|JVI Current Issue|
Pandemic H1N1 (pH1N1) influenza virus emerged from swine in 2009 with an adequate capability to infect and transmit between people. In subsequent years, it has circulated as a seasonal virus and evolved further human-adapting mutations. Mutations in the hemagglutinin (HA) stalk that increase pH stability have been associated with human adaptation and airborne transmission of pH1N1 virus. Yet, our understanding of how pH stability impacts virus-host interactions is incomplete. Here, using recombinant viruses with point mutations that alter the pH stability of pH1N1 HA, we found distinct effects on virus phenotypes in different experimental models. Increased pH sensitivity enabled viruses to uncoat in endosomes more efficiently, manifesting as increased replication rate in typical continuous cell cultures under single-cycle conditions. A more acid-labile HA also conferred a small reduction in sensitivity to antiviral therapeutics that act at the pH-sensitive HA fusion step. Conversely, in primary human airway epithelium cultured at the air-liquid interface, increased pH sensitivity attenuated multicycle viral replication by compromising virus survival in the extracellular microenvironment. In a mouse model of influenza pathogenicity, there was an optimum HA activation pH, and viruses with either more- or less-pH-stable HA were less virulent. Opposing pressures inside and outside the host cell that determine pH stability may influence zoonotic potential. The distinct effects that changes in pH stability exert on viral phenotypes underscore the importance of using the most appropriate systems for assessing virus titer and fitness, which has implications for vaccine manufacture, antiviral drug development, and pandemic risk assessment.
IMPORTANCE The pH stability of the hemagglutinin surface protein varies between different influenza strains and subtypes and can affect the virusrrsquo; ability to replicate and transmit. Here, we demonstrate a delicate balance that the virus strikes within and without the target cell. We show that a pH-stable hemagglutinin enables a human influenza virus to replicate more effectively in human airway cells and mouse lungs by facilitating virus survival in the extracellular environment of the upper respiratory tract. Conversely, after entering target cells, being more pH stable confers a relative disadvantage, resulting in less efficient delivery of the viral genome to the host cell nucleus. Since the balance we describe will be affected differently in different host environments, it may restrict a virusrrsquo; ability to cross species. In addition, our findings imply that different influenza viruses may show variation in how well they are controlled by antiviral strategies targeting pH-dependent steps in the virus replication cycle.
Hand, foot, and mouth disease (HFMD), a highly contagious disease in children, is caused by human enteroviruses, including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and coxsackievirus A6 (CVA6). Although HFMD is usually mild and self-limiting, EV71 infection occasionally leads to fatal neurological disorders. Currently, no commercial antiviral drugs for HFMD treatment are available. Here, numerous sulfonated azo dyes, widely used as food additives, were identified as having potent antiviral activities against human enteroviruses. Among them, brilliant black BN (E151) was able to inhibit all EV71, CVA16, and CVA6 strains tested. In rhabdomyosarcoma cells, the 50% inhibitory concentrations of the dye E151 for various strains of EV71 ranged from 2.39 mmu;M to 28.12 mmu;M, whereas its 50% cytotoxic concentration was 1,870 mmu;M. Food azo dyes, including E151, interacted with the vertex of the 5-fold axis of EV71 and prevented viral entry. Their efficacy in viral inhibition was regulated by amino acids at VP1-98, VP1-145, and/or VP1-246. Dye E151 not only prevented EV71 attachment but also eluted attached viruses in a concentration-dependent manner. Moreover, E151 inhibited the interaction between EV71 and its cellular uncoating factor cyclophilin A. In vivo studies demonstrated that E151 at a dose of 200 mg/kg of body weight/day given on the initial 4 days of challenge protected AG129 mice challenged with 10x the 50% lethal dose of wild-type EV71 isolates. Taken together, these data highlight E151 as a promising antiviral agent against EV71 infection.
IMPORTANCE Human enterovirus 71 (EV71) is one of the causative agents of hand, foot, and mouth disease in children and is responsible for thousands of deaths in the past 20 years. Food azo dyes have been widely used since the nineteenth century; however, their biological effects on humans and microbes residing in humans are poorly understood. Here, we discovered that one of these dyes, brilliant black BN (E151), was particularly effective in inhibiting the infectivity of EV71 in both cell culture and mouse model studies. Mechanistic studies demonstrated that these sulfonated dyes mainly competed with EV71 attachment factors for viral binding to block viral attachment/entry to host cells. As no commercial antiviral drugs against EV71 are currently available, our findings open an avenue to exploit the development of permitted food dye E151 as a potential anti-EV71 agent.
ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human
IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.
The retroviral Gag capsid (Gag-CA) interdomain linker is an unstructured peptide segment connecting structured N-terminal and C-terminal domains. Although the region is reported to play roles in virion morphogenesis and infectivity, underlying molecular mechanisms remain unexplored. To address this issue, we determined biological and molecular phenotypes of HIV-1 CA linker mutants by experimental and in silico approaches. Among the nine linker mutants tested, eight exhibited attenuation of viral particle production to various extents mostly in parallel with a reduction in viral infectivity. Sucrose density gradient, confocal microscopy, and live-cell protein interaction analyses indicated that the defect is accompanied by attenuation of Gag-Gag interactions following Gag plasma membrane targeting in the cells. In silico analyses revealed distinct distributions of interaction-prone hydrophobic patches between immature and mature CA proteins. Molecular dynamics simulations predicted that the linker mutations can allosterically alter structural fluctuations, including the interaction surfaces apart from the mutation sites in both the immature and mature CA proteins. These results suggest that the HIV-1 CA interdomain linker is a cis-modulator of the CA interaction surfaces to optimize efficiency of Gag assembly, virion production, and viral infectivity.
IMPORTANCE HIV-1 particle production and infection are highly ordered processes. Viral Gag proteins play a central role in the assembly and disassembly of viral molecules. Of these, capsid protein (CA) is a major contributor to the Gag-Gag interactions. CA consists of two structured domains, i.e., N-terminal (NTD) and C-terminal (CTD) domains, connected by an unstructured domain named the interdomain linker. While multiple regions in the NTD and CTD are reported to play roles in virion morphogenesis and infectivity, the roles of the linker region in Gag assembly and virus particle formation remain elusive. In this study, we showed by biological and molecular analyses that the linker region functions as an intramolecular modulator to tune Gag assembly, virion production, and viral infectivity. Our study thus illustrates a hitherto-unrecognized mechanism, an allosteric regulation of CA structure by the disordered protein element, for HIV-1 replication.
Sequences derived from parvoviruses (family Parvoviridae) are relatively common in animal genomes, but the functional significance of these endogenous parvoviral element (EPV) sequences remains unclear. In this study, we used a combination of in silico and molecular biological approaches to investigate a fusion gene carried by guinea pigs (genus Cavia) that is partially derived from an EPV. This gene, named enRep-M9l, encodes a predicted polypeptide gene product comprising a partial myosin9-like (M9l) gene fused to a 3' truncated, EPV-encoded replicase. We examined the genomic and phylogenetic characteristics of the EPV locus (enRep) that encodes the viral portions of enRep-M9l, revealing that it derives from an ancient dependoparvovirus (genus Dependoparvovirus) that was incorporated into the guinea pig germ line between approximately 22 and 35 million years ago (MYA). Despite these ancient origins, the regions of the enRep locus that are expressed in the enRep-M9l gene are conserved across multiple species in the family Caviidae (guinea pigs and cavies), consistent with a potential function at the amino acid level. Using molecular biological approaches, we further demonstrated that (i) enRep-M9l mRNA is broadly transcribed in guinea pig cells, (ii) the cloned enRep-M9l transcript can express a protein of the expected size in guinea pig cells in vitro, and (iii) the expressed protein localizes to the cytosol. Our findings demonstrate that, consistent with a functional role, the enRep-M9l fusion gene is evolutionarily conserved, broadly transcribed, and capable of expressing protein.
IMPORTANCE DNA from viruses has been "horizontally transferred" to mammalian genomes during evolution, but the impact of this process on mammalian biology remains poorly understood. The findings of our study indicate that a novel gene has evolved in guinea pigs through fusion of host and virus genes.
The Us11 protein encoded by herpes simplex virus 1 (HSV-1) functions to impair autophagy; however, the molecular mechanisms of this inhibition remain to be fully established. Here, we report that the Us11 protein targets tripartite motif protein 23 (TRIM23), which is a key regulator of autophagy-mediated antiviral defense mediated by TANK-binding kinase 1 (TBK1). In virus-infected cells, the Us11 protein drastically reduces the formation of autophagosomes mediated by TRIM23 or TBK1. This autophagy-inhibitory effect is attributable to the binding of the Us11 protein to the ARF domain in TRIM23. Furthermore, such interaction spatially excludes TBK1 from the TRIM23 complex that also contains heat shock protein 90 (Hsp90). When stably expressed alone in host cells, the Us11 protein recapitulates the observed phenotypes seen in cells infected with the US11-expressing or wild-type virus. Consistent with this, expression of the Us11 protein promotes HSV-1 growth, while expression of TRIM23 restricts HSV-1 replication in the absence of US11. Together, these results suggest that disruption of the TRIM23-TBK1 complex by the Us11 protein inhibits autophagy-mediated restriction of HSV-1 infection.
IMPORTANCE Autophagy is an evolutionarily conserved process that restricts certain intracellular pathogens, including HSV-1. Although HSV-1 is well known to inhibit autophagy, little is known about the precise molecular mechanisms of autophagy inhibition. We demonstrate that the Us11 protein of HSV-1 spatially disrupts the TRIM23-TBK1 complex, which subsequently suppresses autophagy and autophagy-mediated virus restriction. Thus, expression of the Us11 protein facilitates HSV-1 replication. These data unveil new insight into viral escape from autophagy-mediated host restriction mechanisms.
Varicella-zoster virus (VZV) is an alphaherpesvirus that lacks the herpesviral neurovirulence protein ICP34.5. The underlying hypothesis of this project was that inhibitors of autophagy reduce VZV infectivity. We selected the vacuolar proton ATPase inhibitor bafilomycin A1 for analysis because of its well-known antiautophagy property of impeding acidification during the late stage of autophagic flux. We documented that bafilomycin treatment from 48 to 72 h postinfection lowered VZV titers substantially (P lle; 0.008). Because we were unable to define the site of the block in the infectious cycle by confocal microscopy, we turned to electron microscopy. Capsids were observed in the nucleus, in the perinuclear space, and in the cytoplasm adjacent to Golgi apparatus vesicles. Many of the capsids had an aberrant appearance, as has been observed previously in infections not treated with bafilomycin. In contrast to prior untreated infections, however, secondary envelopment of capsids was not seen in the trans-Golgi network, nor were prototypical enveloped particles with capsids (virions) seen in cytoplasmic vesicles after bafilomycin treatment. Instead, multiple particles with varying diameters without capsids (light particles) were seen in large virus assembly compartments near the disorganized Golgi apparatus. Bafilomycin treatment also led to increased numbers of multivesicular bodies in the cytoplasm, some of which contained remnants of the Golgi apparatus. In summary, we have defined a previously unrecognized property of bafilomycin whereby it disrupted the site of secondary envelopment of VZV capsids by altering the pH of the trans-Golgi network and thereby preventing the correct formation of virus assembly compartments.
IMPORTANCE This study of VZV assembly in the presence of bafilomycin A1 emphasizes the importance of the Golgi apparatus/trans-Golgi network as a platform in the alphaherpesvirus life cycle. We have previously shown that VZV induces levels of autophagy far above the basal levels of autophagy in human skin, a major site of VZV assembly. The current study documented that bafilomycin treatment led to impaired assembly of VZV capsids after primary envelopment/de-envelopment but before secondary reenvelopment. This VZV study also complemented prior herpes simplex virus 1 and pseudorabies virus studies investigating two other inhibitors of endoplasmic reticulum (ER)/Golgi apparatus function: brefeldin A and monensin. Studies with porcine herpesvirus demonstrated that primary enveloped particles accumulated in the perinuclear space in the presence of brefeldin A, while studies with herpes simplex virus 1 documented an impaired secondary assembly of enveloped viral particles in the presence of monensin.
Autophagy maintains cellular homeostasis by degrading organelles, proteins, and lipids in lysosomes. Autophagy is involved in the innate and adaptive immune responses to a variety of pathogens. Some viruses can hijack host autophagy to enhance their replication. However, the role of autophagy in porcine reproductive and respiratory syndrome virus (PRRSV) infection is unclear. Here, we show that N-Myc downstream-regulated gene 1 (NDRG1) deficiency induced autophagy, which facilitated PRRSV replication by regulating lipid metabolism. NDRG1 mRNA is expressed ubiquitously in most porcine tissues and most strongly in white adipose tissue. PRRSV infection downregulated the expression of NDRG1 mRNA and protein, while NDRG1 deficiency contributed to PRRSV RNA replication and progeny virus assembly. NDRG1 deficiency reduced the number of intracellular lipid droplets (LDs), but the expression levels of key genes in lipogenesis and lipolysis were not altered. Our results also show that NDRG1 deficiency promoted autophagy and increased the subsequent yields of hydrolyzed free fatty acids (FFAs). The reduced LD numbers, increased FFA levels, and enhanced PRRSV replication were abrogated in the presence of an autophagy inhibitor. Overall, our findings suggest that NDRG1 plays a negative role in PRRSV replication by suppressing autophagy and LD degradation.
IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped single-positive-stranded RNA virus, causes acute respiratory distress in piglets and reproductive failure in sows. It has led to tremendous economic losses in the swine industry worldwide since it was first documented in the late 1980s. Vaccination is currently the major strategy used to control the disease. However, conventional vaccines and other strategies do not provide satisfactory or sustainable prevention. Therefore, safe and effective strategies to control PRRSV are urgently required. The significance of our research is that we demonstrate a previously unreported relationship between PRRSV, NDRG1, and lipophagy in the context of viral infection. Furthermore, our data point to a new role for NDRG1 in autophagy and lipid metabolism. Thus, NDRG1 and lipophagy will have significant implications for understanding PRRSV pathogenesis for developing new therapeutics.
We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.
IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.
Herpesviruses are ubiquitous, and infection by some, like Epstein-Barr virus (EBV), is nearly universal. To persist, EBV must periodically switch from a latent to a replicative/lytic phase. This productive phase is responsible for most herpesvirus-associated diseases. EBV encodes a latency-to-lytic switch protein which, upon activation, sets off a vectorially constrained cascade of gene expression that results in production of infectious virus. While triggering expression of the switch protein ZEBRA is essential to lytic cycle entry, sustaining its expression is equally important to avoid premature termination of the lytic cascade. We report that the viral protein kinase (vPK), encoded by a gene that is kinetically downstream of the lytic switch, sustains expression of ZEBRA, amplifies the lytic cascade, increasing virus production, and, importantly, prevents the abortive lytic cycle. We find that vPK, through a noncanonical site phosphorylation, activates the cellular phosphatidylinositol 3-kinase-related kinase ATM to cause phosphorylation of the heterochromatin enforcer KAP1/TRIM28 even in the absence of EBV genomes or other EBV proteins. Phosphorylation of KAP1 renders it unable to restrain ZEBRA, thereby further derepressing and sustaining its expression to culminate in virus production. This partnership with a host kinase and a transcriptional corepressor enables retrograde regulation by vPK of ZEBRA, an observation that is counter to the unidirectional regulation of gene expression reminiscent of most DNA viruses.
IMPORTANCE Herpesviruses infect nearly all humans and persist quiescently for the life of the host. These viruses intermittently activate into the lytic phase to produce infectious virus, thereby causing disease. To ensure that lytic activation is not prematurely terminated, expression of the virally encoded lytic switch protein needs to be sustained. In studying Epstein-Barr virus, one of the most prevalent human herpesviruses that also causes cancer, we have discovered that a viral kinase activated by the viral lytic switch protein partners with a cellular kinase to deactivate a silencer of the lytic switch protein, thereby providing a positive feedback loop to ensure successful completion of the viral productive phase. Our findings highlight key nodes of interaction between the host and virus that could be exploited to treat lytic phase-associated diseases by terminating the lytic phase or kill cancer cells harboring herpesviruses by accelerating the completion of the lytic cascade.
Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.
IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.
There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37ddeg;C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-AAring; resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.
IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.
The RV144 vaccine trial is the only clinical study to have shown a modest but statistically significant decrease in HIV infection risk. RV144 and the subsequent studies identifying the level of V1V2-specific antibodies as a correlate of reduced infection risk are still controversial despite many papers supporting and expanding the initial study. We address these controversies and summarize active-immunization and passive-immunization experiments in nonhuman primates that support the initial finding.
Despite the fact that the cell surface expression level of HLA-C on both uninfected and HIV-infected cells is lower than those of HLA-A and -B, increasing evidence suggests an important role for HLA-C and HLA-C-restricted CD8+ T cell responses in determining the efficiency of viral control in HIV-1-infected individuals. Nonetheless, HLA-C-restricted T cell responses are much less well studied than HLA-A/B-restricted ones, and relatively few optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C alleles have been defined. Recent improvements in the sensitivity of mass spectrometry (MS)-based approaches for profiling the immunopeptidome present an opportunity for epitope discovery on a large scale. Here, we employed an MS-based immunopeptidomic strategy to characterize HIV-1 peptides presented by a protective allele, HLA-C*12:02. We identified a total of 10,799 unique 8- to 12-mer peptides, including 15 HIV-1 peptides. The latter included 2 previously reported immunodominant HIV-1 epitopes, and analysis of T cell responses to the other HIV-1 peptides detected revealed an additional immunodominant epitope. These findings illustrate the utility of MS-based approaches for epitope definition and emphasize the capacity of HLA-C to present immunodominant T cell epitopes in HIV-infected individuals, indicating the importance of further evaluation of HLA-C-restricted responses to identify novel targets for HIV-1 prophylactic and therapeutic strategies.
IMPORTANCE Mass spectrometry (MS)-based approaches are increasingly being employed for large-scale identification of HLA-bound peptides derived from pathogens, but only very limited profiling of the HIV-1 immunopeptidome has been conducted to date. Notably, a growing body of evidence has recently begun to indicate a protective role for HLA-C in HIV-1 infection, which may suggest that despite the fact that levels of HLA-C expression on both uninfected and HIV-1-infected cells are lower than those of HLA-A/B, HLA-C still presents epitopes to CD8+ T cells effectively. To explore this, we analyzed HLA-C*12:02-restricted HIV-1 peptides presented on HIV-1-infected cells expressing only HLA-C*12:02 (a protective allele) using liquid chromatography-tandem MS (LC-MS/MS). We identified a number of novel HLA-C*12:02-bound HIV-1 peptides and showed that although the majority of them did not elicit T cell responses during natural infection in a Japanese cohort, they included three immunodominant epitopes, emphasizing the contribution of HLA-C to epitope presentation on HIV-infected cells.
