG4B as a Key Host Factor for Enterovirus 71 Proliferation [Virus-Cell Interactions]

  • Virus-encoded proteases play diverse roles in the efficient replication of enterovirus 71 (EV71), which is the causative agent of human hand, foot, and mouth disease (HFMD). However, it is unclear how host proteases affect viral proliferation. Here, we designed activity-based probes (ABPs) based on an inhibitor of the main EV71 protease (3Cpro), which is responsible for the hydrolysis of the EV71 polyprotein, and successfully identified host candidates that bind to the ABPs. Among the candidates, the host cysteine protease autophagy-related protein 4 homolog B (ATG4B), a key component of the autophagy machinery, was demonstrated to hydrolytically process the substrate of EV71 3Cpro and had activity comparable to that of the viral protease. Genetic disruption of ATG4B confirmed that the enzyme is indispensable for viral proliferation in vivo. Our results not only further the understanding of host-virus interactions in EV71 biology but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.

    IMPORTANCE Enterovirus 71 (EV71), one of the major pathogens of human HFMD, has caused outbreaks worldwide. How EV71 efficiently assesses its life cycle with elaborate interactions with multiple host factors remains to be elucidated. In this work, we deconvoluted that the host ATG4B protein processes the viral polyprotein with its cysteine protease activity and helps EV71 replicate through a chemical biology strategy. Our results not only further the understanding of the EV71 life cycle but also provide a sample for the usage of activity-based proteomics to reveal host-pathogen interactions.

  • Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor [Virus-Cell Interactions]

  • When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-aalpha;) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 nef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 nef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-aalpha; in modulating SERINC5 through accumulation at the cell surface.

    IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.

  • Dissecting the Role of DDX21 in Regulating Human Cytomegalovirus Replication [Virus-Cell Interactions]

  • DDX21 regulates the biogenesis of rRNA and transcription of ribonucleoprotein genes. Recently, it has been reported that DDX21 regulates the growth of some RNA viruses through various mechanisms, such as inhibiting viral genome replication, suppressing virion assembly and release, and modulating antiviral immune responses (Chen et al., Cell Host Microbe 15:484nndash;493, 2014,; Dong et al., Biophys Res Commun, 473:648nndash;653, 2016,; and Watanabe et al., PLoS Pathog 5:e1000654, 2009, The relationship between DDX21 and DNA viruses has not yet been explored. In this study, we used human cytomegalovirus (HCMV), a large human DNA virus, to investigate the potential role of DDX21 in DNA virus replication. We found that HCMV infection prevented the repression of DDX21 at protein and mRNA levels. Knockdown of DDX21 inhibited HCMV growth in human fibroblast cells (MRC5). Immunofluorescence and quantitative PCR (qPCR) results showed that knockdown of DDX21 did not affect viral DNA replication or the formation of the viral replication compartment but did significantly inhibit viral late gene transcription. Some studies have reported that DDX21 knockdown promotes the accumulation of R-loops that could restrain RNA polymerase II elongation and inhibit the transcription of certain genes. Thus, we used the DNA-RNA hybrid-specific S9.6 antibody to stain R-loops and observed that more R-loops formed in DDX21-knockdown cells than in control cells. Moreover, an DNA-RNA immunoprecipitation assay showed that more R-loops accumulated on a viral late gene in DDX21-knockdown cells. Altogether, these results suggest that DDX21 knockdown promotes the accumulation of R-loops, which prevents viral late gene transcription and consequently results in the suppression of HCMV growth. This finding provides new insight into the relationship between DDX21 and DNA virus replication.

    IMPORTANCE Previous studies have confirmed that DDX21 is vital for the regulation of various aspects of RNA virus replication. Our research is the first report on the role of DDX21 in HCMV DNA virus replication. We identified that DDX21 knockdown affected HCMV growth and viral late gene transcription. In order to elucidate how DDX21 regulated this transcription, we applied DNA-RNA immunoprecipitation by using the DNA-RNA hybrid-specific S9.6 antibody to test whether more R-loops accumulated on the viral late gene. Consistent with our expectation, more R-loops were detected on the viral late gene at late HCMV infection time points, which demonstrated that the accumulation of R-loops caused by DDX21 knockdown prevented viral late gene transcription and consequently impaired HCMV replication. These results reveal that DDX21 plays an important role in regulating HCMV replication and also provide a basis for investigating the role of DDX21 in regulating other DNA viruses.

  • Clearance of Chikungunya Virus Infection in Lymphoid Tissues Is Promoted by Treatment with an Agonistic Anti-CD137 Antibody [Pathogenesis and Immunity]

  • CD137, a member of the tumor necrosis factor receptor superfamily of cell surface proteins, acts as a costimulatory receptor on T cells, natural killer cells, B cell subsets, and some dendritic cells. Agonistic anti-CD137 monoclonal antibody (MAb) therapy has been combined with other chemotherapeutic agents in human cancer trials. Based on its ability to promote tumor clearance, we hypothesized that anti-CD137 MAb might activate immune responses and resolve chronic viral infections. We evaluated anti-CD137 MAb therapy in a mouse infection model of chikungunya virus (CHIKV), an alphavirus that causes chronic polyarthritis in humans and is associated with reservoirs of CHIKV RNA that are not cleared efficiently by adaptive immune responses. Analysis of viral tropism revealed that CHIKV RNA was present preferentially in splenic B cells and follicular dendritic cells during the persistent phase of infection, and animals lacking B cells did not develop persistent CHIKV infection in lymphoid tissue. Anti-CD137 MAb treatment resulted in T cell-dependent clearance of CHIKV RNA in lymphoid tissue, although this effect was not observed in musculoskeletal tissue. The clearance of CHIKV RNA from lymphoid tissue by anti-CD137 MAb was associated with reductions in the numbers of germinal center B cells and follicular dendritic cells. Similar results were observed with anti-CD137 MAb treatment of mice infected with Mayaro virus, a related arthritogenic alphavirus. Thus, anti-CD137 MAb treatment promotes resolution of chronic alphavirus infection in lymphoid tissues by reducing the numbers of target cells for infection and persistence.

    IMPORTANCE Although CHIKV causes persistent infection in lymphoid and musculoskeletal tissues in multiple animals, the basis for this is poorly understood, which has hampered pharmacological efforts to promote viral clearance. Here, we evaluated the therapeutic effects on persistent CHIKV infection of an agonistic anti-CD137 MAb that can activate T cell and natural killer cell responses to clear tumors. We show that treatment with anti-CD137 MAb promotes the clearance of persistent alphavirus RNA from lymphoid but not musculoskeletal tissues. This occurs because anti-CD137 MAb-triggered T cells reduce the numbers of target germinal center B cells and follicular dendritic cells, which are the primary reservoirs for CHIKV in the spleen and lymph nodes. Our studies help to elucidate the basis for CHIKV persistence and begin to provide strategies that can clear long-term cellular reservoirs of infection.

  • Flexibility in Nucleic Acid Binding Is Central to APOBEC3H Antiviral Activity [Cellular Response to Infection]

  • APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination.

    IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

  • How HIV Nef Proteins Hijack Membrane Traffic To Promote Infection [Minireviews]

  • The accessory protein Nef of human immunodeficiency virus (HIV) is a primary determinant of viral pathogenesis. Nef is abundantly expressed during infection and reroutes a variety of cell surface proteins to disrupt host immunity and promote the viral replication cycle. Nef counteracts host defenses by sequestering and/or degrading its targets via the endocytic and secretory pathways. Nef does this by physically engaging a number of host trafficking proteins. Substantial progress has been achieved in identifying the targets of Nef, and a structural and mechanistic understanding of Nefrrsquo;s ability to command the protein trafficking machinery has recently started to coalesce. Comparative analysis of HIV and simian immunodeficiency virus (SIV) Nef proteins in the context of recent structural advances sheds further light on both viral evolution and the mechanisms whereby trafficking is hijacked. This review describes how advances in cell and structural biology are uncovering in growing detail how Nef subverts the host immune system, facilitates virus release, and enhances viral infectivity.

  • Tracking the Fate of Endogenous Retrovirus Segregation in Wild and Domestic Cats [Genetic Diversity and Evolution]

  • Endogenous retroviruses (ERVs) of domestic cats (ERV-DCs) are one of the youngest feline ERV groups in domestic cats (Felis silvestris catus); some members are replication competent (ERV-DC10, ERV-DC18, and ERV-DC14), produce the antiretroviral soluble factor Refrex-1 (ERV-DC7 and ERV-DC16), or can generate recombinant feline leukemia virus (FeLV). Here, we investigated ERV-DC in European wildcats (Felis silvestris silvestris) and detected four loci: ERV-DC6, ERV-DC7, ERV-DC14, and ERV-DC16. ERV-DC14 was detected at a high frequency in European wildcats; however, it was replication defective due to a single G -ggt; A nucleotide substitution, resulting in an E148K substitution in the ERV-DC14 envelope (Env). This mutation results in a cleavage-defective Env that is not incorporated into viral particles. Introduction of the same mutation into feline and murine infectious gammaretroviruses resulted in a similar Env dysfunction. Interestingly, the same mutation was found in an FeLV isolate from naturally occurring thymic lymphoma and a mouse ERV, suggesting a common mechanism of virus inactivation. Refrex-1 was present in European wildcats; however, ERV-DC16, but not ERV-DC7, was unfixed in European wildcats. Thus, Refrex-1 has had an antiviral role throughout the evolution of the genus Felis, predating cat exposure to feline retroviruses. ERV-DC sequence diversity was present across wild and domestic cats but was locus dependent. In conclusion, ERVs have evolved species-specific phenotypes through the interplay between ERVs and their hosts. The mechanism of viral inactivation may be similar irrespective of the evolutionary history of retroviruses. The tracking of ancestral retroviruses can shed light on their roles in pathogenesis and host-virus evolution.

    IMPORTANCE Domestic cats (Felis silvestris catus) were domesticated from wildcats approximately 9,000 years ago via close interaction between humans and cats. During cat evolution, various exogenous retroviruses infected different cat lineages and generated numerous ERVs in the host genome, some of which remain replication competent. Here, we detected several ERV-DC loci in Felis silvestris silvestris. Notably, a species-specific single nucleotide polymorphism in the ERV-DC14 env gene, which results in a replication-defective product, is highly prevalent in European wildcats, unlike the replication-competent ERV-DC14 that is commonly present in domestic cats. The presence of the same lethal mutation in the env genes of both FeLV and murine ERV provides a common mechanism shared by endogenous and exogenous retroviruses by which ERVs can be inactivated after endogenization. The antiviral role of Refrex-1 predates cat exposure to feline retroviruses. The existence of two ERV-DC14 phenotypes provides a unique model for understanding both ERV fate and cat domestication.

  • A New Family of Small-Molecule CD4-Mimetic Compounds Contacts Highly Conserved Aspartic Acid 368 of HIV-1 gp120 and Mediates Antibody-Dependent Cellular Cytotoxicity [Pathogenesis and Immunity]

  • The HIV-1 envelope glycoprotein (Env) trimer mediates virus entry into cells. The "closed" conformation of Env is resistant to nonneutralizing antibodies (nnAbs). These antibodies mostly recognize occluded epitopes that can be exposed upon binding of CD4 or small-molecule CD4 mimetics (CD4mc). Here, we describe a new family of small molecules that expose Env to nnAbs and sensitize infected cells to antibody-dependent cellular cytotoxicity (ADCC). These compounds have a limited capacity to inhibit virus infection directly but are able to sensitize viral particles to neutralization by otherwise nonneutralizing antibodies. Structural analysis shows that some analogs of this family of CD4mc engage the gp120 Phe43 cavity by contacting the highly conserved D368 residue, making them attractive scaffolds for drug development.

    IMPORTANCE HIV-1 has evolved multiple strategies to avoid humoral responses. One efficient mechanism is to keep its envelope glycoprotein (Env) in its "closed" conformation. Here, we report on a new family of small molecules that are able to "open up" Env, thus exposing vulnerable epitopes. This new family of molecules binds in the Phe43 cavity and contacts the highly conserved D368 residue. The structural and biological attributes of molecules of this family make them good candidates for drug development.

  • Small-Molecule Antiviral {beta}-D-N4-Hydroxycytidine Inhibits a Proofreading-Intact Coronavirus with a High Genetic Barrier to Resistance [Vaccines and Antiviral Agents]

  • Coronaviruses (CoVs) have emerged from animal reservoirs to cause severe and lethal disease in humans, but there are currently no FDA-approved antivirals to treat the infections. One class of antiviral compounds, nucleoside analogues, mimics naturally occurring nucleosides to inhibit viral replication. While these compounds have been successful therapeutics for several viral infections, mutagenic nucleoside analogues, such as ribavirin and 5-fluorouracil, have been ineffective at inhibiting CoVs. This has been attributed to the proofreading activity of the viral 3'-5' exoribonuclease (ExoN). bbeta;-d-N4-Hydroxycytidine (NHC) (EIDD-1931; Emory Institute for Drug Development) has recently been reported to inhibit multiple viruses. Here, we demonstrate that NHC inhibits both murine hepatitis virus (MHV) (50% effective concentration [EC50] = 0.17 mmu;M) and Middle East respiratory syndrome CoV (MERS-CoV) (EC50 = 0.56 mmu;M) with minimal cytotoxicity. NHC inhibited MHV lacking ExoN proofreading activity similarly to wild-type (WT) MHV, suggesting an ability to evade or overcome ExoN activity. NHC inhibited MHV only when added early during infection, decreased viral specific infectivity, and increased the number and proportion of G:A and C:U transition mutations present after a single infection. Low-level NHC resistance was difficult to achieve and was associated with multiple transition mutations across the genome in both MHV and MERS-CoV. These results point to a virus-mutagenic mechanism of NHC inhibition in CoVs and indicate a high genetic barrier to NHC resistance. Together, the data support further development of NHC for treatment of CoVs and suggest a novel mechanism of NHC interaction with the CoV replication complex that may shed light on critical aspects of replication.

    IMPORTANCE The emergence of coronaviruses (CoVs) into human populations from animal reservoirs has demonstrated their epidemic capability, pandemic potential, and ability to cause severe disease. However, no antivirals have been approved to treat these infections. Here, we demonstrate the potent antiviral activity of a broad-spectrum ribonucleoside analogue, bbeta;-d-N4-hydroxycytidine (NHC), against two divergent CoVs. Viral proofreading activity does not markedly impact sensitivity to NHC inhibition, suggesting a novel interaction between a nucleoside analogue inhibitor and the CoV replicase. Further, passage in the presence of NHC generates only low-level resistance, likely due to the accumulation of multiple potentially deleterious transition mutations. Together, these data support a mutagenic mechanism of inhibition by NHC and further support the development of NHC for treatment of CoV infections.

  • Vesicular Stomatitis Virus Transcription Is Inhibited by TRIM69 in the Interferon-Induced Antiviral State [Virus-Cell Interactions]

  • Interferons (IFNs) induce the expression of interferon-stimulated genes (ISGs), many of which are responsible for the cellular antiviral state in which the replication of numerous viruses is blocked. How the majority of individual ISGs inhibit the replication of particular viruses is unknown. We conducted a loss-of-function screen to identify genes required for the activity of alpha interferon (IFN-aalpha;) against vesicular stomatitis virus, Indiana serotype (VSVIND), a prototype negative-strand RNA virus. Our screen revealed that TRIM69, a member of the tripartite motif (TRIM) family of proteins, is a VSVIND inhibitor. TRIM69 potently inhibited VSVIND replication through a previously undescribed transcriptional inhibition mechanism. Specifically, TRIM69 physically associates with the VSVIND phosphoprotein (P), requiring a specific peptide target sequence encoded therein. P is a cofactor for the viral polymerase and is required for viral RNA synthesis, as well as the assembly of replication compartments. By targeting P, TRIM69 inhibits pioneer transcription of the incoming virion-associated minus-strand RNA, thereby preventing the synthesis of viral mRNAs, and consequently impedes all downstream events in the VSVIND replication cycle. Unlike some TRIM proteins, TRIM69 does not inhibit viral replication by inducing degradation of target viral proteins. Rather, higher-order TRIM69 multimerization is required for its antiviral activity, suggesting that TRIM69 functions by sequestration or anatomical disruption of the viral machinery required for VSVIND RNA synthesis.

    IMPORTANCE Interferons are important antiviral cytokines that work by inducing hundreds of host genes whose products inhibit the replication of many viruses. While the antiviral activity of interferon has long been known, the identities and mechanisms of action of most interferon-induced antiviral proteins remain to be discovered. We identified gene products that are important for the antiviral activity of interferon against vesicular stomatitis virus (VSV), a model virus that whose genome consists of a single RNA molecule with negative-sense polarity. We found that a particular antiviral protein, TRIM69, functions by a previously undescribed molecular mechanism. Specifically, TRIM69 interacts with and inhibits the function of a particular phosphoprotein (P) component of the viral transcription machinery, preventing the synthesis of viral messenger RNAs.

  • Authentication Analysis of MT-4 Cells Distributed by the National Institutes of Health AIDS Reagent Program [Commentary]

  • The MT-4 human T-cell line expresses HTLV-1 Tax and is permissive for replication of an HIV-1 gp41 mutant lacking the cytoplasmic tail. MT-4 cells (lot 150048), distributed by the NIH AIDS Reagent Program (NIH-ARP), were found to be Tax deficient and unable to host replication of the gp41-truncated HIV-1 mutant. These findings, together with short tandem repeat profiling, established that lot 150048 are not bona fide MT-4 cells.

  • Functional Identification and Characterization of the Nuclear Egress Complex of a Gammaherpesvirus [Structure and Assembly]

  • The herpesvirus nuclear egress complex (NEC) is composed of two viral proteins. They play key roles in mediating the translocation of capsids from the nucleus to the cytoplasm by facilitating the budding of capsids into the perinuclear space (PNS). The NEC of alphaherpesvirus can induce the formation of virion-like vesicles from the nuclear membrane in the absence of other viral proteins. However, whether the NEC of gammaherpesvirus harbors the ability to do so in mammalian cells remains to be determined. In this study, we first constructed open reading frame 67 (ORF67)-null and ORF69-null mutants of murine gammaherpesvirus 68 (MHV-68) and demonstrated that both ORF67 and ORF69 play critical roles in nuclear egress and hence viral lytic replication. Biochemical and bioimaging analyses showed that ORF67 and ORF69 interacted with each other and were sufficient to induce the formation of virion-like vesicles from the nuclear membrane in mammalian cells. Thus, we designated ORF67 and ORF69 components of MHV-68 NEC. Furthermore, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 through homology modeling and verified their function in nuclear egress, providing insights into the molecular basis of NEC formation in gammaherpesviruses.

    IMPORTANCE Increasing amounts of knowledge indicate that the nuclear egress complex (NEC) is critical for the nuclear egress of herpesvirus capsids, which can be viewed as a vesicle-mediated transport pathway through the nuclear membrane. In this study, we identified open reading frame 67 (ORF67) and ORF69 as components of the NEC in murine gammaherpesvirus 68 (MHV-68) and demonstrated that they efficiently induce virion-like vesicles from the nuclear membrane in mammalian cells. This is the first time that the NEC of a gammaherpesvirus has been found to demonstrate such an essential characteristic. In addition, we identified amino acids critical for mediating the interaction between ORF67 and ORF69 as well as nuclear egress. Notably, these amino acids are conserved in Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), providing a structural basis to design antigammaherpesvirus drugs.

  • The nsp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages [Virus-Cell Interactions]

  • Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2323nndash;521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism.

    IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism.

  • Broad Cross-Species Infection of Cultured Cells by Bat HKU2-Related Swine Acute Diarrhea Syndrome Coronavirus and Identification of Its Replication in Murine Dendritic Cells In Vivo Highlight Its Potential for Diverse Interspecies Transmission [Virus-Cell Interactions]

  • Outbreaks of severe diarrhea in neonatal piglets in Guangdong, China, in 2017 resulted in the isolation and discovery of a novel swine enteric alphacoronavirus (SeACoV) derived from the species Rhinolophus bat coronavirus HKU2 (Y. Pan, X. Tian, P. Qin, B. Wang, et al., Vet Microbiol 211:15nndash;21, 2017). SeACoV was later referred to as swine acute diarrhea syndrome CoV (SADS-CoV) by another group (P. Zhou, H. Fan, T. Lan, X.-L. Yang, et al., Nature 556:255nndash;258, 2018). The present study was set up to investigate the potential species barriers of SADS-CoV in vitro and in vivo. We first demonstrated that SADS-CoV possesses a broad species tropism and is able to infect cell lines from diverse species, including bats, mice, rats, gerbils, hamsters, pigs, chickens, nonhuman primates, and humans. Trypsin contributes to but is not essential for SADS-CoV propagation in vitro. Furthermore, C57BL/6J mice were inoculated with the virus via oral or intraperitoneal routes. Although the mice exhibited only subclinical infection, they supported viral replication and prolonged infection in the spleen. SADS-CoV nonstructural proteins and double-stranded RNA were detected in splenocytes of the marginal zone on the edge of lymphatic follicles, indicating active replication of SADS-CoV in the mouse model. We identified that splenic dendritic cells (DCs) are the major targets of virus infection by immunofluorescence and flow cytometry approaches. Finally, we demonstrated that SADS-CoV does not utilize known CoV receptors for cellular entry. The ability of SADS-CoV to replicate in various cells lines from a broad range of species and the unexpected tropism for murine DCs provide important insights into the biology of this bat-origin CoV, highlighting its possible ability to cross interspecies barriers.

    IMPORTANCE Infections with bat-origin coronaviruses (CoVs) (severe acute respiratory syndrome CoV [SARS-CoV] and Middle East respiratory syndrome CoV [MERS-CoV]) have caused severe illness in humans after "host jump" events. Recently, a novel bat-HKU2-like CoV named swine acute diarrhea syndrome CoV (SADS-CoV) has emerged in southern China, causing lethal diarrhea in newborn piglets. It is important to assess the species barriers of SADS-CoV infection since the animal hosts (other than pigs and bats) and zoonotic potential are still unknown. An in vitro susceptibility study revealed a broad species tropism of SADS-CoV, including various rodent and human cell lines. We established a mouse model of SADS-CoV infection, identifying its active replication in splenic dendritic cells, which suggests that SADS-CoV has the potential to infect rodents. These findings highlight the potential cross-species transmissibility of SADS-CoV, although further surveillance in other animal populations is needed to fully understand the ecology of this bat-HKU2-origin CoV.

  • Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons [Virus-Cell Interactions]

  • Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-B signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species.

    IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

  • Recognizing the Top 25 Peer Reviewers for the Journal of Virology [Editorial]

  • Acknowledgment of Ad Hoc Reviewers [Editorial]

  • Herpes Simplex Virus 1 Replication, Ocular Disease, and Reactivations from Latency Are Restricted Unilaterally after Inoculation of Virus into the Lip [Pathogenesis and Immunity]

  • Ocular herpes simplex keratitis (HSK) is a consequence of viral reactivations from trigeminal ganglia (TG) and occurs almost exclusively in the same eye in humans. In our murine oro-ocular (OO) model, herpes simplex virus 1 (HSV-1) inoculation in one side of the lip propagates virus to infect the ipsilateral TG. Replication here allows infection of the brainstem and infection of the contralateral TG. Interestingly, HSK was observed in our OO model only from the eye ipsilateral to the site of lip infection. Thus, unilateral restriction of HSV-1 may be due to differential kinetics of virus arrival in the ipsilateral versus contralateral TG. We inoculated mice with HSV-1 reporter viruses and then superinfected them to monitor changes in acute- and latent-phase gene expression in TG after superinfection compared to the control (single inoculation). Delaying superinfection by 4 days after initial right lip inoculation elicited failed superinfecting-virus gene expression and eliminated clinical signs of disease. Initial inoculation with thymidine kinase-deficient HSV-1 (TKdel) completely abolished reactivation of wild-type (WT) superinfecting virus from TG during the latent stage. In light of these seemingly failed infections, viral genome was detected in both TG. Our data demonstrate that inoculation of HSV-1 in the lip propagates virus to both TG, but with delay in reaching the TG contralateral to the side of lip infection. This delay is responsible for restricting viral replication to the ipsilateral TG, which abrogates ocular disease and viral reactivations from the contralateral side. These observations may help to understand why HSK is observed unilaterally in humans, and they provide insight into vaccine strategies to protect against HSK.

    IMPORTANCE Herpetic keratitis (HK) is the leading cause of blindness by an infectious agent in the developed world. This disease can occur after reactivation of herpes simplex virus 1 in the trigeminal ganglia, leading to dissemination of virus to, and infection of, the cornea. A clinical paradox is evidenced by the bilateral presence of latent viral genomes in both trigeminal ganglia, while for any given patient the disease is unilateral with recurrences in a single eye. Our study links the kinetics of early infection to unilateral disease phenomenon and demonstrates protection against viral reactivation when kinetics are exploited. Our results have direct implications in the understanding of human disease pathogenesis and immunotherapeutic strategies for the treatment of HK and viral reactivations.

  • Generation of Recombinant Rotavirus Expressing NSP3-UnaG Fusion Protein by a Simplified Reverse Genetics System [Genome Replication and Regulation of Viral Gene Expression]

  • Rotavirus is a segmented double-stranded RNA (dsRNA) virus that causes severe gastroenteritis in young children. We have established an efficient simplified rotavirus reverse genetics (RG) system that uses 11 T7 plasmids, each expressing a unique simian SA11 (+)RNA, and a cytomegalovirus support plasmid for the African swine fever virus NP868R capping enzyme. With the NP868R-based system, we generated recombinant rotavirus (rSA11/NSP3-FL-UnaG) with a genetically modified 1.5-kb segment 7 dsRNA encoding full-length nonstructural protein 3 (NSP3) fused to UnaG, a 139-amino-acid green fluorescent protein (FP). Analysis of rSA11/NSP3-FL-UnaG showed that the virus replicated efficiently and was genetically stable over 10 rounds of serial passaging. The NSP3-UnaG fusion product was well expressed in rSA11/NSP3-FL-UnaG-infected cells, reaching levels similar to NSP3 levels in wild-type recombinant SA11-infected cells. Moreover, the NSP3-UnaG protein, like functional wild-type NSP3, formed dimers in vivo. Notably, the NSP3-UnaG protein was readily detected in infected cells via live-cell imaging, with intensity levels ~3-fold greater than those of the NSP1-UnaG fusion product of rSA11/NSP1-FL-UnaG. Our results indicate that FP-expressing recombinant rotaviruses can be made through manipulation of the segment 7 dsRNA without deletion or interruption of any of the 12 open reading frames (ORFs) of the virus. Because NSP3 is expressed at higher levels than NSP1 in infected cells, rotaviruses expressing NSP3-based FPs may be more sensitive tools for studying rotavirus biology than rotaviruses expressing NSP1-based FPs. This is the first report of a recombinant rotavirus containing a genetically engineered segment 7 dsRNA.

    IMPORTANCE Previous studies generated recombinant rotaviruses that express FPs by inserting reporter genes into the NSP1 ORF of genome segment 5. Unfortunately, NSP1 is expressed at low levels in infected cells, making viruses expressing FP-fused NSP1 less than ideal probes of rotavirus biology. Moreover, FPs were inserted into segment 5 in such a way as to compromise NSP1, an interferon antagonist affecting viral growth and pathogenesis. We have identified an alternative approach for generating rotaviruses expressing FPs, one relying on fusing the reporter gene to the NSP3 ORF of genome segment 7. This was accomplished without interrupting any of the viral ORFs, yielding recombinant viruses that likely express the complete set of functional viral proteins. Given that NSP3 is made at moderate levels in infected cells, rotaviruses encoding NSP3-based FPs should be more sensitive probes of viral infection than rotaviruses encoding NSP1-based FPs.

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    JVI Accepts: Articles Published Ahead of Print

  • Long-term protection of rhesus macaques from Zika virus reinfection [Pathogenesis and Immunity]

  • By the end of the 2016 Zika virus (ZIKV) outbreak, it is estimated that there were up to 100 million infections in the Americas. In approximately one in seven infants born to mothers infected during pregnancy, ZIKV has been linked to microcephaly, developmental delays, or other congenital disorders collectively known as congenital Zika syndrome (CZS) as well as Guillain-Barreeacute; syndrome (GBS) in ZIKV infected adults. It is a global health priority to develop a vaccine against ZIKV that elicits long-lasting immunity; however, the durability of immunity to ZIKV is unknown. Previous studies in mice and nonhuman primates have been crucial in vaccine development but have not defined the duration of immunity generated by ZIKV infection. In this study, we rechallenged five rhesus macaques with ZIKV 22 to 28 months after a primary ZIKV infection. We show that primary ZIKV infection generates high titers of neutralizing antibodies (nAbs) that protect from detectable plasma viremia following rechallenge and persist for at least 22 to 28 months. While additional longitudinal studies are necessary with longer time frames, this study establishes a new experimentally defined minimal length of protective ZIKV immunity.

    Importance ZIKV emerged as a vector-borne pathogen capable of causing illness in infected adults and congenital birth defects in infants born to mothers infected during pregnancy. Despite the drop in ZIKV cases since the 2015-16 epidemic, questions concerning the prevalence and longevity of protective immunity have left vulnerable communities fearful that they may become the center of next ZIKV outbreak. While pre-existing herd immunity in regions of past outbreaks may dampen the potential for future outbreaks to occur, we currently do not know the longevity of protective immunity to ZIKV after a person becomes infected. Here, we establish a new experimentally defined minimal length of protective ZIKV immunity. We show that five rhesus macaques initially infected with ZIKV 22 to 28 months prior to rechallenge elicit a durable immune response that protected from detectable plasma viremia. This work establishes a new minimal length of protective immunity.

  • {beta}-catenin is Required for cGAS/STING Signaling Pathway but Antagonized by HSV-1 US3 Protein [Virus-Cell Interactions]

  • The cGAS/STING-mediated DNA-sensing signal pathway is crucial for interferon (IFN) production and host antiviral responses. Herpes simplex virus type I (HSV-1) is a DNA virus that has evolved multiple strategies to evade host immune responses. Here we demonstrated that the highly conserved beta-catenin (bbeta;-catenin) protein in the Wnt signaling pathway is an important factor to enhance the transcription of type I interferon (IFN-I) in cGAS/STING signaling pathway, and the production of IFN-I mediated by bbeta;-catenin was antagonized by HSV-1 US3 protein via its kinase activity. Infection of US3 deficiency HSV-1 and its kinase dead viruses failed to downregulate IFN-I and IFN-stimulated genes (ISG) production induced by bbeta;-catenin. Consistent with this, absence of bbeta;-catenin enhanced the replication of US3 deficiency HSV-1, but not wide-type HSV-1. The underlying mechanism is that US3 interacted with bbeta;-catenin and hyperphosphorylated it at Thr556 to block the nuclear translocation of bbeta;-catenin. For the first time, HSV-1 US3 was shown to inhibit IFN-I production through hyperphosphorylation of bbeta;-catenin and subvert host antiviral innate immunity.

    IMPORTANCE Although increasing evidences have demonstrated that HSV-1 subverts host immune responses and establishes a lifelong latent infection, the molecular mechanisms by which HSV-1 interrupts antiviral innate immunity, especially cGAS/STING-mediated cellular DNA-sensing signal pathway have not been fully explored. Here we show that bbeta;-catenin promotes cGAS/STING-mediated activation of the IFN pathway which is important for cellular innate immune responses and intrinsic resistance to DNA virus infection. The protein kinase US3 antagonizes the production of IFN by targeting bbeta;-catenin via its kinase activity. Findings in this study reveal a novel mechanism for HSV-1 to evade host antiviral immunity and add new knowledge to understand the interaction between the host and HSV-1 infection.

  • Biochemical reconstitution of HIV-1 assembly and maturation [Structure and Assembly]

  • Assembly of an orthoretrovirus such as HIV-1 requires the coordinated functioning of multiple biochemical activities of the viral Gag protein. These activities include membrane targeting, lattice formation, packaging of the RNA genome, and recruitment of cellular co-factors that modulate assembly. In most previous studies, these Gag activities have been investigated individually, which have provided somewhat limited insight on how they functionally integrate during the assembly process. Here, we report the development of a biochemical reconstitution system that allowed us to investigate how Gag lattice formation, RNA binding, and the assembly factor inositol hexakisphosphate (IP6) synergize to generate immature virus particles in vitro. Results identify an important rate limiting step in assembly and reveal new insights on how RNA and IP6 promote immature Gag lattice formation. The immature virus-like particles can be converted into mature capsid-like particles by simple addition of viral protease, suggesting that it is possible in principle to fully biochemically reconstitute the sequential processes of HIV-1 assembly and maturation from purified components.

    Importance Assembly and maturation are essential steps in the replication of orthoretroviruses such as HIV-1, and are proven therapeutic targets. These processes require the coordinated functioning of the viral Gag protein's multiple biochemical activities. We describe here the development of an experimental system that allows integrative analysis of how Gag's multiple functionalities cooperate to generate a retrovirus particle. Our current studies help to illuminate how Gag synergizes formation of the virus compartment with RNA binding, and how these activities are modulated by the small molecule, IP6. Further development and use of this system should lead to a more comprehensive understanding of the molecular mechanisms of HIV-1 assembly and maturation, and may provide new insights for the development of anti-retroviral drugs.

  • Trypsin treatment unlocks barrier for zoonotic bat coronaviruses infection. [Virus-Cell Interactions]

  • Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with SARS-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate infection of human gut cells, but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that addition of exogenous trypsin also rescues HKU5-CoV, a second bat group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate emergence potential of bat CoVs and offer a means to recover previously unrecoverable zoonotic CoV strains.

    Importance Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

  • Monoclonal antibodies with neutralizing activity and Fc-effector functions against the Machupo virus glycoprotein [Vaccines and Antiviral Agents]

  • Machupo virus (MACV), the causative agent of Bolivian hemorrhagic fever (BHF), is a New World arenavirus that was first isolated in Bolivia from a human spleen in 1963. Due to the lack of a specific vaccine or therapy, this virus is considered a major risk to public health and is classified as a Category A Priority Pathogen by the US National Institutes of Health. In this study, we used DNA vaccination against the MACV glycoprotein precursor complex (GPC) and murine hybridoma technology to generate 25 mouse monoclonal antibodies (mAbs) against the GPC of MACV. Out of 25, five mAbs were found to have potent neutralization activity in vitro against a recombinant vesicular stomatitis virus expressing MACV GPC (VSV-MACV) as well as authentic MACV. Furthermore, the five neutralizing mAbs exhibited strong antibody-dependent cellular cytotoxicity (ADCC) activity in a reporter assay. When tested in vivo using VSV-MACV in a Stat2-/- mouse model, three mAbs significantly lowered viral loads in the spleen. Our work provides valuable insights into epitopes targeted by neutralizing antibodies that could be potent targets for vaccines and therapeutics, and shed light towards the importance of effector functions in immunity against MACV.

    Significance MACV infections are a significant public health concern and lead to high case fatality rates. No specific treatment or vaccine for MACV infections exist. However, cases of Junin virus infection, a related virus, can be treated with convalescent serum. This indicates, that a mAb-based therapy for MACV could be effective. Here, we describe several mAbs that neutralize MACV and could be used for this purpose.