Broad and potent neutralizing antibodies (bnAbs) with multiple epitope specificities evolve in HIV-1-infected children. Herein, we studied two antiretroviral-naive chronically HIV-1 clade C-infected monozygotic pediatric twins, AIIMS_329 and AIIMS_330, with potent plasma bnAbs. Elite plasma neutralizing activity was observed since the initial sampling at 78 months of age in AIIMS_330 and persisted throughout, while in AIIMS_329 it was seen at 90 months of age, after which the potency decreased over time. We evaluated potential viral characteristics associated with the varied immune profiles by generating single genome-amplified pseudoviruses. The AIIMS_329 viruses generated from the 90-month time point were neutralization sensitive to bnAbs and contemporaneous plasma antibodies, while viruses from the 112-month and 117-month time points were resistant to most bnAbs and contemporaneous plasma. AIIMS_329 viruses developed resistance to plasma neutralizing antibodies (nAbs) plausibly by N160 glycan loss and V1 and V4 loop lengthening. The viruses generated from AIIMS_330 (at 90 and 117 months) showed varied susceptibility to bnAbs and autologous contemporaneous plasma antibodies, while the viruses of the 112-month time point, at which the plasma nAb specificities mapped to the V2 glycan, V3 glycan, and CD4 binding site (CD4bs), were resistant to contemporaneous plasma antibodies as well as to most bnAbs. Chimeric viruses were constructed from 90-month-time-point PG9-sensitive AIIMS_329 and AIIMS_330 viruses with swapped V1V2 regions of their respective evolved viruses (at 112 and 117 months), which led to higher resistance to neutralization by PG9 and autologous plasma antibodies. We observed the evolution of a viral pool in the AIIMS_330 donor comprising plasma antibody neutralization-sensitive or -resistant diverse autologous viruses that may have contributed to the development and maintenance of elite neutralizing activity.
IMPORTANCE Herein, we report the longitudinal development of bnAbs in a pair of chronically HIV-1 clade C-infected monozygotic pediatric twins, AIIMS_329 and AIIMS_330, who acquired the infection by vertical transmission. The plasma from both donors, sharing a similar genetic makeup and infecting virus, showed the evolvement of bnAbs targeting common epitopes in the V2 and V3 regions of the envelope, suggesting that bnAb development in these twins may perhaps be determined by specific sequences in the shared virus that can guide the development of immunogens aimed at eliciting V2 and V3 bNAbs. Characterization of the neutralization-sensitive and -resistant viruses coevolving with bNAbs in the contemporaneous AIIMS_330 plasma provides information toward understanding the viral alterations that may have contributed to the development of resistance to bnAbs. Further longitudinal studies in more monozygotic and dizygotic twin pairs will help in delineating the role of host and viral factors that may contribute to the development of bnAbs.
The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses.
IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.
MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) and Marekrrsquo;s disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.
IMPORTANCE Marekrrsquo;s disease virus (MDV) is an alphaherpesvirus associated with Marekrrsquo;s disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.
We previously produced a replication-defective West Nile virus (WNV) lacking NS1 (WNV-NS1) that could propagate at low levels (105 infectious units [IU]/ml) in a 293T cell line expressing wild-type (WT) NS1. This finding indicates the potential of developing WNV-NS1 as a noninfectious vaccine. To explore this idea, we developed an NS1-expressing Vero cell line (VeroNS1) that significantly improved the yield of WNV-NS1 (108 IU/ml). We evaluated the safety and efficacy of WNV-NS1 in mice. WNV-NS1 appeared to be safe, as no replicative virus was found in naive Vero cells after continuous culturing of WNV-NS1 in VeroNS1 cells for 15 rounds. WNV-NS1 was noninfectious in mice, even when IFNARnndash;/nndash; mice were administered a high dose of WNV-NS1. Vaccination with a single dose of WNV-NS1 protected mice from a highly lethal challenge with WT WNV. The antibody response against WNV correlated well with the protection of vaccinated mice. Our study demonstrates the potential of the NS1 trans complementation system as a new platform for flavivirus vaccine development.
IMPORTANCE Many flaviviruses are significant human pathogens that frequently cause outbreaks and epidemics around the world. Development of novel vaccine platforms against these pathogens is a public health priority. Using WNV as a model, we developed a new vaccine platform for flaviviruses. WNV containing a NS1 deletion (WNV-NS1) could be efficiently trans complemented in Vero cells that constitutively expressed WT NS1 protein. A single-dose immunization with WNV-NS1 elicited robust immune responses in mice. The immunized animals were fully protected against pathogenic WNV infection. No adverse effects related to the WNV-NS1 vaccination were observed. The results have demonstrated the potential of the NS1 complementation system as an alternative platform for flavivirus vaccine development, especially for highly pathogenic flaviviruses.
HIV's ability to persist during suppressive antiretroviral therapy is the main barrier to cure. Immune-privileged tissues, such as the testes, may constitute distinctive sites of HIV persistence, but this has been challenging to study in humans. We analyzed the proviral burden and genetics in the blood and testes of 10 individuals on suppressive therapy who underwent elective gender-affirming surgery. HIV DNA levels in matched blood and testes were quantified by quantitative PCR, and subgenomic proviral sequences (nef region) were characterized from single templates. HIV diversity, compartmentalization, and immune escape burden were assessed using genetic and phylogenetic approaches. Diverse proviruses were recovered from the blood (396 sequences; 354 nef-intact sequences) and testes (326 sequences; 309 nef-intact sequences) of all participants. Notably, the frequency of identical HIV sequences varied markedly between and within individuals. Nevertheless, proviral loads, within-host unique HIV sequence diversity, and the immune escape burden correlated positively between blood and testes. When all intact nef sequences were evaluated, 60% of participants exhibited significant blood-testis genetic compartmentalization, but none did so when the evaluation was restricted to unique sequences per site, suggesting that compartmentalization, when present, is attributable to the clonal expansion of HIV-infected cells. Our observations confirm the testes as a site of HIV persistence and suggest that individuals with larger and more diverse blood reservoirs will have larger and more diverse testis reservoirs. Furthermore, while the testis microenvironment may not be sufficiently unique to facilitate the seeding of unique viral populations therein, differential clonal expansion dynamics may be at play, which may complicate HIV eradication.
IMPORTANCE Two key questions in HIV reservoir biology are whether immune-privileged tissues, such as the testes, harbor distinctive proviral populations during suppressive therapy and, if so, by what mechanism. While our results indicated that blood-testis HIV genetic compartmentalization was reasonably common (60%), it was always attributable to differential frequencies of identical HIV sequences between sites. No blood-tissue data set retained evidence of compartmentalization when only unique HIV sequences per site were considered; moreover, HIV immune escape mutation burdens were highly concordant between sites. We conclude that the principal mechanism by which blood and testis reservoirs differ is not via seeding of divergent HIV sequences therein but, rather, via differential clonal expansion of latently infected cells. Thus, while viral diversity and escape-related barriers to HIV eradication are of a broadly similar magnitude across the blood and testes, clonal expansion represents a challenge. The results support individualized analysis of within-host reservoir diversity to inform curative approaches.
Hypoxia and hypoxia inducible factors (HIFs) play important roles in the Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) life cycle. KSHV is the causative agent of Kaposirrsquo;s sarcoma (KS) and other AIDS-related malignancies. Kaposirrsquo;s sarcoma is a highly vascular tumor, which preferentially develops in the lower extremities of the body where blood vessels are often poorly oxygenated. The main cellular responses to hypoxia are mediated mainly by two isoforms of HIF, HIF-1aalpha; and HIF-2aalpha;. HIF-1aalpha; and HIF-2aalpha; have common as well as distinct functions, although they are similar in structure and function. Previously, we showed that the KSHV ORF34 protein binds HIF-1aalpha; and facilitates its degradation through the ubiquitin-proteasome pathway causing negative regulation of HIF-1aalpha;-dependent genes (Haque and Kousoulas, J Virol 87:2164-2173, 2013, https://www.doi.org/10.1128/JVI.02460-12). Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. Coimmunoprecipitation experiments revealed that the ORF34 protein physically interacted with HIF-2aalpha; in transfected as well as in KSHV-infected cells. Utilization of ORF34 truncations revealed that three distinct domains bind HIF-2aalpha; and that both bHLH and PAS domains of HIF-2aalpha; interacted with ORF34. Unlike HIF-1aalpha;, dose-dependent coexpression of ORF34 stabilized the HIF-2aalpha; protein, ensuring HIF-2aalpha;-dependent transcriptional activity. The ORF34 protein enhanced HIF-2aalpha; ubiquitination at the bHLH and PAS domains. The results show that the KSHV ORF34 protein is involved in the KSHV life cycle by regulating the expression of HIF-1aalpha; and HIF-2aalpha; proteins.
IMPORTANCE Hypoxia inducible factor 1aalpha; (HIF-1aalpha;) and HIF-2aalpha; are transcription factors which play important roles in the Kaposirrsquo;s sarcoma-associated herpesvirus (KSHV) latent and lytic gene replication. Herein, we show that the ORF34 gene is involved in the regulation of KSHV lytic gene expression, since deletion of ORF34 resulted in reduced immediate early and early lytic gene expression and blocked late gene expression. In addition, we demonstrate that the KSHV ORF34 protein binds and stabilizes HIF-2aalpha;, in contrast to its role in binding HIF-1aalpha; and causing its degradation via the proteasome pathway. Thus, the KSHV ORF34 protein plays a regulatory role in the KSHV life cycle by regulating HIF-1aalpha; and HIF-2aalpha; expression.
Previous studies in our laboratory characterized a panel of highly mutated HIV-specific conformational epitope-targeting antibodies (Abs) from a panel of HIV-infected long-term nonprogressors (LTNPs). Despite binding HIV envelope protein and having a high number of somatic amino acid mutations, these Abs had poor neutralizing activity. Because of the evidence of antigen-driven selection and the long CDR3 region (21 amino acids [aa]), we further characterized the epitope targeting of monoclonal Ab (MAb) 76-Q3-2C6 (2C6). We confirmed that 2C6 binds preferentially to trimeric envelope and recognizes the clades A, B, and C SOSIP trimers. 2C6 binds gp140 constructs of clades A, B, C, and D, suggesting a conserved binding site that we localized to the ectodomain of gp41. Ab competition with MAb 50-69 suggested this epitope localizes near aa 579 to 613 (referenced to HXB2 gp160). Peptide library scanning showed consistent binding in this region but to only a single peptide. Lack of overlapping peptide binding supported a nonlinear epitope structure. The significance of this site is supported by 2C6 having Ab-dependent cell cytotoxicity (ADCC) against envelope proteins from two clades. Using 2C6 and variants, alanine scanning mutagenesis identified three amino acids (aa 592, 595, and 596) in the overlapping region of the previously identified peptide. Additional amino acids at sites 524 and 579 were also identified, helping explain its conformational requirement. The fact that different amino acids were included in the epitope depending on the targeted protein supports the conclusion that 2C6 targets a native conformational epitope. When we mapped these amino acids on the trimerized structure, they spanned across oligomers, supporting the notion that the epitope targeted by 2C6 lies in a recessed pocket between two gp41 oligomers. A complete understanding of the epitope specificity of ADCC-mediating Abs is essential for developing effective immunization strategies that optimize protection by these Abs.
IMPORTANCE This paper further defines the function and area of the HIV trimeric envelope protein targeted by the monoclonal antibody 2C6. 2C6 binding is influenced by amino acid mutations across two separate gp41 sections of the envelope trimer. This epitope is recognized on multiple clades (variant groups of circulating viruses) of gp41, gp140 trimers, and SOSIP trimers. For the clades tested, 2C6 has robust ADCC. As the target of 2C6 is available in the major clades of HIV and has robust ADCC activity, further definition and appreciation of targeting of antibodies similar to 2C6 during vaccine development should be considered.
Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development.
IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre- to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.
Combating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates using in vitro and in vivo assays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though the in vivo efficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.
IMPORTANCE Anti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.
Accumulated evidence demonstrates that Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, the precise role of PERK in JEV-induced apoptosis and encephalitis remains unknown. Here, we report that JEV infection activates the PERK-ATF4-CHOP apoptosis pathway both in vitro and in vivo. PERK activation also promotes the formation of stress granule, which in turn represses JEV-induced apoptosis. However, PERK inhibitor reduces apoptosis, indicating that JEV-activated PERK predominantly induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway. Among JEV proteins that have been reported to induce ER stress, only JEV NS4B can induce PERK activation. PERK has been reported to form an active molecule by dimerization. The coimmunoprecipitation assay shows that NS4B interacts with PERK. Moreover, glycerol gradient centrifugation shows that NS4B induces PERK dimerization. Both the LIG-FHA and the LIG-WD40 domains within NS4B are required to induce PERK dimerization, suggesting that JEV NS4B pulls two PERK molecules together by simultaneously interacting with them via different motifs. PERK deactivation reduces brain cell damage and encephalitis during JEV infection. Furthermore, expression of JEV NS4B is sufficient to induce encephalitis via PERK in mice, indicating that JEV activates PERK primarily via its NS4B to cause encephalitis. Taken together, our findings provide a novel insight into JEV-caused encephalitis.
IMPORTANCE Japanese encephalitis virus (JEV) infection triggers endoplasmic reticulum (ER) stress and neuron apoptosis. ER stress sensor protein kinase R-like endoplasmic reticulum kinase (PERK) has been reported to induce apoptosis under acute or prolonged ER stress. However, whether the PERK pathway of ER stress response plays important roles in JEV-induced apoptosis and encephalitis remains unknown. Here, we found that JEV infection activates ER stress sensor PERK in neuronal cells and mouse brains. PERK activation induces apoptosis via the PERK-ATF4-CHOP apoptosis pathway upon JEV infection. Among the JEV proteins prM, E, NS1, NS2A, NS2B, and NS4B, only NS4B activates PERK. Moreover, activated PERK participates in apoptosis and encephalitis induced by JEV and NS4B. These findings provide a novel therapeutic approach for JEV-caused encephalitis.
Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor bbeta; (TGF-bbeta;) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-bbeta;1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression.
IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression.
The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.
IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.
The herpesvirus human cytomegalovirus (HCMV) is a leading cause of congenital birth defects. Infection can result in infants born with a variety of symptoms, including hepatosplenomegaly, microcephaly, and developmental disabilities. Microcephaly is associated with disruptions in the neural progenitor cell (NPC) population. Here, we defined the impact of HCMV infection on neural tissue development and calcium regulation, a critical activity in neural development. Regulation of intracellular calcium involves purinergic receptors and voltage-gated calcium channels (VGCC). HCMV infection compromised the ability of both pathways in NPCs as well as fibroblasts to respond to stimulation. We observed significant drops in basal calcium levels in infected NPCs which were accompanied by loss in VGCC activity and purinergic receptor responses. However, uninfected cells in the population retained responsiveness. Addition of the HCMV inhibitor maribavir reduced viral spread but failed to restore activity in infected cells. To study neural development, we infected three-dimensional cortical organoids with HCMV. Infection spread to a subset of cells over time and disrupted organoid structure, with alterations in developmental and neural layering markers. Organoid-derived infected neurons and astrocytes were unable to respond to stimulation whereas uninfected cells retained nearly normal responses. Maribavir partially restored structural features, including neural rosette formation, and dampened the impact of infection on neural cellular function. Using a tissue model system, we have demonstrated that HCMV alters cortical neural layering and disrupts calcium regulation in infected cells.
IMPORTANCE Human cytomegalovirus (HCMV) replicates in several cell types throughout the body, causing disease in the absence of an effective immune response. Studies on HCMV require cultured human cells and tissues due to species specificity. In these studies, we investigated the impact of infection on developing three-dimensional cortical organoid tissues, with specific emphasis on cell-type-dependent calcium signaling. Calcium signaling is an essential function during neural differentiation and cortical development. We observed that HCMV infects and spreads within these tissues, ultimately disrupting cortical structure. Infected cells exhibited depleted calcium stores and loss of ATP- and KCl-stimulated calcium signaling while uninfected cells in the population maintained nearly normal responses. Some protection was provided by the viral inhibitor maribavir. Overall, our studies provide new insights into the impact of HCMV on cortical tissue development and function.
Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker bbeta;-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects.
IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker bbeta;-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.
Zika virus (ZIKV) infection in pregnant women can lead to fetal deaths and malformations. We have previously reported that ZIKV envelope protein domain III (EDIII) is a subunit vaccine candidate with cross-neutralization activity; however, like many other subunit vaccines, its efficacy is limited. To improve the efficacy of this subunit vaccine, we identified a nonneutralizing epitope on ZIKV EDIII surrounding residue 375, which is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We then shielded this epitope with an engineered glycan probe. Compared to the wild-type EDIII, the mutant EDIII induced significantly stronger neutralizing antibodies in three mouse strains and also demonstrated significantly improved efficacy by fully protecting mice, particularly pregnant mice and their fetuses, against high-dose lethal ZIKV challenge. Moreover, the mutant EDIII immune sera significantly enhanced the passive protective efficacy by fully protecting mice against lethal ZIKV challenge; this passive protection was positively associated with neutralizing antibody titers. We further showed that the enhanced efficacy of the mutant EDIII was due to the shielding of the immunodominant nonneutralizing epitope surrounding residue 375, which led to immune refocusing on the neutralizing epitopes. Taken together, the results of this study reveal that an intrinsic limitation of subunit vaccines is their artificially exposed immunodominant nonneutralizing epitopes, which can be overcome through glycan shielding. Additionally, the mutant ZIKV protein generated in this study is a promising subunit vaccine candidate with high efficacy in preventing ZIKV infections in mice.
IMPORTANCE Viral subunit vaccines generally show low efficacy. In this study, we revealed an intrinsic limitation of subunit vaccine designs: artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. More specifically, we identified an epitope on Zika virus (ZIKV) envelope protein domain III (EDIII) that is buried in the full-length envelope protein but becomes exposed in recombinant EDIII. We further shielded this epitope with a glycan, and the resulting mutant EDIII vaccine demonstrated significantly enhanced efficacy over the wild-type EDIII vaccine in protecting animal models from ZIKV infections. Therefore, the intrinsic limitation of subunit vaccines can be overcome through shielding these artificially exposed unfavorable epitopes. The engineered EDIII vaccine generated in this study is a promising vaccine candidate that can be further developed to battle ZIKV infections.
|JVI Accepts: Articles Published Ahead of Print|
HVTN 505 was a phase 2b efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) HIV vaccine regimen. Although the trial was stopped early for lack of overall efficacy, later correlates of risk and sieve analyses generated the hypothesis that the DNA/rAd5 vaccine regimen protected some vaccinees from HIV infection, yet enhanced HIV infection risk for others. Here we assessed whether and how host Fc gamma receptor (FcR) genetic variations influenced the DNA/rAd5 vaccine regimen's effect on HIV infection risk. We found that vaccine receipt significantly increased HIV acquisition compared with placebo receipt among participants carrying the FCGR2C-TATA haplotype (comprising minor alleles of four FCGR2C single nucleotide polymorphism (SNP) sites) (HR=9.79, p=0.035) but not among participants without the haplotype (HR=0.86, p=0.67); the interaction of vaccine and haplotype effect was significant (p=0.034). Similarly, vaccine receipt increased HIV acquisition compared with placebo receipt among participants carrying the FCGR3B-AGA haplotype (comprising minor alleles of the 3 FCGR3B SNPs) (HR=2.78, p=0.058) but not among participants without the haplotype (HR=0.73, p=0.44); again, the interaction of vaccine and haplotype was significant (p-value=0.047). The FCGR3B-AGA haplotype also influenced whether a combined Env-specific CD8+ T-cell polyfunctionality score and IgG response correlated significantly with HIV risk; an FCGR2A SNP and two FCGR2B SNPs influenced whether anti-gp140 antibody-dependent cellular phagocytosis correlated significantly with HIV risk. These results provide further evidence that Fc gamma receptor genetic variations may modulate HIV vaccine effects and immune function after HIV vaccination.
IMPORTANCE By analyzing data from the HVTN 505 efficacy trial of a DNA/recombinant adenovirus 5 (rAd5) vaccine regimen, we found that host genetics, specifically Fc gamma receptor genetic variations, influenced whether receiving the DNA/rAd5 regimen was beneficial, neutral, or detrimental to an individual with respect to HIV-1 acquisition risk. Moreover, Fc gamma receptor genetic variations influenced immune responses to the DNA/rAd5 vaccine regimen. Thus, Fc gamma receptor genetic variations should be considered in the analysis of future HIV vaccine trials and the development of HIV vaccines.
Three RNA viruses related to nodaviruses were previously described to naturally infect the nematode Caenorhabditis elegans and its relative Caenorhabditis briggsae. Here we report on a collection of over 50 viral variants from wild-caught Caenorhabditis. We describe the discovery of a new related virus, the Mělniiacute;k virus, infecting C. briggsae, which similarly infects intestinal cells. In France, a frequent pattern of co-infection of C. briggsae by the Santeuil virus and Le Blanc virus was observed at the level of an individual nematode and even a single cell. We do not find evidence of reassortment between the RNA1 and RNA2 molecules of Santeuil and Le Blanc viruses. However, by studying patterns of evolution of each virus, reassortments of RNA1 and RNA2 among variants of each virus were identified. We develop assays to test the relative infectivity and competitive ability of the viral variants and detect an interaction between host genotype and Santeuil virus genotype, such that the result depends on the host strain.
IMPORTANCE The roundworm Caenorhabditis elegans is a laboratory model organism in biology. We study natural populations of this small animal and its relative C. briggsae and the viruses that infect them. We previously discovered three RNA viruses related to nodaviruses and here describe a fourth one, called the Mělniiacute;k virus. These viruses have a genome composed of two RNA molecules. We find that two viruses may infect the same animal and the same cell. The two RNA molecules may be exchanged between variants of a given viral species. We study the diversity of each viral species and devise an assay of their infectivity and competitive ability. Using this assay, we show that the outcome of the competition also depends on the host.
We describe a novel function for the interferon (IFN)-induced protein 44-like (IFI44L) in negatively modulating innate immune responses induced after virus infections. Furthermore, we show that decreasing IFI44L expression impairs virus production, and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. The mechanism likely involves the interaction of IFI44L with the cellular FK506-binding protein 5 (FKBP5), which in turn interacts with kinases essential for type I and III IFN responses, such as the inhibitor of nuclear factor kappa B (IB) kinases (IKK) aalpha;, bbeta; and . Consequently, binding of IFI44L to FKBP5 decreased interferon regulatory factor 3 (IRF-3) and nuclear factor kappa-B (NF-B) inhibitor (IBaalpha;) mediated phosphorylation by IKK and IKKbbeta;, respectively. According to these results, IFI44L is a good target to treat diseases associated with excessive IFN and/or proinflammatory responses and for reducing viral replication.
IMPORTANCE Excessive innate immune responses could be deleterious for the host, and therefore, negative feedback is needed. Here, we describe a completely novel function for IFI44L in negatively modulating innate immune responses induced after virus infections. In addition, we show that decreasing IFI44L expression impairs virus production and that IFI44L expression negatively modulates the antiviral state induced by an analog of dsRNA or by IFN treatment. IFI44L binds to the cellular protein FKBP5, which in turn interacts with kinases essential for type I and III IFN induction and signaling, such as the kinases IKK- aalpha;, bbeta; and . IFI44L binding to FKBP5 decreased the phosphorylation of IRF-3 and IBaalpha; mediated by IKK and IKKbbeta;, respectively, providing an explanation for the function of IFI44L in negatively modulating IFN responses. Therefore, IFI44L is a candidate target for reducing virus replication.
Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok), and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (mAbs) and 38 Flucelvax-induced mAbs for avidity, cross reactivity and any selectivity towards the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of mAbs targeting epitopes near the receptor-binding domain on HA head (HAI+ mAbs) compared to Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive mAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive ASCs showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but may differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head.
IMPORTANCE There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax that use alternate production strategies, can induce protective influenza specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.
Lymph-borne Friend murine leukemia virus (FrMLV) exploits the sentinel macrophages in the draining popliteal lymph node (pLN) to infect highly permissive innate-like B-1 cells and establish infection in mice. The reason for FrMLV-sensitivity of B-1 cells and their impact on viral spread is unknown. Here we demonstrate that TLR7 sensing and type I interferon (IFN-I) signaling in B-1 cells contributes to FrMLV susceptibility. FrMLV infection in B-1 cell-deficient mice (bumble; IBNS-dysfunctional) was significantly lower than in the wild-type mice and was rescued by adoptive transfer of wild-type B-1 cells. This rescue of FrMLV infection in bumble mice was dependent on intact TLR7 sensing and IFN-I signaling within B-1 cells. Analyses of infected cell types revealed that the reduced infection in bumble mice was predominantly due to compromised virus spread to the B-2 cell population. Our data reveal how FrMLV exploits innate immune sensing and activation in the B-1 cell population for infection and subsequent spread to other lymphocytes.
IMPORTANCE Viruses establish infection in hosts by targeting highly permissive cell types. The retrovirus Friend murine leukemia virus (FrMLV) infects a subtype of B cells called B-1 cells that permit robust virus replication. The reason for their susceptibility had remained unknown. We found that innate sensing of incoming virus and the ensuing type I interferon response within B-1 cells is responsible for their observed susceptibility. Our data provide insights into how retroviruses coevolved with the host to coopt innate immune sensing pathways designed to fight virus infections for establishing infection. Understanding early events in viral spread can inform antiviral intervention strategies that prevent the colonization of a host.
Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally-occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially-expressed in JSRV-infected lung compared to non-infected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and RT-qPCR. A key finding was the activation of anterior-gradient-2, yes-associated protein-1 and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity including genes encoding cytokines, chemokines and complement system proteins. In contrast, there was little evidence for upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.
IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.
Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression and parturition stressors did not lead to overt/recrudescent clinical disease but intermittent viremia and virus shedding were detected up to day 60 post-infection (pi) in all treatment groups following stress stimulation. Notably, real-time PCR, and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for dsRNA showed that SVA RNA persists in tonsillar cells in a double-stranded conformation. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 pi, confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.
IMPORTANCE Persistent viral infection infections have significant implications to disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally- or naturally infected animals long after resolution of the clinical disease. Here we showed that SVA establishes persistent infection in SVA infected animals with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as source of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology suggesting that persistently infected pigs may function as reservoirs for SVA.
Previously, we identified a set of lncRNAs that were differentially expressed in influenza A virus (IAV)-infected cells. In this study, we focused on lnc-MxA, which is upregulated during IAV infection. We found that the overexpression of lnc-MxA facilitates the replication of IAV, while the knockdown of lnc-MxA inhibits viral replication. Further studies demonstrated that lnc-MxA is an interferon-stimulated gene. However, lnc-MxA inhibits the SeV- and IAV-induced activation of IFN-bbeta;. The luciferase assay indicated that lnc-MxA inhibits the activation of the IFN-bbeta; reporter upon stimulation with RIG-I, MAVS, TBK1 or active IRF3 (IRF3-5D). These data indicated that lnc-MxA negatively regulates the RIG-I-mediated antiviral immune response. The CHIP assay showed that the enrichment of IRF3 and p65 at the IFN-bbeta; promoter in lnc-MxA-overexpressing cells was significantly lower than that in control cells, indicating that lnc-MxA interfered with the binding of IRF3 and p65 to the IFN-bbeta; promoter. CHIRP, triplex pulldown and biolayer interferometry assays indicated that lnc-MxA can bind to the IFN-bbeta; promoter. Furthermore, the EMSA assay showed that lnc-MxA can form complexes with the IFN-bbeta; promoter fragment. These results demonstrated that lnc-MxA can form a triplex with the IFN-bbeta; promoter to interfere with the activation of IFN-bbeta; transcription. Using a VSV infection assay, we confirmed that lnc-MxA can repress the RLR-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we revealed that lnc-MxA is an interferon-stimulated gene that negatively regulates the transcription of IFN-bbeta; by forming an RNA-DNA triplex.
IMPORTANCE IAV can be recognized as a nonself molecular pattern by host immune systems and can cause immune responses. However, the intense immune response induced by influenza virus known as a "cytokine storm" can also cause widespread tissue damage (Guo XZJ, Thomas PG. 2017. Seminars in Immunopathology 39:1-10; Yokota S. 2003. Nihon Rinsho 61:1953-1958; Clark IA. 2007. Immunology aamp; Cell Biology 85:271-273). Meanwhile, the detailed mechanisms involved in the balancing of immune responses in host cells are not well understood. Our studies reveal that, as an IFN-inducible gene, lnc-MxA functions as a negative regulator of the antiviral immune response. We uncovered the mechanism by which lnc-MxA inhibits the activation of IFN-bbeta; transcription. Our findings demonstrate that, as an ISG, lnc-MxA plays an important role in the negative feedback loop involved in maintaining immune homeostasis.
The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses against ASP in HIV-1 patients indicate that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows a polarized sub-nuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm, and is detectable on the cell surface even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 co-localize, suggesting that ASP might become incorporated in the membrane of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to anti-gp120 VRC01. Our studies indicate that ASP is an integral protein of the plasma membrane of chronically infected cells stimulated with PMA, and upon viral budding ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle, and suggest that ASP may represent a new therapeutic or vaccine target.
IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite or antisense direction of all other genes. The protein product of this antisense gene, called ASP, is poorly characterized and its role in viral replication remains still unknown. We provide evidence that the antisense protein, ASP of HIV-1 is found within the cell nucleus of unstimulated cells. In addition, we show that after PMA treatment ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrated that ASP is present on the surface of viral particles. Altogether, our studies identify ASP as a new structural component of the HIV-1 virus, and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surface of both infected cells and viral particles might be exploited therapeutically.
Due to the limiting coding capacity for members of the Picornaviridae family of positive-strand RNA viruses, their successful replication cycles require complex interactions with host cell functions. These interactions span from the down-modulation of many aspects of cellular metabolism to the hijacking of specific host functions used during viral translation, RNA replication, and other steps of infection by picornaviruses such as human rhinovirus, coxsackievirus, poliovirus, foot-and-mouth disease virus, enterovirus D-68, and a wide range of other human and non-human viruses. Although picornavirus replicate exclusively in the cytoplasm of infected cells, they have extensive interactions with host cell nuclei and the proteins and RNAs that normally reside in this compartment of the cell. This review will highlight some of the more recent studies that have revealed how picornavirus infections impact RNA metabolism of the host cell post-transcriptionally and how they usurp and modify host RNA binding proteins as well as microRNAs to potentiate viral replication.
Interferon (IFN) production activated by phosphorylated interferon regulatory factor 7 (IRF7) is a pivotal process during host antiviral infection. For viruses, suppressing the host IFN response is beneficial for viral proliferation; in such cases, evoking host-derived IFN negative regulators would be very useful for viruses. Here, we report that the zebrafish rapunzel 5 (RPZ5) protein which activated by virus degraded phosphorylated IRF7 is activated by TBK1, leading to the reduction of IFN production. Upon viral infection, zebrafish rpz5 was significantly upregulated as ifn, in response to the stimulation. Overexpression of RPZ5 blunted the IFN expression induced by both viral and retinoic acid-inducible gene I (RIG-I) likenndash;receptor (RLR) factors. Subsequently, RPZ5 interacted with RLRs but did not affect the stabilization of the proteins in the normal state. Interestingly, RPZ5 degraded the phosphorylated IRF7 under TBK1 activation through K48-linked ubiquitination. Finally, overexpression of RPZ5 remarkably reduced the host cell antiviral capacity. These findings suggest that zebrafish RPZ5 is a negative regulator of phosphorylated IRF7 and attenuates IFN expression during viral infection, providing insight into the IFN balance mechanism in fish.
IMPORTANCE The phosphorylation of IRF7 is helpful for host IFN production to defend against viral infection, thus, it is a potential target for viruses to mitigate the antiviral response. We report that the fish RPZ5 is an IFN negative regulator induced by fish viruses and degrades the phosphorylated IRF7 activated by TBK1, leading to IFN suppression and promoting viral proliferation. These findings reveal a novel mechanism for interactions between the host cell and viruses in the lower vertebrate.
In the host, many RING-domain E3 ligases have been reported to inhibit viral replication through various mechanisms. In a previous screen, we found that the porcine RING finger protein 114 (pRNF114), an RING-domain E3 ubiquitin ligase, inhibits classical swine fever virus (CSFV) replication. This study aimed to clarify the underlying antiviral mechanism of pRNF114 against CSFV. Upon CSFV infection, the pRNF114 mRNA was upregulated both in vitro and in vivo. CSFV replication was significantly suppressed in PK-pRNF114 cells stably expressing pRNF114 by lentivirus-delivered system, whereas CSFV growth was enhanced in PK-15 cells with RNF114 knockout by the CRISPR/Cas9 system. The RING domain of pRNF114, which has the E3 ubiquitin ligase activity, is crucial for its antiviral activity. Mechanistically, pRNF114 interacted with the CSFV NS4B protein through their C-terminal domains, which led to the K27-linked polyubiquitination and degradation of NS4B through a proteasome-dependent pathway. Collectively, these findings indicate that pRNF114 as a critical regulator of CSFV replication and uncover a mechanism by which pRNF114 employs its E3 ubiquitin ligase activity to inhibit CSFV replication.
IMPORTANCE The porcine RING finger protein 114 (pRNF114) is a member of RING-domain E3 ligases. In this study, pRNF114 is a potential anti-CSFV factor and the anti-CSFV effect of pRNF114 depends on its E3 ligase activity. Notably, pRNF114 targets and catalyzes the K27-linked polyubiquitination of the NS4B protein and then promotes proteasome-dependent degradation of NS4B, inhibiting the replication of CSFV. To our knowledge, pRNF114 is the first E3 ligase to be identified as being involved in anti-CSFV activity and targeting NS4B could be a crucial route for antiviral development.
The presence of T cell reservoirs in which HIV establishes latency by integrating into the host genome represents a major obstacle to a HIV cure and has prompted the development of strategies aimed at eradication of HIV from latently infected cells. The "Shock and kill" strategy is one of the most pursued approaches directed towards the elimination of viral reservoirs. Although several Latency-Reversing Agents (LRAs) have shown promising reactivation activity, they have failed to eliminate the cellular reservoir. Here, we evaluated a novel immune-mediated approach to clear the HIV reservoir, based on the combination of innate immune stimulation and epigenetic reprogramming. The combination of the STING agonist cGAMP and the FDA-approved histone deacetylase inhibitor Resminostat resulted in a significant increase in HIV proviral reactivation and specific apoptosis in HIV-infected cells in vitro. A reduction in HIV-harboring cells and in the total amount of HIV-DNA were also observed in CD4+ T central memory (TCM) cells, a primary cell model of latency, where Resminostat alone or together with cGAMP induced high levels of selective cell death. Finally, high levels of cellular-associated HIV-RNA were detected ex vivo in PBMCs and CD4+ T cells from individuals on suppressive ART. Although synergism was not detected in PBMCs with the combination, viral RNA expression was significantly increased in CD4+ T cells. Collectively, these results represent a promising step towards HIV eradication by demonstrating the potential of innate immune activation and epigenetic modulation to reduce the viral reservoir and induce specific death of HIV-infected cells.
One of the challenges associated with HIV-1 infection is that, despite anti-retroviral therapies that reduce the levels of HIV-1 virus to undetectable levels, proviral DNA remains dormant in a sub-population of T lymphocytes. Numerous strategies to eliminate residual virus, so-called llsquo;shock and kill' strategies, have been proposed to reactivate latent virus and to eliminate the reservoir of HIV-1. In the present study, Palermo et al use a combination of small molecules that activate the innate anti-viral cGAS-STING immune response (di-cyclic nucleotide cGAMP) and epigenetic modulators (histone deacetylase inhibitors) that induce reactivation and HIV- infected T cell killing in cell lines, primary T lymphocytes and patient samples. These studies represent a novel strategy towards HIV eradication by reducing the viral reservoir and inducing specific death of HIV-infected cells.
Influenza D virus (IDV) of the Orthomyxoviridae family has a wide host range and a broad geographical distribution. Recent IDV outbreaks in swine, along with serological and genetic evidence of IDV infection in humans have raised concerns regarding the zoonotic potential of this virus. To better study IDV at the molecular level, a reverse genetics system (RGS) is urgently needed, but to date no RGS had been described for IDV. In this study, we rescued the recombinant influenza D/swine/Oklahoma/1314/2011 (D/OK) virus by using a bidirectional seven plasmid-based system, and further characterized rescued viruses in terms of growth kinetics, replication stability, and receptor-binding capacity. Our results collectively demonstrated that RGS-derived viruses resembled the parental viruses for these properties, thereby supporting the utility of this RGS to study IDV infection biology. In addition, we developed an IDV mini-genome replication assay and identified the E697K mutation in PB1 and the L462F mutation in PB2 that directly affected the activity of the IDV ribonucleoprotein complex (RNP), resulting in either attenuated or replication-incompetent viruses. Finally, by using the mini-genome replication assay, we demonstrated that a single nucleotide polymorphism at position 5 of the 3' conserved noncoding region in IDV and ICV resulted in the inefficient cross-recognition of the heterotypic promoter by the viral RNP complex. In conclusion, we successfully developed a mini-genome replication assay and a robust reverse genetics system that can be used to further study replication, tropism, and pathogenesis of IDV.