  • Influenza B viruses exhibit lower within-host diversity than influenza A viruses in human hosts [Genetic Diversity and Evolution]

  • Influenza B virus undergoes seasonal antigenic drift more slowly than influenza A, but the reasons for this difference are unclear. While the evolutionary dynamics of influenza viruses play out globally, they are fundamentally driven by mutation, reassortment, drift, and selection at the level of individual hosts. These processes have recently been described for influenza A virus, but little is known about the evolutionary dynamics of influenza B virus (IBV) during individual infections and transmission events. Here we define the within-host evolutionary dynamics of influenza B virus by sequencing virus populations from naturally-infected individuals enrolled in a prospective, community-based cohort over 8176 person-seasons of observation. Through analysis of high depth-of-coverage sequencing data from samples from 91 individuals with influenza B, we find that influenza B virus accumulates lower genetic diversity than previously observed for influenza A virus during acute infections. Consistent with studies of influenza A viruses, the within-host evolution of influenza B viruses is characterized by purifying selection and the general absence of widespread positive selection of within-host variants. Analysis of shared genetic diversity across 15 sequence-validated transmission pairs suggests that IBV experiences a tight transmission bottleneck similar to that of influenza A virus. These patterns of local-scale evolution are consistent with influenza B virus' slower global evolutionary rate.

    Importance The evolution of influenza virus is a significant public health problem and necessitates the annual evaluation of influenza vaccine formulation to keep pace with viral escape from herd immunity. Influenza B virus is a serious health concern for children, in particular, yet remains understudied compared to influenza A virus. Influenza B virus evolves more slowly than influenza A, but the factors underlying this are not completely understood. We studied how the within-host diversity of influenza B virus relates to its global evolution by sequencing viruses from a community-based cohort. We found that influenza B virus populations have lower within-host genetic diversity than influenza A virus and experience a tight genetic bottleneck during transmission. Our work provides insights into the varying dynamics of influenza viruses in human infection.

  • Inhibition of metalloprotease TRABD2A facilitates the study of HIV-1 replication in resting CD4+ T cells [Letters to the Editor]

  • Although the HIV-1 particle can enter resting CD4+ T cells efficiently in vitro, the replication cycle is suppressed in resting CD4+ T cells unlike in stimulated CD4+ T cells (1-3)....

  • Functional perturbation of mucosal group 3 innate lymphoid and natural killer cells in SHIV/SIV-infected infant rhesus macaques [Pathogenesis and Immunity]

  • Mother-to-child transmission of HIV-1 via breastfeeding is responsible for nearly half of children newly infected with HIV. Although innate lymphoid cells (ILC) and natural killer (NK) cells are found throughout the oral mucosae, the effects of HIV/SHIV in these tissues are largely unknown. To better understand the mechanics of postnatal transmission we performed a comprehensive study of SIV/SHIV-infected infant rhesus macaques (RM) and tracked changes in frequency, trafficking, and function of ILC3 and NK cells using polychromatic flow cytometry and cell stimulation assays in colon, tonsil and oral lymph node samples. Infection led to a 3-fold depletion of ILC3 in the colon and an increase in NK cells in tonsils and oral lymph nodes. ILC3 and NK cells saw alterations in their trafficking repertoires as a result of infection, with increased expression of CD103 in colon NK cells and curtailment of CXCR3, and a significant decrease in aalpha;4bbeta;7 expression in colon ILC3. SPICE analyses revealed that ILC3 and NK cells displayed distinct functional profiles by tissue in naïve samples. Infection perturbed these profiles, with a near total loss of IL-22 production in the tonsil and colon and increase in CD107a, IFN- and TNF-aalpha; from ILC3, and increase in CD107a, MIP-1bbeta; and TNF-aalpha; from NK cells. Collectively, these data reveal that lentiviurus infection alters the frequency, receptor repertoires, and functions of innate cells in the oral and gut mucosa of infants. Further study will be required to delineate the full extent that these changes have on oral and gut homeostasis, SHIV/SIV pathogenesis and oral opportunistic disease.

    Importance Vertical transmission of HIV from mother-to-child accounts for many of the new cases seen worldwide. To date, there is no vaccine to mitigate this transmission and limited research on the effects that lentiviral infection has on the innate immune system in oral tissues of infected children. To fill this knowledge gap, our lab studied infant rhesus macaques to evaluate how acute SIV/SHIV infections impacted ILC3 and NK cells; immune cells critical for mucosal homeostasis and antimicrobial defense. Our data revealed that SIV/SHIV infection led to a depletion of ILC3 and increase of NK cells, and a functional shift from homoestatic to multifunctional proinflammatory. Taken together, we describe how lentiviral infection perturbs the oral and gastrointestinal mucosae of infant macaques through alterations of resident innate immune cells giving rise to chronic inflammation and potentially exacerbating morbidity and mortality in children living with HIV.

  • The Ubiquitin specific Protease Usp7, a novel Merkel cell polyomavirus Large T-Antigen interaction partner, modulates viral DNA replication [Genome Replication and Regulation of Viral Gene Expression]

  • Merkel Cell Polyomavirus (MCPyV) is the major cause for Merkel Cell Carcinoma (MCC), a rare but highly aggressive skin cancer predominantly found in elderly and immunosuppressed patients. The early viral gene products large T-Antigen (LT) and small T-Antigen (sT) are important for efficient viral DNA replication and both contribute to transformation processes. Mainly, these functions are executed through interactions with host factors. We here identify the cellular Ubiquitin-specific-processing protease 7 (Usp7) as a new interaction partner of the MCPyV LT.

    Using GST pulldown experiments, we show that MCPyV LT directly binds to Usp7, and that N- as well as the C-terminal regions of LT bind to the TRAF (tumor necrosis factor receptor-associated) domain of Usp7. We demonstrate that endogenous Usp7 co-precipitates with MCPyV T-antigens and re-localizes to viral DNA replication centers in cells actively replicating MCPyV genomes. We show that Usp7 does not alter ubiquitination levels of the T-antigens; however, Usp7 binding increases the binding affinity of LT to the origin of replication thereby negatively regulating viral DNA replication. Together, these data identify Usp7 as a restriction factor of MCPyV replication. In contrast to other DNA viruses, Usp7 does not affect MCPyV gene expression via its ubiquitination activity, but solely influences MCPyV DNA replication via novel mechanism that modulates binding of LT to viral DNA.

    Importance MCPyV is the only human polyomavirus that is associated with cancer; the majority of Merkel cell cancers have a viral etiology. While much emphasis was investigated to understand the transformation process by MCPyV oncoproteins and cellular factors, we have only limited knowledge on cellular factors participating in the MCPyV life cycle. We here describe Usp7, a cellular deubiquitination enzyme, as a new factor involved in MCPyV replication. Usp7 is known in the context of large DNA tumor viruses, EBV and KSHV, to restrict viral replication. Similar to EBV, where Usp7 binding to EBNA1 increases EBNA1 binding affinity to viral DNA, we find MCPyV LT binding to the origin of replication to be increased in the presence of Usp7 resulting in restriction of viral DNA replication. However, Usp7 induced restriction of MCPyV replication is independent of its enzymatic activity thereby constituting a novel mechanism of Usp7 induced restriction of viral replication.

  • Kaposi's Sarcoma Associated Herpesvirus LANA Modulates the Stability of the E3 Ubiquitin Ligase RLIM [Virus-Cell Interactions]

  • The Kaposi's sarcoma associated herpesvirus (KSHV) encoded LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. In a previous study, we have identified LANA interacting proteins using a protein array screen. Here, we explore the effect of LANA on the stability and activity of RLIM (RING-finger LIM-domain-interacting protein, encoded by the RNF12 gene) a novel LANA interacting protein identified in that protein screen. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1 and the telomeric protein TRF1. Expression of LANA leads to down-regulation of RLIM protein levels. This LANA-mediated RLIM degradation is blocked in the presence of the proteasome inhibitor, MG132. Therefore, the interaction between LANA and RLIM could be detected in co-immunoprecipitation assay only in the presence of MG132 to prevent RLIM degradation. A RING finger mutant RLIM (HH 590, 593 EE) is resistant to LANA mediated degradation, suggesting that LANA promotes RLIM auto-ubiquitination. Interestingly, we found that LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM mediated degradation of others such as LHX3 and TRF1. We also show that transcription regulation by RLIM substrates is modulated by LANA. RLIM substrates are assembled into multi-protein transcription regulator complexes that regulate the expression of many cellular genes. Therefore, our study identified another way KSHV can modulate cellular gene expression.

    IMPORTANCE E3 ubiquitin ligases mark their substrates for degradation and therefore control the cellular abundance of their substrates. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1 and the telomeric protein TRF1. Here we show that the Kaposi's sarcoma associated herpesvirus (KSHV) encoded LANA protein enhances the ubiquitin ligase activity of RLIM leading to enhanced RLIM auto-ubiquitination and degradation. Interestingly, LANA enhanced the degradation of some RLIM substrates, such as LDB1 and LMO2, and prevented RLIM mediated degradation of others such as LHX3 and TRF1. In agreement with protein stability of RLIM substrates, we found that LANA modulates transcription by LHX3-LDB1 complex and suggests additional way LANA can modulate cellular gene expression. Our study adds another way a viral protein can regulate cellular protein stability, by enhancing the auto-ubiquitination and degradation of an E3 ubiquitin ligase.

  • Induction of Kaposi Sarcoma Associated Herpesvirus-encoded Thymidine Kinase (ORF21) by X-Box Binding Protein-1 [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein-1 (XBP-1s). XBP-1s is also present in high amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and thus contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV thymidine kinase (TK), the ORF21 gene product, can enhance production of thymidine triphosphate and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced up-regulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominanty found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and thus contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and ganciclovir in this disease.

    Importance: Spliced X-box binding protein-1 (XBP-1s), part of the unfloded protein response and expressed in developing germinal center B cells, can induce Kaposi sarcoma associated herpesvirus (KSHV) lytic repliction and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of thymidine triphosphate for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused in part by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD .

  • Impact of suboptimal APOBEC3G neutralization on the emergence of HIV drug resistance in humanized mice [Genetic Diversity and Evolution]

  • HIV diversification facilitates immune escape and complicates antiretroviral therapy. In this study, we take advantage of a humanized mouse model to probe the contribution of APOBEC3 mutagenesis to viral evolution. Humanized mice were infected with isogenic HIV molecular clones (HIV-WT, HIV-45G, HIV-SLQ) that differ in their ability to counteract APOBEC3G (A3G). Infected mice remained naïve or were treated with the RT inhibitor lamivudine (3TC). Viremia, emergence of drug resistant variants and quasispecies diversification in the plasma compartment were determined throughout infection. While both HIV-WT and HIV-45G achieved robust infection, over time HIV-45G replication was significantly reduced compared to HIV-WT in the absence of 3TC treatment. In contrast, treatment response differed significantly between HIV-45G and HIV-WT infected mice. Antiretroviral treatment failed in 91% of HIV-45G infected mice while only 36% of HIV-WT infected mice displayed a similar negative outcome. Emergence of 3TC resistant variants and nucleotide diversity were determined by analyzing 155,462 single HIV reverse transcriptase (RT) and 6,985 vif sequences from 33 mice. Prior to treatment, variants with genotypic 3TC resistance (RT-M184I/V) were detected at low levels in over a third of all animals. Upon treatment, the composition of the plasma quasispecies rapidly changed leading to a majority of circulating viral variants encoding RT-184I. Interestingly, increased viral diversity prior to treatment initiation correlated with higher plasma viremia in HIV-45G but not in HIV-WT infected animals. Taken together, HIV variants with suboptimal anti-A3G activity were attenuated in the absence of selection but display a fitness advantage in the presence of antiretroviral treatment.

    IMPORTANCE Both viral (e.g., reverse transcriptase, RT) and host factors (e.g., APOBEC3G (A3G)) can contribute to HIV sequence diversity. This study shows that suboptimal anti-A3G activity shapes viral fitness and drives viral evolution in the plasma compartment of humanized mice.

  • PACSIN2 Interacts with Nonstructural 5A Protein and Regulates Hepatitis C Virus Assembly [Virus-Cell Interactions]

  • Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. By protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family that is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and F-BAR region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCaalpha;)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCaalpha; interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the siRNA-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core interaction. We further showed that inhibition of PKCaalpha; increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating ERK1/2 phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly.

    IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCaalpha; from binding to PACSIN2. Therefore, phosphorylation level of PACSIN2 is decreased in HCV infected cells. We showed that HCV NS5A interrupted PKCaalpha;-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.

  • Expression of the conserved herpesvirus protein kinase (CHPK) of Marek's disease alphaherpesvirus in the skin reveals a mechanistic importance for CHPK during interindividual spread in chickens [Pathogenesis and Immunity]

  • The Herpesviridae encode many conserved genes including the conserved herpesvirus protein kinase (CHPK) that has multifunctional properties. In most cases, herpesviruses lacking CHPK can propagate in cell culture at varying degrees, depending on the virus and cell culture system. However, in the natural animal model system of Marek's disease alphaherpesvirus (MDV) in chickens, CHPK is absolutely required for interindividual spread from chicken-to-chicken. The lack of biological reagents for chicken and MDV has limited our understanding of this important gene during interindividual spread. Here, we engineered epitope-tagged proteins in the context of virus infection in order to detect CHPK in the host. Using immunofluorescence assays and western blotting during infection in cell culture and in chickens, we determined that the invariant lysine 170 (K170) of MDV CHPK is required for interindividual spread, auto phosphorylation of CHPK, and mutation to methionine (M170) results in instability of the CHPK protein. Using these newly generated viruses allowed us to examine the expression of CHPK in infected chickens and these results showed that mutant CHPK localization and late viral protein expression were severely affected in feather follicles wherein MDV is shed, providing important information on the requirement of CHPK for interindividual spread.

    IMPORTANCE Marek's disease in chickens is caused by Gallid alphaherpesvirus 2, better known as Marek's disease alphaherpesvirus (MDV). Current vaccines only reduce tumor formation, but do not block interindividual spread from chicken-to-chicken. Understanding MDV interindividual spread provides important information for the development of potential therapies to protect against Marek's disease, while also providing a reliable natural host in order to study herpesvirus replication and pathogenesis in animals. Here, we studied the conserved Herpesviridae protein kinase (CHPK) in cell culture and during infection in chickens. We determined MDV CHPK is not required for cell-to-cell spread; for disease induction, and for oncogenicity. However, it is required for interindividual spread and mutation of the invariant lysine (K170) results in stability issues and aberrant expression in chickens. This study is important because it addresses the critical role CHPK orthologs play in the natural host.

  • ER reorganization and intracellular retention of CD58 are functionally independent properties of the human cytomegalovirus ER resident glycoprotein UL148 [Virus-Cell Interactions]

  • The human cytomegalovirus (HCMV) endoplasmic reticulum (ER)-resident glycoprotein UL148 is posited to play roles in immune evasion and regulation of viral cell tropism. UL148 prevents cell surface presentation of the immune cell costimulatory ligand CD58 while promoting maturation and virion incorporation of glycoprotein O, a receptor binding subunit for an envelope glycoprotein complex involved in entry. Meanwhile, UL148 activates the unfolded protein response (UPR) and causes large-scale reorganization of the ER. In order to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated six charged-cluster-to-alanine (CCTA) mutants within the UL148 ectodomain, and compared them against wildtype UL148, both in the context of infection studies using recombinant viruses and in ectopic expression experiments, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79-83 (CC3) and 133-136 (CC4), respectively, retained the potential to impede CD58 surface presentation. Of the six mutants, only CC3 retained the capacity to reorganize the ER, but showed a partial phenotype. Wildtype UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which may imply a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses surface presentation of CD58 but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate varying degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that the capacity to cause ER reorganization is unique to HCMV UL148.

    IMPORTANCE In myriad examples, viral gene products cause striking effects on cells, such as activation of stress responses. It can be challenging to decipher how such effects contribute to the biological roles of the proteins. The HCMV glycoprotein UL148 retains CD58 within the ER, thereby preventing it from reaching the cell surface where it functions to stimulate cell-mediated antiviral responses. Intriguingly, UL148 also triggers the formation of large, ER-derived membranous structures, and activates the UPR, a set of signaling pathways involved in adaptation to ER stress. We demonstrate that the potential of UL148 to reorganize the ER and to retain CD58 are separable by mutagenesis and possibly, by evolution, since chimpanzee cytomegalovirus UL148 retains CD58 but does not remodel the ER. Our findings imply that ER reorganization contributes to other roles of UL148, such as modulation of alternative viral glycoprotein complexes that govern the virus' ability to infect different cell types.

  • Mutations in the NA-like protein of bat influenza H18N11 virus enhance virus replication in mammalian cells, mice, and ferrets [Pathogenesis and Immunity]

  • To characterize bat influenza H18N11 virus, we propagated a reverse genetics-generated H18N11 virus in MDCK II cells and detected two adapting mutations in the neuraminidase (NA)-like protein (NA-F144C and NA-T342A, N2 numbering) that increased virus titers in three mammalian cell lines (i.e., Madin-Darby canine kidney, Madin-Darby canine kidney II, and human lung adenocarcinoma Calu-3 cells). In mice, wild-type H18N11 virus replicated only in the lungs of the infected animals, whereas the NA-T342A and NA-F144C/T342A mutant viruses were detected in the nasal turbinates in addition to the lungs. Bat influenza viruses have not been tested for their virulence and organ tropism in ferrets. We detected wild-type and single mutant viruses each possessing NA-F144C or NA-T342A in the nasal turbinates of one or several infected ferret(s), respectively. A mutant virus possessing both NA-F144C and T342A was isolated from both the lung and trachea, suggesting broader organ tropism compared with wild-type virus. However, none of the H18N11 viruses caused symptoms in mice or ferrets. The NA-F144C/T342A double mutation did not substantially affect virion morphology or the release of virions from cells. Collectively, our data demonstrate that propagation of bat influenza H18N11 virus in mammalian cells can result in mammalian-adapting mutations that could increase virus replicative ability and/or organ tropism; overall, however, these viruses did not replicate to high titers throughout the respiratory tract of mice and ferrets.

    IMPORTANCE Bats are reservoirs for several severe zoonotic pathogens. The genomes of influenza A viruses of the H17N10 and H18N11 subtypes were identified in bats, but no live virus has been isolated. The characterization of artificially generated bat influenza H18N11 virus in mammalian cell lines and animal models revealed that this virus can acquire mammalian-adapting mutations that could increase its zoonotic potential; however, the wild-type and mutant viruses did not replicate in the lungs of all infected animals.

  • Kaposi's Sarcoma-associated Herpesvirus Viral Interleukin 6 Signaling Upregulates Integrin Beta 3 Levels and is Dependent on STAT3 [Virus-Cell Interactions]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of two B-cell lymphoproliferative diseases and Kaposi's sarcoma, an endothelial-cell driven cancer. KSHV viral interleukin-6 (vIL-6) is a viral homolog of human IL-6 that is expressed in KSHV-associated malignancies. Previous studies have shown that the expression of the integrin bbeta;3 (ITGB3) subunit is induced upon KSHV infection. Here, we report that KSHV vIL-6 is able to induce the expression of ITGB3 and increase surface expression of the aalpha;Vbbeta;3 integrin heterodimer. We demonstrate using siRNA depletion and inhibitor studies that KSHV vIL-6 can increase ITGB3 by inducing STAT3 signaling. Furthermore, we found that secreted vIL-6, is capable of inducing ITGB3 in endothelial cells in a paracrine manner. Importantly, the ability to induce ITGB3 in endothelial cells seems to be specific to vIL-6 as over-expression of hIL-6 alone did not affect levels of this integrin. Our lab and others have previously shown that vIL-6 can induce angiogenesis, and we investigated whether ITGB3 was involved in this process. We found that siRNA depletion of ITGB3 in vIL-6-expressing endothelial cells resulted in a decrease in adhesion to extracellular matrix proteins. Moreover, depletion of ITGB3 hindered the ability of vIL-6 to promote angiogenesis. In conclusion, we found that vIL-6 can singularly induce ITGB3 and that this induction is dependent on vIL-6 activation of the STAT3 signaling pathway.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of three human malignancies: multicentric Castleman's disease, primary effusion lymphoma, and Kaposi's sarcoma. Kaposi's sarcoma is a highly angiogenic tumor that arises from endothelial cells. It has been previously reported that KSHV infection of endothelial cells leads to an increase of integrin aalpha;Vbbeta;3, a molecule observed to be involved in the angiogenic process of several malignancies. Our data demonstrate that the KSHV protein, viral interleukin-6 (vIL-6) can induce integrin bbeta;3 in an intracellular and paracrine manner. Furthermore, we showed that this induction is necessary for vIL-6-mediated cell adhesion and angiogenesis, suggesting a potential role of integrin bbeta;3 in KSHV pathogenesis and development of Kaposi's sarcoma.

  • The evolutionary dynamics of Oropouche Virus in South America [Genetic Diversity and Evolution]

  • The Amazon basin is home to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the genus Orthobunyavirus, family Peribunyaviridae, that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the OROV genome remain poorly understood. Here we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genomes sequences obtained from patients in Ecuador, representing the first set of genomes from this country. Our results show differing evolutionary processes on the three segments that comprise the viral genome. We infer differing times of the most recent common ancestors (TMRCAs) of the genome segments and propose this can be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, and codons that evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of OROV through a combined phylogenetic and structural approach.

    Importance The emergence and re-emergence of pathogens such as Zika virus (ZIKV), Chikungunya virus (CHIKV) and yellow fever virus (YFV) have drawn attention towards other co-circulating arboviruses in South America. Oropouche virus (OROV) is a poorly-studied pathogen responsible for over a dozen outbreaks since the early 1960s, and represents a public health burden to countries such as Brazil, Panama and Peru. OROV is likely underreported as its symptomatology can be easily confounded with other febrile illnesses (e.g. dengue fever and leptospirosis), and point-of-care testing for the virus is still uncommon. With limited data, there is a need to optimise the information currently available. Analysis of OROV genomes can help us understand how the virus circulates in nature and can reveal the evolutionary forces that shape the genetic diversity of the virus, which has implications for molecular diagnostics and the design of potential vaccines.

  • Polymorphisms in the most oncolytic reovirus strain confer enhanced cell attachment, transcription and single-step replication kinetics. [Genetic Diversity and Evolution]

  • Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strain of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo. This study aimed to determine why the most-oncolytic T3DPL lab strain of reovirus replicates more efficiently in cancer cells than other commonly used laboratory strains, T3DKC and T3DTD. In single step growth curves, T3DPL titers increased at faster rates and produced ~9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3DPL than T3DTD. In conclusion, the most-oncolytic T3DPL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3DPL were identified. First, T3DPL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (lle;3) 1 cell attachment proteins. Second, T3DPL transcribed RNA at superior rates to the less-oncolytic T3D strains, attributed to polymorphisms in M1-encoding mmu;2 protein, as confirmed in an in vitro transcription assay, demonstrating that T3DPL has an inherent transcription advantage that is cell-independent. Accordingly, T3DPL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains, and mechanistically, reveal the importance of rapid onset of infection for reovirus oncolysis.

    Importance: Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently it was discovered that highly related laboratory strains of T3D exhibit large differences in their ability to replicate in cancer cells in vitro, which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3DPL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3DPL binds to cells more efficiency and more-rapidly produces viral RNAs; this increased rate of infection relative to the less-oncolytic strains gives T3DPL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread.

  • Nucleocapsid protein recruitment to replication-transcription complexes plays a crucial role in coronaviral life cycle [Genome Replication and Regulation of Viral Gene Expression]

  • Coronaviruses (CoV) nucleocapsid N proteins are key in incorporating the genomic RNA into the progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used CoV model virus, interacts with non-structural protein 3 (nsp3), a component of the RTCs. These two aspects of CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 but not with other RTCs components using a systematic yeast two-hybrid approach, and identified two distinct regions in the N protein that redundantly mediates this interaction. A selective N protein variant carrying point mutations in these two regions fail to bind nsp3 in vitro, resulting in an inhibition of its recruitment to the RTCs in vivo. Furthermore, opposite to the wild type N protein, this N protein variant impairs the stimulation of gRNA and viral mRNA transcription in vivo and in vitro, which in turn leads to an impairment in MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in CoV life cycle as it is critical for optimal viral RNA synthesis.

    IMPORTANCE Coronaviruses (CoV) have been regarded for a long time as a relatively harmless pathogen for humans. Two severe respiratory tract infection outbreaks caused by SARS-CoV and MERS-CoV, however, have caused high pathogenicity and mortality rates in humans. This highlighted the relevance of being able to control CoV infections. We used a CoV model virus, mouse hepatitis virus (MHV), to investigate the importance of the recruitment of nucleocapsid (N) protein, a central component of CoV virions, to intracellular platforms where CoV replicate, transcribe and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial in promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain both an important aspect of CoV life cycle and reveal an interaction between viral proteins that could be targeted in antiviral therapies.

  • Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5 [Virus-Cell Interactions]

  • Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL) from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleolus of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleolus of transfected cells. Multiple point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed, several NoLS-mutated LEF5s were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production and occlusion body production.

    IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleolus of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

  • Dysregulation of Dual-Specificity Phosphatases by Epstein-Barr Virus LMP1 and Its Impact on Lymphoblastoid Cell Line Survival [Transformation and Oncogenesis]

  • The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet, the underlying mechanisms of how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of mitogen-activated protein kinases (MAPKs), during EBV infection and immortalization. Thirteen DUSPs, including ten typical and three atypical types of DUSPs, were tested. Most of them were down-regulated post EBV infection. Herein, a role of the viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates ERK or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this study provides the first description of the interplay between EB viral genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.

    Importance The infection of ubiquitous Epstein-Barr virus (EBV) is associated with a wide spectrum of lymphomas and carcinomas. It has been well-documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual specificity phosphates (DUSPs) exert great ability in regulating MAPK activities for their abilities to dephosphorylate MAPKs. In this study, we found that DUSPs generally are down-regulated post EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppresses DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation could be continuously activated. Furthermore, DUSP down-regulation contributes greatly to prevent apoptosis of EBV infected cells. To sum up, this study sheds light on a novel molecular mechanism on how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.

  • Chronic Lymphocytic Choriomeningitis infection causes susceptibility to mousepox and impairs Natural Killer cell maturation and function [Pathogenesis and Immunity]

  • Chronic viral infections like those of humans with cytomegalovirus, human immunodeficiency virus (even when under antiretroviral therapy), and hepatitis C virus; or of mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) result in immune dysfunction that predispose to severe infections with unrelated pathogens. It is known that C57BL/6 (B6) mice are resistant to mousepox, a lethal disease caused by the orthopoxvirus ectromelia virus (ECTV) and that this resistance requires Natural Killer (NK) cells and other immune cells. We show that most B6 mice chronically infected with CL13 succumb to mousepox while most of those that recovered from acute infection with the LCMV Armstrong strain (Arm) survive. We also show that B6 mice chronically infected with CL13 and those that recovered from Arm have reduced frequency and number of NK cells. However, at steady state, NK cells in Arm-recovered mice mature normally and, in response to ECTV, get activated, become more mature, proliferate and increase their cytotoxicity in vivo. Conversely, in mice chronically infected with CL13, NK cells are immature and residually activated and, following ECTV infection, they do not mature, proliferate or increase their cytotoxicity. Given the well established importance of NK cells in resistance to mousepox, these data suggests that the NK cell dysfunction caused by CL13 persistence may contribute to the susceptibility of CL13-infected mice to mousepox. Whether chronic infections similarly affect NK cells in humans should be explored.

    Importance Infection of adult mice with the Clone 13 (CL13) strain of lymphocytic choriomeningitis virus (LCMV) is extensively used as a model of chronic infection. In this paper, Alves-Peixoto et al. show that mice chronically infected with CL13 succumb to challenge with ectromelia virus (ECTV, the agent of mousepox), and that Natural Killer (NK) cells in CL13 infected mice are reduced in numbers and have an immature and partially activated phenotype but do respond to ECTV. These data may provide additional clues for why humans chronically infected with certain pathogens are less resistant to viral diseases.

  • HPV E7 Oncoprotein Subverts Host Innate Immunity Via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes [Virus-Cell Interactions]

  • Subversion of innate immunity by oncoviruses, such as human papillomavirus (HPV), favors carcinogenesis because the mechanism(s) of viral immune evasion can also hamper cancer immunosurveillance. Previously, we demonstrated that high-risk (hr) HPVs trigger simultaneous epigenetic silencing of multiple effectors of innate immunity to promote viral persistence. Here, we expand on those observations and show that the HPV E7 oncoprotein upregulates the H3K9-specific methyltransferase, whose action shuts down the host innate immune response. Specifically, we demonstrate that SUV39H1 contributes to chromatin repression at the promoter regions of the viral nucleic acid sensors RIG-I, cGAS and the adaptor molecule STING in HPV-transformed cells. Inhibition of SUV39H1 leads to transcriptional activation of these genes, especially RIG-I, followed by increased IFNbbeta; and 1 production after poly(dA:dT) or RIG-I agonist M8 transfection, Collectively, our findings provide new evidence that the E7 oncoprotein plays a central role in dampening host innate immunity and raise the possibility that targeting the downstream effector SUV39H1 or the RIG-I pathway may be a viable strategy to treat viral and neoplastic disease.

    IMPORTANCE High-risk HPVs are major viral human carcinogens responsible for approximately 5% of all human cancers. The growth of HPV-transformed cells depends on the ability of viral oncoproteins to manipulate a variety of cellular circuits, including those involved in innate immunity. Here, we show that one of these strategies relies on E7-mediated transcriptional activation of the chromatin repressor SUV39H1, which then promotes epigenetic silencing of RIG-I, cGAS and STING genes, thereby shutting down interferon secretion in HPV-transformed cells. Pharmacological or genetic inhibition of SUV39H1 restored the innate response in HPV-transformed cells, mostly through activation of RIG-I signaling. We also show that IFN production upon transfection of poly(dA:dT) or the RIG-I agonist M8 predominantly occurs through RIG-I signaling. Altogether, the reversible nature of the modifications associated with E7-mediated SUV39H1 upregulation provides a rationale for the design of novel anticancer and antiviral therapies targeting these molecules.

  • Genetic diversity, compartmentalization and age of HIV proviruses persisting in CD4+ T cell subsets during long-term combination antiretroviral therapy [Genetic Diversity and Evolution]

  • The HIV reservoir, which comprises diverse proviruses integrated into the genomes of infected, primarily CD4+ T cells, is the main barrier to developing an effective HIV cure. Our understanding of the genetics and dynamics of proviruses persisting within distinct CD4+ T cell subsets however remains incomplete. Using single-genome amplification we characterized subgenomic proviral sequences (nef region) from naive, central memory, transitional memory and effector memory CD4+ T cells from five HIV-infected individuals on long-term cART, and compared these to HIV RNA sequences isolated longitudinally from archived plasma collected prior to cART initiation, yielding HIV datasets spanning a median 19.5 (range 10-20) years per participant. We inferred a distribution of within-host phylogenies for each participant, from which we characterized proviral ages, phylogenetic diversity and genetic compartmentalization between CD4+ T cell subsets. While three of five participants exhibited some degree of proviral compartmentalization between CD4+ T cell subsets, combined analyses revealed no evidence that any particular CD4+ T cell subset harbored the longest-persisting, most genetically diverse and/or most genetically distinctive HIV reservoir. In one participant, diverse proviruses archived within naive T cells were significantly younger than those in memory subsets, while for three others we observed no significant differences in proviral ages between subsets. In one participant, "old" proviruses were recovered from all subsets, and included one sequence, estimated to be 21.5 years old, that dominated (ggt;93%) of their effector memory subset. HIV eradication strategies will need to overcome within- and between-host genetic complexity of proviral landscapes, possibly via personalized approaches.


    The main barrier to HIV cure is the ability of a genetically diverse pool of proviruses, integrated into the genomes of infected CD4+ T cells, to persist despite long-term suppressive combination antiretroviral therapy (cART). CD4+ T cells however constitute a heterogeneous population due to their maturation across a developmental continuum, and the genetic "landscapes" of latent proviruses archived within them remains incompletely understood. We applied phylogenetic techniques, largely novel to HIV persistence research, to reconstruct within-host HIV evolutionary history and characterize proviral diversity in CD4+ T cell subsets in five individuals on long-term cART. Participants varied widely in terms of proviral burden, genetic diversity, and age distribution between CD4+ T cell subsets, revealing that proviral landscapes can differ between individuals and between infected cell types within an individual. Our findings expose each within-host latent reservoir as unique in its genetic complexity, and support personalized strategies for HIV eradication.

  • An Alu element insertion into intron 1 results in the aberrant alternative splicing of APOBEC3G pre-mRNA in northern pig-tailed macaques (Macaca leonina) may reduce APOBEC3G-mediated hypermutation pressure on HIV-1 [Virus-Cell Interactions]

  • APOBEC3 family members, particularly APOBEC3F and APOBEC3G, inhibit the replication and spread of various retroviruses by inducing hypermutation in newly synthesized viral DNA. Viral hypermutation by APOBEC3 is associated with viral evolution, viral transmission and disease progression. In recent years, increasing attention has been paid to targeting APOBEC3G for AIDS therapy. Thus, a controllable model system using species such as macaques, which provide a relatively ideal in vivo system, is needed for the study of APOBEC3-related issues. To appropriately utilize this animal model for biomedical research, the important differences between human and macaque APOBEC3s must be considered. In this study, we found that the ratio of APOBEC3G-mediated/APOBEC3s-mediated HIV-1 hypermutation footprints was much lower in peripheral blood mononuclear cells (PBMCs) from northern pig-tailed macaques than that in PBMCs from humans. Then we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and have resulted from Alu element insertion into the macaque APOBEC3G gene intron 1. This alternative splicing pattern generating an aberrant APOBEC3G mRNA isoform may significantly dilute the full length APOBEC3G and reduce APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques, which was supported by the elimination of other possibilities accounting for this hypermutation difference between the two hosts.

    IMPORTANCE APOBEC3 family members, particularly APOBEC3F and APOBEC3G, are important cellular antiviral factors. Recently, more attention has been paid to targeting APOBEC3G for AIDS therapy. To appropriately utilize macaque animal models for the study of APOBEC3-related issues, it is important that the differences between human and macaque APOBEC3s are clarified. In this study, we identified a novel and conserved APOBEC3G pre-mRNA alternative splicing pattern in macaques, which differed from that in humans and which may reduce the APOBEC3G-mediated hypermutation pressure on HIV-1 in northern pig-tailed macaques (NPMs). Our work provides important information for the proper application of macaque animal models for APOBEC3-related issues in AIDS research and a better understanding of the biological functions of APOBEC3 proteins.

  • Astrovirus Replication Is Inhibited by Nitazoxanide In Vitro and In Vivo [Vaccines and Antiviral Agents]

  • Astroviruses (AstV) are a leading cause of diarrhea especially in the very young, the elderly, and immunocompromised populations. Despite their significant impact on public health, no drug therapies for astrovirus have been identified. In this study we fill this gap in knowledge and demonstrate that the FDA-approved broad-spectrum anti-infective drug nitazoxanide (NTZ) blocks astrovirus replication in vitro with a 50% effective concentration (EC50) of approximately 1.47 mmu;M. It can be administered up to 8 hours post-infection and is effective against multiple human astrovirus serotypes including clinical isolates. Most importantly, NTZ reduces viral shed in vivo, exhibiting its potential as a future clinical therapeutic.

    Importance Human astroviruses (HAstV) are thought to cause between 2 and 9% of acute, non-bacterial diarrhea cases in children worldwide. HAstV infection can be especially problematic in immunocompromised people and infants where the virus has been associated with necrotizing enterocolitis, severe and persistent diarrhea, as well as rare instances of systemic and fatal disease. Yet no antivirals have been identified to treat astrovirus infection. Our study provides the first evidence that nitazoxanide may be an effective therapeutic strategy against astrovirus disease.

  • Herpes simplex virus latency is noisier the closer we look [Gem]

  • During herpes simplex virus (HSV) latency the viral genome is harboured in peripheral neurons in the absence of infectious virus, but with the potential to restart infection. Advances in epigenetics have helped explain how viral gene expression is largely inhibited during latency. Paradoxically at the same time, the view that latency is entirely silent has been eroding. This low-level noise has implications for our understanding of HSV latency and should not be ignored.

  • Specific Anchoring and Local Translation of Poxviral ATI mRNA at Cytoplasmic Inclusion Bodies [Genome Replication and Regulation of Viral Gene Expression]

  • On-site translation of mRNAs provides an efficient means of subcellular protein localization. In eukaryotic cells, the transport of cellular mRNAs to membrane-less sites usually occurs prior to translation and involves specific sequences known as zipcodes that interact with RNA-binding and motor proteins. Poxviruses replicate in specialized cytoplasmic factory regions where DNA synthesis, transcription, translation and virion assembly occur. Some poxviruses embed infectious virus particles outside of factories in membrane-less protein bodies with liquid gel-like properties known as A-type inclusions (ATIs) that are comprised of numerous copies of the viral 150-kDa ATI protein. Here, we demonstrate by fluorescent in situ hybridization that these inclusions are decorated with ATI mRNA. On-site translation is supported by the localization of a translation initiation factor eIF4E and by ribosome-bound nascent chain ribopuromycylation. Nascent peptide-mediated anchoring of ribosome-mRNA translation complexes to the inclusions is suggested by release of the mRNA by puromycin, a peptide chain terminator. Following puromycin washout, re-localization of ATI mRNA at inclusions depends on RNA and protein synthesis but requires neither microtubules nor actin polymerization. Further studies show that the ATI mRNAs remain near the sites of transcription in the factory regions when stop codons are introduced near the N-terminus of the ATI or large truncations are made at the N- or C-termini. Instead of using a zipcode, we propose that ATI mRNA localization is mediated by ribosome-bound nascent ATI polypeptides that interact with ATI protein in inclusions and thereby anchor the complex for multiple rounds of mRNA translation.

    IMPORTANCE Poxvirus genome replication, transcription, translation and virion assembly occur at sites within the cytoplasm known as factories. Some poxviruses sequester infectious virions outside of the factories in inclusion bodies comprised of numerous copies of the 150-kDa ATI protein, which can provide stability and protection in the environment. We provide evidence that ATI mRNA is anchored by nascent peptides and translated at the inclusion sites rather than in virus factories. Association of ATI mRNA with inclusion bodies allows multiple rounds of local translation and prevents premature ATI protein aggregation and trapping of virions within the factory.

  • Two Pioneer Transcription Factors, Krüppel-Like Transcription Factor 4 and Glucocorticoid Receptor, Cooperatively Transactivate the Bovine Herpesvirus 1 ICP0 Early Promoter and Stimulate Productive Infection [Genome Replication and Regulation of Viral Gene Expression]

  • An important site for bovine herpesvirus 1 (BoHV-1) latency is sensory neurons within trigeminal ganglia (TG). The synthetic corticosteroid dexamethasone consistently induces BoHV-1 reactivation from latency. Expression of four Krüppel-like transcription factors (KLF), KLF4, KLF6, PLZF (promyelocytic leukemia zinc finger), and KLF15, are induced in TG neurons early during dexamethasone-induced reactivation. The glucocorticoid receptor (GR) and KLF15 form a feed-forward transcription loop that cooperatively transactivates the BoHV-1 immediate early transcription unit 1 (IEtu1) promoter that drives infected cell protein 0 (bICP0) and bICP4 expression. Since the bICP0 gene also contains a separate early (E) promoter, we tested the hypothesis that GR+KLF family members transactivate the bICP0 E promoter. GR+KLF4, both pioneer transcription factors, cooperated to stimulate bICP0 E promoter activity via a ligand independent manner in mouse neuroblastoma cells (Neuro-2A): furthermore, GR+KLF4 stimulated productive infection. Mutating both 1/2 GR response elements did not significantly reduce GR+KLF4 mediated transactivation of the bICP0 E promoter suggesting a novel mechanism exists for transactivation. GR+KLF15 cooperatively stimulated bICP0 activity less efficiently than GR+KL4: however, KLF6 and PLZF plus GR had little effect on the bICP0 E promoter. GR, KLF4, and KLF15 occupied bICP0 E promoter sequences in transfected Neuro-2A cells. GR and KLF15, but not KLF4, occupied the bICP0 E promoter at late times during productive infection of bovine cells. Collectively, these studies suggest cooperative transactivation of the bICP0 E promoter by two pioneer transcription factors (GR and KLF4) correlates with stimulating lytic cycle viral gene expression following stressful stimuli.

    IMPORTANCE Bovine herpesvirus 1 (BoHV-1), an important bovine pathogen, establishes life-long latency in sensory neurons. Reactivation from latency is consistently induced by the synthetic corticosteroid dexamethasone. We predict increased corticosteroid levels activate the glucocorticoid receptor (GR): consequently, viral gene expression is stimulated by the activated GR. The immediate early transcription unit 1 promoter (IEtu1) drives expression of two viral transcriptional regulatory proteins, infected cell protein 0 (bICP0) and bICP4. Interestingly, a separate early promoter also drives bICP0 expression. Two pioneer transcription factors, GR and Krüppel-like transcription factor 4 (KLF4), cooperatively transactivate the bICP0 early (E) promoter. GR+KLF15 cooperate to stimulate bICP0 E promoter activity, but significantly less relative to GR+KLF4. The bICP0 E promoter contain distinct enhancer-like domains necessary for GR+KLF4 mediated transactivation relative to GR+KLF15. Stress induced pioneer transcription factors are proposed to activate key viral promoters, including the bICP0 E promoter, during early stages of reactivation from latency.

  • A murine model for the study of influenza D virus. [Pathogenesis and Immunity]

  • A novel genus within the Orthomyxoviridae family was identified in the USA and named Influenza D virus (IDV). Bovine have been proposed to be the primary host and three main viral lineages (D/OK-like, D/660-like and D/Japan-like) have been described. Experimental infections were so far performed in swine, ferret, calf and guinea pig, in order to study IDV pathogenesis.

    We developed a murine experimental model to ease the study of IDV pathogenesis and immune response. DBA/2 mice were inoculated with 105 TCID50 of D/bovine/France/5920/2014 (D/OK-like). No clinical signs and weight loss were observed. Viral replication was observed mainly in the upper respiratory tract (nasal turbinates) but also in lower respiratory tract of infected mice, with a peak at 4 days post-infection. Moreover, the virus was also detected in the intestines. All infected mice seroconverted by 14 days post infection. Transcriptomic analyses demonstrated that IDV induced an activation of pro-inflammatory genes such as IFN- and CCL2. Inoculation of NFB-luciferase and Ifnar1-/- mice demonstrated that IDV induced mild inflammation and that type I interferons response was not necessary in IDV clearance. Adaptation of IDV by serial passages in mice was not sufficient to induce disease or increased pathogenesis.

    Taken together, present data and comparisons with the calf model show that our mouse model allows for the study of IDV replication and fitness (before selected viruses may be inoculated on calves) and also of the immune response.


    Influenza D virus (IDV), a new genus of Orthomyxoviridae family, presents a large host range and a worldwide circulation. The pathogenicity of this virus has been studied in the calf model. The mouse model is frequently used to enable a first assessment of a pathogen's fitness, replication and pathogenesis for influenza A and B viruses. We showed that DBA/2 mice are a relevant in vivo model for the study of IDV replication. This model will allow for rapid IDV fitness and replication evaluation and will enable phenotypic comparisons between isolated viruses. It will also allow for a better understanding of the immune response induced after IDV infection.

  • HIV-1 accessory protein Vpr interacts with REAF/RPRD2 to mitigate its antiviral activity. [Virus-Cell Interactions]

  • The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intact vpr is more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.

    Importance For at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1 in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell's protein degradation machinery.

  • The IFNL4 gene is a non-canonical interferon gene with a unique but evolutionarily conserved regulation [Cellular Response to Infection]

  • Interferon lambda 4 (IFNL4) is a recently identified enigmatic member of the interferon lambda family. Genetic data suggest that the IFNL4 gene acts in a pro-viral and anti-inflammatory manner in patients. However, the protein is in vitro indistinguishable from the other members of the interferon lambda family. We have investigated the gene regulation of IFNL4 in detail and found that it differs radically from that of canonical antiviral interferons. Being induced by viral infection is a defining characteristic of interferons, but viral infection or overexpression of members of the interferon regulatory factor (IRF) family of transcription factors only leads to a minute induction of IFNL4. This behavior is evolutionarily conserved and can be reversed by inserting a functional IRF3 binding site into the IFNL4 promoter. Thus, the regulation of the IFNL4 gene is radically different and might explain some of the atypical phenotypes associated with the IFNL4 gene in humans.

    Importance Recent genetic evidence has highlighted how the IFNL4 gene acts in a counterintuitive manner as patients with a non-functional IFNL4 gene exhibit increased clearance of hepatitis C virus but also increased liver inflammation. This suggests that the IFNL4 gene acts in a pro-viral and anti-inflammatory manner. Those surprising but quite clear genetic data have prompted an extensive examination of the basic characteristics of the IFNL4 gene and its gene product IFN-4. We have investigated the expression of the IFNL4 gene and found it to be poorly induced by viral infections. A thorough investigation of the IFNL4 promoter revealed a highly conserved and functional promoter, but also one that lacks the defining characteristic of IFNs, i.e. the ability to be effectively induced by viral infections. We suggest that the unique function of the IFNL4 gene is related to its non-canonical transcriptional regulation.

  • Intact HIV proviruses persist in children 7-9 years after initiation of ART in the first year of life [Genome Replication and Regulation of Viral Gene Expression]

  • In adults starting ART during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. By contrast, a recent report in early treated children failed to detect sequence intact proviruses. We sought to detect and characterize proviral sequences in another cohort of early treated children after 6-9 years on suppressive ART. PBMCs from perinatally-infected children from the CHER study were analysed. Near full-length proviral amplification and sequencing (NFL-PAS) was performed at one time point after 6-9 years on ART. Amplicons with large internal deletions were excluded (llt;9kb). All amplicons gge;9kb were sequenced and analysed through a bio-informatic pipeline to detect indels, frameshifts or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range: 2.0 nndash; 11.1), 733 single NFL-PAS amplicons were generated. Of these, 534(72.9%) had large internal deletions, 174(23.7%) had hypermutation, 15(1.4%) had small internal deletions, 3(1.0%) had deletions in the packaging signal/major splice donor site and 7(1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age; of whom one had two identical intact sequences, suggestive of a cell clone harbouring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably just as in adults, maintained by clonal expansion of cells infected before ART initiation.

    Importance There is limited data on the proviral landscape in early treated, long-term suppressed children, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through near full-length proviral amplification and sequencing (NFL-PAS) we generated 733 NFL-PAS amplicons from 8 children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower than (pllt;0.05) reported for HIV subtype B infected adults treated during early HIV infection. We show that cells harbouring genetically intact HIV proviruses are rare in early treated long-term suppressed children and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs particularly in paediatric studies where sample availability is limited.

  • Viral determinants in H5N1 influenza A virus enable productive infection of HeLa cells. [Virus-Cell Interactions]

  • Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, and sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to growth of human H1N1 and H3N2, and low pathogenic avian influenza (LPAIs) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) virus H5N1 successfully propagated in HeLa cells to levels comparable to a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 growth, suggesting the absence of a host factor(s) required for replication of H1N1, but not H5N1, in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, and deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from H5N1 was necessary and sufficient to enable H1N1 growth. Overall, this study suggests the absence of one or more cellular factors in HeLa cells that results in abortive replication of H1N1, H3N2, and LPAI viruses, but can be circumvented upon introduction of H5N1 NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into virus-host interactions that underlie IAV pathogenesis and tropism.

    IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk for these zoonotic viruses to acquire adaptive mutations to efficiently propagate and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon introduction of H5N1 HA and NP proteins. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 pathogenesis and tropism.

  • Molecular basis of binding between MERS-CoV and CD26s from seven bat species [Structure and Assembly]

  • Continued reports of Middle East respiratory syndrome coronavirus (MERS-CoV) infecting human have occurred since its identification in 2012. MERS-CoV is prone to cause endemic disease in the Middle East, with several dozens of spill-over infections to other continents. It is hypothesized that MERS-CoV originated from bat coronaviruses and that dromedary camels are its natural reservoir. Although gene segments identical to MERS-CoV were sequenced from certain species of bats, and one species experimentally shed the virus, it is still unknown whether other bats can transmit the virus. Here, at the molecular level, we found that all purified bat CD26s (bCD26s) from a diverse range of species interact with the receptor binding domain (RBD) of MERS-CoV, with KD values ranging from several to hundreds of micromolar level. Moreover, all bCD26s expressed in this study mediated the entry of pseudotyped MERS-CoV to receptor-expressing cells, indicating the broad potential engagement of bCD26s as MERS-CoV receptors. Further structural analysis indicated that in the bat receptor, compared to the human receptor, substitutions of key residues and their adjacent amino acids leads to decreased binding affinity to the MERS-RBD. These results add more evidence to the existing belief that bats are the original source of MERS-CoV and suggest that bCD26s in many species can mediate the entry of the virus, which has significant implications for the surveillance and control of MERS-CoV infection.

    Importance In this study, we found that bat CD26s (bCD26s) from different species exhibit large diversities, especially in the region responsible for binding to the receptor binding domain of Middle East respiratory syndrome coronavirus (MERS-CoV). However, they maintain the interaction with MERS-RBD at varied affinities and support the entry of pseudotyped MERS-CoV. These bat receptors polymorphisms seem to confer evolutionary pressure for the adaptation of CD26-binding virus, such as the ancestor of MERS-CoV, and led to the generation of diversified CD26-engaging CoV strains. Thus, our data add more evidence to support that bats are the reservoir of MERS-CoV and similar viruses, as well as further emphasize the necessity to survey MERS-CoV and other CoVs among bats.

  • Breaking the convention: Sialoglycan variants, Co-receptors and Alternative Receptors for Influenza A Virus Entry [Gem]

  • The influenza A virus (IAV) envelope protein haemagglutinin binds aalpha;2,6- or aalpha;2,3-linked sialic acid as host cell receptor. Bat IAV subtypes H17N10 and H18N11 form an exception to this rule and do not bind sialic acid but enter cells via MHC class II complexes. Here, we review current knowledge on IAV receptors with a focus on sialoglycan variants, protein co-receptors and alternative receptors that impact IAV attachment and internalization beyond the well-described sialic acid binding.

  • A MUC16 IgG binding activity selects for a restricted subset of IgG enriched for certain SIV epitope specificities. [Pathogenesis and Immunity]

  • We have recently shown that MUC16, a component of the glycocalyx of some mucosal barriers, has elevated binding to the G0 glycoform of the Fc portion of IgG. Therefore, IgG from chronically infected HIV patients, who typically exhibit increased amounts of G0 glycoforms, showed increased MUC16 binding compared to uninfected controls. Using the rhesus macaque SIVmac251 model, we can compare plasma antibodies before and after chronic infection. We find increased binding of IgG to MUC16 after chronic SIV infection. Antibodies isolated for tight association with MUC16 (MUC16-eluted antibodies) show reduced FcR engagement and antibody-dependent cellular cytotoxicity (ADCC) activity. The glycosylation profile of these IgGs was consistent with a decrease in FcR engagement and subsequent ADCC effector function as they contain a decrease in afucosylated bisecting glycoforms that preferentially bind FcRs. Testing of the SIV antigen specificity of IgG from SIV infected macaques revealed the MUC16-eluted antibodies were enriched for certain specific epitopes including regions of gp41 and gp120. This enrichment of specific antigen responses for fucosylated bisecting glycoforms and subsequent association with MUC16 suggests the immune response has the potential to direct specific epitope responses to localize to the glycocalyx through interaction with this specific mucin.

    Importance Understanding how antibodies are distributed in the mucosal environment is valuable for development of a vaccine to block HIV infection. Here we study an IgG binding activity in MUC16, potentially representing a new IgG effector function that would concentrate certain antibodies within the glycocalyx to trap pathogens before they can reach the underlying columnar epithelial barriers. These studies reveal that rhesus macaque IgG responses during chronic SIV infection generate increased antibodies that bind MUC16 and interestingly these MUC16-tethered antibodies are enriched for binding to certain antigens. Therefore, it may be possible to direct HIV vaccine-generated responses to associate with MUC16 and enhance the antibody's ability to mediate immune exclusion by trapping virions within the glycocalyx and preventing the virus from reaching immune target cells within the mucosa. This concept will ultimately have to be tested in the rhesus macaque model which is shown here to have MUC16 targeted antigen responses.

  • Boosting with AIDSVAX B/E enhances Env constant region 1 and 2 antibody-dependent cellular cytotoxicity breadth and potency [Vaccines and Antiviral Agents]

  • Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce non-neutralizing antibodies (NNAbs) that kill virus-infected cells as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) monoclonal antibodies (mAbs) frequently mediate potent antibody dependent cellular cytotoxicity (ADCC) making them an important vaccine target. Here we explore the effect of delayed and repetitive boosting of RV144 vaccine-recipients with AIDSVAX B/E on the C1C2-specific mAb repertoire. It was found that boosting increased clonal lineage specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific mAb showed that it bound a highly conserved Env gp120 epitope. Thus, boosting to affinity mature these type of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than seen in the RV144 trial.

    SIGNIFICANCE Over one million people become infected with HIV-1 each year making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine-regimen is the only HIV-1 clinical trial to date to demonstrate vaccine-efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine-efficacy. The RV305 HIV-1 vaccine-regimen was a follow-up boost of RV144 vaccine-recipients that occurred 6-8 years after the conclusion of RV144. Our study focused on the effect of delayed boosting in humans on the vaccine-induced Env constant region 1 and 2 (C1C2) nndash; specific antibody repertoire. It was found that boosting with a HIV-1 Env vaccine increased C1C2-specific antibody dependent cellular cytotoxicity potency and breadth.

  • HDV alters the autophagy process to promote its genome replication. [Virus-Cell Interactions]

  • A substantial number of viruses have been demonstrated to subvert autophagy to promote their own replication. Recent publications have reported the proviral effect of autophagy induction on hepatitis B virus (HBV) replication. The hepatitis delta virus (HDV) is a defective virus and an occasional obligate satellite of HBV. However, no previous work has studied the relationship between autophagy and HDV. In this article, we analyze the impact of HBV and HDV replication on autophagy as well as the involvement of the autophagy machinery in HDV lifecycle when produced alone and in combination with HBV. We prove that HBxAg and HBsAg can induce early steps of autophagy but ultimately block the flux. It is worth noting that, the two isoforms of the HDV protein, S-HDAg and L-HDAg, can also efficiently promote autophagosome accumulation and disturb the autophagic flux. Using the CRISPR-Cas9 technology to generate specific knockouts, we demonstrate that the autophagy machinery, specifically the proteins implicated in the elongation step (ATG7, ATG5 and LC3), are important for the release of HBV without affecting the level of intracellular HBV genome. Surprisingly, the knockout of ATG5 and ATG7 decreased the intracellular HDV RNA level in both Huh7 and HepG2.2.15 cells without additional effect on HDV secretion. Therefore, we conclude that HBV and HDV have evolved to utilize the autophagy machinery so as to assist at different steps of their lifecycle.

    IMPORTANCE Hepatitis delta virus is a defective RNA virus that requires hepatitis B virus envelope proteins (HBsAg) to fulfill its lifecycle. Thus, HDV virus can only infect individuals at the same time as HBV (coinfection) or superinfect individuals who are already chronic carriers of HBV. The presence of HDV in the liver accelerates the progression of infection to fibrosis and to hepatic cancer. Since the current treatments against HBV are ineffective against HDV, it is of paramount importance to study the interaction between HBV, HDV and the host factors. This will help unravel new targets whereby a therapy that is capable of simultaneously impeding both viruses could be developed. In this research paper we evidence that the autophagy machinery promotes the replication of HBV and HDV at different steps of their lifecycle. Notwithstanding their contribution to HBV release, autophagy proteins seem to assist HDV intracellular replication but not its secretion.

  • Herpes simplex virus type 1 spread in oligodendrocytic cells is highly dependent on MAL proteolipid [Virus-Cell Interactions]

  • Myelin and lymphocyte protein, MAL, is a tetraspan integral membrane protein that resides in detergent-insoluble membrane fractions enriched in condensed membranes. MAL is expressed in oligodendrocytes, in Schwann cells where it is essential for the stability of myelin, and at the apical membrane of epithelial cells, where it has a critical role in transport. In T lymphocytes MAL is found at the immunological synapse and plays a crucial role in exosome secretion. However, no involvement of MAL in viral infections has been reported so far. Here, we show thatHSV-1 virions travel in association with MAL-positive structures to reach the end of cellular processes, which contact uninfected oligodendrocytes. Importantly, the depletion of MAL led to a significant decrease of infection, with a drastic reduction in the number of lytic plaques in MAL-silenced cells. These results suggest a significant role for MAL in viral spread at cell contacts. The participation of MAL in the cell-to-cell spread of HSV-1 may shed light on the involvement of proteolipids in this process.

    Importance Herpes simplex virus type 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establish latent infections in neurons. HSV-1 may spread from infected to uninfected cells by two main routes: by cell-free virus, or by cell-to-cell spread. In the first case, virions exit into the extracellular space and then infect another cell from the outside. In the second one, the viral transmission occurs through cell-to-cell contacts via a mechanism still poorly understood. A third mode of spread, using extracellular vesicles, also exists. In this study, we demonstrate the important role for a myelin protein, MAL, in the process of cell-to-cell viral spread in oligodendrocytes. We show that MAL is involved in trafficking of virions along cell processes, and that MAL depletion produces a significant alteration in the viral cycle, which reduces cell-to cell spread of HSV-1.

  • The C protein is recruited to measles virus ribonucleocapsids by the phosphoprotein [Genome Replication and Regulation of Viral Gene Expression]

  • Measles virus (MeV), like all viruses of the order Mononegavirales, utilizes a complex consisting of genomic RNA, nucleoprotein, the RNA-dependent RNA polymerase and a polymerase co-factor, the phosphoprotein (P), for transcription and replication. We previously showed that a recombinant MeV that does not express another viral protein, C, has severe transcription and replication deficiencies, including a steeper transcription gradient compared to parental virus and generation of defective-interfering RNA. This virus is attenuated in vitro and in vivo. However, how the molecular mechanism by which C protein operates and whether it is a structural component of the replication complex remained unclear. Here we show that C associates with the ribonucleocapsid and forms a complex that can be purified by immunoprecipitation or ultracentrifugation. In the presence of detergent, the C protein is retained on purified ribonucleocapsids less efficiently than the P protein and the polymerase. The C protein is recruited to the ribonucleocapsid through its interaction with the P protein, as shown by immunofluorescence microscopy of cells expressing different combinations of viral proteins and split luciferase complementation assays. Forty amino-terminal C protein residues are dispensable for the interaction with P, and the carboxyl-terminal half of P is sufficient for the interaction with C. Thus, the C protein, rather than being accessory as qualified in textbooks so far, is a ribonucleocapsid-associated protein that interacts with P, thereby increasing replication accuracy and processivity of the polymerase complex.

    Importance Replication of negative strand RNA viruses relies on two components: a helical ribonucleocapsid and an RNA-dependent RNA polymerase composed of a catalytic subunit, the L protein, and a co-factor, the P protein. We show that the measles virus (MeV) C protein is an additional component of the replication complex. We provide evidence that the C protein is recruited to the ribonucleocapsid by the P protein and map the interacting segments of both C and P proteins. We conclude that the primary function of MeV C is to improve polymerase processivity and accuracy, rather than uniquely to antagonize the type I interferon response. Since most viruses of the Paramyxoviridae family express C proteins, their primary function may be conserved.

  • Structural insight into the African swine fever virus dUTPase reveals a novel folding pattern in the dUTPase family [Structure and Assembly]

  • The African swine fever virus (ASFV) is the deadly pathogen of African swine fever (ASF) that induces high mortality, approaching 100% in domestic pigs, causes enormous losses to the global pig industry and threatens food security. Currently, there is no effective treatment or preventive countermeasure. dUTPases (deoxyuridine 5'-triphosphate pyrophosphatases) are ubiquitous enzymes that are essential for the hydrolysis of dUTP and prevent the misincorporation of dUTP into newly synthesized DNA. Here, we present the crystal structures of the ASFV dUTPase in complex with the product dUMP and cofactor Mg2+ at a resolution of 2.2 angstroms. We observed that a unique "turning point" at G125 plays an unexpected critical role in the swapping region of the C-terminal segment, which is further stabilized by the interactions of the last C-terminal bbeta; strand with the bbeta;1 and bbeta;2 strands, thereby positioning the catalytic motif 5 into the active site of its own subunit instead of into a third subunit. Therefore, the ASFV dUTPase employs a novel two-subunit active site that is different than the classic trimeric dUTPase active site, which is composed of all three subunits. Meanwhile, further results confirmed that the configuration of motifs 1 to 5 has high structural homology with and a catalytic mechanism similar to that of the known trimeric dUTPases. In general, our study expands the information not only on the structural diversity of the conserved dUTPase family but also on the details needed to utilize this dUTPase as a novel target in the treatment of ASF.

    IMPORTANCE African swine fever virus, a large enveloped double-stranded DNA virus, causes a deadly infection in domestic pigs. In addition to Africa, Europe and South America, countries in Asia, such as China, Vietnam, and Mongolia, have suffered the hazards posed by ASFV outbreaks in recent years. Until now, there has been no vaccine for protection from ASFV infection or effective treatments to cure ASF. Here, we solved the crystal structure of the ASFV dUTPase-dUMP-Mg2+ complex. The ASFV dUTPase displays a noncanonical folding pattern that differs from that of the classic homotrimeric dUTPase, in which the active site is composed of two subunits. In addition, several non-conserved residues within the 3-fold axis channel play a vital role in ASFV dUTPase homotrimer stability. Our finding on these unique structural features of the ASFV dUTPase could be explored for the design of potential specific inhibitors that target this unique enzyme.

  • Single amino acid differences between closely related reovirus T3D lab strains alter oncolytic potency in vitro and in vivo. [Genetic Diversity and Evolution]

  • Little is known about how genetic variations in viruses affect their success as therapeutic agents. The Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D from various sources extensively impacts oncolytic activity. The T3DPL laboratory strain showed significantly stronger oncolytic activities in a murine model of melanoma than the T3DTD strain. Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD, and T3D strains obtained from ATCC (T3DATCC) and T3DKC. Reassortant and reverse genetics analysis was used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL. Four single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size, and when combined were able to fully account for differences between T3DPL versus T3DTD. Moreover, we discovered polymorphisms in T3DTD that promoted virus replication and spread in tumors, and generated a new T3DPL/T3DTD hybrid with enhanced plaque size compared to T3DPL. Altogether, single amino acid changes acquired during laboratory virus propagation can have large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.

    Importance: The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their ability to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that four single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

  • Glutaminolysis and glycolysis are essential for optimal replication of Marek's disease virus [Virus-Cell Interactions]

  • Viruses may hijack glycolysis, glutaminolysis or fatty acid bbeta;-oxidation of host cells to provide energy and macromolecules required for efficient viral replication. Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens and modulates metabolism of host cells. Metabolic analysis of MDV-infected chicken embryonic fibroblasts (CEFs) identified elevated levels of metabolites involved in glutamine catabolism such as glutamic acid, alanine, glycine, pyrimidine and creatine. In addition, our results demonstrate that glutamine uptake is elevated by MDV-infected cells in vitro. Although glutamine, but not glucose, deprivation significantly reduced cell viability in MDV-infected cells, both glutamine and glucose were required for virus replication and spread. In the presence of minimum glutamine requirements based on optimal cell viability, virus replication was partially rescued by the addition of the tricarboxylic acid (TCA) cycle intermediate, aalpha;-ketoglutarate, suggesting that exogenous glutamine is an essential carbon source for the TCA cycle to generate energy and macromolecules required for virus replication. Surprisingly, the inhibition of carnitine palmitoyltransferase 1a (CPT1a), which is elevated in MDV-infected cells, by chemical (etomoxir) or physiological (Manoly-CoA) inhibitors did not reduce MDV replication indicating that MDV replication does not require fatty acid bbeta;-oxidation. Taken together, our results demonstrate that MDV infection activates anaplerotic substrate from glucose to glutamine to provide energy and macromolecules required for MDV replication, and optimal MDV replication occurs when the cells do not depend on mitochondrial bbeta;-oxidation.

    Importance Viruses can manipulate host cellular metabolism to provide energy and essential biosynthetic requirements for efficient replication. Marek's disease virus (MDV), an avian alphaherpesvirus, causes a deadly lymphoma in chickens and hijacks host cell metabolism. This study provides evidence for the importance of glycolysis and glutaminolysis, but not fatty acid bbeta;-oxidation, as an essential energy source for the replication and spread of MDV. Moreover, it suggests that in MDV infection, as in many tumour cells, glutamine is used for generation of energetic and biosynthetic requirements of the MDV infection, while glucose is used biosynthetically.

  • Differences in antibody responses against Chelonid alphaherpesvirus 5 suggest differences in virus biology between green turtles from Hawaii and Florida [Pathogenesis and Immunity]

  • Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (Chelonid herpesvirus 5-ChHV5) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by lack of robust serological assays to monitor exposure to ChHV5. This is due in part to inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on ELISA plates in their native form, and assayed for reactivity to two types of antibodies, the full length 7S and 5.7S IgY that has a truncated Fc region. In Florida, turtles were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in Hawaii, we detected strong antibody reactivity mainly from tumored animals with lower antibody response in non-tumored animals, including those from FP Enzootic areas. In Hawaii, turtles actively shedding ChHV5 were more seropositive than non-shedders. In trying to account for differences in serological responses to ChHV5 between Hawaii and Florida, we rejected cross reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and Florida, respectively, might have led to the emergence of biologically different viral strains. While Florida strains apparently spread independent of tumors, transmission of the Hawaiian subtype relies heavily on tumor formation.

    Importance: Fibropapillomatosis (FP) is a tumor disease associated with Chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality for threatened green turtle globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors that are the only sites of viral replication whereas tumored and non-tumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such unusual life history. Our findings along with fundamental differences in life history between Floridian and Hawaiian turtles may partly explain differential dynamics of FP between the two regions.

  • The ESCRT-II subunit EAP20/VPS25 and the Bro1 domain proteins HD-PTP and BROX are individually dispensable for HSV-1 replication [Virus-Cell Interactions]

  • Capsid envelopment during assembly of the neurotropic herpesviruses HSV-1 and PRV in the infected cell cytoplasm is thought to involve the late acting cellular ESCRT components ESCRT-III and VPS4. However HSV-1, unlike members of many other families of enveloped viruses, does not appear to require the ESCRT-I subunit TSG101 or the Bro1 domain-containing protein ALIX to recruit and activate ESCRT-III. Alternative cellular factors that are known to be capable of regulating ESCRT-III function include the ESCRT-II complex and other members of the Bro1 family. We therefore used siRNA to knock down the essential ESCRT-II subunit EAP20/VPS25 and the Bro1 proteins HD-PTP and BROX. We demonstrated reduction in levels of the targeted proteins by western blotting and used quantitative microscopic assays to confirm loss of ESCRT-II and HD-PTP function. We found that in single step replication experiments the final yields of HSV-1 were unchanged following loss of EAP20, HD-PTP or BROX.

    IMPORTANCE HSV-1 is a pathogen of the human nervous system that uses its own, virally-encoded proteins and the normal cellular ESCRT machinery to drive the construction of its envelope. How HSV-1 structural proteins interact with ESCRT components, and which subsets of cellular ESCRT proteins are utilized by the virus remain largely unknown. Here we demonstrate that an essential component of the ESCRT-II complex, and two ESCRT-associated Bro1 proteins, are dispensable for HSV-1 replication.

  • Novel Sulfolobus fuselloviruses with extensive genomic variations [Genetic Diversity and Evolution]

  • Fuselloviruses are among the most widespread and best characterized archaeal viruses. They exhibit remarkable diversity as the list of members of this family is rapidly growing. However, it has yet to be shown how a fuselloviral genome may undergo variation at the levels of both single nucleotides and sequence stretches. Here we report the isolation and characterization of four novel spindle-shaped viruses, named Sulfolobus spindle-shaped viruses 19-22 (SSV19-22), from a hot spring in the Philippines. SSV19 is a member of the genus Alphafusellovirus, whereas SSV20-22 belong to the genus Betafusellovirus. The genomes of SSV20-SSV22 are identical except for the presence of two large variable regions, as well as numerous sites of single nucleotide polymorphism (SNP) unevenly distributed throughout the genomes and enriched in certain regions including the gene encoding the putative end-filament protein VP4. We show that co-infection of the host with SSV20 and SSV22 led to the formation of a SSV21-like virus, presumably through homologous recombination. In addition, a large number of SNPs were identified in DNA sequences retrieved by PCR amplification targeting the SSV20-22 vp4 gene from the original enrichment culture, indicating the enormous diversity of SSV20-22-like viruses in the environment. The high variability of VP4 is consistent with its potential role in host recognition and binding by the virus.

    Importance How a virus survives in the arms race with its host is an intriguing question. In this study, we isolated and characterized four novel fuselloviruses named Sulfolobus spindle-shaped viruses 19-22 (SSV19-22). Interestingly, SSV20-22 differ primarily in two genomic regions and are apparently convertible through homologous recombination during co-infection. Moreover, sites of single nucleotide polymorphism (SNP) were identified throughout the genomes of SSV20-22 and, notably, enriched in certain regions including the gene encoding the putative end-filament protein VP4, which is believed to be involved in host recognition and binding by the virus.