IMPORTANCE Influenza D virus (IDV) is a new type of influenza virus that uses cattle as the primary reservoir and infects multiple agricultural animals. Increased outbreaks in pigs, and serological and genetic evidence of human infection have raised concerns about potential IDV adaptation in humans. Here, we have developed a plasmid-based IDV reverse genetics system that can generate infectious viruses similar in replication kinetics to wild-type viruses following transfection of cultured cells. Further characterization demonstrated that viruses rescued from the described RGS resembled the parental viruses in biological and receptor binding properties. We also developed and validated an IDV minireplicon reporter system that specifically measures viral RNA polymerase activity. In summary, the reverse genetics system and minireplicon reporter assay as described in this study should be of value in identifying viral determinants of cross-species transmission and pathogenicity of novel influenza D viruses.
Particle maturation is a critical step in the HIV-1 replication cycle that requires proteolytic cleavage of the Gag polyprotein into its constitutive proteins: matrix (MA), capsid (CA), nucleocapsid (NC), and p6. Accurate and efficient cleavage of Gag is essential for virion infectivity: inhibitors of the viral protease are potent antivirals, and substitutions in Gag that prevent its cleavage result in reduced HIV-1 infectivity. In a previous study, a mutation inhibiting cleavage at the MA-CA junction was observed to potently inhibit virus infection: incorporation of small amounts of uncleaved MA-CA protein into HIV-1 particles inhibited infectivity by ~95%, and the resulting viral particles exhibited aberrant capsids. Here we report a detailed mechanistic analysis of the HIV-1 particles bearing uncleaved MA-CA protein. We show that the particles contain stable cores and can efficiently saturate host restriction by TRIMCyp in target cells. We further show that MA-CA associates with CA in particles without detectibly affecting the formation of intermolecular CA interfaces. Incorporation of MA-CA did not markedly affect reverse transcription in infected cells, but nuclear entry was impaired, and integration targeting was altered. Additionally, results from mutational analysis of Gag revealed that membrane-binding elements of MA contribute to the antiviral activity of uncleaved MA-CA protein. Our results suggest that small amounts of partially-processed Gag subunits coassemble with CA during virion maturation, resulting in impaired capsid functions.
IMPORTANCE To become infectious, newly formed HIV-1 particles undergo a process of maturation in which the viral polyproteins are cleaved into smaller components. A previous study demonstrated that inclusion of even small quantities of an uncleavable mutant Gag polyprotein resulted in strong reduction in virus infectivity. Here we show that the mechanism of transdominant inhibition by uncleavable Gag involves inhibition of nuclear entry and alteration of viral integration sites. Additionally, the results of mutational analysis suggest that membrane binding activity of Gag is a major requirement for the antiviral activity. These results further define the antiviral mechanism of uncleavable Gag, which may be useful for exploiting this effect to develop new antivirals.
Porcine reproductive and respiratory syndrome virus (PRRSV) is widely prevalent in pigs, resulting in significant economic losses worldwide. A compelling impact of PRRSV infection is severe pneumonia. In the present study, we found that IL-17 was up-regulated by PRRSV infection. Subsequently, we demonstrated that PI3K and p38MAPK signaling pathways were essential for PRRSV-induced IL-17 production as addition of PI3K and p38MAPK inhibitors dramatically reduced IL-17 production. Furthermore, we revealed that deleting C/EBPbbeta; and CREB binding motif in porcine IL-17 promoter abrogated its activation, and knock-down of C/EBPbbeta; and CREB remarkably impaired PRRSV-induced IL-17 production, suggesting that IL-17 expression was dependent on C/EBPbbeta; and CREB. More specifically, we demonstrated that PRRSV nonstructural protein 11 (nsp11) induced IL-17 production, which was also dependent on PI3K-p38MAPK-C/EBPbbeta;/CREB pathways. We then showed that Ser74 and Phe76 amino acids were essential for nsp11 to induce IL-17 production and viral rescue. In addition, IRAK1 was required for nsp11 to activate PI3K and enhance IL-17 expression by interacting with each other. Importantly, we demonstrated that PI3K inhibitor significantly suppressed IL-17 production and lung inflammation caused by HP-PRRSV in vivo, implicating that higher IL-17 level induced by HP-PRRSV might be associated with severe lung inflammation. These findings provide new insights onto the molecular mechanisms of the PRRSV-induced IL-17 production and help us further understand the pathogenesis of PRRSV infection.
IMPORTANCE Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) associated with severe pneumonia has been one of the most important viral pathogens in pigs. IL-17 is a pro-inflammatory cytokine that might be associated with the strong inflammation caused by PRRSV. Therefore, we seek to determine whether PRRSV infection affects IL-17 expression, and if so, it might partially explain the underlying mechanisms for the strong inflammation in HP-PRRSV-infected pigs, especially in lungs. Here we showed that PRRSV significantly induced IL-17 expression and subsequently we dissected the molecular mechanisms about how PRRSV regulated IL-17 production. Furthermore, we showed that Ser74 and Phe76 in nsp11 were indispensable for IL-17 production and viral replication. Importantly, we demonstrated that PI3K inhibitor impaired IL-17 production and alleviated lung inflammation caused by HP-PRRSV infection. Our findings will help us for a better understanding of PRRSV pathogenesis.
Alternative splicing of HIV-1 mRNAs increases viral coding potential and controls the levels and timing of gene expression. HIV-1 splicing is regulated in part by heterogeneous nuclear ribonucleoproteins (hnRNPs) and their viral target sequences, that typically repress splicing when studied outside their native viral context. Here, we determined the location and extent of hnRNP binding to HIV-1 mRNAs and their impact on splicing in a native viral context. Notably, hnRNPA1, hnRNPA2 and hnRNPB1 bound to many dispersed sites across viral mRNAs. Conversely, hnRNPH1 bound to a few discrete purine-rich sequences, a finding that was mirrored in vitro. HnRNPH1 depletion and mutation of a prominent viral RNA hnRNPH1 binding site decreased use of splice acceptor A1, causing a deficit in Vif expression and replicative fitness. This quantitative framework for determining the regulatory inputs governing alternative HIV-1 splicing revealed an unexpected splicing enhancer role for hnRNPH1 through binding to its target element.
IMPORTANCE Alternative splicing of HIV-1 mRNAs is an essential, yet quite poorly understood step of virus replication that enhances the coding potential of the viral genome and allows temporal regulation of viral gene expression. Although HIV-1 constitutes an important model system for general studies of the regulation of alternative splicing, the inputs that determine the efficiency with which splice sites are utilized remain poorly defined. Our studies provide an experimental framework to study an essential step of HIV-1 replication more comprehensively and in much greater detail than was previously possible and reveal novel cis acting elements regulating HIV-1 splicing.
An earlier report showed that HSV-1 expresses two miRNAs, miR-H28 and miR-H29, late in the infectious cycle. The mRNAs are packed in exosomes and in recipient cells restrict transmission of virus from infected to uninfected cells. We now report that (i) miR-H28 induces the synthesis of IFN both in infected cells and cells transfected with miR-H28, (ii) In infected cells IFN accumulates concurrently with viral proteins, (iii) IFN was produced in HEp-2 cells derived from cancer tissue and 293T cells derived from normal tissues, (iv) HSV-1 replication is affected by the exposure to IFN before infection but not during or after infection. The results presented in this report support the growing body of evidence that HSV-1 encodes function designed to reduce the spread of infection from infected to uninfected cells possibly in order to maximize transmission of virus from infected to uninfected individuals.
In this report we show that IFN is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN to curtail its spread from infected to uninfected cells. In essence this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.
Dengue virus (DENV) infection causes serious clinical symptoms, including Dengue hemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Vascular permeability change is the main feature of the diseases, and the abnormal expression of pro-inflammation cytokines is the important cause of vascular permeability change. However, the mechanism underlying vascular permeability induced by DENV is not fully elucidated. Here we reveal a distinct mechanism by which DENV infection promotes the NLRP3 inflammasome activation and interleukin-1 beta (IL-1bbeta;) release to induce endothelial permeability and vascular leakage in mice. DENV M protein interacts with NLRP3 to facilitate the NLRP3 inflammasome assemble and activation, which lead to the induction of pro-inflammation cytokine IL-1bbeta; activation and release. Notably, M can induce vascular leakage in mice tissues through activating NLRP3 inflammasome and IL-1bbeta;. More importantly, inflammatory cell infiltration and tissue injuries are induced by M in WT mice tissues, but they are not affected by M in NLRP3 knock-out (NLRP3-/-) mice tissues, and Evans blue intensities in WT mice tissues are significantly higher as compared with NLRP3-/- mice tissues, demonstrating an essential role of NLRP3 in M-induced vascular leakages in mice. Therefore, we propose that upon DENV infection, M interacts with NLRP3 to facilitate the inflammasome activation and IL-1bbeta; secretion, which lead to induction of endothelial permeability and vascular leakage in mice tissues. The important role of the DENV-M-NLRP3-IL-1bbeta; axis in the induction of vascular leakage provides new insights into the mechanisms underlying DENV pathogenesis and DENV-associated DHF and DSS development.
IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen and its infection is prevalent in over 100 tropical and sub-tropical countries or regions with approximately 2.5 billion people at risk. DENV infection induces a spectrum of clinical symptoms ranging from classical dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Therefore, it is important to understand the mechanisms underlying DENV pathogenesis. In this study, the authors reveal that the DENV membrane protein (M) interacts with the host NLRP3 protein to promote the NLRP3 inflammasome activation, which leads to the activation and release of pro-inflammation cytokine, interleukin-1 beta (IL-1bbeta;). More importantly, the authors demonstrate that M protein can induce vascular permeability and vascular leakage and NLRP3 is required for M-induced vascular leakage in mice tissues. Collectively, this study reveals a distinct mechanism underlying DENV pathogeneses and provides new insights into the development of therapeutic agent for DENV-associated diseases.
Lassa virus is genetically diverse with several lineages circulating in West Africa. This study aimed at describing the sequence variability of Lassa virus across Nigeria and inferring its spatio-temporal evolution. We sequenced and isolated 77 Lassa virus strains from 16 Nigerian States. The final dataset, including previous works, comprised metadata and sequences of 219 unique strains sampled between 1969 and 2018 in 22 States. Most of this data originated from Lassa fever patients diagnosed at Irrua Specialist Teaching Hospital, Edo State, Nigeria. The majority of sequences clustered with the main Nigerian lineages II and III, while few sequences formed a new cluster related to Lassa virus strains from Hylomyscus pamfi. Within lineages II and III, seven and five sub-lineages, respectively, were distinguishable. The phylogeographic analysis suggests an origin of lineage II in the southeastern part of the country around Ebonyi State and a main vector of dispersal towards the west across the Niger River, through Anambra, Kogi, Delta, and Edo into Ondo State. The frontline of virus dispersal appears to be in Ondo. Minor vectors are directed northeast towards Taraba and Adamawa and south towards Imo and Rivers. Lineage III might have spread from northern Plateau State into Kaduna, Nasarawa, Federal Capital Territory, and Bauchi. One sub-lineage moved south and crossed the Benue River into Benue State. This study provides a geographic mapping of lineages and phylogenetic clusters in Nigeria at higher resolution. In addition, we estimated the direction and timeframe of virus dispersal in the country.
IMPORTANCE Lassa virus is the causative agent of Lassa fever, a viral hemorrhagic fever with a case fatality rate of approximately 30% in Africa. Previous studies disclosed a geographical pattern in the distribution of Lassa virus strains and a westward movement of the virus across West Africa during evolution. Our study provides a deeper understanding of the geography of genetic lineages and sub-lineages of the virus in Nigeria. In addition, we modeled how the virus spread in the country. This knowledge allows predicting into which geographical areas the virus might spread in future and prioritizing areas for Lassa fever surveillance. Our study not only aimed at generating Lassa virus sequences from across Nigeria, but also to isolate and conserve the respective viruses for future research. Both isolates and sequences are important for the development and evaluation of medical countermeasures to treat and prevent Lassa fever, such as diagnostics, therapeutics, and vaccines.
The innate immune response is vital for host defense and must be tightly controlled, but the mechanisms responsible for its negative regulation have not been fully understood. The cell growth-regulating nucleolar protein (LYAR) has been found to promote replication of multiple viruses in our previous study. Here, we report that LYAR acts as a negative regulator of the innate immune responses. We find that LYAR expression is induced by interferon-bbeta; (IFN-bbeta;) during virus infection. Further studies show that LYAR interacts with the phosphorylated IFN regulatory factor 3 (IRF3) to impede the DNA binding capacity of IRF3, thereby suppressing the transcription of IFN-bbeta; and the downstream IFN-stimulated genes (ISGs). In addition, LYAR inhibits nuclear factor-B (NF-B) mediated expression of proinflammatory cytokines. In summary, our study reveals the mechanism of LYAR modulating IFN-bbeta; mediated innate immune responses by targeting phosphorylated IRF3, which not only helps us to better understand the mechanisms of LYAR-regulated virus replication but also uncovers a novel role of LYAR in the host innate immunity.
IMPORTANCE IFN-I plays a critical role in the antiviral innate immune responses that protect host against virus infection. The negative regulators of IFN-I are not only important for fine-tuning the antiviral responses to pathogens but also for preventing excessive inflammation. Identification of negative regulators and study of their modulation in innate immune responses will lead to new strategies for the control of both viral and inflammatory diseases. Here, we report for the first time that LYAR behaves as a repressor of the host innate immune responses. We demonstrate that LYAR negatively regulates IFN-bbeta; mediated immune responses by inhibiting the DNA binding ability of IRF3. Our study reveals a common mechanism of LYAR promoting different virus replication and improves the knowledge of the host negative regulation of innate immune responses.
The plant-pathogenic virus, tomato spotted wilt virus (TSWV), encodes a structural glycoprotein (GN) that, like with other bunyavirus/vector interactions, serves a role in viral attachment and possibly entry into arthropod vector host cells. It is well documented that Frankliniella occidentalis is one of nine competent thrips vectors of TSWV transmission to plant hosts. However, the insect molecules that interact with viral proteins, such as GN, during infection and dissemination in thrips vector tissues are unknown. The goals of this project were to identify TSWV-interacting proteins (TIPs) that interact directly with TSWV GN and to localize expression of these proteins in relation to virus in thrips tissues of principal importance along the route of dissemination. We report here the identification of six TIPs from first instar larvae (L1), the most acquisition-efficient developmental stage of the thrips vector. Sequence analyses of these TIPs revealed homology to proteins associated with the infection cycle of other vector-borne viruses. Immunolocalization of the TIPs in L1s revealed robust expression in the midgut and salivary glands of F. occidentalis, the tissues most important during virus infection, replication and plant-inoculation. The TIPs and GN interactions were validated using protein-protein interaction assays. Two of the thrips proteins, endocuticle structural glycoprotein and cyclophilin, were found to be consistent interactors with GN. These newly discovered thrips protein-GN interactions are important for a better understanding of the transmission mechanism of persistent propagative plant viruses by their vectors, as well as for developing new strategies of insect pest management and virus resistance in plants.
Thrips-transmitted viruses cause devastating losses to numerous food crops worldwide. For negative-sense RNA viruses that infect plants, the arthropod serves as a host as well by supporting virus replication in specific tissues and organs of the vector. The goal of this work was to identify thrips proteins that bind directly to the viral attachment protein and thus may play a role in the infection cycle in the insect. Using the model plant bunyavirus, tomato spotted wilt virus (TSWV), and the most efficient thrips vector, we identified and validated six TSWV-interacting proteins from Frankliniella occidentalis first instar larvae. Two proteins, an endocuticle structural glycoprotein and cyclophilin, were able to interact directly with the TSWV attachment protein, GN, in insect cells. The TSWV GN-interacting proteins provide new targets for disrupting the viral disease cycle in the arthropod vector and could be putative determinants of vector competence.
Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ~70-150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here we report the first complete knockout (KO) of viral ORF57 gene from all ~100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination of multiple rounds of cell selection and single cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ. We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells which contain only a few copies of KSHV genome. The ORF57-KO genome was stable in iSLK/Bac16 cells and upon lytic induction, could be partially rescued to express viral lytic gene ORF59 and production of infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high-copy numbers.
IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology could be applied to knock out the ORF57 gene from all ~100 copies of KSHV genome in PEL cells by co-expression of two gRNAs and Cas9 from a single expression vector in combination of single cell cloning. The gene knockout efficiency in this system could be evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology could also mediate ORF57 inversion in situ in the targeted site of KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57-KO genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of virus genome and to understand their contributions to viral genetics and virus life cycle.
Vaccines aimed at inducing T cell responses to protect against human immunodeficiency virus (HIV) infection have been under development for more than 15 years. Replication defective adenovirus (rAd) vaccine vectors are at the forefront of this work and tested extensively in the simian immunodeficiency virus (SIV) challenge macaque model. Vaccination with rAd vectors coding for SIV gag or other non-envelope proteins induce T cell responses that control virus load but disappointingly are unsuccessful so far in preventing infection and attention has turned to inducing antibodies to the envelope. However, here we report that Mauritian cynomologus macaques, Macaca fascicularis (MCM), vaccinated against unmodified gag alone with a DNA prime followed by a rAd boost exhibit increased protection from infection by repeated intrarectal challenge with low dose SIVmac251. There was no evidence of infection followed by eradication. A significant correlation was observed between cytokine expression by CD4 T cells and delayed infection. Vaccination with gag fused to the ubiquitin gene or fragmented, designed to increase CD8 magnitude and breadth, did not confer resistance to challenge or enhance immunity. On infection a significant reduction in peak virus load was observed in all vaccinated animals including those vaccinated with modified gag. These findings suggest that a non-persistent viral vector vaccine coding for internal virus proteins may be able to protect against HIV-1 infection. The mechanisms are probably distinct from antibody-mediated virus neutralization or cytotoxic CD8 cell killing of virus infected cells and may be mediated in part by CD4 T cells.
IMPORTANCE The Simian Immunodeficiency Virus (SIV) macaque model represents the best animal model for testing new HIV-1 vaccines. Previous studies employing replication defective adenovirus (rAd) vectors that transiently express SIV internal proteins induced T cell responses that controlled virus load but did not protect against virus challenge. However, we show for the first time that SIV gag delivered in a DNA prime followed by a boost with rAd vector confers resistance to SIV intrarectal challenge. Other partially successful SIV/HIV-1 protective vaccines induce antibody to envelope and neutralise the virus or mediate antibody dependent cytotoxicity. Induction of CD8 T cells which do not prevent initial infection but eradicate infected cells before infection becomes established have also shown some success. By contrast the vaccine described here mediates resistance by a different mechanism from that described above which may reflect CD4 T cell activity. This could indicate an alternative approach for HIV-1 vaccine development.