  • The HSV-1 immediate early protein ICP22 is a functional mimic of a cellular J-protein [Virus-Cell Interactions]

  • Molecular chaperones and co-chaperones are the most abundant cellular effectors of protein homeostasis, assisting protein folding and preventing aggregation of misfolded proteins. We have previously shown that HSV-1 infection results in the drastic spatial reorganization of the cellular chaperone Hsc70 into nuclear domains called VICE (Virus Induced Chaperone Enriched) domains and that this recruitment is dependent on the viral immediate early protein ICP22. In this paper, we present several lines of evidence supporting the notion that ICP22 functions as a virally encoded co-chaperone (J-protein/Hsp40) functioning together with its Hsc70 partner to recognize and manage aggregated and misfolded proteins. We show that ICP22 results in (i) nuclear sequestration of non-native proteins, (ii) reduction of cytoplasmic aggresomes in cells expressing aggregation-prone proteins, (iii) thermoprotection against heat-inactivation of firefly luciferase and (iv) Sequence homology analysis indicated that ICP22 contains an N-terminal J-domain and a C-terminal substrate binding domain, similar to type II cellular J-proteins. ICP22 may, thus, be functionally similar to J-protein/Hsp40 co-chaperones that function together with their HSP70 partners to prevent aggregation of non-native proteins. This is not the first example of a virus hijacking a function of a cellular chaperone, as SV40 T Antigen was previously shown to contain a J-domain; however, this the first known example of the acquisition of a functional J-like protein by a virus and suggests that HSV has taken advantage of the adaptable nature of J-proteins to evolve a multi-functional co-chaperone that functions with Hsc70 to promote lytic infection.

    IMPORTANCE Viruses have evolved a variety of strategies to succeed in a hostile environment. The HSV-1 immediate early protein ICP22 plays several roles in the virus life cycle including down-regulation of cellular gene expression, up-regulation of late viral gene expression, inhibition of apoptosis, prevention of aggregation of non-native proteins and the recruitment of a cellular heat shock protein, Hsc70, to nuclear domains. We present evidence that ICP22 functionally resembles a cellular J-protein/HSP40 family co-chaperone, interacting specifically with Hsc70. We suggest that HSV has taken advantage of the adaptable nature of J-proteins to evolve a multi-functional co-chaperone that functions with Hsc70 to promote lytic infection.

  • Cytoplasmic parvovirus capsids recruit importin beta for nuclear delivery [Virus-Cell Interactions]

  • Parvoviruses are an important platform for gene and cancer therapy. Their cell entry and the following steps including nuclear import are inefficient limiting their use in therapeutic applications. Two models exist on parvoviral nuclear entry: classical import of the viral capsid using nuclear transport receptors of the importin (karyopherin) family, or direct attachment of the capsid to the nuclear pore complex leading to local disintegration of the nuclear envelope. Here, by laser scanning confocal microscopy and in situ proximity ligation analysis combined with co-immunoprecipitation we showed that infection requires importin bbeta;-mediated access into the nuclear pore complex and nucleoporin 153-mediated interactions on the nuclear side. Importin bbeta;-capsid interaction continued within the nucleoplasm, which suggests that a mixed model of nuclear entry in which the classical nuclear import across the nuclear pore complex is accompanied by transient ruptures of the nuclear envelope allowing also passive entry of importin bbeta;-capsid complexes into the nucleus.

    IMPORTANCE Parvoviruses are small DNA viruses that deliver their DNA into the post-mitotic nuclei, which is an important step for parvoviral gene and cancer therapies. Limitations in virus-receptor interaction or endocytic entry do not fully explain the low transduction/infection efficiency, indicating a bottleneck after virus entry into the cytoplasm. We thus investigated the transfer of parvovirus capsids from the cytoplasm to the nucleus showing that the nuclear import of parvovirus capsid follows a unique strategy, which differs from classical nuclear import and that of other viruses.

  • Genome-wide transcriptome analysis of human papillomavirus type 16 infected primary keratinocytes reveals subtle perturbations mostly due to E7 protein expression [Cellular Response to Infection]

  • It is established that the host cell transcriptome of natural lesions, organotypic rafts, and HPV-imortalized keratinocytes is altered in the presence of Human Papillomavirus (HPV) genomes. However, the establishment of HPV-harboring cell lines requires selection and immortalization, which makes it impossible to distinguish between alterations directly induced by HPV or indirectly by the need for immortalization or selection. To address direct effects of HPV infection on the host cell transcriptome, we have used our recently established infection model that allows efficient infection of primary keratinocytes with HPV16 virions. We observed only a small set of genes to be deregulated at the transcriptional level at seven days post infection (dpi), most of which fall into the category regulated by pocket proteins pRb, p107 and p130. Furthermore, cell cycle genes were not deregulated in cells infected with a virus lacking E7 despite the presence of episomal genome and viral transcripts. These findings imply that the majority of transcriptional changes are due to E7 protein impairing pocket protein function. Additional pathways, such as the Fanconi anemia-BRCA pathway, became perturbed only after long-term culturing of infected cells. When grown as organotypic raft cultures, keratinocytes infected with wild-type but not E7 mutant virus perturbed transcriptional regulations of pathways previously identified in natural lesions and in rafts derived from immortalized keratinocytes. We conclude that the HPV infection model provides a valuable tool to distinguish immediate transcriptional alterations from those induced by persistent infection and the need for selection and immortalization.

    IMPORTANCE In order to establish infection and complete the viral lifecycle, HPV needs to alter the transcriptional program of host cells. Until recently, studies were restricted to keratinocytes-derived cell lines immortalized by HPV due to the lack of experimental systems to efficiently infect primary keratinocytes. Need for selection and immortalization made it impossible to distinguish between alterations induced by HPV and secondary adaptation due to selection and immortalization. With our recent establishment of an ECM-to-cell transfer system allowing efficient infection of primary keratinocytes, we were able to identify transcriptional changes attributable to HPV16 infection. Most perturbed genes fall into the class of S phase genes, which are regulated by pocket proteins. Indeed, infection with viruses lacking E7 abrogated most transcriptional changes. It is important to note that many transcriptional alterations thought to be important for the HPV life cycle are actually late events that may reflect immortalization and possibly disease progression.

  • CpG dinucleotides inhibit HIV-1 replication through zinc finger antiviral protein (ZAP)-dependent and -independent mechanisms [Virus-Cell Interactions]

  • CpG dinucleotides are suppressed in the genomes of many vertebrate RNA viruses, including HIV-1. The cellular antiviral protein ZAP binds CpGs and inhibits HIV-1 replication when they are introduced in the viral genome. However, it is not known if ZAP-mediated restriction is the only mechanism driving CpG suppression. To determine how CpG dinucleotides affect HIV-1 replication, we increased their abundance in multiple regions of the viral genome and analyzed the effect on RNA expression, protein abundance and infectious virus production. We found that the antiviral effect of CpGs does not correlate with their abundance. Interestingly, CpGs inserted into some regions of the genome sensitize the virus to ZAP antiviral activity more efficiently than other regions and this sensitivity can be modulated by interferon treatment or ZAP overexpression. Furthermore, the sensitivity of the virus to endogenous ZAP correlated with its sensitivity to the ZAP cofactor KHNYN. Finally, we show that CpGs in some contexts can also inhibit HIV-1 replication by ZAP-independent mechanisms and one of these is the activation of a cryptic splice site at the expense of a canonical splice site. Overall, we show that the location and sequence context of the CpG in the viral genome determines its antiviral activity.

    IMPORTANCE Some RNA virus genomes are suppressed in the nucleotide combination of a cytosine followed by a guanosine (CpG), indicating that they are detrimental for the virus. The antiviral protein ZAP binds viral RNA containing CpGs and prevents the virus from multiplying. However, it remains unknown how the number and position of CpGs in viral genomes affect restriction by ZAP and whether CpGs have other antiviral mechanisms. Importantly, manipulating the CpG content in viral genomes could help create new vaccines. HIV-1 shows marked CpG suppression and, by introducing CpGs into its genome, we show that ZAP efficiently targets a specific region of the viral genome, the number of CpGs does not predict the magnitude of antiviral activity and that CpGs can inhibit HIV-1 gene expression through a ZAP-independent mechanism. Overall, the position of CpGs in the HIV-1 genome determines the magnitude and mechanism through which they inhibit the virus.

  • Guinea pig transferrin receptor 1 mediates cellular entry of Junin virus and other pathogenic New World arenaviruses [Virus-Cell Interactions]

  • Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Juniiacute;n virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 plaque-forming units of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs.

    Importance JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold-standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.

  • Broadly inhibiting anti-neuraminidase monoclonal antibodies induced by trivalent influenza vaccine and H7N9 infection in humans [Vaccines and Antiviral Agents]

  • The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time-window as seen in serological studies and the analysis of many murine monoclonal antibodies (mAbs). We report three broadly reactive human mAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody isolated from a child with acute mild H7N9 infection inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.

    Importance Antibodies to the influenza NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that for HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy, and that efficacy of influenza vaccines could be enhanced by ensuring appropriate content of NA antigen.

  • A live-attenuated influenza vaccine (LAIV) elicits enhanced heterologous protection when the internal genes of the vaccine are matched to the challenge virus [Vaccines and Antiviral Agents]

  • Influenza A virus (IAV) causes significant morbidity and mortality, despite the availability of viral vaccines. The efficacy of live attenuated influenza vaccines (LAIVs) has been especially poor in recent years. One potential reason is that the master donor virus (MDV), on which all LAIVs are based, contains either the internal genes of the 1960 A/Ann Arbor/6/60 or the 1957 A/Leningrad/17/57 H2N2 viruses (i.e., they diverge considerably from currently circulating strains). We previously showed that introduction of the temperature sensitive (ts) residue signature of the AA/60 MDV into a 2009 pandemic A/California/04/09 H1N1 virus (Cal/09) results in only 10-fold in vivo attenuation in mice. We have previously shown that the ts residue signature of the Russian A/Leningrad/17/57 H2N2 LAIV (Len LAIV) more robustly attenuates the prototypical A/Puerto Rico/8/1934 (PR8) H1N1 virus. In this work, we therefore introduced the ts signature from Len LAIV into Cal/09. This new Cal/09 LAIV is ts in vitro, highly attenuated (att) in mice, and protects from a lethal homologous challenge. In addition, when our Cal/09 LAIV with PR8 HA and NA was used to vaccinate mice, it provided enhanced protection against a wild type Cal/09 challenge relative to a PR8 LAIV with the same attenuating mutations. These findings suggest it may be possible to improve the efficacy of LAIVs by better matching the sequence of the MDV to currently circulating strains.

    Importance Seasonal influenza infection remains a major cause of disease and death, underscoring the need for improved vaccines. Among current influenza vaccines, the live attenuated influenza vaccine (LAIV) is unique in its ability to elicit T cell immunity to the conserved internal proteins of the virus. Despite this, LAIV has shown limited efficacy in recent years. One possible reason is that the conserved, internal genes of all current LAIVs derive from virus strains that were isolated between 1957 and 1960, and that - as a result - do not resemble currently circulating influenza viruses. We have therefore developed and tested a new LAIV, based on a currently circulating pandemic strain of influenza. Our results show that this new LAIV elicits improved protective immunity when compared to a more conventional LAIV.

  • Lack of B Lymphocytes Enhances CD8 T cell Mediated Resistance Against Respiratory Viral Infection but Compromises Memory Formation [Cellular Response to Infection]

  • Following a respiratory virus infection, CXCR3hiCX3CR1lo and CXCR3loCX3CR1hi CD8 T cells localize to different compartments within the lung and play an important role in host resistance, but mechanisms governing their optimal generation are poorly defined. We serendipitously found that B cell deficient (mmu;MT-/-) mice were highly resistant to lethal infection with a virulent poxvirus strain and depletion of CD8 T cells rendered these mice susceptible to infection. B cells were not required for the expansion of virus-specific CD8 T cells but a greater proportion of activated CD8 T cells acquired an effector-like CXCR3loCX3CR1hi phenotype in absence of B cells. After recovery from infection, CD8 T cells in mmu;MT-/- mice contracted normally but failed to survive and seed the memory pool in both the lungs and spleen. These findings reveal a previously unappreciated role for B cells in regulating the balance between CD8 T cell mediated resistance against respiratory viral infection and memory development.

    IMPORTANCE B cells play critical role in host resistance against many respiratory viral infections. However, the role of B cells beyond antibody producing cells is less well defined. In this report, we made a surprising observation that mice lacking B cells were more resistant to respiratory infection with vaccinia virus compared to wild-type mice. This enhanced resistance was mediated by CD8 T cells because when we depleted CD8 T cells in B cell deficient mice, these mice were unable to survive the infection. Interestingly, CD8 T cells in B cell deficient mice were skewed more towards effector phenotype and less towards memory phenotype which resulted in severely compromised memory CD8 T cell development. Thus, our study shows a novel role of B cells as regulators of CD8 T cell mediated host resistance and memory CD8 T cell formation during respiratory viral infection.

  • Naturally occurring and engineered alphaviruses sensitive to PKR show restricted translation in mammalian cells, an increased sensitivity to interferon and a marked oncotropism [Cellular Response to Infection]

  • Alphaviruses are insect-borne viruses that alternate between replication in mosquitoes and vertebrate species. Adaptation of some alphaviruses to vertebrate hosts has involved the acquisition of an RNA structure (DLP; Downstream LooP) in viral subgenomic mRNAs that confers translational resistance to PKR-mediated eIF2aalpha; phosphorylation. Here, we found that in addition to promoting eIF2-independent translation of viral subgenomic mRNAs, presence of the DLP structure also increased the resistance of alphavirus to type I interferon (IFN). Aura virus (AURAV), an ecologically isolated relative of Sindbis virus (SV) that is poorly adapted to replication in vertebrate cells, displayed a non-functional DLP structure and dramatic sensitivity to type I IFN. Our data suggest that an increased resistance to IFN emerged during translational adaptation of alphavirus mRNA to vertebrate hosts, reinforcing the role that dsRNA-activated protein kinase (PKR) plays as both a constitutive and IFN-induced antiviral effector. Interestingly, a mutant SV lacking the DLP structure (SV-DLP) and AURAV both showed a marked oncotropism for certain tumor cell lines that have defects in PKR expression and/or activation. AURAV selectively replicated in and killed some cell lines derived from human hepatocarcinoma (HCC) that lacked PKR response to infection or poly (I:C) transfection. The oncolytic activities of SV-DLP and AURAV were also confirmed using tumor xenografts in mice, showing tumor regression activities comparable to wild type SV. Our data show that translation of alphavirus subgenomic mRNAs plays a central role in IFN susceptibility and cell tropism, suggesting an unanticipated oncolytic potential that some naive arboviruses may have in virotherapy.

    Importance. Interferons (IFNs) induce the expression of a number of antiviral genes that protect the cells of vertebrates against viruses and other microbes. The susceptibility of cells to viruses greatly depends on the level and activity of these antiviral effectors, but also on the ability of viruses to counteract this antiviral response. Here, we found that the level of one of the main IFN effectors in the cell, the dsRNA-activated protein kinase (PKR), greatly determines the permissiveness of cells to alphaviruses that lack mechanisms to counteract its activation. These naive viruses also showed a hypersensitivity to IFN, suggesting that acquisition of IFN resistance (even partial) has probably been involved in expanding the host range of alphaviruses in the past. Interestingly, some of these naive viruses showed a marked oncotropism for some tumor cell lines derived from human hepatocarcinoma (HCC), opening the possibility of their use in oncolytic therapy to treat human tumors.

  • Lethal hemorrhagic disease and clinical illness associated with the elephant EEHV1 virus are caused by primary infection: Implications for the detection of diagnostic proteins. [Pathogenesis and Immunity]

  • Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, both in captivity and in the wild. Most deaths associated with this virus are caused by two chimeric variants of EEHV1 (EEHV1A and EEHV1B), while two other EEHVs endemic within Asian elephants (EEHV4 and EEHV5) have been recognized but cause death less often. Whether lethal EEHV infections are due to primary infection or reactivation of latent virus remains unknown, and knowledge of the anti-EEHV antibody levels in young elephants is limited. To close these gaps, we sought to develop a serologic assay capable of distinguishing among infections with different EEHV types using a luciferase immunoprecipitation system (LIPS) for antibody profiling and a panel of conserved EEHV recombinant proteins and proteins unique to EEHV1. The results show that elephants dying from EEHV1 hemorrhagic disease or ill from EEHV infection were seronegative for the EEHV species that caused this disease or illness, indicating that these events were associated with primary infection rather than reactivation of latent virus. We also demonstrated that waning of EEHV1-specific antibodies can occur in the first 2 years of life, when a threshold protective level of antibody may be needed to prevent severe EEHV1-related disease. Use of the LIPS assay to identify putative "diagnostic" proteins would be a valuable asset in determining the EEHV immune status of young elephants and responses to candidate EEHV vaccines in the future.

    Importance Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a long standing question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of these viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHV types and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.

  • LANA and hnRNP A1 regulate the translation of LANA mRNA through G-quadruplexes [Genome Replication and Regulation of Viral Gene Expression]

  • During the latent phase, Kaposi Sarcoma associated Herpes Virus (KSHV) maintains itself inside the host by escaping the host immune surveillance mechanism through restricted protein expression. Latency Associated Nuclear Antigen (LANA), the most abundantly expressed protein, is essential for viral persistence as it plays important roles in latent viral DNA replication and efficient segregation of the viral genome to the daughter cells following cell division. KSHV evades immune detection by maintaining the levels of LANA protein below a threshold required for detection by the host immune system but sufficient to maintain the viral genome. LANA achieves this by controlling its expression through regulation of its promoters and by inhibiting its presentation through interaction with the proteins of class I and class II MHC pathways. Here, we identified a mechanism of LANA expression and restricted immune recognition through formation of G-quadruplex in LANA mRNA. We show that the formation of these stable structures in LANA mRNA inhibits its translation to control antigen presentation, which was supported by treatment of cells with TMPyP4, a G-quadruplex stabilizing ligand. We identified heterogenous ribonucleoprotein A1 (hnRNP A1) as a G-quadruplex unwinding helicase, which unfolds these stable secondary structures to regulate LANA translation.

    Importance LANA, the most abundantly expressed protein during latency, is a multi-functional protein, which is absolutely required for the persistence of KSHV in the host cell. Even though the functions of LANA in aiding pathogenesis of the virus have been extensively studied but the mechanism of how LANA escapes host's immune surveillance is not fully understood. This study sheds light on the autoregulatory role of LANA to modulate its expression and immune evasion through formation of G-quadruplexes in its mRNA. We used G-quadruplex stabilizing ligand to define the inhibition in LANA expression and presentation on the cell surface through MHC class I. We defined the auto regulatory role of LANA and identified a cellular RNA helicase, hnRNP A1 in regulating the translation of LANA mRNA. This interaction of hnRNP A1 with LANA mRNA could be exploited for controlling KSHV latency.

  • Genome-wide mutagenesis of hepatitis C virus reveals genome ability to overcome detrimental mutations [Genetic Diversity and Evolution]

  • To gain an insight into the impact of mutations on viability of hepatitis C virus (HCV) genome, we created a set of full-genome mutant libraries, differing from the parent sequence as well as each other by using a random mutagenesis approach; the proportion of mutations increased across these libraries with declining template amount or dATP concentration. Replication efficiency of full-genome mutant libraries ranged between 71 and 329 foci forming unit (FFU) per 105 Huh7.5 cells. Mutant libraries with low proportions of mutations demonstrated low replication capability, whereas those with high proportions of mutations had their replication capability restored. Hepatoma cells transfected with selected mutant libraries, low (4 mutations per 10,000 bp copied), moderate (33 mutations) and high (66 mutations) proportions of mutations, and their progeny were subjected to serial passage. Predominant virus variants (mutants) from these mutant libraries (Mutantl, Mutantm and Mutanth, respectively) were evaluated for changes in growth kinetics and particle-to-FFU-unit ratio, virus protein expression and modulation of host cell protein synthesis. Compared to the parent, Mutantm and Mutantl produced ggt;3.0 log higher extracellular progeny per ml and, Mutanth produced progeny at a rate 1.0-log lower. More than 80% of mutations were in nonstructural part of mutant genomes, majority were nonsynonymous, and a moderate to large proportion were in the conserved regions. Our results suggest that HCV genome has the ability to overcome lethal/deleterious mutations because of the high reproduction rate, but highly selected for random, beneficial mutations.

    IMPORTANCE Hepatitis C virus (HCV) in vivo displays high genetic heterogeneity, which is partly due to the high reproduction and random substitutions during error-prone genome replication. It is difficult to introduce random substitutions in vitro because of limitations in inducing mutagenesis from the 5' end to 3'end of the genome. Our study has overcome this limitation. We synthesized full-length genomes with few to several random mutations in the background of a HCV clone that can recapitulate all steps of life cycle. Our study provides evidence for the capability of HCV genome to overcome deleterious mutations and remain viable. Mutants emerged from the libraries had diverse phenotype profiles compared to parent and putative adaptive mutations mapped to segments of conserved nonstructural genome. We demonstrate the potential utility of our system for the study of sequence variation that ensure the survival and adaptation of HCV.

  • HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function [Transformation and Oncogenesis]

  • Kaposi's sarcoma associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an acquired immunodeficiency syndrome (AIDS)-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency viruses (HIV)-infected AIDS patients has been ascribed to HIV-1 and KSHV interaction, focusing on secretory proteins. HIV-1 secreted protein HIV-Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV-Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long non-coding RNA (lncRNA) that interacts with HIV-Tat. We found that LINC00313 overexpression inhibits cell migration, invasion and tube formation, and this suppressive effect was relieve by HIV-Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components and this interaction was disrupted by HIV-Tat suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and HIV-Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV-Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV-Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313 that is up-regulated during KSHV reactivation interacts with HIV-Tat to promote endothelial cell motility. These results demonstrate that a lncRNA serves as a novel connector in HIV-KSHV interactions.

    IMPORTANCE KS is a prevalent tumor associated with two distinct viral infections, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the viral-viral interaction associated with KS formation has focused on secretory factors. HIV-Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA up-regulated during KSHV lytic reactivation, as a novel HIV-Tat interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially through interacting with chromatin remodeling complex PRC2 and down-regulation of cell migration regulating genes. Interaction between HIV-Tat and LINC00313 contributed to dissociation of PRC2 from LINC00313 and disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV co-infection.

  • The impact of antiretroviral therapy duration on the HIV-1 infection of T-cells within anatomic sites [Cellular Response to Infection]

  • Understanding the impact of antiretroviral therapy (ART) duration on HIV-infected cells is critical for developing successful curative strategies. To address this issue, we conducted a cross-sectional/inter-participant genetic characterization of HIV-1 RNA from pre- and on-therapy plasma and HIV-1 DNA from CD4+ T-cell subsets derived from peripheral blood (PB), lymph node (LN) and gut tissues of 26 participants after 3-17.8 years of ART. Our studies revealed in four acute/early participants who had paired PB and LN samples a substantial reduction in the proportion of HIV-infected cells per year on therapy within the LN. Extrapolation to all 12 acute/early participants estimated a much smaller reduction in the proportion of HIV-1 infected cells within LN per year on therapy that was similar to the participants treated during chronic infection. LN-derived effector memory T-cells (TEM) contained HIV-1 DNA that was genetically identical to viral sequences derived from pre- and on-therapy plasma samples. The proportion of identical HIV-1 DNA sequences increased within PB-derived TEM cells. However, the infection frequency of TEM cells in PB was stable, indicating cellular proliferation that compensates for T-cell loss over time contributes to HIV-1 persistence. This study suggests that ART reduces HIV-infected T-cells, and that clonal expansion of HIV-infected cells maintains viral persistence. Importantly, LN-derived TEM cells are capable of encoding infectious HIV-1, and should be targeted by future curative strategies.

    Importance HIV-1 persists as an integrated genome in CD4+ memory T-cells during effective therapy, and cessation of current treatments results in resumption of viral replication. To date, the impact of antiretroviral therapy duration on the HIV-infected CD4+ T-cells and the mechanisms of viral persistence in different anatomic sites is not clearly elucidated. In the current study, we found treatment duration was associated with a reduction of HIV-infected T-cells. Our genetic analyses revealed that CD4+ effector memory T-cells (TEM) derived from the lymph node appear to contain provirus which is genetically identical to plasma-derived virions. Moreover, we found that cellular proliferation counterbalances the decay of HIV-infected cells throughout therapy. The contribution of cellular proliferation to viral persistence is particularly significant in TEM cells. Our study emphasizes the importance of HIV-1 intervention and provides new insights into the location of memory T-cells infected with HIV-1 DNA which is capable of contributing to viremia.

  • Puumala and Andes hantaviruses cause transient PKR-dependent formation of stress granules [Virus-Cell Interactions]

  • Virus infection frequently triggers host cell stress signaling resulting in translational arrest and as a consequence many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs), but only in a limited proportion of infected cells. Our data indicates some cell type and hantavirus species specific variability in SG prevalence and shows SG formation to be dependent on the activation of PKR. Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like ER kinase (PERK).

    Importance: Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

  • Mutations in Hypervariable Domain of Venezuelan Equine Encephalitis Virus nsP3 Protein Differentially Affect Viral Replication [Virus-Cell Interactions]

  • Venezuelan equine encephalitis virus (VEEV) is one of the important human and animal pathogens. It forms replication enzyme complexes (RCs) containing viral nonstructural proteins (nsPs) that mediate synthesis of virus-specific RNAs. The assembly and associated functions of RC also depend on the presence of a specific set of host proteins. Our study demonstrates that the hypervariable domain (HVD) of VEEV nsP3 interacts with the members of the FXR family of cellular proteins and binds also the SH3 domain-containing proteins CD2AP and SH3KBP1. Interactions with FXR family members are mediated by the C-terminal repeating peptide of HVD. A single short, minimal motif identified in this study is sufficient for driving efficient VEEV replication in the absence of HVD interactions with other host proteins. The SH3 domain-containing proteins bind to another fragment of VEEV HVD. They can promote viral replication in the absence of FXR-HVD interactions, albeit less efficiently. VEEV replication can be also switched from FXR-dependent to chikungunya virus-specific, G3BP-dependent mode. The described modifications of VEEV HVD have strong impact on viral replication in vitro and its pathogenesis. Their effects on viral pathogenesis depend on the mouse age and genetic background of the virus.

    IMPORTANCE Replication of alphaviruses is determined by specific sets of cellular proteins, which mediate assembly of viral replication complexes. Some of these critical host factors interact with the hypervariable domain (HVD) of alphavirus nsP3. In this study, we have explored binding sites of host proteins, which are specific partners of nsP3 HVD of Venezuelan equine encephalitis virus. We also defined the roles of these interactions in viral replication both in vitro and in vivo. Mechanistic understanding of binding of CD2AP, SH3KBP1 and FXR protein family members to VEEV HVD uncovers important aspects of alphavirus evolution, determines new targets for development of alphavirus-specific drugs and directions for viral attenuation and vaccine development.

  • Modulation of Voltage-Gated Sodium Channel (VGSC) Activity in Human Dorsal Root Ganglion (DRG) Neurons by Herpesvirus Quiescent Infection [Cellular Response to Infection]

  • The molecular mechanisms of pain associated with alphaherpesvirus latency are not clear. We hypothesize that the voltage-gated sodium channels (VGSC) on the dorsal root ganglion (DRG) neurons controlling electrical impulses may have abnormal activity during viral latent infection and reactivation. We used HSV-1 to infect the human DRG-derived neuronal cell line HD10.6 to study the viral latency establishment, maintenance and reactivation as well as changes of VGSC functional expression. Differentiated cells exhibited robust tetrodotoxin (TTX) sensitive sodium currents and the acute infection significantly reduced the VGSC functional expression within 24 hours, and completely abolished the VGSC activity within 3 days. A quiescent state of infection mimicking latency can be achieved in the presence of acyclovir (ACV) for 7 days followed by 5 days of ACV washout and then the viruses can remain dormant for another three weeks. It was noted that during the HSV-1 latency establishment, the loss of the VGSC activity caused by HSV-1 infection could not be blocked by ACV treatment. However, neurons with continued treatment of ACV for another 4 days showed a gradual recovery of VGSC functional expression. Furthermore, the latent neurons exhibited higher VGSC activity in comparison to controls. The overall regulation of VGSC by HSV-1 during its quiescent infection was proved by increased transcription and possible translation of Nav1.7. Together these observations demonstrated a very complex pattern of electrophysiological changes during HSV infection of DRG neurons, which may have implication for understanding the mechanisms of virus-mediated pain linked to latency and reactivation.

    Importance: The reactivation of the herpesvirus, most commonly VZV and PRV, may cause cranial nerve disorder and unbearable pain. Clinical studies also reported that HSV-1 caused postherpetic neuralgia and chronic occipital neuralgia in humans. The current work meticulously studies the functional expression profile changes of VGSC during the process of HSV-1 latency establishment and reactivation using a human dorsal root ganglion neuron HD 10.6 cells as an in vitro model. Our results indicated that VGSC activity was eliminated upon infection but steadily recovered during the latency establishment and the latent neurons exhibited even higher VGSC activity. This finding advances our knowledge of how ganglion neurons generated uncharacteristic electrical impulses due to abnormal VGSC functional expression influenced by the latent virus.

  • Murine coronavirus infection activates AhR in an IDO1-independent manner contributing to cytokine modulation and pro-viral TiPARP expression [Cellular Response to Infection]

  • The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus, and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a pro-viral manner during MHV infection. We also show that MHV replication induced expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated expression levels of cytokines induced by infection, specifically IL-1bbeta;, IL-10, and TNFaalpha;, consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in WT and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors including the pro-viral factor, TiPARP, and to modulation of cytokine gene expression and identify a previously unappreciated role for AhR signaling in CoV pathogenesis.

    Importance Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

  • Epigenetic Upregulation of Chicken MicroRNA-16-5p Expression in DF-1 Cells following Infection with Infectious Bursal Disease Virus (IBDV) Enhances IBDV-induced Apoptosis and Viral Replication [Cellular Response to Infection]

  • MicroRNAs (miRNAs, miR) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, and can play important roles in the host response to pathogenic infection. Although infectious bursal disease virus (IBDV)-induced apoptosis in host cells has been established, the underlying molecular mechanism is not completely unraveled. Here we show that infection of DF-1 cells by IBDV induced gga-miR-16-5p (chicken miR-16-5p) expression via demethylation of the pre-miR-16-2 (gga-miR-16-5p precursor) promoter. We found that ectopic expression of gga-miR-16-5p in DF-1 cells enhanced IBDV-induced apoptosis by directly targeting cellular anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), facilitating IBDV replication in DF-1 cells. On the contrary, inhibition of endogenous miR-16-5p markedly suppressed apoptosis associated with enhanced Bcl-2 expression, arresting viral replication in DF-1 cells. Furthermore, infection of DF-1 cells with IBDV reduced Bcl-2 expression, and this reduction could be abolished by inhibition of gga-miR-16-5p expression. Moreover, transfection of DF-1 cells with gga-miR-16-5p mimics enhanced IBDV-induced apoptosis associated with increased cytochrome c release, caspase-9/3 activation, and inhibition of caspase-3 decreased IBDV growth in DF-1 cells. Thus, epigenetic upregulation of gga-miR-16-5p expression by IBDV infection enhances IBDV-induced apoptosis by targeting cellular anti-apoptotic protein Bcl-2, facilitating IBDV replication in host cells.

    Importance Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive disease in young chickens, causing severe economic losses to stakeholders across the globe. Although IBD virus (IBDV)-induced apoptosis in host has been established, the underlying mechanism is not very clear. Here we show that infection of DF-1 cells by IBDV upregulated gga-miR-16-5p expression via demethylation of the pre-miR-16-2 promoter. Overexpression of gga-miR-16-5p enhanced IBDV-induced apoptosis associated with increased cytochrome c release, caspase-9 and -3 activation. Importantly, we found that IBDV infection induced expression of gga-miR-16-5p that triggered apoptosis by targeting Bcl-2, favoring IBDV replication, while inhibition of gga-miR-16-5p in IBDV-infected cells restored Bcl-2 expression, slowing down viral growth, indicating that IBDV induces apoptosis by epigenetic upreulation of gga-miR-16-5p expression. These findings uncover a novel mechanism employed by IBDV for its own benefit, which may be used as a potential target for intervening IBDV infection.

  • The G protein-coupled receptor FFAR2 promotes internalization during influenza A virus entry [Virus-Cell Interactions]

  • Influenza A virus (IAV) co-opts numerous host factors to complete its replication cycle. Here, we identified free fatty acid receptor 2 (FFAR2) as a cofactor for IAV entry into host cells. We found that downregulation of FFAR2 or Ffar2 expression significantly reduced the replication of IAV in A549 or RAW 264.7 cells. The treatment of A549 cells with FFAR2 siRNA or the FFAR2 pathway agonists 4-CMTB and compound 58 [Cmp 58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide], dramatically inhibited the nuclear accumulation of viral NP protein at early timepoints post infection, indicating that FFAR2 functions in the early stage of IAV replication cycle. FFAR2 downregulation had no effect on the expression of sialic acid (SA) receptors on the cell membrane, the attachment of IAV to the SA receptors, or the activity of the viral ribonucleoprotein (RNP) complex. Rather, the amount of internalized IAVs was significantly reduced in FFAR2 knocked-down, 4-CMTB-, or Cmp 58-treated A549 cells. Further studies showed that FFAR2 associated with bbeta;-arrestin1 and that bbeta;-arrestin1 interacted with the bbeta;2-subunit of the AP-2 complex (AP2B1), the essential adaptor of the clathrin-mediated endocytosis pathway. Notably, siRNA knockdown of either bbeta;-arrestin1 or AP2B1 dramatically impaired IAV replication, and AP2B1 knockdown or treatment with Barbadin, an inhibitor targeting the bbeta;-arrestin1/AP2B1 complex, remarkably decreased the amount of internalized IAVs. Moreover, we found that FFAR2 interacted with three GPCR kinases (i.e., GRK2, GRK5, and GRK6) whose downregulation inhibited IAV replication. Together, our findings demonstrate that the FFAR2 signaling cascade is important for the efficient endocytosis of IAV into host cells.

    IMPORTANCE To complete its replication cycle, IAV hijacks the host endocytosis machinery to invade cells. However, the underlying mechanisms of how IAV is internalized into host cells remain poorly understood, emphasizing the need to elucidate the role of host factors in IAV entry into cells. In this study, we identified FFAR2 as an important host factor for the efficient replication of both low pathogenic and highly pathogenic IAV. We revealed that FFAR2 facilitates the internalization of IAV into target cells during the early stage of infection. Upon further characterization of the role of FFAR2-associated proteins in virus replication, we found that the FFAR2nndash;bbeta;-arrestin1nndash;AP2B1 signaling cascade is important for the efficient endocytosis of IAV. Our findings thus further our understanding of the biological details of IAV entry into host cells and establish FFAR2 as a potential target for antiviral drug development.

  • Engineering Newcastle disease virus as oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines [Vaccines and Antiviral Agents]

  • Newcastle Disease Virus (NDV) is an attractive candidate for oncolytic immunotherapy due to its ability to replicate in tumor cells and potentially overcome the inherently immunosuppressive nature of the tumor microenvironment. The advent of checkpoint blockade immunotherapy over the past few years represents a paradigm shift in cancer therapy. However, the prevalence of severe immune-related adverse events with CTLA-4 and PD1 pathway blockade in clinical studies especially in combination therapy groups, is a cause for concern. Immunotherapies with cytokines have also been extensively explored but they have been associated with adverse events in clinical trials. Oncolytic vectors engineered to express checkpoint blockade antibodies and cytokines could provide an avenue for reducing the clinical toxicity associated with systemic therapy by concentrating the immunomodulatory payload at the site of disease. In this study, we engineered six different recombinant viruses, NDVs expressing checkpoint inhibitors (rNDV-anti-PD1 and rNDV-anti-PDL1), superagonists (rNDV-anti-CD28) and immunocytokines, where these antibodies are fused to an immunostimulatory cytokine such as IL12 (rNDV-anti-CD28-mIL12, rNDV-anti-PD1-mIL12 and rNDV-anti-PDL1-mIL12). These six engineered viruses induced tumor control and survival benefit both in a highly aggressive unilateral and bilateral B16-F10 murine melanoma model, indicative of an abscopal effect. The data represent a strong proof-of-concept, on which further clinical evaluation could build.

    IMPORTANCE Checkpoint inhibitor therapy has shown tremendous efficacy but also frequent and often severe side effectsmmdash;especially when multiple drugs of this class are used simultaneously. Similarly, many investigational immunotherapy agents, which have shown promise in animal models, have failed in clinical trials due to dose-limiting toxicity when administered systemically. This study utilized a murine melanoma model to evaluate the efficacy of intratumoral injections of recombinant NDVs engineered to express multiple immunotherapeutic proteins with well-documented side effects in humans. Our results indicate that intratumoral administration of these recombinant NDVs, particularly when combined with systemic CTLA4 checkpoint inhibition, exerts a robust effect in treated and non-treated tumors indicative of a systemic anti-tumoral response. The intratumoral delivery of rNDVs expressing immunotherapeutic proteins may be an effective method of targeting immune cell populations most relevant for anti-tumoral immunity and allowing to restrict the use of systemic immunotherapy agents.

  • Experimental evolution generates novel oncolytic vesicular stomatitis viruses with improved replication in virus-resistant pancreatic cancer cells [Genome Replication and Regulation of Viral Gene Expression]

  • Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against various cancers. We have shown that pancreatic ductal adenocarcinoma (PDAC) cell lines exhibit great diversity in susceptibility and permissibility to VSV. Here, using a directed evolution approach with our two previously described oncolytic VSV recombinants, VSV-p53wt and VSV-p53-CC, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines. VSV-p53wt and VSV-p53-CC encode VSV matrix protein (M) with M51 mutation (M-M51) and one of two versions of a functional human tumor suppressor p53 fused to a far-red fluorescent protein eqFP650. Each virus was serially passaged 32 times (accounts for more than 60 viral replication cycles) on either SUIT-2 (moderately resistant to VSV) or MIA PaCa-2 (highly permissive to VSV) human PDAC cell lines. While no phenotypic changes were observed for MIA PaCa-2-passaged viruses, both SUIT-2-passaged VSV-p53wt and VSV-p53-CC showed improved replication in SUIT-2 and AsPC-1, another human PDAC cell line also moderately resistant to VSV, while remaining highly attenuated in non-malignant cells. Surprisingly, two identical VSV glycoprotein (G) mutations, K174E and E238K, were identified in both SUIT-2-passaged viruses. Additional experiments indicated that the acquired G mutations improved VSV replication at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no mutations were found in the M-M51 protein and no deletions or mutations were found in the p53 or eqFP650 portions of virus-encoded transgenes in any of the passaged viruses, demonstrating long-term genomic stability of complex VSV recombinants encoding large transgenes.

    IMPORTANCE Vesicular stomatitis virus (VSV) based oncolytic viruses are promising agents against pancreatic ductal adenocarcinoma (PDAC). However, some PDAC cell lines are resistant to VSV. Here, using directed viral evolution approach, we generated novel oncolytic VSVs with an improved ability to replicate in virus-resistant PDAC cell lines, while remaining highly attenuated in non-malignant cells. Two independently evolved VSVs obtained 2 identical VSV glycoprotein mutations, K174E and E238K. Additional experiments indicated that these acquired G mutations improved VSV replication at least in part due to improved virus attachment to SUIT-2 cells. Importantly, no deletions or mutations were found in the virus-encoded transgenes in any of the passaged viruses. Our findings demonstrate long-term genomic stability of complex VSV recombinants encoding large transgenes, and support further clinical development of oncolytic VSV recombinants as safe therapeutics for cancer.

  • Amphipathic helices of cellular proteins can replace the helix in M2 of Influenza A virus with only small effects on virus replication [Structure and Assembly]

  • M2 of influenza virus functions as proton channel during virus entry. In addition, an amphipathic helix in its cytoplasmic tail plays a role during budding. It targets M2 to the assembly site where it inserts into the inner membrane leaflet to induce curvature that causes virus scission. Since vesicularisation of membranes can be performed by a variety of amphiphilic peptides we used reverse genetics to investigate whether they can substitute for M2's helix.

    Virus could not be generated if M2's helix was deleted or replaced by a peptide predicted not to form an amphiphilic helix. In contrast, viruses could be rescued if the M2 helix was exchanged by helices known to induce membrane curvature. Infectious virus titers were marginally reduced if M2 contains the helix of the amphipathic lipid packing sensor, from the Epsin N-Terminal Homology domain or the non-natural membrane inducer RW16. Transmission EM of infected cells did not reveal unequivocal evidence that virus budding or membrane scission was disturbed in any of the mutants. Instead, individual virus mutants exhibit other defects in M2, such as reduced surface expression, incorporation into virus particles and ion channel activity. The protein composition and specific infectivity was also altered for mutant virions. We conclude that the presence of an amphiphilic helix in M2 is essential for virus replication, but other helices can replace its basic (curvature-inducing) function.

    Importance Influenza is unique among enveloped viruses since it does not rely on the cellular ESCRT-machinery for budding. Instead viruses encode their own scission machine, the M2 protein. M2 is targeted to the edge of the viral assembly site where it inserts an amphiphilic helix into the membrane to induce curvature. Cellular proteins utilize a similar mechanism for scission of vesicles. We show that the helix of M2 can be replaced by helices from cellular proteins with only small effects on virus replication. No evidence was obtained that budding is disturbed, but individual mutants exhibit other defects in M2 which explain the reduced virus titers. In contrast, no virus could be generated if the helix of M2 is deleted or replaced by irrelevant sequences. These experiments support the concept that M2 requires an amphiphilic helix to induce membrane curvature, but its biophysical properties are more important than the amino acid sequence.

  • HIV-1 co-receptor usage and variable loop contact impacts V3 loop bnAb susceptibility [Pathogenesis and Immunity]

  • In clinical trials, HIV-1 broadly neutralizing antibodies (bnAbs) effectively lower plasma viremia and delay virus reemergence. Presence of less neutralization susceptible strains prior to treatment decreases the efficacy of these antibody-based treatments, but neutralization sensitivity often cannot be predicted by sequence analysis alone. We find that phenotypically confirmed CXCR4- as compared to exclusive CCR5-utilizing strains are less neutralization sensitive, especially to variable loop 3 (V3) directed bnAbs in some but not all cases. Homology modeling suggests that the primary V3 loop bnAb epitope is equally accessible among CCR5- and CXCR4-using strains although variants that exclusively use CXCR4 have V3 loop protrusions that interfere with CCR5 receptor interactions. Homology modeling also shows that among some but not all envelopes with decreased neutralization sensitivity, V1 loop orientation interferes with V3 loop directed bnAb binding. Thus, there are likely different structural reasons for the co-receptor usage restriction and the differential bnAb susceptibility. Importantly, we show that individuals harboring envelopes with higher likelihood of using CXCR4 or greater predicted V1 loop interference have faster virus rebound and lower maximum decrease in plasma viremia respectively after treatment with a V3 loop bnAb. Knowledge about receptor usage and homology models may be useful in developing future algorithms that predict treatment efficacy with V3 loop bnAbs.

    IMPORTANCE The efficacy of HIV-1 broadly neutralizing antibody (bnAb) therapies may be compromised by the pre-existence of less susceptible variants. Sequence-based methods are needed to predict pre-treatment variants' neutralization sensitivity. HIV-1 strains that exclusively use the CXCR4 as compared to the CCR5 receptor are less neutralization susceptible, especially to variable loop 3 (V3) loop bnAbs in some but not all instances. While the inability to utilize the CCR5 receptor maps to a predicted protrusion in the envelope V3 loop, this viral determinant does not directly influence V3 loop bnAb sensitivity. Homology modeling predicts contact between the envelope V1 loop and antibody impacts V3 loop bnAb susceptibility in some cases. Among pre-treatment envelopes, increased probability of using CXCR4 and greater predicted V1 interference is associated with faster virus rebound and lower decrease in plasma virus level respectively after V3 loop bnAb treatment. Receptor usage information and homology models may be useful for predicting V3 loop bnAb therapy efficacy.

  • Intact Viral Particle Counts Measured by Flow Virometry Provides Insight into the Infectivity and Genome Packaging Efficiency of the Moloney Murine Leukemia Virus [Genome Replication and Regulation of Viral Gene Expression]

  • Murine leukemia viruses (MLVs) have long been used as a research model to further our understanding of retroviruses. These simple gammaretroviruses have been studied extensively in various facets of science for nearly half a century, yet we have surprisingly little quantitative information about some of the basic features of these viral particles. These include parameters such as the genome packaging efficiency and the number of particles required for a productive infection. The reason for this knowledge gap relies primarily on the technical challenge of accurately measuring intact viral particles from infected cell supernatants. Virus infected cells are well known to release soluble viral proteins, defective viruses and extracellular vesicles (EVs) harboring viral proteins that may mimic viruses, all of which can skew virus titer quantifications. Flow virometry, also known as nanoscale flow cytometry or simply small particle flow cytometry, is an emerging analytical method enabling high throughput single-virus phenotypic characterizations. By utilizing the viral envelope glycoprotein (Env) and monodisperse light scattering characteristics as discerning parameters of intact virus particles, here we analyzed the basic properties of Moloney MLV (M-MLV). We show that less than 24% of total p30 capsid protein measured in infected cell supernatants is associated with intact viruses. We calculate that about one in five M-MLV particles contain a viral RNA genome pair and that individual intact particle infectivity is about 0.4%. These findings provide new insights into the characteristics of an extensively studied prototypical retrovirus, while highlighting the benefits of flow virometry for the field of virology.

    IMPORTANCE Gammaretroviruses, or more specifically murine leukemia viruses (MLVs), have been a longstanding model for studying retroviruses. Although being extensively analysed and dissected for decades, several facets of MLV biology are still poorly understood. One of the primary challenges has been enumerating total intact virus particles in a sample. While several analytical methods can precisely measure virus protein amounts, MLVs are known to induce the secretion of soluble and vesicle-associated viral proteins that can skew these measurements. With recent technological advances in flow cytometry, it is now possible to analyze viruses down to 90nm in diameter with an approach called flow virometry. The technique has the added benefit of being able to discriminate viruses from extracellular vesicles and free viral proteins in order to confidently provide an intact viral particle count. Here we used flow virometry to provide new insights into the basic characteristics of Moloney MLV.

  • A CD4+ T cell/NK cell axis of {gamma}-herpesvirus control [Pathogenesis and Immunity]

  • CD4+ T cells are essential to control herpesviruses. Murid Herpesvirus-4 (MuHV-4)-driven lung disease in CD4+ T cell-deficient mice provides a well studied example. Protective CD4+ T cells have been hypothesized to kill infected cells directly. However removing MHC class II (MHCII) from LysM+ or CD11c+ cells increased MuHV-4 replication not in those cells but in type 1 alveolar epithelial cells, which lack MHCII, LysM or CD11c. Disrupting MHCII in infected cells had no effect. Therefore CD4+ T cells engaged uninfected presenting cells, and protected indirectly. Mice lacking MHCII in LysM+ or CD11c+ cells maintained systemic anti-viral CD4+ T cell responses, but recruited fewer CD4+ T cells into infected lungs. NK cell infiltration was also reduced, and NK cell depletion normalized infection between MHCII-deficient and control mice. Therefore NK cell recruitment seemed to be an important component of CD4+ T cell-dependent protection. Disrupting viral CD8+ T cell evasion made this defence redundant, suggesting that it is important mainly to control CD8-evasive pathogens.

    Importance Gamma-herpesviruses are widespread and cause cancers. CD4+ T cells are a key defence. We found that they defend indirectly, engaging uninfected presenting cells and recruiting innate immune cells to attack infected targets. This segregation of CD4+ T cells from immediate contact with infection helps the immune system to cope with viral evasion. Priming this defence by vaccination offers a way to protect against gamma-herpesvirus-induced cancers.

  • The Novel Nuclear Targeting and BFRF1-interacting Domains of BFLF2 are Essential for Efficient EBV Virion Release [Virus-Cell Interactions]

  • Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it co-localizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in co-expression cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 are critical for their proper function. The N-terminal sequence of BFLF2 is less conserved to that of alpha- and beta herpesvirus homologs. Here we found BFLF2 a.a. 2-102 is required for both nuclear targeting and its interaction with BFRF1. Co-immunoprecipitation and confocal analysis indicated that a.a 82-106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, R52) and several non-continuous arginine and histidine residues within a.a. 59-80 function together as a non-canonical NLS, which can be transferred onto YFP-LacZ for nuclear targeting, in an importin bbeta;-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction, and is restored by trans-complementation of wild type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.

    Importance Although EBV BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N-terminus of BFRF2. We showed that a.a. 82-106 are the major region required for BFLF2 to interact with BFRF1. However, the Co-IP data and GST-pull down experiments revealed that multiple regions of both proteins contribute to reciprocal interaction. Different from the canonical NLS in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within a.a. 82-106 of BFLF2 are required for virion secretion.

  • VAMP8 contributes to TRIM6-mediated type-I interferon antiviral response during West Nile virus infection [Virus-Cell Interactions]

  • Several members of the tripartite motif (TRIM) family of E3 ubiquitin ligases regulate immune pathways including the antiviral type I interferon (IFN-I) system. Previously, we demonstrated that TRIM6 is involved in IFN-I induction and signaling. In the absence of TRIM6, optimal IFN-I signaling is reduced, allowing increased replication of interferon-sensitive viruses. Despite having evolved numerous mechanisms to restrict the vertebrate host's IFN-I response, West Nile virus (WNV) replication is sensitive to pre-treatment with IFN-I. However, the regulators and products of the IFN-I pathway that are important in regulating WNV replication are incompletely defined. Consistent with WNV's sensitivity to IFN-I, we found that in TRIM6 knockout (TRIM6-KO) A549 cells WNV replication is significantly increased and IFN-I induction and signaling is impaired compared to wild-type (wt) cells. IFNbbeta; pre-treatment was more effective in protecting against subsequent WNV infection in wt cells as compared to TRIM6-KO, indicating that TRIM6 contributes to the establishment of an IFN-induced antiviral response against WNV. Using next generation sequencing, we identified VAMP8 as a potential factor involved in this TRIM6-mediated antiviral response. VAMP8 knockdown resulted in reduced Jak1 and STAT1 phosphorylation and impaired induction of several ISGs following WNV infection or IFNbbeta; treatment. Furthermore, VAMP8-mediated STAT1 phosphorylation required the presence of TRIM6. Therefore, the VAMP8 protein is a novel regulator of IFN-I signaling, and its expression and function is dependent on TRIM6 activity. Overall, these results provide evidence that TRIM6 contributes to the antiviral response against WNV and identified VAMP8 as a novel regulator of the IFN-I system.

    IMPORTANCE WNV is a mosquito-borne flavivirus that poses threat to human health across large discontinuous areas throughout the world. Infection with WNV results in febrile illness, which can progress to severe neurological disease. Currently, there are no approved treatment options to control WNV infection. Understanding the cellular immune responses that regulate viral replication is important in diversifying the resources available to control WNV. Here we show that the elimination of TRIM6 in human cells results in an increase in WNV replication and alters the expression and function of other components of the IFN-I pathway through VAMP8. Dissecting the interactions between WNV and host defenses both informs basic molecular virology and promotes the development of host- and viral-targeted antiviral strategies.

  • Near atomic resolution cryo-electron microscopy structures of cucumber leaf spot virus and red clover necrotic mosaic virus; evolutionary divergence at the icosahedral three-fold axes. [Structure and Assembly]

  • Members of the Tombusviridae family have highly similar structures and yet there are important differences among them in host, transmission, and capsid stabilities. Viruses in the Tombusviridae family have ssRNA genomes with T=3 icosahedral protein shells with a maximum diameter of ~340AAring;. Each capsid protein is comprised of three domains: R (RNA binding), S (shell), and P(protruding). Between the R and S domain is the llsquo;armrrsquo; region that studies have shown to play a critical role in assembly. To better understand how the details of structural differences and similarities influence the Tombusviridae viral lifecycles, the structures of cucumber leaf spot virus (CLSV, genus Aureusvirus) and red clover necrotic mosaic (RCNMV, genus Dianthovirus) were determined to resolutions of 3.2AAring; and 2.9AAring;, respectively, with cryo-electron microscopy and image reconstruction methods. While the shell domains had homologous structures, the stabilizing interactions at the icosahedral 3-fold axes and the R domains differed greatly. The heterogeneity in the R domains among the Tombusviridae family are likely correlated with differences in the size and characteristics of the corresponding genomes. We propose that the changes in the R domain/RNA interactions evolved different arm domain interactions at the bbeta;-annuli. For example, RCNMV has the largest genome and it appears to have created the necessary space in the capsid by evolving the shortest R domain. The resulting loss in RNA/R domain interactions may have been compensated for by increased inter-subunit bbeta;-strand interactions at the icosahedral 3-fold axes. Therefore, the R and arm domains may have co-evolved to package different genomes within the conserved and rigid shell.

    Importance Members of the Tombusviridae family have nearly identical shells and yet they package genomes that range from 4.6kb (monopartite) to 5.3 kb (bi-partite) in size. To understand how this genome flexibility occurs within a rigidly conserved shell, we determined the high resolution cryo-EM structures of cucumber leaf spot virus and red clover necrotic mosaic virus. In response to genomic size differences, it appears that the ssRNA binding (R) domain of the capsid diverged evolutionarily in order to recognize the different genomes. The next region, the llsquo;armrrsquo;, seems to have also co-evolved with the R domain to allow for particle assembly via interactions at the icosahedral 3-fold axes. In addition, there are differences at the icosahedral 3-fold axes with regard to metal binding that are likely important for transmission and the viral life cycle.

  • HA stability regulates H1N1 influenza virus replication and pathogenicity in mice by modulating type I interferon responses in dendritic cells [Pathogenesis and Immunity]

  • Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated mechanisms by which a destabilizing HA mutation, Y17H (activation pH 6.0), attenuates virus replication and pathogenicity in DBA/2 mice, compared to wild-type (WT; activation pH 5.5). Extracellular lung pH was measured to be near neutral (pH 6.9nndash;7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and Raw264.7 cells when inoculated at an MOI of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrownndash;derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced replication in murine airway mNEC and mTEC cultures and attenuated virus replication, virus spread, severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in greater activation of antiviral responses, including type I IFN. These studies reveal HA stability may regulate pathogenicity by modulating IFN responses.

    Importance Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza. Understanding HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

  • Three Amino Acid Changes In Avian Coronavirus Spike Protein Allows Binding To Kidney Tissue [Virus-Cell Interactions]

  • Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here we elucidate the determinants for kidney tropism by studying interactions between the receptor binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the non-nephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by pre-incubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by ELISA, demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99-159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110-112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor binding site for QX is located at a different location on the spike than that of M41.

    Importance: Infectious bronchitis virus is the causative agent of Infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV in two pathotypes: non-nephropathogenic (example IBV-M41) and nephropathogenic viruses (including IBV-QX). Here we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field.

  • Elucidating the interactions between influenza virus polymerase and host factor ANP32A [Virus-Cell Interactions]

  • The avian origin influenza A viral polymerase is restricted in human cells. This restriction has been associated with species differences in host factor ANP32A. Avian ANP32A supports the activity of an avian origin polymerase. However, the avian origin polymerase is incompatible with human ANP32A. Avian ANP32A proteins harbour an additional 33 amino acids compared to human ANP32A proteins, which are crucial for their ability to support the avian origin influenza polymerase. Here, we elucidate the interactions between ANP32A proteins and the influenza A viral polymerase using split luciferase complementation assays, co-immunoprecipitation and in situ split Venus interaction assays. We show greater interaction between chicken ANP32A and the viral polymerase compared to human ANP32A and visualise these interactions in situ in the cell nucleus. We demonstrate that the 33 amino acids of chicken ANP32A and the PB2 627 domain of viral polymerase complex both contribute to this enhanced interaction. Finally, we show how these interactions are affected by the presence of viral RNA and the processivity of the polymerase, giving insights into the way that ANP32A might act during virus infection.

    Importance Successful zoonotic transmission of influenza A virus into humans can lead to pandemics in an immunologically naïve population. Host encoded ANP32A proteins are required to support influenza A polymerase activity, and species differences in ANP32A can restrict host range of influenza. Understanding how ANP32A proteins support the viral polymerase and how differences in ANP32A affect the ability of the polymerase to co-opt these proteins will enhance our understanding of viral replication and species restriction as well as suggesting targeted antiviral approaches to treat influenza infection.

  • Foot-and-mouth disease virus capsid protein VP1 interacts with host ribosomal protein SA to maintain the activation of MAPKs signal pathway and promote virus replication [Virus-Cell Interactions]

  • Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease, a highly contagious, economically important viral disease. The structural protein VP1 plays significant roles during FMDV infection. Here, we identified that VP1 interacted with host ribosomal protein SA (RPSA). RPSA is a viral receptor for dengue virus and classical swine fever virus infections. However, the incubation of susceptible cells using the anti-RPSA antibodies did not block the infection of FMDV. Overexpression of porcine RPSA in the insusceptible cells could not trigger FMDV infection, suggesting RPSA was not responsible for FMDV entry and infection. On the contrary, we found that overexpression of RPSA suppressed FMDV replication, and knockdown of RPSA enhanced FMDV replication. We further determined that FMDV infection activated mitogen-activated protein kinases (MAPKs) pathway, and demonstrated that MAPKs pathway activation was critically important for FMDV replication. RPSA negatively regulated MAPKs pathway activation during FMDV infection, showing an antiviral function. FMDV VP1 interacted with RPSA to abrogate RPSA-mediated suppressive role on MAPKs pathway activation. Together, our study indicated that the MAPKs pathway activation was required for FMDV replication, and host RPSA played a negatively regulatory role on MAPKs pathway activation to suppress FMDV replication. FMDV VP1 bound to RPSA to promote viral replication by repressing RPSA-mediated function and maintaining the activation of MAPKs signal pathway.

    IMPORTANCE Identification of virus-cell interactions is essential for making strategies to limit virus replication and refine the models of virus replication. This study demonstrated that FMDV utilized the MAPKs pathway for viral replication. The host RPSA protein inhibited FMDV replication by suppressing the activation of MAPKs pathway during FMDV infection. FMDV VP1 bound to RPSA to repress RPSA-mediated regulatory effect on MAPKs pathway activation. This work revealed an important implication of MAPKs pathway for FMDV infection and identified a novel mechanism by which FMDV VP1 has evolved to interact with RPSA and maintain the activation of MAPKs pathway, elucidating new information regarding the signal reprogramming of host cells by FMDV.

  • Kaposi's sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34 [Genome Replication and Regulation of Viral Gene Expression]

  • Kaposi's sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34, transcription of K8.1 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production.

    IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therfore, we generated a ORF66-deficient BAC clone and evaluated its viral replication. Results showed that ORF66 played a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing ORF66 function in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacted with the transcription start site of K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C- terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among bbeta;- and -herpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

  • The Capsid Protein of Semliki Forest Virus Antagonizes RNAi in Mammalian Cells [Virus-Cell Interactions]

  • RNA interference (RNAi) is a conserved antiviral immune defence in eukaryotes, and numerous viruses have been found to encode viral suppressors of RNAi (VSRs) to counteract antiviral RNAi. Alphaviruses are a large group of positive-stranded RNA viruses that maintain their transmission and life cycles in both mosquitoes and mammals. However, there is little knowledge about how alphaviruses antagonize RNAi in both host organisms. In this study, we identified that Semliki Forest virus (SFV) capsid protein can efficiently suppress RNAi in both insect and mammalian cells by sequestrating dsRNA and siRNA. More importantly, when the VSR activity of SFV capsid was inactivated by reverse genetics, the resulting VSR-deficient SFV mutant showed severe replication defects in mammalian cells, which could be rescued by blocking the RNAi pathway. Besides, capsid protein of Sindbis virus (SINV) also inhibited RNAi in cells. Together, our findings show that SFV uses capsid protein as VSR to antagonize RNAi in infected mammalian cells, and this mechanism is probably used by other alphaviruses, which shed new light on the knowledge of SFV and alphavirus.

    Importance Alphaviruses are a genus of positive-stranded RNA viruses and include numerous important human pathogens, such as Chikungunya virus, Ross River virus, Western equine encephalitis virus, etc, which create the emerging and re-emerging public health threat worldwide. RNA interference (RNAi) is one of the most important antiviral mechanisms in plants and insects. Accumulating evidence has provided strong support for the existence of antiviral RNAi in mammals. In response to antiviral RNAi, viruses have evolved to encode viral suppressors of RNAi (VSRs) to antagonize the RNAi pathway. It is unclear if alphaviruses encode VSRs that can suppress antiviral RNAi during their infection in mammals. In this study, we first uncovered that capsid protein encoded by Semliki Forest virus (SFV), a prototypic alphavirus, had a potent VSR activity that can antagonize antiviral RNAi in the context of SFV infection in mammalian cells, and this mechanism is probably used by other alphaviruses.

  • Human Cytomegalovirus Alters Host Cell Mitochondrial Function During Acute Infection [Cellular Response to Infection]

  • Human cytomegalovirus (HCMV) is a large DNA herpesvirus that is highly prevalentin the human population. HCMV can result in severe direct and indirect pathologies under immunosuppressed conditions and is the leading cause of birth defects related to infectious disease. Currently, the effect of HCMV infection on host cell metabolism as an increase in glycolysis during infection has been defined. We have observed that oxidative phosphorylation is also increased. We have identified morphological and functional changes to host mitochondria during HCMV infection. The mitochondrial network undergoes fission events after HCMV infection. Interestingly, the network does not undergo fusion. At the same time mitochondrial mass and membrane potential increase. The electron transport chain (ETC) functions at an elevated rate resulting in the release of increased reactive oxygen species. Surprisingly, despite the stress applied to the host mitochondria, the network is capable of responding to and meeting the increased bioenergetic and biosynthetic demands placed on it. When mitochondrial DNA is depleted from the cells, we observed severe impairment of viral replication. Mitochondrial DNA encodes many of the ETC components. These findings suggest that the host cell ETC is essential to HCMV replication. Our studies suggest the host cell mitochondria may be a therapeutic target.

    IMPORTANCE Human cytomegalovirus (HCMV) is a herpesvirus present in up to 85% of some populations. Like all herpesviruses, HCMV infection is for life. There is no vaccine currently available, neutralizing antibody therapies are ineffective and current antivirals have limited long-term efficacy due to side effects and potential for viral mutation and resistance. The significance of this research is in understanding how HCMV manipulates the host mitochondria to support bioenergetic and biosynthetic requirements for replication. Despite a large genome, HCMV relies exclusively on host cells for metabolic functions. By understanding the dependency of HCMV on the mitochondria, we could exploit these requirements and develop novel antivirals.

  • Genetic diversity of Collaborative Cross mice controls viral replication, clinical severity and brain pathology induced by Zika virus infection, independently of Oas1b. [Pathogenesis and Immunity]

  • The explosive spread of Zika virus (ZIKV) has been associated with major variations in severe disease and congenital afflictions among infected populations, suggesting an influence of host genes. We investigated how genome-wide variants could impact susceptibility to ZIKV infection in mice. We first describe that the susceptibility of Ifnar1 knockout mice is largely influenced by their genetic background. We then show that the broad genetic diversity of Collaborative Cross mice, of which receptor to type I interferon (IFNAR) was blocked by anti-IFNAR antibody, expressed phenotypes ranging from complete resistance to severe symptoms and death with large variations in the peak and rate of decrease of plasma viral load, in brain viral load, in brain histopathology and in viral replication rate in infected cells. Differences in susceptibility between CC strains were correlated between Zika, Dengue and West Nile viruses. We identified highly susceptible and resistant mouse strains as new models to investigate the mechanisms of human ZIKV disease and other flavivirus infections. Genetic analyses revealed that phenotypic variations are driven by multiple genes with small effects, reflecting the complexity of ZIKV disease susceptibility in human population. Notably, our results rule out a role of the Oas1b gene in the susceptibility to ZIKV. Altogether, this study emphasizes the role of host genes in the pathogeny of ZIKV infection and lays the foundation for further genetic and mechanistic studies.

    IMPORTANCE In recent outbreaks, ZIKV has infected millions of people and induced rare but potentially severe complications, including Guillain-Barreeacute; syndrome and encephalitis in adults. While several viral sequence variants were proposed to enhance the pathogenicity of ZIKV, the influence of host genetic variants in mediating the clinical heterogeneity remains mostly unexplored. We have addressed this question using a mouse panel which models the genetic diversity of human population and a ZIKV strain from a recent clinical isolate. Through a combination of in vitro and in vivo approaches, we demonstrate that multiple host genetic variants determine viral replication in infected cells, and clinical severity, kinetics of blood viral load and brain pathology in mice. We describe new mouse models expressing high susceptibility or resistance to ZIKV and to other flaviviruses. These models will facilitate the identification and mechanistic characterization of host genes that influence ZIKV pathogenesis.

  • Identifying the cellular interactome of Epstein-Barr virus lytic regulator Zta reveals cellular targets contributing to viral replication. [Genome Replication and Regulation of Viral Gene Expression]

  • The human gamma herpesvirus Epstein-Barr virus (EBV; HHV4), infects most adults and is an important contributor to the development of many types of lymphoid and epithelial cancers. Essential contributions of viral genes to viral replication are known, but the potential contributions of cell genes are less well delineated. A key player is the viral protein Zta (BZLF1, ZEBRA, Z). This sequence-specific DNA-binding protein can disrupt EBV latency by driving the transcription of target genes and by interacting with the EBV lytic origin of replication. Here we used an unbiased proteomics approach to identify the Zta-interactome in cells derived from a Burkitt's lymphoma. Isolating Zta and associated proteins from Burkitt's lymphoma cells undergoing EBV replication, followed by Tandem Mass Tag (TMT) mass spectrometry resulted in the identification of thirty-nine viral and cellular proteins within the Zta interactome. An association of Zta with the cellular protein NFATc2 was validated in independent experiments. Furthermore, the ability of Zta to attenuate the activity of an NFAT-dependent promoter was shown which suggests a functional consequence for the association. The expression of Zta is itself regulated through NFAT activity, suggesting that Zta may contribute to a feed-back loop that would limit its own expression, thus aiding viral replication by preventing the known toxic effects of Zta overexpression.

    Data are available: ProteomeXchange PXD013727.

    Importance: Epstein-Barr virus, infects most people across the world and causes several kinds of cancer. Zta is an important viral protein that makes the virus replicate by binding to its DNA and turning on the expression of some genes. We used a sensitive, unbiased approach to isolate and identify viral and cellular proteins that physically interact with Zta. This revealed thirty-nine viral and cellular proteins. We found that one protein termed NFATc2, was already known to be important for a very early step in viral replication. We identify that once this step has occurred, Zta reduces the effectiveness of NFATc2 and suggest that this is important to prevent cells from dying before viral replication is complete and the mature virus is released from the cells.

  • Merkel Cell Polyomavirus downregulates N-myc downstream regulated gene-1 (NDRG1) leading to cellular proliferation and migration [Virus-Cell Interactions]

  • Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, a viral oncogene required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analysed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream regulated gene-1 (NDRG1) in NIKs-MCPyV and hTERT-MCPyV human keratinocytes (HK) as compared to their controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV HK or MCC cells resulted in decrease of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of CDK2 and cyclinD1 proteins. Overall NDRG1 plays an important role in MCPyV induced cellular proliferation.

    Importance Merkel Cell Carcinoma was first described in 1972 as a neuroendocrine tumor of skin most of which in 2008, was reported to be caused by a PyV named Merkel Cell Polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus induced carcinogenesis. However, it is still a new field and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV as opposed to other PyVs in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies thereby providing hope for management of patients suffering from this highly aggressive cancer.

  • Advances towards curing HIV-1 infection from tissue reservoirs [Minireviews]

  • A disease of more than 39.6 million people world-wide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts towards a sterilizing or functional cure. On the horizon is research concentrated in multiple separate, but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including check point inhibitors, and gene editing. Here we review the concept of targeting the HIV-1 tissue reservoirs with an emphasis on the central nervous system and describe relevant new work in functional cure research and strategies for HIV-1 eradication.

  • Integrin {beta}1 promotes peripheral entry by rabies virus [Virus-Cell Interactions]

  • Rabies virus (RABV) is a widespread pathogen that causes fatal disease in humans and animals. It is suggested that multiple host factors are involved in RABV host entry. Here, we showed that RABV uses integrin bbeta;1 (ITGB1) for cellular entry. RABV infection was drastically decreased after ITGB1 siRNA knockdown and moderately increased after ITGB1 overexpression in cells. ITGB1 directly interacts with RABV glycoprotein. Upon infection, ITGB1 is internalized into cells and transported to late endosomes together with RABV. The infectivity of cell-adapted RABV in cells and street RABV in mice was neutralized by ITGB1 ectodomain soluble protein. The role of ITGB1 in RABV infection depends on interaction with fibronectin in cells and mice. We found that Arg-Gly-Asp (RGD) peptide and antibody to ITGB1 significantly blocked RABV infection in cells in vitro, and street RABV infection in mice via intramuscular inoculation but not the intracerebral route. ITGB1 also interacts with nicotinic acetylcholine receptor, which is the proposed receptor for peripheral RABV infection. Our findings suggest that ITGB1 is a key cellular factor for RABV peripheral entry, and could be a potential therapeutic target for post-exposure treatment against rabies.

    IMPORTANCE Rabies is a severe zoonotic disease caused by rabies virus (RABV). However, the nature of RABV entry remains unclear, which has hindered the development of therapy for rabies. It is suggested that modulations of RABV glycoprotein and multiple host factors are responsible for RABV invasion. Here, we showed that integrin bbeta;1 (ITGB1) directly interacts with RABV glycoprotein, and both proteins are internalized together into host cells. Differential expression of ITGB1 in mature muscle and cerebral cortex of mice led to A-4 (ITGB1 special antibody) and RGD peptide (competitive inhibitor for interaction between ITGB1 and fibronectin) blocked street RABV infection via intramuscular but not intracerebral inoculation in mice, suggesting that ITGB1 plays a role in RABV peripheral entry. Our study revealed this distinct cellular factor in RABV infection, which may be an attractive target for therapeutic intervention.