During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) UL34 and UL31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro. Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein UL25, but not to the other capsid proteins tested (i.e., VP5, VP23 and UL17) and that HSV-1 NEC binding nucleocapsids was mediated by NEC interaction with UL25. UL31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and UL25. We also showed that alanine substitution of UL31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles, that were similar in size and morphology to primary envelopes, in the perinuclear space. These results suggested that NEC binding via UL31 R281 and D282 to nucleocapsids, probably to UL25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.
Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis has not been reported thus far. In this study, we have presented data showing that two amino acids in the membrane distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.
Human immunodeficiency virus (HIV-1) entry into cells is mediated by the viral envelope glycoprotein (Env) trimer, which consists of three gp120 exterior glycoproteins and three gp41 transmembrane glycoproteins. When gp120 binds sequentially to the receptors, CD4 and CCR5, on the target cell, the metastable Env trimer is triggered to undergo entry-related conformational changes. PF-68742 is a small molecule that inhibits the infection of a subset of HIV-1 strains by interfering with an Env function other than receptor binding. Determinants of HIV-1 resistance to PF-68742 map to the disulfide loop and fusion peptide of gp41. Of the four possible PF-68742 stereoisomers, only one, MF275, inhibited the infection of CD4-positive, CCR5-positive cells by some HIV-1 strains. MF275 inhibition of these HIV-1 strains occurred after CD4 binding, but before the formation of the gp41 six-helix bundle. Unexpectedly, MF275 activated the infection of CD4-negative, CCR5-positive cells by several HIV-1 strains resistant to the inhibitory effects of the compound in CD4-positive target cells. In contrast to CD4 complementation by CD4-mimetic compounds, activation of CD4-independent infection by MF275 did not depend upon the availability of the gp120 Phe 43 cavity. Sensitivity to inhibitors indicates that MF275-activated virus entry requires formation/exposure of the gp41 heptad repeat (HR1) as well as CCR5 binding. MF275 apparently activates a virus entry pathway parallel to that triggered by CD4 and CD4-mimetic compounds. Strain-dependent divergence in Env conformational transitions allows different outcomes, inhibition or activation, in response to MF275. Understanding the mechanisms of MF275 activity should assist efforts to optimize its utility.
Envelope glycoprotein (Env) spikes on the surface of human immunodeficiency virus (HIV-1) bind target cell receptors, triggering changes in the shape of Env. We studied a small molecule, MF275, that also induced shape changes in Env. The consequences of MF275 interaction with Env depended on the HIV-1 strain, with infection by some viruses inhibited and infection by other viruses enhanced. These studies reveal the strain-dependent diversity of HIV-1 Envs as they undergo shape changes in proceeding down the entry pathway. Appreciation of this diversity will assist attempts to develop broadly active inhibitors of HIV-1 entry.
Human cytomegalovirus (HCMV) enters primary CD34+ hematopoietic progenitor cells by macropinocytosis where it establishes latency in part because its tegument transactivating protein, pp71, remains associated with endosomes and is therefore unable to initiate productive, lytic replication. Here we show that multiple HCMV strains also enter cell line models used to study latency by macropinocytosis and endocytosis. In all latency models tested, tegument-delivered pp71 was found co-localized with endosomal markers and not associated with the seven other cytoplasmic localization markers tested. Like the capsid-associated pp150 tegument protein, we detected capsid proteins initially associated with endosomes but later in the nucleus. Inhibitors of macropinocytosis and endocytosis reduced latent viral gene expression and precluded reactivation. Importantly, we utilized electron microscopy to observe entry by macropinocytosis and endocytosis, providing additional visual corroboration to our functional studies. Our demonstration that HCMV enters cell line models for latency in a manner indistinguishable from its entry into primary cells illustrates the utility of these cell lines for probing the mechanisms, host genetics, and small molecule-mediated inhibition of HCMV entry into the cell types where it establishes latency.
Primary cells cultured in vitro currently provide the highest available relevance for examining molecular and genetic requirements for the establishment, maintenance, and reactivation of HCMV latency. However, their expense, heterogeneity, and intransigence to both long-term culture and molecular or genetic modification create rigor and reproducibility challenges for HCMV latency studies. There are several cell line models for latency not obstructed by deficiencies inherent in primary cells. However, many researchers view cell line studies of latency as physiologically irrelevant because of the perception that these models display numerous and significant differences from primary cells. Here we show that the very first step in a latent HCMV infection, entry of the virus into cells, occurs in cell line models indistinguishably from how it occurs in primary CD34+ hematopoietic progenitor cells. Our data argue that experimental HCMV latency in cell lines and primary cells is much more similar than it is different.
Adeno-associated viruses (AAV) are helper-dependent parvoviruses that have been developed into promising gene therapy vectors. Many studies including a recent unbiased genomic screen have identified host factors essential for AAV cell entry, but no genome-wide screens that address inhibitory host factors have been reported. Here, we utilize a novel CRISPR screen to identify AAV restriction factors in a human hepatocyte cell line. The major hit from our gain-of-function screen is the apical polarity determinant Crumbs 3 (Crb3). Knockout (KO) of Crb3 enhances AAV transduction, while overexpression exerts the opposite effect. Further, Crb3 appears to restrict AAV transduction in a serotype and cell type-specific manner. Particularly, for AAV serotype 9 and a rationally engineered AAV variant, we demonstrate that increased availability of galactosylated glycans on the surface of Crb3 KO cells, but not the universal AAV receptor (AAVR) leads to increased capsid attachment and enhanced transduction. We postulate that Crb3 could serve as a key molecular determinant that restricts the availability of AAV glycan attachment factors on the cell surface by maintaining apical-basal polarity and tight junction integrity.
Adeno-associated viruses (AAVs) have recently emerged at the forefront as gene therapy vectors; however, our understanding of host factors that influence AAV transduction in different cell types is still evolving. In the present study, we perform a genome scale CRISPR knock out screen to identify cellular host factors that restrict AAV infection in hepatocyte cultures. We discover that Crumbs 3, which determines cellular polarity also influences the distribution of certain carbohydrate attachment factors on the cell surface. This in turn affects the ability of virions to bind and enter the cells. This work underscores the importance of cell polarity in AAV transduction and provides a potential molecular basis for the differential infectious mechanism(s) in cell culture versus organ systems.
Human cytomegalovirus (HCMV) replication requires host metabolism. Infection alters the activity in multiple metabolic pathways, including increasing fatty acid elongation and lipid synthesis. The virus-host interactions regulating the metabolic changes associated with replication are essential to infection. While multiple host factors, including kinases and transcription factors, important to metabolic changes that occur following HCMV infection have been identified, little is known about the viral factors required to alter metabolism. In this study, we tested the hypothesis that pUL37x1 is important to the metabolic remodeling that is necessary for HCMV replication using a combination of metabolomics, lipidomics, and metabolic tracers to measure fatty acid elongation. We observed that fibroblast cells infected with wild-type (WT) HCMV had similar levels of metabolites as those infected with a mutant virus lacking the UL37x1 gene, subUL37x1. However, we found that relative to WT-infected cells subUL37x1-infected cells had reduced levels of two host proteins that were previously demonstrated to be important for lipid metabolism during HCMV infectionmmdash;fatty acid elongase 7 (ELOVL7) and ER-stress related kinase PERK. Moreover, we observed that HCMV infection results in an increase in phospholipids with very long-chain fatty acid tails (PL-VLCFAs) that contain 26 or more carbons in one of their two tails. The levels of many PL-VLCFAs were lower in subUL37x1-infected cells compared to WT-infected cells. Overall, we conclude that although pUL37x1 is not necessary for network-wide metabolic changes associated with HCMV-infection, it is important to the remodeling of a subset of metabolic changes that occur during infection.
Human cytomegalovirus (HCMV) is a common pathogen that asymptomatically infects most people and establishes a lifelong infection. However, HCMV can cause end-organ disease that results in death in the immunosuppressed and is a leading cause of birth defects. HCMV infection depends on host metabolism, including lipid metabolism. However, the viral mechanisms for remodeling metabolism are poorly understood. In this study, we demonstrate that the viral UL37x1 protein (pUL37x1) is important for infection-associated increases in lipid metabolism, including fatty acid elongation to produce very long-chain fatty acids (VLCFAs). Further, we found that HCMV infection results in a significant increase in phospholipids, particularly those with VLCFA tails (PL-VLCFAs). We found that pUL37x1 was important for the high levels of fatty acid elongation and PL-VLCFAs accumulation that occurs in HCMV-infected cells. Our findings identify a viral protein that is important for changes to lipid metabolism that occurs following HCMV infection.
Influenza is a global public health problem. Current seasonal influenza vaccines have highly variable efficacy and hence attempts to develop broadly protective universal influenza vaccines with durable protection are underway. While much attention is given to the virus-related factors contributing to inconsistent vaccine responses, host-associated factors are often neglected. Growing evidences suggest that host factors including age, biological sex, pregnancy, and immune history play important roles as modifier of influenza virus vaccine efficacy. We hypothesize that host genetics, the hormonal milieu, and gut microbiota contribute to host-related differences in influenza virus vaccine efficacy. This review highlights the current insights and future perspectives into host-specific factors that impact influenza vaccine-induced immunity and protection. Consideration of the host factors that affect influenza vaccine-induced immunity might improve influenza vaccines by providing empirical evidence for optimizing or even personalizing vaccine type, dose, and use of adjuvants for current seasonal and future universal influenza vaccines.
Equine infectious anemia virus (EIAV) is an equine lentivirus similar to HIV-1, targets to host immune cells and causes life-long infection in horses. The Chinese live EIAV vaccine is attenuated from long-term passaging of a high virulent strain in vitro. The parent pathogenic strain (EIAVDLV34) induces a host inflammatory storm to cause severe pathological injury of animals. However, the vaccine strain (EIAVDLV121) induces a high level of apoptosis to eliminate the infected cells. To investigate how these processes are regulated, we performed a comparative proteomics analysis and functional study in equine monocyte-derived macrophages (eMDMs), and found that divergent mitochondrial protein expression profiles caused by EIAV strains with different virulence lead to disparate mitochondrial function, morphology and metabolism. This in turn promoted distinct transformation of macrophage inflammatory polarization and intrinsic apoptosis. In EIAVDLV34 infected cells, a high level of glycolysis and increased mitochondrial fragmentation were induced, resulting in M1-polarized pro-inflammatory type transformation of macrophages and subsequently producing a strong inflammatory response. Following infection with EIAVDLV121, the infected cells were transformed into M2-polarized anti-inflammatory macrophages by inhibition of glycolysis. In this case, decrease of mitochondrial membrane potential and impairment of electronic respiratory chain led to increased levels of apoptosis and ROS. These results are correlated with the viral pathogenicity loss and may help to understand the key mechanism of lentiviral attenuation.
Following viral infection, the working pattern and function of the cell can be transformed through the impact on mitochondria. It still unknown how mitochondrial response changes in the cells infected with viruses in the process of virulence attenuation. EIAVDLV121 is the only effective lentiviral vaccine for large-scale use in world. EIAVDLV34 is a parent pathogenic strain. Unlike EIAVDLV34-induced inflammation storms, EIAVDLV121 can induce high levels of apoptosis. For the first time, we found that, after altering mitochondrial protein expression profile, EIAVDLV34 infected cells are transformed into M1-polarized type macrophages to cause inflammatory injury and the intrinsic apoptosis pathway is activated in EIAVDLV121 infected cells. These studies shed light on how the mitochondrial protein expression profile change from cells infected by pathogenic or attenuated lentivirus strains to drive different cellular response, especially from inflammation to apoptosis.
Cleavage of influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity and spread. We previously demonstrated in vitro that the transmembrane protease TMPRSS2 cleaves influenza A and B virus (IAV/IBV) HA possessing a monobasic cleavage site. Subsequent studies revealed that TMPRSS2 is crucial for activation and pathogenesis of H1N1pdm and H7N9 IAV in mice. In contrast, activation of H3N2 IAV and IBV was found to be independent of TMPRSS2 expression and supported by as-yet undetermined protease(s).
Here, we investigated the role of TMPRSS2 in proteolytic activation of IAV and IBV in three human airway cell culture systems: primary human bronchial epithelial cells (HBEC), primary type II alveolar epithelial cells (AECII) and Calu-3 cells. Knockdown of TMPRSS2 expression was performed using a previously described antisense peptide-conjugated phosphorodiamidate morpholino oligomer, T-ex5, that interferes with splicing of TMPRSS2 pre-mRNA, resulting in the expression of enzymatically inactive TMPRSS2. T-ex5 treatment produced efficient knockdown of active TMPRSS2 in all three airway cell culture models and prevented proteolytic activation and multiplication of H7N9 IAV in Calu-3 cells and H1N1pdm, H7N9 and H3N2 IAV in HBEC and AECII. T-ex5 treatment also inhibited activation and spread of IBV in AECII, but did not affect IBV activation in HBEC and Calu-3 cells.
This study identifies TMPRSS2 as the major HA-activating protease of IAV in human airway cells and IBV in type II pneumocytes and as a potential target for the development of novel drugs to treat influenza infections.
Influenza A and B viruses (IAV/IBV) cause significant morbidity and mortality during seasonal outbreaks. Cleavage of the viral surface glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. Inhibition of relevant proteases provides a promising therapeutic approach that may avoid the development of drug resistance. HA of most influenza viruses is cleaved at a monobasic cleavage site and a number of proteases have been shown to cleave HA in vitro. This study demonstrates that the transmembrane protease TMPRSS2 is the major HA-activating protease of IAV in primary human bronchial cells and of both IAV and IBV in primary human type II pneumocytes. It further reveals that human and murine airway cells can differ in their HA-cleaving protease repertoire. Our data will help drive the development of potent and selective protease inhibitors as novel drugs for influenza treatment.
Herpes Simplex Virus type 1 (HSV-1) infects mucosal epithelial cells and establishes life-long infections in sensory neurons. Following reactivation, the virus is transferred anterograde to the initial site of infection or to sites innervated by infected neurons, causing vesicular lesions. Upon immunosuppression frequent HSV-1 reactivation can cause severe diseases such as blindness and encephalitis. Autophagy is a process whereby cell components are recycled, but it also serves as a defense mechanism against pathogens. HSV-1 is known to combat autophagy through the functions of the 134.5 protein, which prevents formation of the autophagophore by binding to Beclin-1, a key factor involved in the elongation of the isolation membrane, and by redirecting the protein phosphatase 1 aalpha; (PP1aalpha;) to dephosphorylate the translation initiation factor 2aalpha; (eIF2aalpha;) to prevent host translational shutoff. Other viral proteins that counteract innate immunity negatively impact autophagy. Here, we present a novel strategy of HSV-1 to evade the host, through the down-regulation of the autophagy adaptor protein sequestosome (p62/SQSTM1) and of the mitophagy adaptor optineurin (OPTN). This down-modulation occurs during the early steps of the infection. We also found that the Infected Cell Protein 0 (ICP0) of the virus mediates the down-modulation of the two autophagy adaptors in a mechanism independent of its E3 ubiquitin ligase activity. Cells depleted of either p62 or OPTN could mount greater antiviral responses, whereas cells expressing exogenous p62 displayed decreased virus yields. We conclude that down-regulation of p62/SQSTM1 and OPTN is a viral strategy to counteract the host.
Autophagy is a homeostatic mechanism of cells to recycle components, as well as a defense mechanism to get rid of pathogens. Strategies that HSV-1 has developed to counteract autophagy have been described and involve inhibition of autophagosome formation or indirect mechanisms. Here, we present a novel mechanism that involves down-regulation of two major autophagy adaptor proteins, sequestosome 1 (p62/SQSTM1) and optineurin (OPTN). These findings generate the question: Why does the virus target two major autophagy adaptors if it has mechanisms to block autophagosome formation? P62/SQSTM1 and OPTN proteins have pleiotropic functions, including regulation of innate immunity, inflammation, protein sorting and chromatin remodeling. The decrease in virus yields in the presence of exogenous p62/SQSTM1 suggests that these adaptors have an antiviral function. Thus, HSV-1 could have developed multiple strategies to incapacitate autophagy to ensure replication. Alternatively, the virus could target another antiviral function of these proteins.
Feline infectious peritonitis (FIP) is one of the most important infectious diseases in cats and is caused by feline coronavirus (FCoV). Tissue culture-adapted type I FCoV shows reduced FIP induction in experimental infections, which complicates the understanding of FIP pathogenesis caused by type I FCoV. We previously found that the type I FCoV strain C3663 efficiently induces FIP in specific pathogen free cats through the naturally infectious route. In this study, we employed a bacterial artificial chromosome-based reverse genetics system to gain more insights into FIP caused by the C3633 strain. We successfully generated recombinant virus (rC3663) from Fcwf-4 cells transfected with infectious cDNA that showed similar growth kinetics to the parental virus. Next, we constructed a reporter C3663 virus carrying the nanoluciferase (Nluc) gene to measure viral replication with high sensitivity. The inhibitory effects of different compounds against rC3663-Nluc could be measured within 24 h post-infection. Furthermore, we found that A72 cells derived from canine fibroblasts permit FCoV replication without apparent cytopathic effects. Thus, our reporter virus is useful for uncovering the infectivity of type I FCoV in different cell lines, including canine-derived cells. Surprisingly, we uncovered aberrant viral RNA transcription of rC3663 in A72 cells. Overall, we succeeded in obtaining infectious cDNA clones derived from type I FCoV that retained its virulence. Our recombinant FCoVs are powerful tools for increasing our understanding of the viral life cycle and pathogenesis of FIP-inducing type I FCoV.
Feline coronavirus (FCoV) is one of the most significant coronaviruses, because this virus induces feline infectious peritonitis (FIP), which is lethal disease in cats. Tissue culture-adopted type I FCoV often loses pathogenicity, which complicates research on type I FCoV-induced feline infectious peritonitis (FIP). Since we previously found that the type I FCoV strain C3663 efficiently induces FIP in specific pathogen free cats, we established a reverse genetics system for the C3663 strain to obtain recombinant viruses in the present study. By using a reporter C3663 virus, we were able to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf-4 cells and infectivity in a canine-derived cell line. Interestingly, one canine cell line, A72, permitted FCoV replication but with low efficiency and aberrant viral gene expression.