  • Identification of chicken CD74 as a novel attachment cellular receptor for infectious bursal disease virus in bursa B lymphocytes [Virus-Cell Interactions]

  • Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family, causing severe immunosuppressive disease in chickens. The major capsid VP2 protein is responsible for the binding of IBDV to the host cell and its cellular tropism. In order to find the potential interaction proteins with IBDV VP2, LC/MS assay was conducted and host protein chicken CD74 protein was identified. Here, we investigate the role of chicken CD74 in IBDV attachment. Co-immunoprecipitation assays indicated that the extracellular domain of CD74 interacted with the VP2 protein of multiple IBDV strains. Knockdown and Overexpression experiments showed that CD74 promotes viral infectivity. Confocal assay showed that CD74 overexpression allows the attachment of IBDV and subvirus-like particles (SVPs) to the cell surface of non-permissive cells, and qPCR analysis further confirmed the attachment function of CD74. Anti-CD74 antibody, soluble CD74, depletion of CD74 by small interfering RNA (siRNA), and CD74 knockdown in the IBDV-susceptible DT40 cell line significantly inhibited IBDV binding, suggesting a pivotal role of this protein in virus attachment. These findings demonstrate that CD74 is a novel important receptor for IBDV attachment to the chicken B lymphocyte cell line DT40.

    IMPORTANCE CD74 plays a pivotal role in the correct folding and functional stability of MHC II molecules and in the presentation of antigenic peptides, acting as a regulatory factor in the antigen presentation process. In our study, we demonstrated a novel role of CD74 during IBDV infection, showing that chicken CD74 plays a significant role in IBDV binding to target B cells by interacting with the viral VP2 protein. This is the first report demonstrating that CD74 is involved as a novel attachment receptor in the IBDV life cycle in target B cells, thus contributing new insight into host-pathogen interactions.

  • Vpu of a simian immunodeficiency virus isolated from greater spot-nosed monkey antagonizes human BST-2 via two AxxxxxxxW motifs [Virus-Cell Interactions]

  • BST-2/CD317/tetherin is a host transmembrane protein that potently inhibits human immunodeficiency virus type 1 (HIV-1) virion release by tethering the nascent virions to the plasma membrane. Vpu is an accessory protein encoded by HIV-1 as well as some simian immunodeficiency viruses infecting wild chimpanzees, gorillas or monkeys (SIVcpz, SIVgor, or SIVgsn/SIVmon/SIVmus, respectively). HIV-1 Vpu directly binds to and downregulates human BST-2. The antagonism is highly species-specific because amino acid sequences of BST-2 are different among animal species. Here we show that Vpus from several SIVcpz, SIVgsn, SIVmon, or SIVmus isolates fail to antagonize human BST-2. Only Vpu from an SIVgsn isolate (SIVgsn-99CM71 [SIVgsn71]) was able to antagonize human BST-2 as well as BST-2 of its natural host greater spot-nosed monkey (GSN). This SIVgsn Vpu interacted with human BST-2, downregulated cell surface human BST-2 expression, and facilitated HIV-1 virion release in the presence of human BST-2. While the unique 14AxxxxxxW22 motif in the transmembrane domain of HIV-1NL4-3Vpu was reported to be important for antagonizing human BST-2, we show here two AxxxxxxxW motifs exist in SIVgsn71 Vpu (A22W30 and A25W33). Only the A22W30 motif was needed for SIVgsn71 Vpu to antagonize GSN BST-2, suggesting that the mechanism of this antagonism resembles that of HIV-1NL4-3 Vpu against human BST-2. Interestingly, SIVgsn71 Vpu requires two AxxxxxxxW (A22W30 and A25W33) motifs to antagonize human BST-2, suggesting a yet undefined way that SIVgsn71 Vpu works against human BST-2. These results imply an evolutionary impact of primate BST-2 on lentiviral Vpu.

    Importance Genetic alterations conferring a selective advantage in protecting from life-threating pathogens will be maintained during evolution. In fact, amino acid sequences of BST-2 are varied among primate animals and their susceptibility to viral proteins are species-specific, suggesting that such genetic diversity has arisen through the evolutionarily controlled balance between the host and pathogens. The M (main) group of HIV-1 is thought to be derived from SIVcpz, which utilizes Nef, but not Vpu, to antagonize chimpanzee BST-2. SIVcpz Nef is, however, unable to antagonize human BST-2 and consequently Vpu was chosen again as an antagonist against human BST-2 in the context of HIV-1. Studies on how Vpu lost and acquired this ability, together with the distinct mechanisms by which SIVgsn71 Vpu binds to and downregulates human or GSN BST-2, may help to understand the evolution of this lentiviral protein as a result of host-pathogen interactions.

  • Finally, a role befitting Astar: the strongly conserved, unessential microvirus A* proteins ensure the product fidelity of packaging reactions. [Structure and Assembly]

  • In microviruses, 60 copies of the positively-charged DNA binding protein J guide the single-stranded (ss) DNA genome into the icosahedral capsid. Consequently, ~12% of the genome is icosahedrally ordered within virions. Although the internal volume of the X174, G4, and aalpha;3 capsids are nearly identical, their genome length varies widely from 5386 (X174) to 6067 (aalpha;3). As genome size increases, the J protein's length and charge decreases. The X174 J protein is 37 amino acids long and has a charge of +12, whereas the 23 residue G4 and aalpha;3 proteins have respective +6 and +8 charges. While the large X174 J protein can substitute for the smaller ones, the converse is not true. Thus, the smallest genome, X174, requires the more stringent J protein packaging guide. To investigate this further, a chimeric virus (XG4J) was generated by replacing the indigenous X174 J gene with that of G4. The resulting mutant, XG4J, was not viable on the level of plaque formation without X174 J gene complementation. During uncomplemented infections, capsids dissociated during packaging or quickly thereafter. Those that survived were significantly less stable and infectious than wild-type. Complementation-independent XG4J variants were isolated. They contained duplications that increased genome size by as much as 3.8%. Each duplication started at nucleotide 991, creating an additional DNA substrate for the unessential but highly conserved A* protein. Accordingly, XG4J viability and infectivity was also restored by the exogenous expression of a cloned A* gene.

    Importance Double-stranded (ds) DNA viruses typically package their genomes into a preformed capsid. By contrast, ssRNA viruses assemble their coat proteins around their genomes via extensive nucleotide-protein interactions. ssDNA viruses appear to blend both strategies, using nucleotide-protein interactions to organize their genomes into preformed shells, likely by a controlled process. Chaotic genome-capsid associations could inhibit packaging or genome release during the subsequent infection. This process appears to be partially controlled by the unessential A* protein, a shorter version of the essential A protein that mediates rolling circle DNA replication. Protein A* may elevate fitness by ensuring the product fidelity of packaging reactions. This phenomenon may be widespread in ssDNA viruses that simultaneously synthesize and package DNA with rolling circle and rolling circle-like DNA replication proteins. Many of these viruses encode smaller, unessential and/or functionally undefined in-frame versions of A/A*-like proteins.

  • Rapid dissemination and monopolization of viral populations in mice revealed using a panel of barcoded viruses [Genetic Diversity and Evolution]

  • The gastrointestinal tract presents a formidable barrier for pathogens to initiate infection. Despite this barrier, enteroviruses, including coxsackievirus B3 (CVB3), successfully penetrate the intestine to initiate infection and spread systemically prior to shedding in stool. However, the effect of the gastrointestinal barrier on CVB3 population dynamics is relatively unexplored, nor are the selective pressures acting on CVB3 in the intestine well-characterized. To examine viral population dynamics in orally infected mice, we produced over 100 CVB3 viruses harboring unique nine nucleotide "barcodes." Using this collection of barcoded viruses, we found diverse viral populations throughout each mouse within the first day post-infection, but by 48 hours the viral populations were dominated by less than three barcoded viruses in intestinal and extra-intestinal tissues. Using light-sensitive viruses to track replication status, we found diverse viruses had replicated prior to loss of diversity. Sequencing whole viral genomes from samples later in infection did not reveal detectable viral adaptations. Surprisingly, orally inoculated CVB3 was detectable in pancreas and liver as soon as 20 minutes post inoculation, indicating rapid systemic dissemination. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues. These results underscore a complex dynamic between dissemination and clearance for an enteric virus.

    Importance Enteric viruses initiate infection in the gastrointestinal tract but can disseminate to systemic sites. However, the dynamics of viral dissemination are unclear. In this study, we created a library of 135 barcoded coxsackieviruses to examine viral population diversity across time and space following oral inoculation of mice. Overall, we found that the broad population of viruses disseminates early, followed by monopolization of mouse tissues with three or fewer pool members at later time points. Interestingly, we detected virus in systemic tissues such as pancreas and liver just 20 minutes post-oral inoculation. These results suggest rapid dissemination of diverse viral populations, followed by a major restriction in population diversity and monopolization in all examined tissues.

  • Human papillomavirus type 16 E5 inhibits interferon signaling and supports episomal viral maintenance [Virus-Cell Interactions]

  • Human papillomaviruses (HPVs) infect keratinocytes of stratified epithelia. Long-term persistence of infection is a critical risk factor for the development of HPV-induced malignancies. Through the action of its oncogenes, HPV evades host immune responses to facilitate its productive life cycle. In this work, we have discovered a previously unknown function of the HPV16 E5 oncoprotein in the suppression of interferon responses. This suppression is focused on keratinocyte-specific IFN and is mediated through E5-induced changes in growth factor signaling pathways as identified through phosphoproteomics analysis. The loss of E5 in keratinocytes maintaining the complete HPV16 genome results in the de-repression of IFNK transcription and subsequent JAK/STAT-dependent upregulation of several ISGs at both mRNA and protein levels. We also establish a link between the loss of E5 and the subsequent loss of genome maintenance and stability, resulting in increased genome integration.

    Importance Persistent human papillomavirus infections can cause a variety of significant cancers. The ability of HPV to persist depends on evasion of the host immune system. In this study, we show that the HPV16 E5 protein can suppress an important aspect of the host immune response. In addition, we find that the E5 protein is important for helping the virus avoid integration into the host genome, which is a frequent step along the pathway to cancer development.

  • USP7-Dependent Regulation of TRAF Activation and Signaling by a Viral Interferon Regulatory Factor Homologue [Virus-Cell Interactions]

  • Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs 1-4), all of which are expressed during lytic replication and inhibit a variety of antiviral signaling pathways. Viral IRFs 1, 2 and 3, are also expressed during latency in primary effusion lymphoma (PEL) cells, and vIRF-1 and vIRF-3 have been reported to promote PEL cell viability. Viral IRFs 1, 3 and 4 are known to interact with ubiquitin-specific protease 7 (USP7); interactions of vIRF-1 and vIRF-3 with USP7 promote PEL cell viability and regulate productive replication. Here, we report that vIRF-2 also targets USP7, utilizing a PSTS motif matching the USP7 N-terminal domain-binding A/PxxS consensus, but uniquely requires catalytic-domain residues for intracellular interaction. In functional and mechanistic analyses, tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling and associated polyubiquitination of TRAFs 3 and 6, specifically, were regulated negatively by USP7 and positively by vIRF-2-USP7 interaction, the latter competing for USP7-TRAF association. Using depletion, depletion-complementation, and targeted mutagenesis approaches, vIRF-2 was determined to promote latent PEL cell viability, likely independently of USP7 interaction, while lytic replication was inhibited by vIRF-2, in part or whole via USP7 interaction. Together, our data identify a new molecular determinant of USP7 recognition, TRAF3/6-specific targeting by the deubiquitinase, associated activation of these TRAFs by vIRF-2, and activities of vIRF-2 and vIRF-2-USP7 interaction in HHV-8 latent and lytic biology.

    IMPORTANCE Human herpesvirus 8-encoded IRF homologues were the first to be identified in a virus. Through inhibitory interactions with cellular IRFs and other mediators of antiviral signaling, the vIRFs are believed to be essential for productive replication, and also for latency in particular cell types. The deubiquitinase USP7 is a regulator of key cellular pathways, modulates HHV-8 latent and lytic infection, and is targeted by vIRFs 1, 3 and 4. Here, we report that vIRF-2 also interacts with USP7, via a means distinguishable from USP7 interactions with other vIRFs and other proteins, that this interaction modulates antiviral signaling via disruption of USP7 interactions with innate immune signaling proteins TRAF3 and TRAF6, and that vIRF-2 targeting of USP7 regulates HHV-8 productive replication. The presented data are the first to identify vIRF-2 targeting of USP7 and its role in HHV-8 biology, expanding our understanding of the repertoire and importance of virus-host interactions.

  • The amino acid mutations A286V and T437M in the nucleoprotein attenuate H7N9 viruses in mice [Pathogenesis and Immunity]

  • The low pathogenic H7N9 influenza viruses that emerged in 2013 acquired an insertion of four amino acids in their hemagglutinin cleavage site and thereby became highly pathogenic to chickens in 2017. Previous studies indicate that these highly pathogenic H7N9 viruses are virulent in chickens but have distinct pathotypes in mice. A/chicken/Guangdong/SD098 (CK/SD098) is avirulent with a mouse lethal dose (MLD50) greater than 7.5 log10 50% egg infectious dose (EID50), whereas A/chicken/Hunan/S1220 (CK/S1220) is virulent in mice with an MLD50 of 3.2 log10 EID50. In this study, we explored the genetic determinants that contribute to the difference in virulence between these two H7N9 viruses by generating a series of reassortants and mutants in the CK/S1220 virus background and testing their virulence in mice. We found that the reassortant CK/1220-SD098-NP carrying the nucleoprotein (NP) of CK/SD098 was avirulent in mice, with an MLD50 greater than 107.5 EID50. The NP proteins of these two viruses differ by two amino acids at positions 286 and 437. We further demonstrated that the two amino acid mutations A286V and T437M of NP independently slow down the process of NP import to and export from the nucleus, and thus jointly impaired the viral life cycle and attenuated the virulence of these H7N9 viruses in mice. Our study identified new virulence determinants in NP and provided novel targets for the development of live attenuated vaccine and antiviral drugs against influenza viruses.

    Importance The H7N9 influenza viruses that emerged in China in 2013 have caused over 1,500 human infections, with a mortality of nearly 40%. The viruses were initially low pathogenic but became highly pathogenic in chickens at the beginning of 2017 and caused severe disease outbreaks in poultry. Several studies suggest that the highly pathogenic H7N9 viruses have increased virulence in mammals; however, the genetic basis of the virulence of H7N9 viruses in mammals is not fully understood. Here, we found that two amino acids, 286A and 437T, in NP are prerequisites for the virulence of H7N9 viruses in mice, and that the mutations A286V and T437M collectively eliminate the virulence of H7N9 viruses in mice. Our study further demonstrated that the virulence of influenza virus is polygenic trait, and the newly identified virulence-related residues in NP may provide new targets for attenuated influenza vaccine and antiviral drug development.

  • Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis [Vaccines and Antiviral Agents]

  • To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low mmu;M to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.

    Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses nndash; well-known as well as (re-)emerging species nndash; has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.

  • Cellular Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Mediates Nuclear Import of Human Bocavirus 1 NP1 Protein and Modulates Viral Capsid Protein Expression [Genome Replication and Regulation of Viral Gene Expression]

  • Human bocavirus 1 (HBoV1), belonging to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid proteins (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed two host mRNA processing factors DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6, aka CFlm68), a subunit of the cleavage factor Im complex (CFlm), interact with HBoV1 NP1 independent of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased capsid protein expression but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication.

    IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens to cause acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein NP1 that plays an important role in maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor CPSF6 that directly interacts with NP1, mediates nuclear import of NP1, and plays a role in maturation of capsid proteins-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism how a viral small nonstructural protein facilitates the multiple regulations of viral gene expression and replication, and reveals a novel target for potent anti-viral drug development.

  • Optimization of the codon-pair usage of human respiratory syncytial virus paradoxically resulted in reduced viral replication in vivo and reduced immunogenicity [Pathogenesis and Immunity]

  • We subjected various ORFs in the genome of respiratory syncytial virus (RSV) to codon-pair optimization (CPO) by increasing the content of codon pairs that are over-represented in the human genome, without changing overall codon usage and amino acid sequences. CPO has the potential to increase the expression of the encoded protein(s). Four viruses were made: Max A (CPO of NS1, NS2, N, P, M, and SH ORFs); Max B (CPO of G and F), Max L (CPO of L), and Max FLC (CPO of all ORFs except M2-1 and M2-2). Because of the possibility of increased viral replication, each CPO virus was attenuated by the inclusion of a codon-deletion mutation (1313) and a missense mutation (I1314L) in the L polymerase. CPO had no effect on multi-cycle virus replication in vitro, temperature sensitivity, or specific infectivity. Max A and L nndash; which in common had CPO of one or more ORFs of proteins of the polymerase complex nndash; exhibited global increases in viral protein synthesis. Max B alone exhibited decreased protein synthesis, and it alone had reduced single-cycle virus replication in vitro. All CPO RSVs exhibited marginal reduction in replication in mice and hamsters. Surprisingly, the CPO RSVs induced lower levels of serum RSV-neutralizing antibodies in hamsters. This reduced immunogenicity might reflect reduced viral replication and possibly also the decrease in CpG and UpA dinucleotides as immune stimulators. Overall, our study describes paradoxical effects of CPO of an RNA virus on viral replication and the adaptive humoral immune response.

    IMPORTANCE Using computer algorithms and large-scale DNA synthesis, one or more ORFs of a microbial pathogen can be recoded by different strategies that involve the introduction of up to thousands of nucleotide changes without affecting amino acid coding. This approach has been used mostly to generate deoptimized viruses used as vaccine candidates. However, the effects of the converse approach of generating optimized viruses are still largely unknown. Here, various ORFs in the genome of respiratory syncytial virus (RSV) were codon-pair optimized (CPO) by increasing the content of codon pairs that are over-represented in the human genome. CPO did not affect RSV replication in multicycle replication experiments in vitro. However, replication was marginally reduced in two rodents models. In hamsters, CPO RSVs induced lower levels of serum RSV-neutralizing antibodies. Thus, CPO of an RNA virus for a mammalian host have paradoxical effects on virus replication and the adaptive humoral immune response.

  • Complex interactions between cohesin and CTCF in regulation of KSHV lytic transcription. [Genome Replication and Regulation of Viral Gene Expression]

  • CTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with both proteins previously reported to act as restrictive factors for lytic cycle transcription and virion production. In this study, we examined the interdependence of CTCF and cohesin binding to the KSHV genome. ChIP-seq analyses revealed that cohesin binding to the KSHV genome is highly CTCF dependent whereas CTCF binding does not require cohesin. Further, depletion of CTCF leads to almost complete dissociation of cohesin from sites at which they colocalize. Thus, previous studies which examined the effects of CTCF depletion actually represent concomitant depletion of both CTCF and cohesin components. Analysis of the effects of single and combined depletion indicate that CTCF primarily activates KSHV lytic transcription whereas cohesin has primarily inhibitory effects. Further, CTCF or cohesin depletion was found to have regulatory effects on cellular gene expression relevant for control of viral infection, with both proteins potentially facilitating expression of multiple genes important in the innate immune response to viruses. Thus, CTCF and cohesin have both positive and negative effects on KSHV lytic replication as well as effects on the host cell that enhance antiviral defenses.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to Kaposi's sarcoma and several lymphoproliferative diseases. KSHV, like other herpesviruses intermittently reactivates from latency and enters a lytic cycle in which numerous lytic mRNAs and proteins are produced, culminating in infectious virion production. These lytic proteins may also contribute to tumorigenesis. Reactivation from latency is controlled by processes that restrict or activate transcription of KSHV lytic genes. KSHV gene expression is modulated by binding of host cell proteins CTCF and the cohesin complex to the KSHV genome. These proteins bind to and modulate chromatin conformation, thereby regulating transcription. We have analyzed the interdependence of binding CTCF and cohesin and demonstrate that while CTCF is required for cohesin binding to KSHV, they have very distinct effects, with cohesin primarily restricting KSHV lytic transcription. Further, we show that cohesin and CTCF also exert effects on the host cell that promote antiviral defenses.

  • Genome-scale Phylogeny and Evolutionary Analysis of Ross River virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977 - 2014. [Genetic Diversity and Evolution]

  • Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages: the Eastern, the Western and the supposedly extinct North-Eastern lineage have been classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analysed 106 RRV complete coding sequences which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977 nndash; 2014) and during the substantial Pacific Islands RRV epidemic (1979 nndash; 1980). Our final dataset composed isolates sampled over 59 years (1959 nndash; 2018), from a range of locations. Four distinct genotypes were defined, with the newly described Genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal co-circulation of distinct genetic groups. Bayesian Markov Chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history, and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis.

    IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia, and causes a significant burden of suffering to infected individuals, as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history has so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust, whole genome analysis has not yet been conducted. This study generated 94 novel near whole genome sequences to investigate the evolutionary history of RRV, to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance and potential future vaccine design.

  • X4-tropic Latent HIV-1 is Enriched in Peripheral Follicular Helper T Cells and is Correlated with Disease Progression [Pathogenesis and Immunity]

  • Follicular helper T (TFH) cells have been shown to support productive human immunodeficiency virus-1 (HIV-1) replication and to serve as a key component of the latent viral reservoir. However, the viral characteristics of this latent reservoir and the clinical relevance of this reservoir remain unclear. In this study, we assessed the tropic composition of latent viruses from peripheral TFH (pTFH), non-TFH memory and naïve CD4+ T cells from individuals with HIV-1-infections on suppressive combined antiretroviral therapy (cART). X4-tropic latent HIV-1 was preferentially enriched in pTFH cells compared to the other two subsets. Interestingly, the ratio of X4-tropic latent HIV-1 in pTFH cells was not only robustly and inversely correlated with blood CD4+ T cell counts across patients but was also prognostic of CD4+ T cell recovery in individuals on long-term cART. Moreover, patients with higher X4-tropic latent HIV-1 ratios in pTFH cells showed greater risks of opportunistic coinfections. These findings reveal the characteristics of latent HIV-1 in TFH cells and suggest that the ratio of X4-tropic latent HIV-1 in pTFH cells is a valuable indicator for disease progression and cART efficacy.

    Importance TFH cells have been shown to harbor a significant amount of latent HIV-1; however, the viral characteristics of this reservoir and its clinical relevance remain largely unknown. In this study, we demonstrate that X4-tropic latent HIV-1 is preferentially enriched in pTFH cells, which also accurately reflects the viral tropism shift. The ratio of X4-tropic proviruses in pTFH cells but not in other memory CD4+ T cell subsets is inversely and closely correlated with blood CD4+ T cell counts and CD4+ T cell recovery rates with cART. Our data suggest that the ratio of X4-tropic provirus in peripheral TFH cells can be easily measured and reflects disease progression and treatment outcomes during cART.

  • An uncultivated virus infecting a symbiotic Nanoarchaeota in the hot springs of Yellowstone National Park [Virus-Cell Interactions]

  • The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses have been described that infect the Nanoarchaeota. Here we identify a virus infecting Nanoarchaeota using a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6kb circular DNA genome encoding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single celled organism that is itself an ectobiont of another single celled organism.

    Importance Here we present evidence for the first virus to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. Based on annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family further demonstrating the remarkable genetic diversity of archaeal viruses.

  • Cross-reactive CD8 T-cell responses elicited by Ad5-based HIV-1 vaccines contributed to early viral evolution in vaccine recipients who became infected [Vaccines and Antiviral Agents]

  • Because of HIV's vast sequence diversity, the ability of the CD8 T-cell response to recognize several variants of a single epitope is an important consideration for vaccine design. Cross recognition of viral epitopes by CD8 T cells is associated with viral control during HIV-1 infection, but little is known about CD8 cross-reactivity in the context of HIV-1 vaccination. Here, we evaluated vaccine-induced CD8 cross-reactivity in two preventative HIV-1 vaccine efficacy trials, the MRKAd5 and DNA/rAd5 studies. Cross-reactive CD8 responses elicited by vaccination were similar in magnitude and frequency to those induced during acute HIV-1 infection. Although responses directed against variant epitopes were less avid than responses to vaccine-matched epitopes, we did not detect any difference in response polyfunctionality (the proportion of cells producing multiple effector molecules). And, while depth, or the frequency of cross-reactive responses, did not correlate with viral loads in recipients who became infected, cross-reactivity did appear to influence early viral evolution. In comparing viral sequences of placebo versus vaccine recipients, we found that viral sequences from vaccinees encoded CD8 epitopes with more substitutions and greater biochemical dissimilarity. In other words, breakthrough sequences of vaccinees would be less cross-recognized by vaccine-induced responses. Additionally, vaccine-induced CD8 T cells poorly cross-recognized variant epitopes encoding HLA-I associated adaptations, further supporting our conclusion that these responses play a role in driving early HIV-1 viral evolution.

    Importance HIV-1 has exceptionally high sequence diversity, much of which is found within CD8 epitopes. Therefore, the ability of CD8 T cells to recognize multiple versions of a single epitope could be important for an effective vaccine. Here, we show that two previously tested vaccines induced a similar level of CD8 cross-reactivity to that seen in acute HIV-1 infection. Although this cross-reactivity did not seem to affect viral control in vaccine recipients who became infected, we identified several ways in which CD8 cross-reactivity appeared to influence HIV-1 viral evolution. First, we saw that strains isolated from infected vaccine recipients would likely be poorly cross-recognized by the vaccine-induced response. Secondly, we saw that adapted CD8 epitopes were poorly cross-recognized in both vaccination and infection. Collectively, we believe these results show that CD8 cross-reactivity could be an important consideration in future HIV-1 vaccine design.

  • What a difference a gene makes - identification of virulence factors of Cowpox virus [Pathogenesis and Immunity]

  • Cowpox virus (CPXV) is a zoonotic Orthopoxvirus (OPV) that causes spill-over infections from their animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality when compared to the mildly virulent laboratory strain Brighton Red (BR). RatPox09 encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing its high virulence. To address this hypothesis, we employed several BR/RatPox09 chimeric viruses using Red-mediated mutagenesis to generate BR knock-in mutants with single or multiple insertions of the respective RatPox09 genes based on BR. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses; and transcriptomic analyses confirmed the expression profile of genes adjacent to the ones modified be unaltered. While in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching that of RatPox09. The insertion of gCPXV0284 encoding the BTB domain protein D7L also increased the virulence of BR, while the other two ORFs failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model.

    Importance With the cessation of vaccination against smallpox, and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats when compared to the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

  • A poly-uridine insertion in the 3'-untranslated region of classical swine fever virus activates immunity and reduces viral virulence in piglets [Vaccines and Antiviral Agents]

  • Low virulent classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Little data is available on the molecular determinants of CSFV virulence. The aim of present study is to assess a possible role for CSFV virulence of a unique uninterrupted 36-uridine (poly-U) sequence found in the 3'-untranslated region (3'UTR) of the low virulent CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3'UTR (vPdR-36U), and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of twenty 5-day-old piglets were infected with vPdR-36U and vPdR-5U, respectively. Ten contact piglets were added to each group. Disease progression, virus replication and immune responses were monitored during 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. 90% of the piglets infected with vPdR-36U seroconverted while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient IFN-aalpha; responses in the serum after one week of infection while the vPdR-36U-infected piglets showed sustained IFN-aalpha; levels during the first two weeks. Taken together, these data show that the 3'UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.

    IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3'-untranslated region (3'UTR) plays an important role in Flavivirus replication, the present study showed for the first time that a long poly-uridine sequence acquired in the 3'UTR by an endemic CSFV isolate can activate immunity, control viral replication and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants are also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

  • Differential Expression of Immune Checkpoint Molecules on CD8+ T Cells Specific for Immunodominant and Subdominant Herpes Simplex Virus 1 Epitopes [Pathogenesis and Immunity]

  • Herpes simplex virus type 1 (HSV-1) causes a lifelong infection of neurons that innervate barrier sites like the skin and mucosal surfaces like the eye. After primary infection of the cornea, the virus enters latency within the trigeminal ganglion (TG), from which it can reactivate throughout the life of the host. Viral latency is maintained, in part, by virus-specific CD8+ T cells that nonlethally interact with infected neurons. When CD8+ T cell responses are inhibited, HSV-1 can reactivate and these recurrent reactivation events can lead to blinding scarring of the cornea. In the C57BL/6 mouse, CD8+ T cells specific to the immunodominant epitope from glycoprotein B maintain functionality throughout latency while CD8+ T cells specific to subdominant epitopes undergo functional impairment that is associated with the expression of the inhibitory checkpoint molecule programmed death 1 (PD-1). Here, we investigate the checkpoint molecule, T-cell immunoglobulin and mucin-domain containing-3 (Tim-3), which has traditionally been associated with CD8+ T cell exhaustion. Unexpectedly, we found that Tim-3 was preferentially expressed on highly functional ganglionic CD8+ T cells during acute and latent HSV-1 infection. This, paired with data that show that Tim-3 expression on CD8+ T cells in the latently infected TG is influenced by viral gene expression, suggests that Tim-3 is an indicator of recent T cell stimulation, rather than functional compromise, in this model. We conclude that Tim-3 expression is not sufficient to define functional compromise during latency; however, it may be useful in identifying activated cells within the TG during HSV-1 infection.

    IMPORTANCE Without an effective means of eliminating HSV-1 from latently infected neurons, efforts to control the virus have centered on preventing viral reactivation from latency. Virus-specific CD8+ T cells within the infected TG have been shown to play a crucial role in inhibiting viral reactivation and with a portion of these cells exhibiting functional impairment, checkpoint molecule immunotherapies have presented a potential solution to enhancing the anti-viral response of these cells. In pursuing this potential treatment strategy, we found that Tim-3 (often associated with CD8+ T cell functional exhaustion) is not upregulated on impaired cells but is instead upregulated on highly functional cells that have recently received antigenic stimulation. These findings support a role for Tim-3 as a marker of activation rather than exhaustion in this model, and we provide additional evidence for the hypothesis that there is persistent viral gene expression in the HSV-1 latently infected TG.

  • A Recombinant Rhesus Monkey Rhadinovirus Deleted of Glycoprotein L Establishes Persistent Infection of Rhesus Macaques and Elicits Conventional T Cell Responses [Pathogenesis and Immunity]

  • A replication-competent, recombinant strain of rhesus monkey rhadinovirus (RRV) expressing the Gag protein of SIVmac239 was constructed in the context of a glycoprotein L (gL) deletion mutation. Deletion of gL detargets the virus from Eph family receptors. The ability of this gL-minus Gag recombinant RRV to infect, persist, and elicit immune responses was evaluated after intravenous inoculation of two Mamu-A*01+ RRV-naïve rhesus monkeys. Both monkeys responded with an anti-RRV antibody response, and quantitation of RRV DNA in peripheral blood mononuclear cells (PBMC) by real time PCR revealed levels similar to those in monkeys infected with recombinant gL+ RRV. Comparison of RRV DNA levels in sorted CD3+ vs. CD20+ vs. CD14+ PBMC subpopulations indicated infection of the CD20+ subpopulation by the gL-minus RRV. This contrasts with results obtained with transformed B cell lines in vitro where deletion of gL resulted in markedly reduced infectivity. Over a period of 20 weeks Gag-specific CD8+ T cell responses were documented by major histocompatibility complex class-I (MHC-I) tetramer staining. Vaccine-induced CD8+ T cell responses, which were predominantly directed against the Mamu-A*01-restricted Gag(181-189)CM9 epitope, could be inhibited by blockade of MHC-I presentation. Our results indicate that gL and the interaction with Eph family receptors is dispensable for the colonization of the B cell compartment following high-dose infection by the intravenous route, which suggests the existence of alternative receptors. Further, gL-minus RRV elicits cellular immune responses that are predominantly canonical in nature.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with a substantial disease burden in sub-Saharan Africa, often in the context of human immunodeficiency virus (HIV) infection. The related rhesus monkey rhadinovirus (RRV) has shown potential as a vector to immunize monkeys with antigens from the simian immunodeficiency virus (SIV), the macaque model for HIV. KSHV and RRV engage cellular receptors from the Eph family via the viral gH/gL glycoprotein complex. We have now generated a recombinant RRV that expresses the SIV Gag antigen and does not express gL. This recombinant RRV was infectious by the intravenous route, established persistent infection in the B cell compartment, and elicited strong immune responses to the SIV Gag antigen. These results argue against a role of gL and Eph family receptors for B cell infection by RRV in vivo and have implications for the development of a live-attenuated KSHV vaccine or vaccine vector.

  • Reverse Genetics System for a Human Group A Rotavirus [Genome Replication and Regulation of Viral Gene Expression]

  • Group A Rotavirus (RV) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established an entirely plasmid-based reverse genetics system for simian RV strain SA11. Although that system was robust enough to generate reassortant RVs, including human RV gene segments, and enabled better understanding of the biological differences between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we established a plasmid-based reverse genetics system for G4P[8] human RV strain Odelia. This technology was used to generate a panel of monoreassortant viruses between human and simian RV strains for all of the eleven gene segments demonstrating full compatibility between human and simian RV strains. Furthermore, we generated recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological function of the interaction between NSP1 and its target protein bbeta;-transducin repeat-containing protein (bbeta;-TrCP) during viral replication. While the NSP1 truncation mutant lacking the C-terminal 13 amino acids displayed lower bbeta;-TrCP degradation activity, it replicated as efficiently as the wild-type virus. By contrast, the truncation mutant lacking the C-terminal 166 amino acids of NSP1 replicated poorly, suggesting that the C-terminal region of NSP1 plays critical roles in viral replication. The system reported herein will allow generation of engineered recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate development of novel therapeutics and vaccines.

    IMPORTANCE Reverse genetics, an approach used to generate viruses from cloned cDNA, has increased our understanding of virus biology. Worldwide research led to the development of an entirely plasmid-based reverse genetics system for the simian RV laboratory strain. Although the technique allows generation of gene-modified recombinant RVs, biological differences between animal and human RVs mean that reverse genetics systems for human RV strains are still needed. Here, we describe a reverse genetics system for the high-yield human RV strain Odelia, which replicates efficiently and is suitable for in vitro molecular studies. Monoreassortant viruses between simian and human RV strains and NSP1 mutant viruses generated by the rescue system enabled study of the biological functions of viral gene segments. This human RV reverse genetics system will facilitate study of RV biology and development of vaccines and vectors.

  • Surfeit 4 contributes to the replication of hepatitis C virus using double membrane vesicles [Genome Replication and Regulation of Viral Gene Expression]

  • A number of positive strand RNA viruses such as hepatitis C virus (HCV) and poliovirus use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, LC-MS/MS, and siRNA methods revealed that Surfeit 4 (Surf4), which encodes an integral membrane protein, involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 as a novel cofactor that is involved in the replication of positive strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive strand RNA virus replication.

    Importance Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive strand RNA viruses.

  • Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering [Virus-Cell Interactions]

  • Mumps virus (MuV), an enveloped negative strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers), like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42-) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42-) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN, without affecting its cell surface expression, glycan-receptor binding and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain, as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry.

    IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling, but sometimes leads to orchitis, oophoritis and neurological complications such as meningitis, encephalitis and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry, but provide useful information about a possible target for anti-viral drugs.

  • Weak cis and trans interactions of the hemagglutinin with receptors trigger fusion proteins of neuropathogenic measles virus isolates [Virus-Cell Interactions]

  • Measles virus (MeV) is an enveloped RNA virus bearing two envelope glycoproteins, the hemagglutinin (H) and fusion (F) proteins. Upon receptor binding, the H protein triggers conformational changes of the F protein, causing membrane fusion and subsequent virus entry. MeV may persist in the brain, infecting neurons and causing fatal subacute sclerosing panencephalitis (SSPE). Since neurons do not express either of the MeV receptors, signaling lymphocytic activation molecule (SLAM, also called CD150) and nectin-4, how MeV propagates in neurons is unknown. Recent studies have shown that specific substitutions in the F protein found in MeV isolates from SSPE patients are critical for MeV neuropathogenicity, by rendering the protein unstable and hyperfusogenic. Recombinant MeVs possessing the F proteins with such substitutions can spread in primary human neurons and in the brains of mice and hamsters, and induce cell-cell fusion in cells lacking SLAM and nectin-4. We here show that receptor-blind mutant H proteins that have decreased binding affinities to receptors can support membrane fusion mediated by hyperfusogenic mutant F proteins, but not the wild-type F protein, in cells expressing the corresponding receptors. The results suggest that weak interactions of the H protein with certain molecules (putative neuron receptors) may trigger hyperfusogenic F proteins in SSPE patients. Notably, where cell-cell contacts are ensured, the weak cis interaction of the H protein with SLAM on the same cell surface could also trigger hyperfusogenic F proteins. Some enveloped viruses may exploit such cis interactions with receptors to infect target cells, especially in cell-to-cell transmission.

    Importance Measles virus (MeV) may persist in the brain, causing incurable subacute sclerosing panencephalitis (SSPE). Because neurons, the main target in SSPE, do not express receptors for wild-type (WT) MeV, how MeV propagates in the brain is a key question for the disease. Recent studies have demonstrated that specific substitutions in the MeV fusion (F) protein are critical for neuropathogenicity. Herein, we show that weak cis and trans interactions of the MeV attachment protein with receptors that are not sufficient to trigger the WT MeV F protein, can trigger the mutant F proteins from neuropathogenic MeV isolates. Our study not only provides an important clue to understand MeV neuropathogenicity, but also reveals a novel viral strategy to expand cell tropism.

  • Ribosomal protein L13 promotes IRES-driven translation of FMDV in helicase DDX3-dependent manner [Virus-Cell Interactions]

  • Internal ribosome entry site (IRES)-driven translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5' cap structure. In the current study, we identified the ribosomal protein RPL13 as a critical regulator of IRES-driven translation of foot-and-mouth disease virus (FMDV), but found that it is not essential for cellular global translation. RPL13 is also a determinant for translation and infection of Seneca Valley virus (SVV) and classical swine fever virus (CSFV), and this suggests that its function may also be conserved in unrelated IRES-containing viruses. We further showed that depletion of DEAD-box helicase DDX3 disrupts binding of RPL13 to the FMDV IRES, whereas the reduction in RPL13 expression impairs the ability of DDX3 to promote IRES-driven translation directly. DDX3 cooperates with RPL13 to support the assembly of 80S ribosomes for optimal translation initiation of viral mRNA. Finally, we demonstrated that DDX3 affects the recruitment of the eukaryotic initiation factor eIF3 subunits e and j to viral IRES. This work provides the first connection between DDX3 and eIF3e/j, and recognition of the role of RPL13 in modulating viral IRES-dependent translation. This previously uncharacterized process may be involved in selective mRNA translation.

    IMPORTANCE Accumulating evidence has unveiled the roles of ribosomal proteins (RPs) belonging to the large 60S subunit in regulating selective translation of specific mRNAs. The translation specificity of the large subunit RPs in this process is thought-provoking, given the role they play canonically in catalyzing peptide-bond formation. Here we have identified the ribosomal protein L13 (RPL13) as a critical regulator of IRES-driven translation during FMDV infection. Our study supports a model whereby the FMDV IRESs recruit helicase DDX3 recognizing RPL13 to facilitate IRES-driven translation, with the assistance of eIF3e, j. A better understanding of these specific interactions surrounding IRES-mediated translation initiation could have important implications for the selective translation of viral mRNA and thus for the development of effective viral prevention.

  • Neutralization breadth and potency of single-chain variable fragments derived from broadly neutralizing antibodies targeting multiple epitopes on the HIV-1 envelope [Vaccines and Antiviral Agents]

  • Passive administration of HIV-directed broadly neutralizing antibodies (bNAbs) can prevent infection in animal models and human efficacy trials are underway. Single-chain variable fragments (scFv), comprised of only the variable regions of antibody heavy and light chains, are smaller molecules that may offer advantages over full-length IgG. We designed and expressed scFv of HIV bNAbs prioritized for clinical testing that target the V2-apex (CAP256-VRC26.25), V3-glycan supersite (PGT121), CD4 binding site (3BNC117) and MPER (10E8v4). The use of either a 15 or 18 amino acid Glycine-Serine linker between the heavy and light chain fragments provided adequate levels of scFv expression. When tested against a 45 multi-subtype virus panel, all four scFv retained good neutralizing activity although there was variable loss of function compared to the parental IgG antibodies. For CAP256-VRC26.25, there was a significant 138-fold loss of potency that was in part related to differential interaction with charged amino acids at positions 169 and 170 in the V2 epitope. Potency was reduced for the 3BNC117 (13-fold) and PGT121 (4-fold) scFv among viruses lacking the N276 and N332 glycans respectively, and in viruses with a longer V1 loop for PGT121. This suggested that scFv interacted with their epitopes in subtly different ways, with variation at key residues affecting scFv neutralization more than the matched IgGs. Remarkably, the scFv of 10E8v4 maintained breadth of 100% with only a minor reduction in potency. Overall scFv of clinically relevant bNAbs had significant neutralizing activity indicating that they may be suitable for passive immunization to prevent HIV-1 infection.

    Importance: Monoclonal antibodies have been isolated against conserved epitopes on the HIV trimer and are being investigated for passive immunization. Some of the challenges associated with full-sized antibody proteins may be overcome by using single-chain variable fragments (scFv). These smaller forms of antibodies can be produced more efficiently, may show fewer off-target effects with increased tissue penetration and are more adaptable to vectored-mediated expression compared to IgG. Here we demonstrate that scFv of four HIV-directed bNAbs (CAP256-VRC26.25, PGT121, 3BNC117 and 10E8v4) had significant neutralizing activity against diverse global strains of HIV. Loss of potency and/or breadth was shown to be due to increased dependence of the scFv on key residues within the epitope. These smaller antibody molecules with functional activity in the therapeutic range may be suitable for further development as passive immunity for HIV prevention.

  • HIV-1 matrix trimerization-impaired mutants are rescued by matrix substitutions that enhance envelope glycoprotein incorporation [Structure and Assembly]

  • The matrix (MA) domain of HIV-1 Gag plays key roles in virus assembly by targeting the Gag precursor to the plasma membrane and directing the incorporation of the viral envelope (Env) glycoprotein into virions. This latter function appears to be in part dependent on trimerization of the MA domain of Gag during assembly, as disruption of the MA trimer interface impairs Env incorporation. Conversely, many MA mutations that impair Env incorporation can be rescued by compensatory mutations in the trimer interface. In this study, we sought to investigate further the biological significance of MA trimerization by isolating and characterizing compensatory mutations that rescue MA trimer interface mutants with severely impaired Env incorporation. By serially propagating MA trimerization-defective mutants in T cell lines, we identified a number of changes in MA, both within, and distant from, the trimer interface. The compensatory mutations located within or near the trimer interface restored Env incorporation and particle infectivity, and permitted replication in culture. The structure of the MA lattice was interrogated by measuring the cleavage of the murine leukemia virus (MLV) transmembrane Env protein by the viral protease in MLV Env-pseudotyped HIV-1 particles bearing the MA mutations, and by performing crystallographic studies of in vitro assembled MA lattices. These results demonstrate that rescue is associated with structural alterations in MA organization and rescue of MA domain trimer formation. Our data highlight the significance of the trimer interface of the MA domain of Gag as a critical site of protein-protein interaction during HIV-1 assembly, and establish the functional importance of trimeric MA for Env incorporation.

    Statement of Significance The immature Gag lattice is a critical structural feature of assembling HIV-1 particles, which is primarily important for virion formation and release. While Gag forms a hexameric lattice, driven primarily by the capsid domain, the MA domain additionally trimerizes where three Gag hexamers meet. MA mutants that are defective for trimerization are deficient for Env incorporation and replication, suggesting a requirement for trimerization of the MA domain of Gag in Env incorporation. This study used a gain-of-function, forced viral evolution approach to rescue HIV-1 mutants that are defective for MA trimerization. Compensatory mutations that rescue virus replication do so by restoring Env incorporation and MA trimer formation. This study supports the importance of MA domain trimerization in HIV-1 replication, and the potential of the trimer interface as a therapeutic target.

  • SOS and IP modifications predominantly affect the yield but not other properties of SOSIP.664 HIV-1 Env glycoprotein trimers [Vaccines and Antiviral Agents]

  • Soluble, recombinant native-like (NL) envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design, designated BG505 SOSIP.664, incorporates an inter-subunit disulfide bond (SOS) to covalently link the gp120 and gp41 ectodomain (gp41ECTO) subunits and a point substitution, I559P (IP) to further stabilize the gp41ECTO components. Without the SOS and IP changes, proteolytically cleaved trimers tend to disintegrate into their constituent gp120 and gp41ECTO subunits. We show, however, that NL trimers lacking the SOS and/or IP changes can be affinity purified in amounts sufficient for analyses of their antigenicity and thermal stability. In general, these trimer variants have highly comparable properties to the fully stabilized SOSIP.664 version. We conclude that the major effect of the SOS and IP changes is to substantially increase trimer stability during and after the expression process, thereby allowing useful amounts to be produced. However, once the trimers have been purified, the SOS and IP changes have only subtle impacts on thermostability and the antigenicity of bNAb and other epitopes.

    Importance Recombinant trimeric proteins based on HIV-1 env genes are being developed for vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. One vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. A commonly used design is designated SOSIP.664, a term reflecting the sequence changes that are used to stabilize the trimers and allow their production in practically useful amounts. Here, we show that these stabilizing changes act to increase trimer yield during the biosynthesis process within the producer cell, but have little impact on the properties of purified trimers.

  • CD80 plays a critical role in increased inflammatory responses in HSV-1 infected mouse corneas [Pathogenesis and Immunity]

  • We recently reported that HSV-1 infection suppresses CD80 but not CD86 expression in vitro and in vivo. This suppression required the HSV-1 ICP22 gene. We also reported that overexpression of CD80 by HSV-1 exacerbated corneal scarring in BALB/c mice. We now show that this recombinant virus (HSV-CD80) expressed high levels of CD80 both in vitro in cultured rabbit skin cells and in vivo in infected mouse corneas. CD80 protein was detected on the surface of infected cells. Virulence of the recombinant HSV-CD80 virus was similar to that of the parental strain and replication of HSV-CD80 was similar to that of control virus in vitro and in vivo. Transcriptome analysis detected 75 known HSV-1 genes in the cornea of mice infected with HSV-CD80 or parental virus on day 4 post infection. Except for significantly higher CD80 expression in HSV-CD80 infected mice, HSV-1 gene expression was similar in corneas from HSV-CD80 infected and parental virus infected mice. The number of CD8+ T cells was higher, and CD4+ T cells lower, in corneas of HSV-CD80 infected mice than mice infected with parental virus. HSV-CD80 infected mice displayed a transient increase in DCs. Transcriptome analysis revealed mild differences in dendritic cell maturation, IL1-signaling pathways, and increased expression of Interferon Induced Protein with Tetratricopeptide Repeats 2 (Ifit2). Together, these results suggest that increased CD80 levels promote increased CD8+ T cells leading to exacerbated eye disease in HSV-1 infected mice.

    Importance HSV-1 ocular infections are the leading cause of corneal blindness. Eye disease is the result of prolonged immune response to the replicating virus. HSV-1, on the other hand, has evolved several mechanisms to evade clearance by the host immune system. We described a novel mechanism of HSV-1 immune evasion via ICP22 dependent downregulation of the host T cell co-stimulatory molecule CD80. However, the exact role of CD80 in HSV-1 immune pathology is not clear. In this study, we show that eye disease is independent of HSV-1 replication, and that viral expression of CD80 has a detrimental role in corneal scarring, likely by increasing CD8+ T cell recruitment and activation.

  • Human enterovirus group B viruses rely on vimentin dynamics for efficient processing of viral non-structural proteins [Virus-Cell Interactions]

  • We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with IDPN did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the non-structural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, thus leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, lower amounts of non-structural proteins did not result from proteasomal degradation, but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1, but had less effect on 2A. The results suggest that, the vimentin dynamics regulate viral non-structural protein synthesis while having no effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and non-structural proteins of enteroviruses, having consequences on host cell survival.

    Importance A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and non-structural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the non-structural proteins orchestrate the take-over of the host cell and its functions. Here we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and the host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages.

  • The Tryptophan-Rich Motif of HIV-1 Gp41 Can Interact with the N-Terminal Deep Pocket Site: New Insights into the Structure and Function of Gp41 and Its Inhibitors [Virus-Cell Interactions]

  • Refolding of the HIV-1 gp41 N- and C-terminal heptad repeats (NHR and CHR) into a six-helix bundle (6-HB) juxtaposes viral and cellular membranes for fusion. The CHR-derived peptide T20 is the only clinically approved viral fusion inhibitor and has potent anti-HIV activity; however, its mechanism of action is not fully understood. In this study, we surprisingly found that T20 disrupted the aalpha;-helical conformation of the NHR-derived peptide N54 through its C-terminal tryptophan-rich motif (TRM), and that synthetic short-peptides containing the TRM sequence, TRM8 and TRM12, disrupted the N54 helix in a dose-dependent manner. Interestingly, TRM8 efficiently interfered with the secondary structures of three overlapped NHR peptides (N44, N38, and N28) and interacted with N28 that mainly contains the deep NHR pocket-forming sequence in a high affinity, suggesting that TRM targeted the NHR pocket site to mediate the disruption. Different from the TRM8, the short-peptide corresponding to the pocket-binding domain (PBD) of the CHR helix had no such disruptive effect and the CHR peptide C34 could form a stable 6-HB with the NHR helix; however, addition of the TRM to the C-terminus of C34 resulted in a peptide (C46) that destroyed the NHR helix. Although the TRM peptides alone had no anti-HIV activity and could not block the formation of 6-HB conformation, substitution of the TRM for the PBD in C34 resulted in a mutant inhibitor (C34TRM) with high binding and inhibitory capacities. Combined, the present data inform a new mode of action of T20 and the structure-function relationship of gp41.

    IMPORTANCE The HIV-1 Env glycoprotein mediates membrane fusion and is conformationally labile. Despite extensive efforts, the structural property of the native fusion protein gp41 is largely unknown and the mechanism of action of the gp41-derived fusion inhibitor T20 remains elusive. Here, we report that T20 and its C-terminal tryptophan-rich motif (TRM) can efficiently impair the conformation of the gp41 N-terminal heptad repeat (NHR) coiled coil, which is through the interaction with the deep NHR pocket site. The TRM sequence has been verified to possess ability to substitute the pocket-binding domain of C34, a fusion inhibitor peptide with high anti-HIV potency. Therefore, our studies have not only facilitated to understand the mechanism of action of T20 and develop novel HIV-1 fusion inhibitors but also provided new insights into the structural property of the pre-fusion state gp41.

  • The C. elegans RIG-I homolog DRH-1 mediates the Intracellular Pathogen Response upon viral infection [Cellular Response to Infection]

  • Mammalian RIG-I-like receptors detect viral dsRNA and 5' triphosphorylated RNA to activate transcription of interferon genes and promote antiviral defense. The C. elegans RIG-I-like receptor DRH-1 promotes defense through antiviral RNA interference, but less is known about its role in regulating transcription. Here we describe a role for DRH-1 in directing a transcriptional response in C. elegans called the Intracellular Pathogen Response (IPR), which is associated with increased pathogen resistance. The IPR includes a set of genes induced by diverse stimuli including intracellular infection and proteotoxic stress. Previous work suggested that the proteotoxic stress caused by intracellular infections might be the common trigger of the IPR, but here we demonstrate that different stimuli act through distinct pathways. Specifically, we demonstrate that DRH-1/RIG-I is required for inducing the IPR in response to Orsay virus infection, but not in response to other triggers like microsporidian infection or proteotoxic stress. Furthermore, DRH-1 appears to be acting independently of its known role in RNAi. Interestingly, expression of the replication competent Orsay virus RNA1 segment alone is sufficient to induce most of the IPR genes in a manner dependent on RNA-dependent RNA polymerase activity and on DRH-1. Altogether, these results suggest that DRH-1 is a pattern-recognition receptor that detects viral replication products to activate the IPR stress/immune program in C. elegans.

    Importance C. elegans lacks homologs of most mammalian pattern recognition receptors, and how nematodes detect pathogens is poorly understood. We show that the C. elegans RIG-I homolog DRH-1 mediates induction of the Intracellular Pathogen Response (IPR), a novel transcriptional defense program, in response to infection by the natural C. elegans viral pathogen Orsay virus. DRH-1 appears to act as a pattern-recognition receptor to induce the IPR transcriptional defense program by sensing the products of viral RNA-dependent RNA polymerase activity. Interestingly, this signaling role of DRH-1 is separable from its previously known role in antiviral RNAi. In addition, we show that there are multiple host pathways for inducing the IPR, shedding light on the regulation of this novel transcriptional immune response.

  • Limited intra-host diversity and background evolution accompany 40 years of canine parvovirus host adaptation and spread [Genome Replication and Regulation of Viral Gene Expression]

  • Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and micro-scale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ~99% identical in nucleotide sequence, with only a limited number (llt;40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intra-host level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few sub-consensus single nucleotide variants (SNVs) above ~0.5%, and experimental passages demonstrate that substantial pre-existing genetic variation is not necessarily required for rapid host receptor driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intra-host diversity are not necessarily required for virus host adaptation.

    IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and inter-host diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intra-host evolutionary processes of a ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained onward transmission does not necessarily depend on a high level of intra-host diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

  • Recombinant rotaviruses rescued by reverse genetics reveal the role of NSP5 hyperphosphorylation in the assembly of viral factories [Genome Replication and Regulation of Viral Gene Expression]

  • Rotavirus (RV) replicates in round-shaped cytoplasmic viral factories, although how they assemble remains unknown.

    During RV infection, NSP5 undergoes hyperphosphorylation, which is primed by the phosphorylation of a single serine residue. The role of this post-translational modification in the formation of viroplasms and its impact on the virus replication remain obscure. Here we investigated the role of NSP5 during RV infection by taking advantage of a modified fully tractable reverse genetics system. A trans-complementing cell line stably producing NSP5 was used to generate and characterise several recombinant rotaviruses (rRVs) with mutations in NSP5. We demonstrate that an rRV lacking NSP5 was completely unable to assemble viroplasms and to replicate, confirming its pivotal role in rotavirus replication.

    A number of mutants with impaired NSP5 phosphorylation were generated to further interrogate the function of this post-translational modification in the assembly of replication-competent viroplasms. We showed that the rRV mutant strains exhibited impaired viral replication and the ability to assemble round-shaped viroplasms in MA104 cells. Furthermore, we have investigated the mechanism of NSP5 hyper-phosphorylation during RV infection using NSP5 phosphorylation-negative rRV strains, as well as MA104-derived stable transfectant cell lines expressing either wt NSP5 or selected NSP5 deletion mutants. Our results indicate that NSP5 hyper-phosphorylation is a crucial step for the assembly of round-shaped viroplasms, highlighting the key role of the C-terminal tail of NSP5 in the formation of replication-competent viral factories. Such a complex NSP5 phosphorylation cascade may serve as a paradigm for the assembly of functional viral factories in other RNA viruses.

    IMPORTANCE Rotavirus (RV) double-stranded RNA genome is replicated and packaged into virus progeny in cytoplasmic structures termed viroplasms. The non-structural protein NSP5, which undergoes a complex hyperphosphorylation process during RV infection, is required for the formation of these virus-induced organelles. However, its roles in viroplasm formation and RV replication have never been directly assessed due to the lack of a fully tractable reverse genetics (RG) system for rotaviruses. Here we show a novel application of a recently developed RG system by establishing a stable trans-complementing NSP5-producing cell line required to rescue rotaviruses with mutations in NSP5. This approach allowed us to provide the first direct evidence of the pivotal role of this protein during RV replication. Furthermore, using recombinant RV mutants we shed light on the molecular mechanism of NSP5 hyperphosphorylation during infection and its involvement in the assembly and maturation of replication-competent viroplasms.

  • Pharmacological modulation of the Wnt/{beta}-catenin pathway inhibits proliferation and promotes differentiation of long-lived memory CD4+ T-cells in ART-suppressed SIV-infected macaques [Pathogenesis and Immunity]

  • The major obstacle to human immunodeficiency type 1 (HIV-1) eradication is a reservoir of latently-infected cells that persists despite long-term antiretroviral therapy (ART) and is maintained through cellular proliferation. Long-lived memory CD4+ T-cells with high self-renewal capacity such as central memory T-cells (CM) and T memory stem cells (SCM) are major contributors to the viral reservoir in HIV-infected individuals on ART. The Wnt/bbeta;-catenin signaling pathway regulates the balance between self-renewal and differentiation of SCM and CM T-cells and pharmacological manipulation of this pathway offers an opportunity to interfere with the proliferation of latently-infected cells. Here, we evaluated in vivo a novel approach to inhibit self-renewal of SCM and CM CD4+ T-cells in the rhesus macaque (RM) model of SIV infection. We used an inhibitor of the Wnt/bbeta;-catenin pathway, PRI-724, that blocks the interaction between the co-activator CREB binding protein (CBP) and bbeta;-catenin, resulting in the cell fate decision to differentiate rather than proliferate. Our study shows that PRI-724 treatment of ART-suppressed SIVmac251-infected RMs resulted in decreased proliferation of SCM and CM T-cells and modified the SCM and CM CD4+ T-cell transcriptome towards a profile of more differentiated memory T-cells. However, short-term treatment with PRI-724 alone did not significantly reduce the size of the viral reservoir. This work demonstrates for the first time that stemness pathways of long-lived memory CD4+ T-cells can be pharmacologically modulated in vivo, thus establishing a novel strategy to target HIV persistence.

    Importance Long-lasting CD4+ T-cell subsets, such as central memory and stem cell memory CD4+ T-cells, represent critical reservoirs for HIV persistence despite suppressive antiretroviral therapy. These cells possess stem cell-like properties of enhanced self-renewal/proliferation and proliferation of latently-infected memory CD4+ T-cells plays a key role in maintaining the reservoir over time. Here, we evaluated an innovative strategy targeting the proliferation of long-lived memory CD4+ T-cells to reduce the viral reservoir stability. Using the rhesus macaque model, we tested a pharmacological inhibitor of the Wnt/bbeta;-catenin signaling pathway that regulates T-cell proliferation. Our study shows that the administration of the inhibitor PRI-724 decreased the proliferation of SCM and CM CD4+ T-cells and promoted a transcriptome enriched in differentiation genes. Although the viral reservoir size was not significantly reduced by PRI-724 treatment alone, we demonstrate the potential to pharmacologically modulate the proliferation of memory CD4+ T-cells as a strategy to limit HIV persistence.

  • Distinct roles of interferon alpha and beta in controlling chikungunya virus replication and modulating neutrophil-mediated inflammation [Pathogenesis and Immunity]

  • Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-bbeta; (IFN-bbeta;-KO mice or mice treated with an IFN-bbeta; blocking antibody) or IFN-aalpha; (IRF7-KO mice or mice treated with a pan IFN-aalpha; blocking antibody). Mice lacking either IFN-aalpha; or IFN-bbeta; developed more severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-aalpha; sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-bbeta;-KO mice displayed minimal differences in viral burden within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-bbeta; had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-bbeta;-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with the IFN-aalpha; limiting early viral replication and dissemination and IFN-bbeta; modulating neutrophil-mediated inflammation.

    IMPORTANCE Type I interferons (IFN) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-aalpha; and IFN-bbeta; both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-aalpha; limits CHIKV replication and dissemination, whereas IFN-bbeta; protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

  • Regulation of the Ebola virus VP24 protein by SUMO [Virus-Cell Interactions]

  • Some viruses take advantage of ubiquitin or ubiquitin-like proteins conjugation to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data expose that the minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced capability to block the type I interferon pathway and to inhibit IFN-mediated STAT1-nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and a significantly diminished stability. Using GST-pull down assay we found that VP24 also interacts with SUMO in a non-covalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to down-modulate the IFN pathway and in the monoubiquitination of the protein. We identified the SUMO deubiquitinating enzyme USP7 as an interactor and negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.

    IMPORTANCE The Ebola virus VP24 protein plays a critical role in the virus escape from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable for therapeutics. Here we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

  • Structural glycoprotein E2 of classical swine fever virus interacts with host protein dynactin subunit 6 (DCTN6) during the virus infectious cycle [Virus-Cell Interactions]

  • The E2 protein in classical swine fever virus (CSFV) is the major virus structural glycoprotein and an essential component of the viral particle. E2 has been shown to be involved in several functions including virus adsorption, induction of protective immunity and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6), which is a component of the cell dynactin complex, as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, co-immunoprecipitation and proximity ligation assay. E2 residues critical to mediate the protein-protein interaction with DCTN6 were mapped by a reverse yeast-two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2DCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for viral replication and virulence in swine. CSFV E2DCTN6v shows reduced replication than the parental virus in an established swine cell line, SK6, and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2DCTN6v remained clinically normal during the 21-day observational period sugesting that the ability of CSFV E2 to bind host DCTN6 protein efficiently during the infection may play a role in virus virulence.

    Importance Structural glycoprotein E2 is an important component of CSFV due to the involvement in many virus activities, particularly virus-host interactions. Herein is the description and characterization of the protein-protein interaction between E2 and swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV was created harboring mutations disrupting E2-DCTN6 interaction. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It is shown that the same amino acid substitutions abrogating E2-DCTN6 interaction in vitro constitute a critical factor in virus virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

  • Yellow fever virus re-emergence and spread in Southeast Brazil, 2016-2019 [Genetic Diversity and Evolution]

  • The recent re-emergence of yellow fever virus (YFV) in Brazil has raised serious concerns due to the virus' rapid dissemination in the southeastern region. To better understand YFV genetic diversity and dynamics during the recent outbreak in southeastern Brazil we generated 18 complete and near-complete genomes from the peak of the epidemic curve from non-human primates (NHPs) and human infected cases across Espiiacute;rito Santo and Rio de Janeiro states. Genomic sequencing of 18 YFV genomes revealed the estimated timing, source and likely routes of yellow fever virus transmission and dispersion during one of the largest outbreaks ever registered in Brazil. We showed that during the recent epidemic YFV was re-introduced from Minas Gerais to Espiiacute;rito Santo and Rio de Janeiro states multiple times between 2016 to 2019. The analysis of data from portable sequencing could identify the corridor of spread of YFV. These findings reinforce that continued genomic surveillance strategies can provide information on virus genetic diversity and transmission dynamics that might assist in the understanding arbovirus epidemics.

    IMPORTANCE Arbovirus infections in Brazil including yellow fever, dengue, zika and chikungunya result in considerable morbidity and mortality and are pressing public health concerns. However, our understanding of these outbreaks is hampered by limited availability genomic data. In this study, we investigated the genetic diversity and spatial distribution of YFV during the current outbreak by analyzing genomic data from areas in southeastern Brazil not covered by other previous studies. To gain insights into the routes of YFV introduction and dispersion, we tracked the virus by sequencing YFV genomes sampled from non-human primates and infected patients from the southeastern region. Our study provides an understanding of how YFV initiates transmission in new Brazilian regions and illustrates that genomics in field can augment traditional approaches to infectious disease surveillance and control.

  • Isolation of Yasminevirus, the first member of Klosneuvirinae isolated in coculture with Vermamoeba vermiformis, demonstrates an extended arsenal of translational apparatus components [Genetic Diversity and Evolution]

  • The family of giant viruses is still expanding, and evidence of a translational machinery is emerging in the virosphere. The Klosneuvirinae group of giant viruses was first reconstructed from in silico studies, and then a unique member was isolated, Bodo saltans virus. Here, we describe the isolation of a new member in this group using coculture with the free-living amoeba Vermamoeba vermiformis. This giant virus, called Yasminevirus, has a 2.1-MB linear double-stranded DNA genome encoding 1541 candidate proteins with a GC content estimated at 40.2%. Yasminevirus possesses a nearly complete translational machinery with a set of 70 tRNAs associated with 45 codons and recognizing 20 amino acids, 20 aminoacyl-tRNA synthetases (aaRSs) recognizing 20 aa, as well as several translation factors and elongation factors. At the genome scale, evolutionary analyses placed this virus in the Klosneuvirinae group of giant viruses. Rhizome analysis demonstrated that the genome of Yasminevirus is mosaic with ~34% of genes having their closer homologues in other viruses, followed by ~13.2% in eukaryota, ~7.2% in bacteria and less than 1% in archaea. Among giant virus sequences, Yasminevirus shared 87% of viral hits with Klosneuvirinae. This description of Yasminevirus sheds light on the Klosneuvirinae group in a captivating quest to understand the evolution and diversity of giant viruses.

    Importance Yasminevirus is an icosahedral double-stranded DNA virus isolated from sewage water by amoeba coculture. Here, its structure and replicative cycle are described in the amoeba Vermamoeba vermiformis, and genomic and evolutionary studies are reported. This virus belongs to the Klosneuvirinae group of giant viruses, representing the second isolated and cultivated giant virus in this group and is the first isolated using a coculture procedure. Extended translational machinery pointed Yasminevirus among the quasi-autonomous giant viruses with the most complete translational apparatus of the known virosphere.

  • Phytovirome Analysis of Wild Plant Populations: Comparison of Double-Stranded RNA and Virion-Associated Nucleic Acid Metagenomic Approaches [Genetic Diversity and Evolution]

  • Metagenomic studies have indicated that the diversity of plant viruses was until recently far underestimated. As important components of ecosystems, there is a need to explore the diversity and richness of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time. Two viral sequence enrichment approaches, double-stranded RNA (dsRNA) and Virion-associated nucleic acids (VANA), have been used and compared here for the description of the virome of complex plant pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. A novel bioinformatics strategy was used to assess viral richness not only at family level but also by determining Operational Taxonomic Units (OTU) following the clustering of conserved viral domains. A large viral diversity, dominated by novel dsRNA viruses was detected in all sites while a large between sites variability limited the ability to draw clear conclusion on the impact of cultivation. A trend for a higher diversity of dsRNA viruses was nevertheless detected in unmanaged sites (118 vs 77 unique OTUs). The dsRNA-based approach consistently revealed a broader and more comprehensive diversity for RNA viruses than the VANA approach, whatever the assessment criterion. In addition, dissimilarity analyses indicated both approaches to be largely reproducible, but not necessarily convergent. These findings illustrate features of phytoviromes in various ecosystems and a novel strategy for precise virus richness estimation. These results allow to reason methodological choices in phytovirome studies and, likely in other viromes study where RNA viruses are the focal taxa.

    IMPORTANCE There are today significant knowledge gaps on phytovirus populations and on the drivers impacting them, but also on the comparative performance methodological approaches for their study. We used and compared two viral sequences enrichment approaches, double-stranded RNAs (dsRNA) and virion-associated nucleic acids (VANA) for phytovirome description in complex pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. Viral richness was assessed by determining Operational Taxonomic Units (OTU) following the clustering of conserved viral domains. There is some limited evidence of an impact of cultivation on viral populations. These results provide data allowing to reason the methodological choices in virome studies. For researchers primarily interested in RNA viruses, the dsRNA approach is advised because it consistently provided a more comprehensive description of the analysed phytoviromes, but it understandably underrepresented DNA viruses and bacteriophages.

  • Zika virus infection, reproductive organ targeting, and semen transmission in the male olive baboon. [Pathogenesis and Immunity]

  • ZIKA virus (ZIKV) infection in pregnant women is a serious threat to the development and viability of the fetus. The primary mode of ZIKV transmission to humans is through mosquito bites, but sexual transmission has also been well documented in humans. However, little is known of the short and long-term effects of ZIKV infection of the human male reproductive system. This study examines the effects of Zika virus (ZIKV) infection on the male reproductive organs, semen and the immune response of the olive baboon (Papio anubis). Nine mature male baboons were infected with ZIKV (French Polynesian strain) subcutaneously. Six animals were euthanized at 41 days, while 3 animals were euthanized at 10/11 days post infection (dpi). Viremia and clinical evidence of infection was present in all 9 baboons. ZIKV RNA was present in semen in 5/9 baboons. ZIKV was present in the testes of 2/3 males euthanized at 10/11 dpi, but 0/6 at 41 dpi. Immunofluorescence of testes suggested ZIKV in sperm progenitor cells, macrophage penetration of seminiferous tubules and increased TNFaalpha;, particularly in vascular walls. This data demonstrates that male olive baboons approximate the male human ZIKV response including viremia, adaptive immune response and persistent ZIKV in semen. While gross testicular pathology was not seen, the demonstrated breach of testes-blood barrier and targeting of spermatogenic precursors suggests possible long-term implications in ZIKV infected primates.

    IMPORTANCE Zika virus (ZIKV) is an emerging flavivirus spread through mosquitoes and sexual contact. ZIKV infection during pregnancy can lead to severe fetal outcomes including miscarriage, fetal death, pre-term birth, intra-uterine growth restriction and fetal microcephaly collectively known as Congenital Zika Syndrome (CZS). Therefore, it is important to understand how this virus spreads as well as the resulting pathogenesis in translational animal models that faithfully mimic ZIKV infection in humans. Such models will contribute to the future development of efficient therapeutics and prevention mechanisms. Through our previous work in olive baboons, we have developed a non-human primate model that is permissible to ZIKV infection and transfers the virus vertically from mother to fetus, modeling human observations. The current study contributes to the understanding of the ZIKV infection in male baboon reproductive tissues and begins to elucidate how this may affect fertility, reproductive capacity, and sexual transmission of the virus.

  • Hemagglutinin cleavability, acid-stability and temperature dependence optimize influenza B virus for replication in human airways [Virus-Cell Interactions]

  • Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAV (A/H1N1 and A/H3N2) and two IBV viruses (B/Victoria and B/Yamagata lineage) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA, by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved present in human respiratory epithelium. Compared to IAV, the IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH proved similar for IBV and human-adapted IAVs (one exception being HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33ddeg;C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host-adaptation during prolonged circulation of this respiratory virus in the human population.