Immune complex (IC) vaccines have been successfully used to increase immune responses against various pathogens, including HIV-1. Additionally, IC vaccines can induce qualitatively different antibody responses with distinct antigenic specificities compared to the same antigens used alone. Here we measured the HIV-1-specific antibody response in female New Zealand White rabbits after immunization with ICs made from BG505 SOSIP.664 trimers (BG505 trimers) and three different rabbit monoclonal antibodies (mAbs) with varying neutralization profiles. Two of the mAbs were specific for a hole in the glycan shield of the BG505 trimer while the third, which bound less avidly, was specific for determinants at the gp41/gp120 interface. We found that immunizing with one of the glycan hole-specific ICs resulted in lower levels of trimer-binding antibodies compared to vaccination with the uncomplexed trimer and that ICs made using either of the glycan hole-specific mAbs resulted in lower rates of anti-trimer antibody decay. We conclude that ICs based on mAbs that bound to the immunodominant glycan hole epitope likely diverted antibody responses, to some extent, away from this site and to other regions of the trimer. However, this outcome was not accompanied by a widening of the breadth or an increase in the potency of neutralizing antibody responses compared with uncomplexed trimers.
IMPORTANCE Immunodominant epitopes may suppress immune responses to more desirable determinants, such as those that elicit potentially protective neutralizing antibody responses. To overcome this problem, we attempted to mask immunodominant glycan holes by immunizing rabbits with immune complexes (ICs) consisting of the BG505 SOSIP.664 gp140 trimer and monoclonal antibodies that target the glycan holes. We found that IC vaccination likely diverted antibody responses, to some extent, away from glycan holes and toward other regions of the trimer. IC vaccination resulted in a slower decay of HIV-1-specific antibodies than did immunization with uncomplexed trimer. We did not observe a widening of the breadth or an increase in the potency of neutralizing antibody responses compared to uncomplexed trimers. Our results suggest that selective epitope dampening of BG505 trimers by ICs is rather ineffective. However, IC vaccination may represent a novel means of increasing the duration of vaccine-induced antibody responses.
Adenovirus (AdV)-based vectors are popular experimental vaccine vectors, but despite their ability to induce strong immune responses, their application is impeded by wide-spread pre-existing immunity against many AdV types that can impair or even abrogate induction of transgene-specific immune responses. Therefore, the development of vectors based on AdV types with low seroprevalence is important for effective AdV-based immunization in humans.
We investigated the immunization efficacy of vectors based on AdV types 48 and 50 in the ovalbumin (ova) model as well as the Friend retrovirus (FV) model, which allows testing the protective effect of vaccine-induced immunity. Using ova-encoding vectors, we found a significantly lower induction of ova-specific CD8+ T cells and antibody responses by Ad48- and Ad50-based vectors compared to Ad5. Similarly, we found a reduced induction of FV-specific CD8+ T cell responses in Ad48- and Ad50.Leader-Gag-immunized mice compared to Ad5; however, some of those mice were able to control the FV infection, and protection correlated with the level of neutralizing antibodies 10 days after FV challenge. Analyses of AdV-specific antibodies and CD8+ T cells induced by the individual AdV types revealed a high level of cross-reactivity, and the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice.
Our results show that immunity induced by Ad48- and Ad50-based vectors is reduced compared to Ad5, and is sufficient only in some of the immunized mice to control FV infection. A high level of cross-reactivity suggests that AdV pre-immunity must be considered even when applying rare AdV based vectors.
AdV-based vectors are important tools for the development of vaccines against a wide range of pathogens. While AdV vectors are generally considered safe and highly effective, their application can be severely impaired by pre-existing immunity due to wide-spread seroprevalence of some AdV types. The characterization of different AdV types with regard to immunogenicity and efficacy in challenge models is of great importance for the development of improved AdV-based vectors that allow for efficient immunization despite anti-AdV immunity. We show that immunity induced by an Ad48-based vector is inferior to Ad5, but can still mediate control of an FV infection in highly FV-susceptible mice. However, the efficacy of Ad48-based immunization was impaired in Ad5 pre-immune mice. Importantly, we found cross-reactivity of both humoral and cellular immune responses raised by the individual AdV types, suggesting that switching to a different AdV type may not be sufficient to circumvent pre-existing anti-AdV immunity.
A variety of strains of vaccinia virus (VACV) have been used as recombinant vaccine vectors with the aim of inducing robust CD8+ T cell immunity. Whilst much of the pioneering work was done with virulent strains, such as Western Reserve (WR), attenuated strains such as Modified Vaccinia Ankara (MVA) are more realistic vectors for clinical use. To unify this literature, side-by-side comparisons of virus strains are required. Here we compare the form of antigen that supports optimal CD8+ T cell responses for VACV strains WR and MVA using equivalent constructs. We found that for multiple antigens, minimal antigenic constructs (epitope minigenes) that prime CD8+ T cells via the direct presentation pathway elicited optimal responses from both vectors, which was surprising because it contradicts the prevailing view in the literature for MVA. We then went on to explore the discrepancy between current and published data for MVA, finding evidence that the expression locus and in some cases the presence of the viral thymidine kinase may influence the ability of this strain to prime optimal responses from antigens that require direct presentation. This extends our knowledge of the design parameters for VACV vectored vaccines, especially those based on MVA.
Recombinant vaccines based on vaccinia virus and particularly attenuated strains such as MVA are in human clinical trials, but due to the complexity of these large vectors much remains to be understood about the design parameters that alter their immunogenicity. Previous work had found that MVA vectors should be designed to express stable protein in order to induce robust immunity by CD8+ (cytotoxic) T cells. Here we find that the primacy of stable antigen is not generalisable to all designs of MVA and may depend where a foreign antigen is inserted into the MVA genome. This unexpected finding suggests that there is an interaction between genome location and the best form of antigen for optimal T cell priming in MVA, and so possibly other vaccine vectors. It also highlights that our understanding of antigen presentation by even the best studied of vaccine vectors remains incomplete.
Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from a fast cytolysis causing echovirus 1 (EV1) with non-structural proteins from Coxsackievirus B5 (CVB5) showing persistent infection in RD cells. The results showed that the chimera behaved similar to the parental EV1 leading to efficient cytolysis in both permissive A549 and semi-permissive RD cells. In contrast to EV1 and chimera, CVB5 replicated slower in permissive cells and showed persistent infection in semi-permissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 virus batches constantly contained significant amounts of empty capsids, also in comparison to its close relative CVB3. During successive passaging of batch containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increased amounts of empty particles and lowering amounts of infective particles is dictated by the CVB5 structural proteins leading to slowing down the infection between passages. Furthermore, the key factor for persistent infection is the low amount of infective particles produced, not the high number of empty particles accumulating.
IMPORTANCE Enteroviruses cause several severe diseases with lytic infections that lead to rapid cell death but also persistent infections that are more silent, and lead to chronic states. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera between the structural proteins of echovirus 1 and non-structural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to production of high number of empty viruses (empty capsids), that do not contain genetic material and are unable to continue the infection. Coinciding with high number of empty capsids, also the amount of infective virions decreased. This characteristic property was not observed in the constructed chimeravirus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infection are essential in understanding the virus caused pathologies.
The matrix (MA) domains of HIV-1 precursor Gag (PrGag) proteins direct PrGag proteins to plasma membrane (PM) assembly sites where envelope (Env) protein trimers are incoporated into virus particles. MA targeting to PM sites is facilitated by its binding to phosphatidylinositol-(4,5)-bisphosphate (PI[4,5]P2), and MA binding to cellular RNAs appears to serve a chaperone function that prevents MA from associating with intracellular membranes prior to arrival at the PI(4,5)P2-rich PM. Investigations have shown genetic evidence of an interaction between MA and the cytoplasmic tails (CTs) of Env trimers that contributes to Env incorporation into virions, but demonstrations of direct MA-CT interactions have proven more difficult. In direct binding assays, we show here that MA binds to Env CTs. Using MA mutants, matrix-capsid (MACA) proteins, and MA proteins incubated in the presence of inositol polyphosphate, we show a correlation between MA trimerization and CT binding. RNA ligands with high affinities for MA reduced MA-CT binding levels, suggesting that MA-RNA binding interferes with trimerization and/or directly or indirectly blocks MA-CT binding. Rough mapping studies indicate that C-terminal CT helices are involved in MA binding, and are in agreement with cell culture studies with replication-competent viruses. Our results support a model in which full-length HIV-1 Env trimers are captured in assembling PrGag lattices by virtue of their binding to MA trimers.
The mechanism by which HIV-1 envelope (Env) protein trimers assemble into virus particles is poorly understood, but involves an interaction between Env cytoplasmic tails (CTs) and the matrix (MA) domain of the structural precursor Gag (PrGag) proteins. We show here that direct binding of MA to Env CTs correlates with MA trimerization, suggesting models where MA lattices regulate CT interactions and/or MA-CT trimer-trimer associations increase the avidity of MA-CT binding. We also show that MA binding to RNA ligands impairs MA-CT binding, potentially by interference with MA trimerization, and/or directly or allosterically blocking MA-CT binding sites. Rough mapping implicated CT C-terminal helices in MA binding, in agreement with cell culture studies on MA-CT interactions. Our results indicate that targeting HIV-1 MA-CT interactions may be a promising avenue for antiviral therapy.
Early HIV-1 treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in chronically SHIV-infected macaques, intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, here we used LSEVh-LS-F, a bispecific bnAb targeting CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically-infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.
IMPORTANCE Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since the early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for the treatment failure in the acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques, respectively. This study revealed the correlation between the fast clearance of infused bnAbs and the treatment failure during the acute period of infection.
Mouse hepatitis virus (MHV) uses its N-terminal domain (NTD) of viral spike (S) protein to bind the host receptor, mouse carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a), and mediate virus entry. Our previous crystal structure study of MHV NTD/mCEACAM1a complex (1) reveals that there are 14 residues in NTD interacting with the receptor. However, their contribution to receptor binding and virus entry has not been fully investigated. Here we analyzed 13 out of 14 contact residues by mutagenesis, and identified I22 essential for receptor binding and virus entry. Unexpectedly, we found that G29 was critical for the conformational changes of S protein triggered either by receptor binding or high pH. Substitution of G29 with A, D, F, K, M, and T, to different extents, caused spontaneous dissociation of S1 from S protein, resulting in enhancement of high pH triggered receptor-independent syncytia (RIS) formation in 293T cells, compared to WT. By contrast, replacement of G29 with P, a turn prone residue with strict conformation, hindered virus entry and conformational changes of S protein triggered by either receptor binding or pH 8.0, suggesting that the structural turn around G29 and its flexibility are critical. Finally, stabilization of NTD by G29P almost had no effect on pH-independent RIS induced by Y320A mutation in C-terminal domain (CTD) of S1 subunit, indicating that there might be absence of crosstalk between NTD and CTD during conformational changes of S protein. Our study will aid better understanding the mechanism how conformational changes of S protein is triggered.
IMPORTANCE Binding of MHV S protein to the receptor, mCEACAM1a, triggers the conformational changes of S proteins, leading to formation of six-helix bundle and viral and cellular membrane fusion. However, the mechanism by which the conformational change of S protein is initiated after receptor binding has not been determined. In this study, we showed that, while replacement of G29, a residue at the edge of receptor binding interface and the center of structural turn after bbeta;1-sheet of S protein, with D or T triggered spontaneous conformational change of S protein and pH-independent RIS, G29P mutation significantly impeded the conformational changes of S proteins triggered either by receptor binding and pH 8.0. We reason that this structural turn might be critical for conformational change of S protein and altering this structural turn could initiate the conformational changes of S protein, leading to membrane fusion.
With a yearly death toll of 880,000, Hepatitis B Virus (HBV) remains a major health problem worldwide despite an effective vaccine and well-tolerated effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long term antiviral therapy and its integration nndash; though no longer able to support viral replication nndash; destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products and thus, is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity, but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.
We have previously demonstrated that Epstein-Barr virus DNA increases the production of the pro-inflammatory cytokine IL-17A in mice. This property may contribute to the established association between EBV and autoimmune diseases. The objective of the current study was to elucidate mechanisms through which EBV DNA modulates IL-17A levels in mice. To determine whether endosomal Toll-Like Receptors (TLRs) played a role in this pathway, the expression of TLR3, 7 or 9 was assessed by real-time RT-PCR in mouse spleens after injection of EBV DNA. Moreover, specific inhibitors were used for these TLRs in mouse Peripheral Blood Mononuclear Cells (PBMCs) cultured with EBV DNA and in mice injected with this viral DNA; IL-17A levels were then assessed using Enzyme Linked Immunosorbent Assay. The expression of the endosomal receptors TLR3, 7, and 9 was increased in mice injected with EBV DNA. When mouse immune cells were cultured with EBV DNA and a TLR3, 7 or 9 inhibitor or when mice were injected with the viral DNA along with either of these inhibitors, a significant decrease in IL-17A levels was detected. Therefore, endosomal TLRs are involved in the EBV DNA-mediated triggering of IL-17A production in mice. Targeting these receptors in EBV positive subjects with autoimmunity may be useful pending investigations assessing whether they play a similar role in humans.
IMPORTANCE The Epstein-Barr virus is a pathogen that causes persistent infection with potential consistent viral DNA shedding. The enhancement of production of pro-inflammatory cytokines by viral DNA itself may contribute to autoimmune disease development or exacerbation. In this project we identified that endosomal Toll-like receptors are involved in triggering pro-inflammatory mediators in response to viral DNA. Pathways and receptors involved may serve as future therapeutic targets in autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.
Vesicular Stomatitis Indiana Virus (VSIV), formerly known as Vesicular Stomatitis Virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons. Interferons induce an antiviral state by stimulating the expression of hundreds of interferon stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis, but helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley Fever Virus and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.
IMPORTANCE Vesicular Stomatitis Indiana Virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here we show that TRIM69 is an antiviral defence that can potently and specifically block VSIV infection.
Non-segmented negative-strand RNA viruses including measles virus (MeV), a member of the Paramyxoviridae family, are assumed to replicate in cytoplasmic inclusion bodies. These cytoplasmic viral factories are not membrane-bound and serve to concentrate the viral RNA replication machinery. Although inclusion bodies are a prominent feature in MeV-infected cells, their biogenesis and regulation are not well understood. Here we show that infection with MeV triggers inclusion body formation via liquid-liquid phase separation (LLPS), a process underlying the formation of membraneless organelles. We find that the viral nucleoprotein (N) and phosphoprotein (P) are sufficient to trigger MeV phase separation, with the C-terminal domains of the viral N and P proteins playing a critical role in the phase transition. We provide evidence suggesting that the phosphorylation of P and dynein-mediated transport facilitate the growth of these organelles, implying they may have key regulatory roles in the biophysical assembly process. In addition, our findings support the notion that these inclusions change from liquid to gel-like structures as a function of time after infection, leaving open the intriguing possibility that the dynamics of these organelles can be tuned during infection to optimally suit the changing needs during the viral replication cycle. Our study provides novel insight into the process of formation of viral inclusion factories, and taken together with earlier studies, suggest that Mononegavirales broadly have evolved to utilize LLPS as a common strategy to assemble cytoplasmic replication factories in infected cells.
Measles virus remains a pathogen of significant global concern. Despite an effective vaccine, outbreaks continue to occur and globally ~100,000 measles-related deaths are seen annually. Understanding the molecular basis of virus-host interactions that impact the efficiency of virus replication is essential for the further development of prophylactic and therapeutic strategies. Measles virus replication occurs in the cytoplasm in association with discrete bodies, though little is known of the nature of the inclusion body structures. We recently established that the cellular protein WDR5 enhances MeV virus growth and is enriched in cytoplasmic viral inclusion bodies that include viral proteins responsible for RNA replication. Here we show that MeV N and P proteins are sufficient to trigger the formation of WDR5-containing inclusion bodies; that these structures display properties characteristic of phase-separated liquid organelles; and, that P phosphorylation together with the host dynein motor affect the efficiency of the liquid-liquid phase separation process.
Influenza A virus (IAV) non-structural protein 1 (NS1), a potent antagonist of host immune response, is capable of interacting with RNA and a wide range of cellular proteins. NS1 consists of an RNA-binding domain (RBD) and an effector domain (ED) separated by a flexible linker region (LR). H5N1-NS1 has a characteristic 5-residue deletion in the LR with either G (minor group) or E (major group) at the 71st position, and non-H5N1-NS1 contains E71 with an intact linker. Based on the orientation of ED with respect to RBD, previous crystallographic studies have shown that minor group H5N1-NS1(G71), a non-H5N1-NS1 (H6N6-NS1(E71)), and the LR-deletion mutant H6N6-NS1(80-84/E71) mimicking the major group H5N1-NS1, exhibit llsquo;openrrsquo;, llsquo;semi-openrrsquo;, and llsquo;closedrrsquo; conformations, respectively, suggesting that NS1 exhibits strain-dependent conformational preference. Here we report the first crystal structure of a naturally occurring H5N1-NS1(E71) and show that it adopts an llsquo;openrrsquo; conformation similar to the minor group of H5N1-NS1 (H5N1-NS1(G71)). We also show that H6N6-NS1(80-84/E71) under a different crystallization condition and H6N6-NS1(80-84/G71) also exhibit llsquo;openrrsquo; conformations, suggesting NS1 can adopt an llsquo;openrrsquo; conformation irrespective of E or G at the 71st position. Our single-molecule FRET analysis to investigate the conformational preference of NS1 in solution showed that all NS1 constructs predominantly exist in llsquo;openrrsquo; conformation. Further, our co-immunoprecipitation and binding studies showed that they all bind to cellular factors with similar affinity. Taken together, our studies suggest that NS1 exhibits strain-independent structural plasticity that allows it to interact with a wide variety of cellular ligands during viral infection.
IAV is responsible for several pandemics over the last century and continues to infect millions annually. The frequent rise in drug-resistant strains necessitates exploring novel targets for developing antiviral drugs that can reduce the global burden of influenza infection. Because of its critical role in the replication and pathogenesis of IAV, non-structural protein 1 (NS1) is a potential target for developing antivirals. Previous studies suggested that NS1 adopts strain-dependent llsquo;openrrsquo;, llsquo;semi-openrrsquo;, and llsquo;closedrrsquo; conformations. Here we show, based on three crystal structures, that NS1 irrespective of strain differences can adopt llsquo;openrrsquo; conformation. We further show that NS1 from different strains primarily exists in llsquo;openrrsquo; conformation in solution and binds to cellular proteins with similar affinity. Together, our findings suggest that conformational polymorphism facilitated by a flexible linker is intrinsic to NS1, and this may be the underlying factor allowing NS1 to bind several cellular factors during IAV replication.