    IMPORTANCE Influenza epidemics are caused by influenza A (IAV) and influenza B (IBV) viruses. IBV causes substantial disease, however it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We here resolve how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid-stability and a preference for 33ddeg;C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans, and may have broader relevance for understanding the biology and evolution of respiratory viruses.

  • In vivo validation of the viral barcoding of SIVmac239 and the development of new barcoded SIV and subtype B and C SHIVs [Genetic Diversity and Evolution]

  • Genetically barcoded viral populations are powerful tools for evaluating overall viral population structure as well as assessing the dynamics and evolution of individual lineages in vivo over time. Barcoded viruses are generated by inserting a small, genetically unique tag into the viral genome, which is retained in progeny virus. We recently reported barcoding the well-characterized molecular clone SIVmac239, resulting in a synthetic swarm (SIVmac239M) containing approximately 10,000 distinct viral clonotypes for which all genetic differences where within a 34-base barcode that can be tracked using next generation deep sequencing. Here, we assessed the population size, distribution, and authenticity of individual viral clonotypes within this synthetic swarm using samples from 120 rhesus macaques infected intravenously. The number of replicating barcodes in plasma correlated with infectious inoculum dose, and the primary viral growth rate was similar in all infected animals regardless of inoculum size. Overall, 97% of detectable clonotypes in the viral stock were identified in the plasma of at least one infected animal. Additionally, we prepared a second generation barcoded SIVmac239 (SIVmac239M2), with over 16-times the number of barcoded variants of the original stock, and an additional barcoded stock with suboptimal nucleotides corrected (SIVmac239Opt5M). We also generated four barcoded stocks from subtype B and C SHIV clones. These new SHIV clones may be particularly valuable models to evaluate Env-targeting approaches to study viral transmission or viral reservoir clearance. Overall, this work further establishes the reliability of the barcoded virus approach and highlights the feasibility of adapting this technique to other viral clones.

    IMPORTANCE We recently developed and published a description of a barcoded simian immunodeficiency virus that has a short random sequence inserted directly into the viral genome. This allows for the tracking of individual viral lineages with high fidelity and ultradeep sensitivity. This virus was used to infect 120 rhesus macaques and we report here the analysis of the barcodes of these animals during primary infection. We found that the vast majority of barcodes were functional in vivo. We then expanded the barcoding approach in a second generation SIVmac239 (SIVmac239M2), with over 16-times the number of barcoded variants of the original stock, and a barcoded stock of SIVmac239Opt5M that has 5 changes from wild type SIVmac239. We also generated 4 barcoded stocks from subtype B and C SHIV clones each containing an HIV-1 envelope. These virus models are functional and can be useful for studying viral transmission and HIV cure/reservoir research.

  • Inherited chromosomally integrated HHV-6 demonstrates tissue-specific RNA expression in vivo that correlates with increased antibody immune response. [Pathogenesis and Immunity]

  • Human herpesvirus-6A and 6B (HHV-6A, HHV-6B) are human viruses capable of chromosomal integration. Approximately 1% of the human population carry one copy of HHV-6A/B integrated into every cell in their body, referred to as inherited chromosomally integrated human herpesvirus 6A/B (iciHHV-6A/B). Whether iciHHV-6A/B is transcriptionally active in vivo and how it shapes the immunological response is still unclear. Here, we screened DNA-Seq and RNA-Seq data for 650 individuals available through the Genotype-Tissue Expression (GTEx) project and identified 2 iciHHV-6A and 4 iciHHV-6B positive candidates. When corresponding tissue-specific gene expression signatures were analyzed, low levels HHV-6A/B gene expression was found across multiple tissues, with the highest levels of gene expression in the brain (specifically for HHV-6A), testis, esophagus, and adrenal gland. U90 and U100 were the most highly expressed HHV-6 genes in both iciHHV-6A and iciHHV-6B individuals. To assess whether tissue-specific gene expression from iciHHV-6A/B influences the immune response, a cohort of 15,498 subjects was screened and 85 iciHHV-6A/B+ subjects were identified. Plasma samples from iciHHV-6A/B+ and age and sex matched controls were analyzed for antibodies to control antigens (CMV, EBV, FLU) or HHV-6A/B antigens. Our results indicate that iciHHV-6A/B+ subjects have significantly more antibodies against the U90 gene product (IE1) relative to non-iciHHV-6 individuals. Antibody responses against EBV and FLU antigens or HHV-6A/B gene products either not expressed or expressed at low levels, such as U47, U57 or U72, were identical between controls and iciHHV-6A/B+ subjects. CMV seropositive individuals with iciHHV-6A/B+ have more antibodies against CMV pp150, relative to CMV seropositive controls. These results argue that spontaneous gene expression from integrated HHV-6A/B leads to an increase in antigenic burden that translates into a more robust HHV-6A/B specific antibody response.

    IMPORTANCE HHV-6A/B are human herpesviruses that have the unique property of being able to integrate into the telomeric regions of human chromosomes. Approximately 1% of the world's population carries integrated HHV-6A/B genome in every cell of their body. Whether viral genes are transcriptionally active in these individuals is unclear. By taking advantage of a unique tissue-specific gene expression dataset, we show the majority of tissues from iciHHV-6 individuals do not show HHV-6 gene expression. Brain and testes showed the highest tissue-specific expression of HHV-6 genes in two separate datasets. Two HHV-6 genes, U90 (immediate early 1 protein) and U100 (glycoproteins Q1 and Q2), were found to be selectively and consistently expressed across several human tissues. Expression of U90 translates into an increase in antigen-specific antibody response in iciHHV-6A/B+ subjects relative to controls. Future studies will be needed to determine the mechanism of gene expression, the effects of these genes on human gene transcription networks and the pathophysiological impact of having increased viral protein expression in tissue in conjunction with increased antigen-specific antibody production.

  • Multi-functional roles of the N-terminal region of HIV-1SF2Nef are mediated by three independent protein interaction sites [Pathogenesis and Immunity]

  • HIV-1 Nef promotes virus spread and disease progression by altering host cell transport and signaling processes through interaction with multiple host cell proteins. An N-terminal region in HIV-1 Nef encompassing residues 12-39 has been implicated in many Nef activities including disruption of CD4 T lymphocyte polarization and homing to lymph nodes, antagonism of SERINC5 restriction to virion infectivity, downregulation of cell surface CD4 and MHC-I, release of Nef-containing extracellular vesicles, and phosphorylation of Nef by recruitment of the Nef-associated kinase complex (NAKC). How this region mediates these pleiotropic functions is unclear. Characterizing a panel of alanine mutants spanning the N-terminal region to identify specific functional determinants revealed this region to be dispensable for effects of HIV-1SF2Nef on T cell actin organization and chemotaxis, retargeting of the host cell kinase Lck to the trans-Golgi network, and incorporation of Nef into extracellular vesicles. MHC-I downmodulation was specific to residue M20 and inhibition of T cell polarization by Nef required the integrity of the entire region. In contrast, downmodulation of cell surface CD4 and SERINC5 antagonism were mediated by a specific motif encompassing residues 32-39 that was also essential for efficient HIV replication in primary CD4 T lymphocytes. Finally, Nef phosphorylation via association with NAKC was mediated by two EP repeats within residues 24-29 but was dispensable for other functions. These results identify the N-terminal region as a multi-functional interaction module for at least three different host cell ligands that mediate independent functions of HIV-1SF2Nef to facilitate immune evasion and virus spread.

    IMPORTANCE HIV-1 Nef critically determines virus spread and disease progression in infected individuals by acting as protein-interaction adaptor via incompletely defined mechanisms and ligands. Residues 12-39 near the N-terminus of Nef have been described as interaction platform for the Nef-associated kinase complex (NAKC) and were recently identified as essential determinants for a broad range of Nef activities. Here we report a systematic mapping of this amino acid stretch that revealed the presence of three independent interaction motifs with specific ligands and activities. While downmodulation of cell surface MHC-I depends on M20, two EP repeats are the minimal binding site for NAKC and residues 32 to 39 mediate antagonism of the host cell restriction factor SERINC5 as well as downmodulation of cell surface CD4. These results reveal the N-terminal region of HIV-1SF2Nef as a versatile and multifunctional protein interaction module that exerts essential functions of the pathogenicity factor via independent mechanisms.

  • COPII Vesicle Transport is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms [Virus-Cell Interactions]

  • Many viruses that replicate in the cytoplasm dramatically remodel and stimulate the accumulation of host cell membranes for efficient replication by poorly understood mechanisms. For rotavirus, a critical step in virion assembly requires the accumulation of membranes adjacent to virus replication centers called viroplasms. Early electron microscopy studies describe viroplasm-associated membranes as "swollen" endoplasmic reticulum (ER). We previously demonstrated that rotavirus infection initiates cellular autophagy, and that membranes containing the autophagy marker protein LC3 and the rotavirus ER-synthesized transmembrane glycoprotein NSP4 traffic to viroplasms, suggesting that NSP4 must exit the ER. This study aimed to address the mechanism of NSP4 exit from the ER and determine whether the viroplasm-associated membranes are ER-derived. We report that (i) NSP4 exits the ER in COPII vesicles, resulting in disrupted COPII vesicle transport and ER exit sites; (ii) COPII vesicles are hijacked by LC3 II, which interacts with NSP4; and (iii) NSP4/LC3 II-containing membranes accumulate adjacent to viroplasms. Additionally, the ER transmembrane proteins SERCA and calnexin were not detected in viroplasm-associated membranes providing evidence that the rotavirus maturation process of "budding" occurs through autophagy-hijacked COPII vesicle membranes. These findings reveal a new mechanism for rotavirus maturation dependent on intracellular host protein transport and autophagy for the accumulation of membranes required for virus replication.

    Importance In a morphogenic step that is exceedingly rare for non-enveloped viruses, immature rotavirus particles assemble in replication centers called viroplasms, and bud through cytoplasmic cellular membranes to acquire the outer capsid proteins for infectious particle assembly. Historically, the intracellular membranes used for particle budding were thought to be endoplasmic reticulum (ER) because the rotavirus nonstructural protein NSP4, which interacts with the immature particles to trigger budding, is synthesized as an ER transmembrane protein. This current study shows that NSP4 exits the ER in COPII vesicles, and that the NSP4-containing COPII vesicles are hijacked by the cellular autophagy machinery, which mediates the trafficking of NSP4 to viroplasms. Changing the paradigm for rotavirus maturation, we propose that the cellular membranes required for immature rotavirus particle budding are not an extension of the ER, but are COPII-derived autophagy isolation membranes.

  • The ESCRT-0 Protein HRS Interacts with the HTLV-2 Antisense Protein APH-2 and Suppresses Viral Replication [Virus-Cell Interactions]

  • The divergent clinical outcomes of human T cell leukemia viruses type 1 and 2 (HTLV-1, HTLV-2) infections has been attributed to functional differences in their antisense proteins. In contrast to HTLV-1 HBZ, the role of APH-2 in HTLV-2 infection is poorly understood. In previous studies, we identified the endosomal sorting complex required for transport (ESCRT) 0 subunit HRS as a novel interaction partner of APH-2 but not HBZ. HRS is a master regulator of endosomal protein sorting for lysosomal degradation and is hijacked by many viruses to promote replication. However, no studies so far have shown a link between HTLVs and HRS. In this study we sought to characterize the interaction between HRS and APH-2 and investigate the impact of HRS on the lifecycle of HTLV-2. We confirmed a direct specific interaction between APH-2 and HRS and show that the CC2 domain of HRS and the N-terminal domain of APH-2 mediate their interaction. We demonstrate that HRS recruits APH-2 to early endosomes possibly furnishing an entry route into the endo/lysosomal pathway. We demonstrate that inhibition of this pathway using either Bafilomycin or HRS overexpression substantially extends the half-life of APH-2 and stabilizes Tax2B expression levels. We found that HRS enhances Tax2B mediated LTR activation, while depletion of HRS enhances HTLV-2 production and release indicating that HRS may have a negative impact on HTLV-2 replication. Overall our study provides important new insights into the role of the ESCRT-0 HRS protein and by extension the ESCRT machinery and the endo/lysosomal pathway in HTLV-2 infection.

    Importance: While the antisense protein of HTLV-2 (APH-2) is the only viral protein consistently expressed in infected carriers, its role in HTLV-2 infection is poorly understood. In this study we characterized the interaction between ESCRT 0 component HRS and APH-2 and explored the role of HRS in HTLV-2 replication. HRS is a master regulator of protein sorting for lysosomal degradation, a feature that is manipulated by several viruses to promote replication. Unexpectedly we found that HRS targets APH-2 and possibly Tax2B for lysosomal degradation and has an overall negative impact on HTLV-2 replication and release. The negative impact of interactions between HTLV-2 regulatory proteins and HRS and by extension the ESCRT machinery may represent an important strategy used by HTLV-2 to limit virus production and promote persistence, features that may contribute to the limited pathogenic potential of this infection.

  • Evaluating the intactness of persistent viral genomes in SIV-infected rhesus macaques initiating antiretroviral therapy within one year of infection [Genetic Diversity and Evolution]

  • The major obstacle to more definitive treatment for HIV infection is the early establishment of virus that persists despite long term combination antiretroviral therapy (cART) and can cause recrudescent viremia if cART is interrupted. Previous studies of HIV DNA that persists despite cART indicated that only a small fraction of persistent viral sequences was intact. Experimental SIV-infections of nonhuman primates (NHPs) are essential models for testing interventions designed to reduce the viral reservoir. We studied viral genomic integrity of virus that persists during cART under conditions typical of many NHP reservoir studies, specifically with cART started within one-year post infection and continued for at least 9 months. The fraction of persistent DNA in SIV-infected NHPs starting cART during acute or chronic infection was assessed with a multi-amplicon, real-time PCR assay ("tile assay") spanning the viral genome combined with near full length (nFL) single genome sequencing. The tile assay is used to rapidly screen for major deletions with nFL sequence analysis used to identify additional potentially inactivating mutations. PBMC from animals starting cART within one month of infection, sampled at least 9 months after cART initiation, contained at least 80% intact genomes, whereas animals starting cART 1-year post infection and treated for one year contained intact genomes only 47% of the time. The most common defect identified was large deletions with the remaining defects caused by APOBEC-mediated mutations, frame shift mutations, and inactivating point mutations. Overall, this approach can be used to assess the intactness of persistent viral DNA in NHPs.

    Importance Molecularly defining the viral reservoir that persists despite antiretroviral therapy and can lead to rebound viremia if antiviral therapy is removed is critical for testing interventions aimed at reducing this reservoir. In HIV infection in humans, with delayed treatment initiation and extended treatment duration, persistent viral DNA has been shown to be dominated by nonfunctional genomes. Using multiple real-time PCR assays across the genome combined with near full genome sequencing, we defined SIV genetic integrity after 9 to 18 months of combination anti-retroviral therapy in rhesus macaques starting therapy within one year of infection. When starting therapy within a month of infection, the vast majority of persistent DNA was intact and presumptively functional. Starting therapy within one year increased the non-intact fraction of persistent viral DNA. The approach described here allows for rapid screening of viral intactness and is a valuable tool for assessing the efficacy of novel reservoir-reducing interventions.

  • NS5 from Dengue virus serotype 2 can adopt a conformation analogous to its ZIKV and JEV homologues. [Structure and Assembly]

  • Flavivirus non-structural protein 5 (NS5) contains a N-terminal methyltransferase domain (MTase) and a C-terminal polymerase domain (RdRp) fused through a nine-amino acid linker. While the individual NS5 domains are structurally conserved, in the full-length protein, their relative orientations fall into two classes: NS5 from JEV and ZIKV adopt one conformation, while NS5 from DENV3 another. Here, we report a crystallographic structure of NS5 from DENV2 in a conformation similar to the extended one seen in JEV and ZIKV NS5 crystal structures, albeit looser. Substituting DENV2 NS5 linker with DENV1, -3, -4, JEV and ZIKV NS5 linkers had modest or minimal effects on in vitro DENV2 MTase and RdRp activities. Heterotypic DENV NS5 linkers attenuated DENV2 replicon growth in cells, whilst JEV and ZIKV NS5 linkers abolished replication. Thus, JEV and ZIKV linkers likely hindered essential DENV2 NS5 interactions with other viral or host proteins within the virus replicative complex. Overall, this work sheds light on the dynamics of the multifunctional flavivirus NS5 protein and its interdomain linker. Targeting the NS5 linker is a possible strategy for producing attenuated flavivirus strains for vaccine design.

    IMPORTANCE Flaviviruses include important human pathogens such as dengue or Zika virus. NS5 is a non-structural protein essential for flavivirus RNA replication, with dual MTase and RdRp enzyme activity and thus constitutes a major drug target. Insights into NS5 structure, dynamics and evolution should inform the development of antiviral inhibitors and vaccine design. We found that NS5 from DENV2 can adopt a conformation resembling NS5 from JEV and ZIKV. Substitution of DENV2 NS5 linker with JEV and ZIKV NS5 linkers abolished DENV2 replication in cells, without significantly impacting in vitro DENV2 NS5 enzymatic activities. We propose that heterotypic Flavivirus NS5 linkers impeded DENV2 NS5 protein-protein interactions that are essential for virus replication.

  • B cell-intrinsic SHP1 expression promotes gammaherpesvirus-driven germinal center response and the establishment of chronic infection. [Pathogenesis and Immunity]

  • Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections in the majority of adults worldwide. Chronic gammaherpesvirus infection has been implicated in both lymphomagenesis and, somewhat controversially, autoimmune disease development. Pathogenesis is largely associated with the unique ability of gammaherpesviruses to usurp B cell differentiation, specifically the germinal center response, to establish long-term latency in memory B cells. The host tyrosine phosphatase SHP1 is known as a brake to immune cell activation and is downregulated in several gammaherpesvirus-driven malignancies. However, here we demonstrate that B cell- but not T cell-intrinsic SHP1 expression supports gammaherpesvirus-driven germinal center response and the establishment of viral latency. Furthermore, B cell intrinsic SHP1 deficiency cooperated with gammaherpesvirus infection to increase the levels of dsDNA reactive antibodies at the peak of viral latency. Thus, in spite of decreased SHP1 levels in gammaherpesvirus-driven B cell lymphomas, B cell-intrinsic SHP1 expression plays a proviral role during the establishment of chronic infection, suggesting that the gammaherpesvirus-Shp1 interaction is more nuanced and is modified by the stage of infection and pathogenesis.

    Importance. Gammaherpesviruses establish life long infection in a majority of adults wordwide and are associated with a number of malignancies, including B cell lymphomas. These viruses infect naïve B cells and manipulate B cell differentiation to achieve a life-long infection of memory B cells. Germinal center stage of B cell differentiation is important both as an amplifier of viral latent reservoir and the target of malignant transformation. In this study we demonstrate that expression of tyrosine phosphatase SHP1, a negative regulator that normally limits activation and proliferation of hematopoietic cells, enhances gammaherpesvirus-driven germinal center response and the establishment of chronic infection. The results of this study uncover an intriguing beneficial interaction between gammaherpesviruses that are presumed to profit from B cell activation and a cellular phosphatase that is traditionally perceived as a negative regulator of the same processes.

  • Hemagglutinin-dependent tropism of H5N1 and H7N9 influenza viruses to human endothelial cells is determined by reduced stability of the HA which allows the virus to cope with inefficient endosomal acidification and constitutively expressed IFITM3 [Virus-Cell Interactions]

  • Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harbouring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I and K582I, which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1-virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium, enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells.

    IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having relatively low pH optimum of fusion, cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells and extracellular pH. Given significant variation of HA stability among animal, human and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.

  • Mouse cytomegalovirus m153 protein stabilizes expression of the inhibitory NKR-P1B ligand, Clr-b [Pathogenesis and Immunity]

  • Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germline-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand, Clr-b (encoded by Clec2d), on infected cells. Here, we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using m153-deficient MCMV mutants (m144-m158; m153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during m153-mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of Clec2d transcription, imaging experiments reveal that the m153 and Clr-b proteins only minimally co-localize within the same subcellular compartments, and tagged versions of the proteins were refractory to co-immunoprecipitation under mild detergent conditions. Surprisingly, the m153 mutant possesses enhanced virulence in vivo, independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlights a unique mechanism by which MCMV modulates NK ligand expression.

    IMPORTANCE Cytomegaloviruses are bbeta;-herpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are amongst the first immune cells that respond to CMV infection and use germline-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand, Clr-b, a molecule commonly lost on stressed cells ("missing-self"). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy.

  • Identification of key amino acids in the PB2 and M1 proteins of H7N9 influenza virus that affect its transmission in guinea pigs [Genetic Diversity and Evolution]

  • Efficient human-to-human transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission are still not fully understood. In this study, we compared the respiratory droplet transmissibility of four H7N9 viruses that are genetic closely related and found that these viruses have dissimilar transmissibility in guinea pigs: A/Anhui/1/2013 (AH/1) transmitted efficiently, whereas the other three viruses did not transmit. The three non-transmissible viruses have one to eight amino acid differences compared with the AH/1 virus. To investigate which of these amino acids is important for transmission, we used reverse genetics to generate a series of reassortants and mutants in the AH/1 background and tested their transmissibility in guinea pigs. We found that the NA of the non-transmissible virus A/chicken/Shanghai/S1053/2013 had low enzymatic activity that impaired the transmission of AH/1 virus, and three amino acid mutationsmmdash;-V292I and K627E in PB2 and D156E in M1mmdash;-independently abolished the transmission of the AH/1 virus. We further found that an NA reassortant and three single-amino-acid mutants replicated less efficiently than the AH/1 virus in A549 cells, and that the amino acid at position 156 of M1 affected the morphology of H7N9 viruses. Our study identifies key amino acids in PB2 and M1 that play important roles in H7N9 influenza virus transmission and provides new insights into the transmissibility of influenza virus.

    Importance Efficient transmission is a prerequisite for a novel influenza virus to cause an influenza pandemic; however, the genetic determinants of influenza virus transmission remain poorly understood. H7N9 influenza viruses, which emerged in 2013 in China, have caused over 1,560 human infection cases, showing clear pandemic potential. Previous studies have shown that the H7N9 viruses differ in their transmissibility in animal models. Here we found two amino acids in PB2 (292V and 627K) and one in M1 (156D) that are extremely important for H7N9 virus transmission. Of note, PB2 292V and M1 156D appear in most H7N9 viruses, and the PB2 627K mutation could easily occur when the H7N9 virus replicates in humans. Our study thus identifies new amino acids that are important for influenza virus transmission and suggests that just a few key amino acid changes can render the H7N9 virus transmissible in mammals.

  • Infrequent HIV infection of circulating monocytes during antiretroviral therapy [Pathogenesis and Immunity]

  • Whereas HIV persists in tissue macrophages during antiretroviral therapy (ART), the role of circulating monocytes as HIV reservoirs remains controversial. Three magnetic bead selection methods and flow cytometry cell-sorting were compared for their capacity to yield pure CD14+ monocyte populations. Cell sorting by flow cytometry provided the purest population of monocytes (median CD4+ T-cell contamination = 0.06%), and the levels of CD4+ T-cell contamination were positively correlated with the levels of integrated HIV DNA in the monocyte populations. Using cell-sorting by flow cytometry, we assessed longitudinally the infection of monocytes and other cell subsets in a cohort of 29 Thai HIV-infected individuals. Low levels of HIV DNA were detected in a minority of monocyte fractions obtained before and after one year of ART (27% and 33%, respectively), whereas HIV DNA was readily detected in CD4+ T cells from all samples. Additional samples (2-5 years of ART) were obtained from 5 individuals in whom monocyte infection was previously detected. Whereas CD4+ T cells were infected at high levels at all time points, monocyte infection was inconsistent and absent in at least one longitudinal sample from 4/5 individuals. Our results indicate that infection of monocytes is infrequent and highlight the importance of using flow cytometry cell-sorting to minimize contamination by CD4+ T cells.

    IMPORTANCE The role of circulating monocytes as persistent HIV reservoirs during ART is still controversial. Several studies have reported persistent infection of monocytes in virally suppressed individuals, however others failed to detect HIV in this subset. These discrepancies are likely explained by the diversity of the methods used to isolate monocytes and to detect HIV infection. In this study, we show that only flow-cytometry cell-sorting yields a highly pure population of monocytes largely devoid of CD4 contaminants. Using this approach in a longitudinal cohort of HIV-infected individuals before and during ART, we demonstrate that HIV is rarely found in monocytes from untreated and treated HIV-infected individuals. This study highlights the importance of using methods that yield highly pure populations of cells as flow cytometry cell sorting to minimize and control for CD4+ T-cell contamination.

  • Herpes Simplex Virus 1 induces brain inflammation and multifocal demyelination in cotton rats Sigmodon hispidus [Pathogenesis and Immunity]

  • Demyelinating CNS disorders like multiple sclerosis (MS) and acute disseminated encephalomyelitis (ADEM) have been difficult to study and treat due to the lack of understanding of their etiology. Numerous cases point to the link between HSV infection and multifocal CNS demyelination in humans, however, convincing evidence from animal models has been missing. In this work we found that HSV-1 infection of cotton rats Sigmodon hispidus via a common route (lip abrasion) can cause multifocal CNS demyelination and inflammation. Remyelination occurred shortly after demyelination in HSV-1-infected cotton rats, but could be incomplete, resulting in "scars", further supporting an association between HSV-1 infection and multifocal demyelinating disorders. Virus was detected sequentially in the lip, trigeminal ganglia, and the brain of infected animals. Brain pathology developed primarily on the ipsilateral side of the brainstem, in the cerebellum, and contralateral side of the forebrain/midbrain, suggesting that the changes may ascend along the trigeminal lemniscus pathway. Neurologic defects occasionally detected in infected animals (e.g., defective whisker touch and blink responses, compromised balance) could be representative of the brainstem/cerebellum dysfunction. Immunization of cotton rats with a split HSV-1 vaccine protected animals against viral replication and brain pathology, suggesting that vaccination against HSV-1 may protect against demyelinating disorders.

    IMPORTANCE Our work demonstrates for the first time a direct association between infection with herpes simplex virus 1, a ubiquitous human pathogen generally associated with facial cold sores, and multifocal brain demyelination in an otherwise normal host, cotton rat Sigmodon hispidus. For a long time demyelinating diseases were considered to be autoimmune in nature and were studied by indirect methods, such as immunizing animals with myelin components or feeding them toxic substances that induce demyelination. Treatment against demyelinating diseases has been elusive, partially because of their unknown etiology. This work provides the first experimental evidence for the role of HSV-1 as the etiologic agent of multifocal brain demyelination in a normal host and suggests that vaccination against HSV-1 can help to combat demyelinating disorders.

  • Interviral recombination between plant, insect and fungal RNA viruses: The role of intracellular Ca++/Mn++ pump [Virus-Cell Interactions]

  • Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including plant viruses, tomato bushy stunt virus, carnation Italian ringspot virus, turnip crinkle virus-associated RNA, and insect (+)RNA viruses, Flock House virus and Nodamura virus, and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are (-)strand recombinant RNAs and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication-competent in cell-free extracts, in yeast and in the plant Nicotiana benthamiana. We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.

    Importance: Viruses with RNA genomes are abundant and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis and adaptation to their hosts. The authors utilized the previously identified intracellular Ca++/Mn++ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal dsRNA virus and an insect virus, in yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca++/Mn++ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

  • Differential modulation of innate immune responses in human primary cells by influenza A viruses carrying human or avian non-structural 1 proteins [Cellular Response to Infection]

  • The influenza A virus (IAV) non-structural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFN), pro-inflammatory cytokines and promote the adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFNaalpha; and IFNbbeta;) and type III (IFN1-3) than viruses expressing avian IAV NS1 proteins (H5N1, H7N9 and H7N2), but the differences observed in the expression of pro-inflammatory cytokines like TNFaalpha; or IL-6 were not significant. In addition, using Imaging Flow Cytometry, we found that human and avian NS1 segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in Normal Human Bronchial Epithelial cells and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells as compared to NS1 from human viruses which could contribute to the severe disease induced by avian IAV in humans.

    Importance Influenza A viruses (IAV) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAV circulating in animals occasionally cross the species barrier infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to transmit among humans and initiate a pandemic. The non-structural (NS) 1 protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.

  • Palmitoylation of the hepatitis C virus NS2 regulates its subcellular localization and NS2-NS3 autocleavage [Genome Replication and Regulation of Viral Gene Expression]

  • Hepatitis C virus (HCV) nonstructural protein 2 (NS2) is a multifunctional protein implicated in both HCV RNA replication and virus particle assembly. NS2-encoded cysteine protease is responsible for an autoprocessing of NS2-NS3 precursor, an essential step in HCV RNA replication. NS2 also promotes HCV particle assembly by recruiting the envelope protein 2 (E2) to the virus assembly sites located at the detergent-resistant membranes (DRM). However, the fundamental mechanism regulating multiple functions of NS2 remains unclear. In this study, we discovered that NS2 is palmitoylated at the position 113 cysteine residue (NS2/C113) when expressed by itself in cells and during infectious HCV replication. Blocking NS2 palmitoylation by introducing NS2/C113S mutation reduced NS2-NS3 autoprocessing and impaired HCV RNA replication. Replication of NS2/C113S mutant was restored by inserting encephalomyocarditis virus internal ribosome entry site (EMCV IRES) between NS2 and NS3 to separate these two proteins independent of NS2-mediated autoprocessing. These results suggest that NS2 palmitoylation is critical for HCV RNA replication by promoting the NS2-NS3 autoprocessing. NS2/C113S mutation also impaired infectious HCV assembly, DRM localization of NS2 and E2, and colocalization of NS2 with Core and endoplasmic reticulum lipid raft-associated protein 2 (Erlin-2). In conclusion, our study revealed that two major functions of NS2 involved in HCV RNA replication and virus assembly, including NS2-NS3 autoprocessing and E2 recruitment to the DRM, are regulated by the palmitoylation at NS2/C113. Since S-palmitoylation is reversible, NS2 palmitoylation likely allows NS2 to fine-tune both HCV RNA replication and infectious particle assembly.

    IMPORTANCE Chronic infection with hepatitis C virus (HCV) is a major cause of severe liver diseases responsible for nearly 400,000 deaths per year. HCV NS2 protein is a multifunctional regulator of HCV replication involved in both viral genome replication and infectious virus assembly. However, the underlying mechanism that enables this protein to participate in multiple steps of HCV replication remains unknown. In this study, we discovered that NS2 palmitoylation is the master regulator of its multiple functions, including NS2-mediated self-cleavage and HCV envelope protein recruitment to the virus assembly sites, which in turn promote HCV RNA replication and infectious particle assembly, respectively. This newly revealed information suggests that NS2 palmitoylation could serve as a promising target to inhibit both HCV RNA replication and virus assembly, inspiring a new avenue for host-targeting strategies against HCV infection.

  • PDZ Domain-Containing Protein NHERF-2 is a Novel Target of Human Papillomavirus type 16 (HPV-16) and HPV-18 [Transformation and Oncogenesis]

  • Cancer-causing HPV E6 oncoproteins have a Class I PDZ-binding motif (PBM) on their C-terminus, which plays critical roles that are related to HPV life cycle and HPV-induced malignancies. E6 oncoproteins use these PBMs to interact with, and target for proteasome-mediated degradation, a plethora of cellular substrates that contain PDZ domains and which are involved in the regulation of various cellular pathways. In this study, we show that both HPV-16 and HPV-18 E6 can interact with Na+/H+ exchange regulatory factor 2 (NHERF-2), a PDZ domain-containing protein, which among other cellular functions also behaves as a tumor suppressor regulating endothelial proliferation. The interaction between the E6 oncoproteins and NHERF-2 is PBM-dependent and results in proteasome-mediated degradation of NHERF-2. We further confirmed this effect in cells derived from HPV-16 and HPV-18 positive cervical tumors, where we show that NHERF-2 protein turnover is increased in the presence of E6. Finally, our data indicate that E6-mediated NHERF-2 degradation results in p27 downregulation and cyclin D1 upregulation, leading to accelerated cellular proliferation. To our knowledge, this is the first report to demonstrate that E6 oncoproteins can stimulate cell proliferation by indirectly regulating p27 via targeting a PDZ domain-containing protein.

    IMPORTANCE This study links HPV-16 and HPV-18 E6 oncoproteins to the modulation of cellular proliferation. The PDZ domain-containing protein NHERF-2 is a tumor suppressor, shown to regulate endothelial proliferation, and here we demonstrate that NHERF-2 is targeted by HPV E6 for proteasome-mediated degradation. Interestingly, this indirectly affects p27, cyclin D1 and CDK4 protein levels and consequently affects cell proliferation. Hence, this study provides information that will improve our understanding of the molecular basis for HPV E6 function, and it also highlights the importance of the PDZ domain-containing protein NHERF2 and its tumor suppressive role in regulating cell proliferation.

  • Conserved CxnC motifs in Kaposi's sarcoma-associated herpesvirus ORF66 are required for viral late gene expression and are essential for its interaction with ORF34 [Genome Replication and Regulation of Viral Gene Expression]

  • Late gene transcription in the beta- and gammaherpesviruses depends on a set of virally-encoded transcriptional activators (vTAs) that hijack the host transcriptional machinery and direct it to a subset of viral genes that are required for completion of the viral replication cycle and capsid assembly. In Kaposi's sarcoma-associated herpesvirus (KSHV), these vTAs are encoded by ORF18, ORF24, ORF30, ORF31, ORF34, ORF66. Assembly of the vTAs into a complex is critical for late gene transcription, and thus deciphering the architecture of the complex is central to understanding its transcriptional regulatory activity. Here, we generated an ORF66-null virus and confirmed that it fails to produce late genes and infectious virions. We show that ORF66 is incorporated into the vTA complex primarily through its interaction with ORF34, which is dependent upon a set of four conserved cysteine-rich motifs in the C-terminal domain of ORF66. While both ORF24 and ORF6-6 occupy the canonical K8.1 late gene promoter, their promoter occupancy requires the presence of the other vTAs, suggesting that sequence-specific, stable binding requires assembly of the entire complex on the promoter. Additionally, we find that ORF24 expression is impaired in the absence of a stable vTA complex. This work extends our knowledge about the architecture of the KSHV vPIC and suggests that it functions as a complex to recognize late gene promoters.

    IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that is the causative agent of multiple human cancers. Release of infectious virions requires production of capsid proteins and other late genes, whose production are transcriptionally controlled by a complex of six virally-encoded proteins that hijack the host transcription machinery. It is poorly understood how this complex assembles or what function five of its six components play in transcription. Here, we demonstrate that ORF66 is an essential component of this complex in KSHV and that its inclusion in the complex depends upon its C-terminal domain, which contains highly conserved cysteine-rich motifs reminiscent of zinc finger motifs. Additionally, we examine assembly of the viral pre-initiation complex at late gene promoters and find that while sequence-specific binding of late gene promoters requires ORF24, it additionally requires a fully assembled viral pre-initation complex.

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