Long-acting antiretrovirals could provide a useful alternative to daily oral therapy for HIV-1 infected individuals. Building on a bi-specific molecule with adnectins targeting CD4 and gp41, a potential long-acting biologic, GSK3732394, was developed with three independent and synergistic modes of HIV entry inhibition that potentially could be self-administered as a long-acting subcutaneous injection. Starting with the bi-specific inhibitor, an alpha-helical peptide inhibitor was optimized as a linked molecule to the anti-gp41 adnectin, with each separate inhibitor exhibiting at least single digit nanomolar (or lower) potency and a broad spectrum. Combination of the two adnectins and peptide activities into a single molecule was shown to have synergistic advantages in potency, resistance barrier and in the ability to inhibit HIV-1 infections at low levels of CD4 receptor occupancy, showing that GSK3732394 can work in trans on a CD4+ T cell. Addition of a human serum albumin molecule prolongs the half-life in a human CD4 transgenic mouse, suggesting that it may have potential as a long acting agent. To show that, GSK3732394 was highly effective in a humanized mouse model of infection. GSK3732394 is currently in human studies.
IMPORTANCE There continue to be significant unmet medical needs for patients with HIV-1 infection. One way to improve adherence and decrease the likelihood of drug-drug interactions in HIV-1 infected patients is through the development of long acting biologic inhibitors. Building on a bi-specific inhibitor approach targeting CD4 and gp41, a tri-specific molecule was generated with three distinct antiviral activities. The linkage of these three biologic inhibitors creates synergy that offer a series of advantages to the molecule. The addition of human serum albumin to the tri-specific inhibitor could allow it to function as a long acting self-administered treatment for patients with HIV infection. This molecule is currently in early clinical trials.
The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent amongst the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a non-enveloped arbovirus, causing haemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical and microscopic techniques, we examine the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilising BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.
IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organisation; prominent members include heat shock protein 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host-factors is of primary importance for the development of intervention strategies. Our data identify and show that heat-shock protein 90, but not heat-shock protein 70, stabilizes Bluetongue virus proteins, safeguarding them from proteasomal degradation.
Replication of many (+)RNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. A non-structural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here we systematically investigated the conserved elements of GBF1 to understand what determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that Arf activating property, but not the primary structure of the Sec7 domain is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, dependent not only on direct interaction of the protein with the viral protein 3A, but also on determinants located in the non-catalytic C-terminal domains of GBF1. Such double targeting mechanism explains the previous observations of remarkable tolerance of different levels of GBF1-3A interaction by the virus, and likely constitutes an important element of resilience of viral replication.
Importance. Enteroviruses are a vast group of viruses associated with diverse human diseases but only two of them could be controlled with vaccines, and effective anti-viral therapeutics are lacking. Here we investigated in details the contribution of a cellular protein GBF1 in the replication of poliovirus, a representative enterovirus. GBF1 supports functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our resuts demonstrate that the virus requires a limited subset of the normal GBF1 functions, and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad spectrum anti-viral therapeutics.
Mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cellular metabolism. In nutrient-rich environments, mTORC1 kinase activity stimulates protein synthesis to meet cellular anabolic demands. In nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes multiple proteins that stimulate mTORC1 activity or subvert autophagy, but precise roles for mTORC1 in different stages of KSHV infection remain incompletely understood. Here, we report that during latent and lytic stages of KSHV infection, chemical inhibition of mTORC1 caused eIF4F disassembly and diminished global protein synthesis, which indicated that mTORC1-mediated control of translation initiation was largely intact. We observed that mTORC1 was required for synthesis of the RTA lytic switch protein and reactivation from latency, but once early lytic gene expression had begun, mTORC1 is not required for genome replication, late gene expression or release of infectious progeny. Moreover, mTORC1 control of autophagy was dysregulated during lytic replication, whereby chemical inhibition of mTORC1 prevented ULK1 phosphorylation but did not affect autophagosome formation or rates of autophagic flux. Together these findings suggest that mTORC1 is dispensable for viral protein synthesis and viral control of autophagy during lytic infection, and that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.
All viruses require host cell machinery to synthesize viral proteins. A host cell protein complex known as mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of protein synthesis. In nutrient-rich conditions, mTORC1 is active and promotes protein synthesis to meet cellular anabolic demands. In nutrient-poor conditions or under stress, mTORC1 is rapidly inhibited, global protein synthesis is arrested, and a cellular catabolic process known as autophagy is activated. Kaposi's sarcoma-associated herpesvirus (KSHV) stimulates mTORC1 activity and utilizes host machinery to synthesize viral proteins. However, we discovered that mTORC1 activity was largely dispensable for viral protein synthesis, genome replication and release of infectious progeny. Likewise, during lytic replication, mTORC1 was no longer able to control autophagy. These findings suggest that KSHV undermines mTORC1-dependent cellular processes during the lytic cycle to ensure efficient viral replication.
Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm in transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that hnRNP A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNA1 maps within the amino terminus (aa 19-40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. Furthermore, we uncovered that the N-terminal 21 amino acids (aa 19-40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41-60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, this study provides mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.
IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remains largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified a NES-containing nucleocytoplasmic shuttling domain (aa 19-40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulates nucleocytoplasmic shuttling of the ARV p17 protein.
Influenza viruses express two surface glycoproteins, the hemagglutinin (HA) and the neuraminidase (NA). Anti-NA antibodies protect from lethal influenza virus challenge in the mouse model and correlate inversely with virus shedding and symptoms in humans. Consequently, the NA is a promising target for influenza virus vaccine design. Current seasonal vaccines, however, poorly induce anti-NA antibodies, partly because of the immunodominance of the HA over the NA when both glycoproteins are closely associated. To address this issue, we here investigated whether extending the stalk domain of the NA could render it more immunogenic on virus particles. Two recombinant influenza viruses based on the H1N1 strain A/Puerto Rico/8/1934 (PR8) were rescued with NA stalk domains extended by 15 or 30 amino acids. Formalin-inactivated viruses expressing wild type NA or the stalk-extended NA variants were used to vaccinate mice. Compared to the wild type PR8 virus, the virus with the 30 amino acid stalk extension induced significantly higher anti-NA IgG responses characterized by increased in vitro antibody-dependent cellular cytotoxicity (ADCC) activity, while anti-HA IgG levels were unaffected. Similarly, extending the stalk domain of the NA of a recent H3N2 virus enhanced the induction of anti-NA IgGs in mice. Based on these results we hypothesize that the subdominance of the NA can be modulated if the protein is modified such that it surpasses the height of the HA on the viral membrane. Extending the stalk domain of NA may help to enhance its immunogenicity in influenza virus vaccines without compromising antibody responses to HA.
IMPORTANCE The efficacy of influenza virus vaccines could be improved by enhancing the immunogenicity of the NA protein. One of the reasons for its poor immunogenicity is the immunodominance of the HA over the NA in many seasonal influenza virus vaccines. Here we demonstrate that, in the mouse model, extending the stalk domain of the NA protein can enhance its immunogenicity on virus particles and overcome the immunodominance of the HA without affecting antibody responses to the HA. The antibody repertoire is broadened by the extended NA and includes additional ADCC-active antibodies. Our findings may assist in the efforts towards more effective influenza virus vaccines.
BLT (bone marrow, liver, thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with HIV-1. We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein utilizing a rapid prime-boost protocol of PLGA microparticles and a replication-defective HSV recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide IFN-+ T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1 infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although pre-challenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these post-challenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5 log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including IL-12p70 and MIP-1aalpha;, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization, however additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy.
IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small animal model for pre-clinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable pre-challenge responses, it did increase the magnitude and kinetics of post-challenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.
Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytia formation in cultured human cells. Based on sequence alignment, NSP1-1 from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multi-cycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.
IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.
Human immunodeficiency virus type 1 (HIV-1) has evolved elaborate ways to evade immune cell recognition, including downregulation of classical HLA class I (HLA-I) from the surface of infected cells. Recent evidence identified HLA-E, a non-classical HLA-I, as an important part of the antiviral immune response to HIV-1. Changes in HLA-E surface levels and peptide presentation can prompt both CD8+ T-cell and natural killer (NK)-cell responses to viral infections. Previous studies reported unchanged or increased HLA-E levels on HIV-1nndash;infected cells. Here, we examined HLA-E surface levels following infection of CD4+ T cells with primary HIV-1 strains and observed that a subset downregulates HLA-E. Two primary strains of HIV-1 inducing the strongest reduction in surface HLA-E expression were chosen for further testing. Expression of single Nef or Vpu proteins in T-cell lines as well as tail-swap experiments exchanging the cytoplasmic tail of HLA-A2 with HLA-E demonstrated that Nef modulated HLA-E surface levels in a cytoplasmic tail-dependent manner. Furthermore, infection of primary CD4+ T cells with HIV-1 mutants showed that lack of functional Nef (and Vpu to some extent) impaired HLA-E downmodulation. Taken together, this study demonstrates for the first time that HIV-1 can downregulate HLA-E surface levels on infected primary CD4+ T cells, potentially rendering them less vulnerable to CD8+ T-cell recognition but at increased risk of NKG2A+ NK-cell killing.
IMPORTANCE For almost two decades, it was thought that HIV-1 selectively downregulated the highly expressed HLA-I molecules HLA-A and HLA-B from the cell surface in order to evade cytotoxic T-cell recognition, while leaving HLA-C and HLA-E molecules unaltered. It was stipulated that, thereby, HIV-1 infection maintains inhibition of NK cells via inhibitory receptors that bind HLA-C and HLA-E. This concept was recently revised when a study showed that primary HIV-1 strains reduce HLA-C surface levels, whereas the cell-line adapted HIV-1 strain NL4-3 lacks this ability. Here, we demonstrate that infection with distinct primary HIV-1 strains results in a significant downregulation of surface HLA-E levels. Given the increasing evidence for HLA-E as important modulator of CD8+ T-cell and NKG2A+ NK-cell function, this finding has substantial implications for future immunomodulatory approaches aimed at harnessing cytotoxic cellular immunity against HIV.
Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulating the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.
IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level, and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which a RNA virus interrupts host DNA replication. Lastly, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.
Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cells interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection, MOI) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOI, resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOI. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread.
IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics by seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for the defense against the virus, and therefore it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells function as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.
Cytomegalovirus (CMV) is a ubiquitous bbeta;-herpesvirus that infects many different cell types. Human (H)CMV has been found in several solid tumors and it has been hypothesized that it may promote cellular transformation or exacerbate tumor growth. Paradoxically, in some experimental situations, murine (M)CMV infection delays tumor growth. We previously showed that wild-type MCMV delayed the growth of poorly immunogenic B16 melanomas via an undefined mechanism. Here we show that MCMV delayed the growth of these immunologically "cold" tumors by recruiting and modulating tumor-associated macrophages. Depletion of monocytic phagocytes with clodronate completely prevented MCMV from delaying tumor growth. Mechanistically, our data suggest that MCMV recruits new macrophages to the tumor via the virus-encoded chemokine MCK2, and viruses lacking this chemokine were unable to delay tumor growth. Moreover, MCMV infection of macrophages drove them toward an M1-like state. Importantly, adaptive immune responses were also necessary for MCMV to delay tumor growth as the effect was substantially blunted in Rag-deficient animals. However, viral spread was not needed and a spread-defective MCMV strain was equally effective. In most mice, the anti-tumor effect of MCMV was transient. Although the recruited macrophages persisted, tumor regrowth correlated with a loss of viral activity in the tumor. However, an additional round of MCMV infection further delayed tumor growth, suggesting that tumor growth delay was dependent on active viral infection. Together, our results suggest that MCMV infection delayed the growth of an immunologically "cold" tumor by recruiting and modulating macrophages in order to promote anti-tumor immune responses.
Importance Cytomegalovirus (CMV) is an exciting new platform for vaccines and cancer therapy. Although CMV may delay tumor growth in some settings, there is also evidence that CMV may promote cancer development and progression. Thus, defining the impact of CMV on tumors is critical. Using a mouse model of melanoma, we previously found that murine (M)CMV delayed tumor growth and activated tumor-specific immunity, although the mechanism was unclear. We now show that MCMV delayed tumor growth through a mechanism that required monocytic phagocytes and a viral chemokine that recruited macrophages to the tumor. Furthermore, MCMV infection altered the functional state of macrophages. Although the effects of MCMV on tumor growth were transient, we found that repeated MCMV injections sustained the anti-tumor effect suggesting that active viral infection was needed. Thus, MCMV altered tumor growth by actively recruiting macrophages to the tumor where they were modulated to promote anti-tumor immunity.
Cowpea mosaic virus (CPMV) is a plant virus that has been developed for multiple biomedical and nanotechnology applications, including immunotherapy. Two key platforms are available: virus nanoparticles (VNPs) based on the complete CMPV virion including the genomic RNA, and virus-like nanoparticles (VLPs) based on the empty CPMV (eCPMV) virion. It is unclear whether these platforms differ in terms of immunotherapeutic potential. We therefore compared their physicochemical properties and immunomodulatory activity following in situ vaccination of an aggressive ovarian tumor mouse model (ID8-Defb29/Vegf-A). In physicochemical terms, CPMV and eCPMV were very similar and both significantly increased the survival of tumor-bearing mice and showed promising antitumor efficacy. However, they demonstrated distinct yet overlapping immunostimulatory effects due to the presence of virus RNA in the wild-type particles, indicating their suitability for different immunotherapeutic strategies. Specifically, we found that the formulations had similar effects on most secreted cytokines and immune cells but the RNA-containing CPMV particles were uniquely able to boost the populations of potent antigen presenting cells, such as tumor infiltrating neutrophils and activated dendritic cells. Our results will facilitate the development of CPMV and eCPMV as immunotherapeutic vaccine platforms with tailored responses.
The engagement of antiviral effector responses caused by viral infection is essential when using viruses or virus-like particles (VLPs) as an immunotherapeutic agent. Here, we compare the chemophysical properties and immunostimulatory properties of wild type cowpea mosaic virus (CPMV, RNA-containing) and eCPMV (RNA-free VLPs) produced from two expression systems (agrobacterium-based plant expression system and baculovirus-insect cell expression). CPMV and eCPMV could each be developed as novel adjuvants to overcome immunosuppression and thus promote tumor regression in ovarian cancer (and other tumor types). To our knowledge, this is the first study to define the immunotherapeutic differences between CPMV and eCPMV, which is essential for the further development of biomedical applications for plant viruses and the selection of rational combinations of immunomodulatory reagents.
Polyamines are small polycationic molecules with flexible carbon chains that are found in all eukaryotic cells. Polyamines are involved in the regulation of many host processes and have been shown to be implicated in viral replication. Depletion of polyamine pools in cells with FDA approved drugs restricts replication of diverse RNA viruses. Viruses can exploit host polyamines to facilitate nucleic acid packaging, transcription, and translation, but other mechanisms remain largely unknown. Picornaviruses, including Coxsackievirus B3 (CVB3), are sensitive to depletion of polyamines and remain a significant public health threat. We employed CVB3 as a model system to investigate a potential pro-viral role for polyamines using a forward screen. Passaging CVB3 in polyamine depleted cells generated a mutation in capsid protein VP3 at residue 234. We show this mutation confers resistance to polyamine depletion. Through attachment assays, we demonstrate that polyamine depletion limits CVB3 attachment to susceptible cells, which is rescued by incubating virus with polyamines. Further, the capsid mutant rescues this inhibition in polyamine depleted cells. More divergent viruses also exhibited reduced attachment to polyamine depleted cells, suggesting that polyamines may facilitate attachment of diverse RNA viruses. These studies inform additional mechanisms of action for polyamine-depleting pharmaceuticals with implications for potential antiviral therapies.
IMPORTANCE Enteroviruses are significant human pathogens that can cause severe disease. These viruses rely on polyamines, small positively-charged molecules, for robust replication, and polyamine depletion limits infection in vitro and in vivo. The mechanisms by which polyamines enhance enteroviral replication are unknown. Here, we describe how Coxsackievirus B3 (CVB3) utilizes polyamines to attach to susceptible cells and initiate infection. Using a forward genetic screen, we identified a mutation in a receptor-binding amino acid that promotes infection of polyamine-depleted cells. These data suggest that inhibiting polyamine biosynthesis pharmacologically may combat virus infection by preventing virus attachment to susceptible cells.
The rotavirus polymerase, VP1, mediates all stages of viral RNA synthesis within the confines of subviral particles and while associated with the core shell protein, VP2. Transcription [positive-strand (+) RNA synthesis] by VP1 occurs within double-layered particles (DLPs), while genome replication [double-stranded (ds) RNA synthesis] by VP1 occurs within assembly intermediates. VP2 is critical for VP1 enzymatic activity; yet the mechanism by which the core shell protein triggers polymerase function remains poorly understood. Structural analyses of transcriptionally-competent DLPs show that VP1 is located beneath the VP2 core shell and sits slightly off-center from each of the icosahedral fivefold axes. In this position, the polymerase is contacted by the core shell at 5 distinct surface-exposed sites, comprising VP1 residues 264-267, 547-550, 614-620, 968-980, and 1022-1025. Here, we sought to test the functional significance of these VP2 contact sites on VP1 in regard to polymerase activity. We engineered 19 recombinant (r) VP1 proteins that contained single- or multi-point alanine mutations within each individual contact site and assayed them for the capacity to synthesize dsRNA in vitro in the presence of rVP2. Three rVP1 mutants (E265A/L267A, R614A, and D971A/S978A/I980A) exhibited diminished in vitro dsRNA synthesis. Despite their loss-of-function phenotypes, the mutants did not show major structural changes in silico, and they maintained their overall capacity to bind rVP2 in vitro via their non-mutated contact sites. These results move towards a mechanistic understanding of rotavirus replication and identify precise VP2-binding sites on the polymerase surface that are critical for its enzymatic activation.
Rotaviruses are important pathogens that cause severe gastroenteritis in the young of many animals. The viral polymerase, VP1, mediates all stages of viral RNA synthesis, and it requires the core shell protein, VP2, for its enzymatic activity. Yet, there are several gaps in knowledge about how VP2 engages and activates VP1. Here, we probed the functional significance of 5 distinct VP2 contact sites on VP1 that were revealed through previous structural studies. Specifically, we engineered alanine amino acid substitutions within each of the 5 VP1 regions, and assayed the mutant polymerases for the capacity to synthesize RNA in the presence of VP2 in a test tube. Our results identified residues within 3 of the VP2 contact sites that are critical for robust polymerase activity. These results are important because they enhance an understanding of a key step of the rotavirus replication cycle.
Several therapeutic strategies targeting Epstein-Barr virus (EBV)-associated tumors involve upregulation of viral lytic gene expression. Evidence has been presented that the unfolded protein response (UPR) leads to EBV lytic gene expression. Clofoctol, an antibacterial antibiotic, has been reported to upregulate the UPR in prostate cancer cell lines and to slow their growth. We investigated the effects of clofoctol on an EBV-Burkitt lymphoma cell line and confirmed upregulation of all three branches of the UPR and activation of EBV lytic viral gene expression. While immediate early, early and late EBV RNAs were all upregulated, immediate early and early viral proteins but not late viral proteins were expressed. Furthermore, infectious virions were not produced. Clofoctol in combination with a PERK inhibitor led to expression of late viral proteins. The effects of clofoctol on EBV lytic upregulation are not limited to lymphoid tumor cell lines but also occur in naturally infected epithelial gastric cancer and nasopharyngeal cancer cell lines. An agent that upregulates lytic viral protein expression but doesn't lead to production of infectious virions may have particular value for lytic induction strategies in the clinical setting.
IMPORTANCE Epstein-Barr virus is associated with many different cancers. In these cancers the viral genome is predominantly latent i.e. most viral genes are not expressed, most viral proteins are not synthesized, and new virions are not produced. Some strategies for treating these cancers involve activation of lytic viral gene expression. We identify an antibacterial antibiotic clofoctol as an activator of EBV lytic viral RNA and protein expression that does not lead to virion production.
Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS), nasopharyngeal carcinoma (NPC43) and B cells (M81) led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit, CK2bbeta;, suggesting that CK2bbeta; might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2bbeta;, but not the CK2aalpha; catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 associated with Zp in reporter assays and this was increased by additional CK2bbeta;. Together the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2bbeta;-mediated interaction.
IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor, c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.
Globoside (Gb4) is considered the primary receptor of parvovirus B19 (B19V), however, its expression does not correlate well with attachment and restricted tropism of the virus. The N-terminal of VP1 (VP1u) of B19V interacts with as-yet-unknown receptor required for virus internalization. In contrast to Gb4, the VP1u cognate receptor is expressed exclusively in cells that B19V can internalize. With the aim to clarify the role of Gb4 as B19V receptor, we knocked out the gene B3GalNT1 encoding for the enzyme globoside synthase in UT7/Epo cells. Consequently, B3GalNT1 transcripts and Gb4 became undetectable in the knockout (KO) cells without affecting cell viability and proliferation. Unexpectedly, virus attachment, internalization, and nuclear targeting were not disturbed in the KO cells. However, NS1 transcription failed and consequently, genome replication and capsid protein expression were abrogated. The block could be circumvented by transfection with a B19V infectious clone, indicating that Gb4 is not required after the generation of viral dsDNA with resolved inverted terminal repeats. While in wild-type cells (WT), occupation of the VP1u cognate receptor with recombinant VP1u disturbed virus binding and blocked the infection, antibodies against Gb4 had no significant effect. In a mixed population of WT and KO cells, B19V selectively infected WT cells. This study demonstrates that Gb4 does not have the expected receptor function as it is dispensable for virus entry, however, is essential for productive infection, explaining the resistance of the rare individuals lacking Gb4 to B19V infection.
Caliciviruses are single stranded RNA viruses with 180 copies of capsid protein comprising the T=3 icosahedral capsids. The main capsid feature is a pronounced protruding (P) domain dimer formed by adjacent subunits on the icosahedral surface while the shell domain forms a tight icosahedral sphere around the genome. While the P domain in the crystal structure of human Norwalk virus (genotype I.1) was tightly associated with the shell surface, the cryo-EM structures of several members of the Calicivirus family (mouse norovirus, rabbit hemorrhagic disease virus, and human norovirus genotype II.10) revealed a llsquo;floatingrrsquo; P domain that hovers above the shell by nearly 10-15AAring; in physiological buffers. Since this unusual feature is shared among, and unique to, the Calicivirus family, it suggests an important biological role. Recently, we demonstrated that bile salts enhance cell attachment to the target cell and increase the intrinsic affinity between the P domain and receptor. Presented here are the cryo-EM structures of MNV-1 in the presence of bile salts (~3AAring;) and the receptor CD300lf (~8AAring;). Surprisingly, bile salts cause the rotation and contraction of the P domain onto the shell surface. This both stabilizes the P domain and appears to allow for a higher degree of saturation of receptor onto the virus. Together, these results suggest that, as the virus moves into the gut and the associated high concentrations of bile, the entire capsid face undergoes a conformational change to optimize receptor avidity while the P domain itself undergoes smaller conformational changes to improve receptor affinity.
IMPORTANCE Mouse norovirus and several other members of the Calicivirus family have been shown to have a highly unusual structure with the receptor binding protruding (P) domain only loosely tethered to the main capsid shell. Recent studies demonstrated that bile salts enhance the intrinsic P domain/receptor affinity and is necessary for cell attachment. Presented here are the high resolution cryo-EM structures of apo MNV, MNV/bile salt, and MNV/bile salt/receptor. Bile salts cause a 90ddeg; rotation and collapse of the P domain onto the shell surface that may increase the number of available receptor binding sites. Therefore, bile salts appear to be having several effects on MNV. Bile salts shift the structural equilibrium of the P domain towards a form that binds the receptor and away from one that binds antibody. They may also cause the entire P domain to optimize receptor binding while burying a number of potential epitopes.
Vaccination is widely used to generate protective immunity to influenza. CD4+ T cells contribute in diverse ways to protective immunity, most notably for provision of help for production of neutralizing antibodies. Several recent reports have suggested that influenza infection elicits CD4+ T cells whose specificity only partially overlaps with those elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4+ T cell repertoire, we have evaluated protein vaccine- vs. infection-generated CD4+ T cell epitopes. Our studies revealed that all the infection-elicited CD4+ T cell epitope specificities are also elicited by protein vaccination, although immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus, and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4+ memory T cells are recalled and boosted after infection and provide early help to accelerate HA-specific antibody responses. The early CD4+ T cell response and HA-specific antibody production is associated with lowered viral titers during the infection challenge. Our data lend confidence in current protein-based vaccines ability to elicit influenza-specific CD4+ T cells that can potentiate protective immunity upon influenza virus infection.
Importance. Most current and new influenza vaccine candidates consist of single or combinations of influenza virus proteins. For these vaccines to elicit CD4+ T cells that can be recalled after infection, the peptides epitopes should be shared between the two modes of confrontation. There have recently been questions raised regarding the relatedness of epitope selection by influenza infection and protein vaccination. However, the studies reported here show that protein-based vaccines overlap with those elicited by infection and that CD4+ T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection.
The RV144 HIV-1 vaccine trial showed a strong association between anti-gp70 V1V2 scaffold (V1V2) and anti-V2 hotspot peptide (V2 HS) antibody responses and reduced risk of HIV infection. Accordingly, a primary goal for HIV vaccines is to enhance the magnitude and breadth of V1V2 and V2 HS antibody responses in addition to neutralizing antibodies. Here, we tested the immunogenicity and efficacy of HIV-1 C.1086 gp140 boosts administered sequentially after priming with CD40L-adjuvanted DNA-SHIV and boosting with modified vaccinia Ankara (MVA)-SHIV vaccines in rhesus macaques. The DNA/MVA vaccination induced robust vaccine-specific CD4 and CD8 T cell responses with a polyfunctional profile. Two gp140 booster immunizations induced very high levels (~2mg/ml) of gp140 binding antibodies in serum with strong reactivity directed against the homologous (C.1086) V1V2, V2 HS, V3 and gp41 immunodominant (gp41 ID) proteins. However, the vaccine induced antibody showed 10-fold (peak) and 32-fold (prechallenge) weaker binding to the challenge virus (SHIV1157ipd3N4) V1V2 and failed to bind to the challenge virus V2 HS due to a single amino acid change. Point mutations in the immunogen V2 HS to match the V2 HS in the challenge virus significantly diminished the binding of vaccine-elicited antibodies to membrane-anchored gp160. Both vaccines failed to protect from infection following repeated SHIV1157ipd3N4 intrarectal challenges. However, only the protein boosted animals showed enhanced viral control. These results demonstrate that C.1086 gp140 protein immunizations administered following DNA/MVA vaccination do not significantly boost heterologous V1V2 and V2HS responses, and fail to enhance protection against heterologous SHIV challenge.
IMPORTANCE - HIV, the virus that causes AIDS, is responsible for millions of infections and deaths annually. Despite intense research for the past 25 years, there remains no safe and effective vaccine available. The significance of this work is in identifying the pros and cons of adding a protein boost to an already well-established DNA/MVA HIV vaccine that is currently being tested in the clinic. Characterizing the effects of the protein boost can allow researchers going forward to design vaccines that generate responses that will be more effective against HIV. Our results in rhesus macaques show that boosting with a specific HIV envelope protein does not significantly boost antibody responses that were identified as immune correlates for protection in a moderately successful RV144 HIV vaccine trial in humans, and highlight the need for the development of improved HIV envelope immunogens.
Comparative examination of viral and host homologs reveals novel mechanisms governing downstream signaling effectors of both cellular and viral origin. The vaccinia B1 protein kinase is involved in promoting multiple facets of the virus life cycle and is a homolog of three conserved cellular enzymes called VRKs (vaccinia related kinases). Recent evidence indicates that B1 and VRK2 mediate a common pathway that is largely uncharacterized but appears independent of previous VRK substrates. Interestingly, separate studies described a novel role for B1 in inhibiting vaccinia B12, which otherwise impedes an early event in the viral lifecycle. Herein, we characterize the B1/VRK2 signaling axis to better understand their shared functions. First, we demonstrate that vaccinia virus uniquely requires VRK2 for viral replication in the absence of B1 as compared to other DNA viruses. Employing loss of function analysis, we demonstrate that vaccinia dependence on VRK2 is only observed in the presence of B12, suggesting that B1 and VRK2 share a pathway controlling B12. Moreover, we substantiate a B1/VRK2/B12 signaling axis by examining B1 and VRK2 co-precipitation of B12. Employing execution point analysis, we reveal that virus replication proceeds normally through early protein translation and uncoating, but stalls at replication factory formation in the presence of B12 activity. Finally, structure/function analysis of B1 and VRK2 demonstrate that enzymatic activity is essential for B1 or VRK2 to inhibit B12. Together, these data provide novel insights into B1/VRK signaling co-regulation and support a model in which these enzymes modulate B12 in a phosphorylation dependent manner.
Constraints placed on viral genome size require that these pathogens must employ sophisticated, yet parsimonious, mechanisms to effectively integrate with host cell signaling pathways. Poxviruses are no exception and employ several methods to balance these goals including encoding single proteins that impact multiple downstream pathways. This study focuses on the vaccinia B1 protein kinase, an enzyme that promotes virus replication at multiple phases of the viral lifecycle. Herein, we demonstrate that in addition to its previously characterized functions, B1 inhibits vaccinia B12 via a phosphorylation-dependent mechanism and that this function of B1 can be complemented by the cellular B1 homolog, VRK2. Combined with previous data implicating functional overlap between B1 and an additional cellular B1 homolog, VRK1, these data provide evidence of how poxviruses can be multifaceted in their mimicry of cellular proteins through the consolidation of functions of both VRK1 and VRK2 within the viral B1 protein kinase.
A relatively stable and flexible capsid is critical to viral life cycle. However, the capsid dynamics and trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation of PCV2 virions by genome labelling and the use of three monoclonal antibodies against native capsid of PCV2. Genome labelling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65ddeg;C causing all antibodies binding incompetence, viral genome was released without capsid disassembly. Antibodies binding experiments with PCV2 virions showed that residues 186 to 192 was concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment, then exposed in PK-15 cytosol and 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking was not significantly affected in monocytic 3D4/31 cells treated with nocodazole by microtubule depolymerization. These findings demonstrated that nuclear-targeting of viral capsids involved conformational changes, PCV2 genome was released from assembled capsid, the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.
IMPORTANCE Circovirus is the smallest virus known to be autonomously replicable. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of PCV2. Here, we report the release of the PCV2 genome from assembled capsid, the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.
Porcine reproductive and respiratory syndrome (PRRS) is one of the most important infectious diseases affecting the global pig industry. Previous studies from our and other groups showed that cholesterol 25-hydroxylase (CH25H), a multi-transmembrane endoplasmic reticulum-associated enzyme, catalyzes the production of 25-hydroxycholesterol (25HC) and inhibits PRRS virus (PRRSV) replication. However, PRRSV infection also actively decreases porcine CH25H (pCH25H) expression through unidentified mechanism(s). In this study, we found that the ubiquitin-proteasome pathway plays a major role in pCH25H degradation during PRRSV infection, and that the PRRSV-encoded envelope (E) protein interacts with pCH25H. PRRSV E protein degraded pCH25H via ubiquitination, and the ubiquitination site was at pCH25H Lys28. Interestingly, PRRSV E protein appeared to specifically degrade pCH25H but not human CH25H, likely because of a Lys28Arg substitution in the human orthologue. As expected, ubiquitin-mediated degradation by E protein attenuated the antiviral effect of pCH25H by downregulating 25HC production. In addition, we found that knockdown of pCH25H decreased E protein-induced inflammatory cytokine expression and that pCH25H overexpression had the opposite effect. These findings suggested that regulation of pCH25H expression was associated with E protein-induced inflammatory responses. Taken together, our results and those of previous studies of the anti-PRRSV effects of CH25H highlight the complex interplay between PRRSV and pCH25H.
Cholesterol 25-hydroxylase (CH25H) has received significant attention due to its broad antiviral activity, which it mediates by catalyzing the production of 25-hydroxycholesterol (25HC). Most studies have focused on the antiviral mechanisms of CH25H; however, whether viruses also actively regulate CH25H expression has not yet been reported. Previous studies demonstrated that porcine CH25H (pCH25H) inhibits PRRSV replication not only via production of 25HC, but also by ubiquitination and degradation of viral nonstructural protein 1aalpha;. In this study, we expanded on previous work and found that PRRSV actively degrades pCH25H through the ubiquitin-proteasome pathway. PRRSV envelope protein, a viral structural protein, is involved in this process. This study reveals a novel interaction mechanism between virus and host during PRRSV infection.
Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTV viruses have only been reported in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within Open Reading Frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (llt;100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread.
Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade.
Endogenous retroviruses (ERV) are found throughout vertebrate genomes and failure to silence their activation can have deleterious consequences on the host. Mutation and subsequent disruption of ERV loci is therefore an indispensable component of the cell-intrinsic defenses that maintain the integrity of the host genome. Abundant in vitro and in silico evidence have revealed that APOBEC3 cytidine-deaminases, including human APOBEC3G (hA3G) can potently restrict retrotransposition; yet in vivo data demonstrating such activity is lacking, since no replication competent human ERV have been identified. In mice deficient for Toll-like receptor 7 (TLR7), transcribed ERV loci can recombine and generate infectious ERV. In this study, we show that ectopic expression of hA3G can prevent the emergence of replication-competent, infectious ERV in Tlr7-/- mice. Mice encode one copy of Apobec3 in their genome. ERV reactivation in Tlr7-/- mice was comparable in the presence and absence of Apobec3. In contrast, expression of a human APOBEC3G transgene abrogated emergence of infectious ERV in the Tlr7-/- background. No ERV RNA was detected in the plasma of hA3G+Apobec3-/-Tlr7-/- mice, and infectious ERV virions could not be amplified through co-culture with permissive cells. These data reveal that hA3G can potently restrict active ERV in vivo, and suggest that expansion of the APOBEC3 locus in primates may have helped to provide for the continued restraint of ERV in the human genome.
Although APOBEC3 proteins are known to be important antiviral restriction factors in both mice and humans, their roles in the restriction of endogenous retroviruses (ERV) has been limited to in vitro studies. Here, we report that human APOBEC3G expressed as a transgene in mice prevents the emergence of infectious ERV from endogenous loci. This study reveals that APOBEC3G can powerfully restrict active retrotransposons in vivo and demonstrates how transgenic mice can be used to investigate host mechanisms that inhibit retrotransposons and reinforce genomic integrity.
One large open reading frame (ORF) encodes for ten potyviral proteins. We compared accumulation of cylindrical inclusion protein (CI) from the middle, coat protein (CP) from the 3'end and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5'RLUC was expressed from an rluc gene inserted between P1 and helper component proteinase (HCPro) cistrons and 3'RLUC from the gene inserted between RNA polymerase and CP cistrons. Viral protein and RNA accumulation was quantitated 1) when expressed from PVA RNA in the presence of ectopically expressed viral protein genome-linked (VPg) and auxiliary proteins and 2) at different time points during natural infection. The rate and timing of 3'RLUC and CP accumulation were found to be different from those of 5'RLUC and CI. Ectopic expression of VPg boosted PVA RNA, 3'RLUC and, together with HCPro, CP accumulation whereas 5'RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3'RLUC accelerated towards the end of infection. 5'RLUC accumulation, which was pronounced already at 2 days post infection, increased moderately and stabilized to a constant level by day 5 whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during the early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism how this occurs.
SIGNIFICANCE STATEMENT: The results of this study suggest that the dynamics of potyviral protein accumulation is regulated differentially from the 3'end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.
The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), III (D). Alanine substitution of the motif I residues (AA-E-D, four nucleotide substitutions) in murine hepatitis virus (MHV) and SARS-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2 or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to MHV-ExoN-AA. In contrast, a low passage (P10) MHV-ExoN-AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multi-protein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants.
Coronaviruses encode an exoribonuclease (ExoN) that is important for viral replication, fitness, and virulence, yet coronaviruses with a defective ExoN (ExoN-AA) have not reverted under diverse experimental conditions. In this study, we identify multiple impediments to MHV-ExoN-AA reversion. We show that ExoN-AA reversion is possible but evolutionarily unfavorable. Instead, compensatory mutations outside of ExoN-AA motif I are more accessible and beneficial than partial reversion. We also show that coevolution between replicase proteins over long-term passage partially compensates for ExoN-AA motif I but renders the virus inhospitable to a reverted ExoN. Our results reveal the evolutionary basis for the genetic stability of ExoN-inactivating mutations, illuminate complex functional and evolutionary relationships between coronavirus replicase proteins, and identify potential mechanisms for stabilization of ExoN-AA coronavirus mutants.
Superinfection exclusion (SIE) or "cross protection" phenomena have been documented for plant viruses for nearly a century and are widespread among taxonomically diverse viruses, but little information is available about SIE of plant negative-strand RNA viruses. Here we demonstrate that SIE by sonchus yellow net nucleorhabdovirus virus (SYNV) is mediated by the viral matrix (M) protein, a multi-functional protein involved in transcription regulation, virion assembly and virus budding. We show that fluorescent protein-tagged SYNV variants display mutual exclusion/cross protection in Nicotiana benthamiana plants. Transient expression of the SYNV M protein, but not other viral proteins, interfered with SYNV local infections. In addition, SYNV M deletion mutants failed to exclude superinfection by wild-type SYNV. An SYNV minireplicon reporter gene expression assay showed that the M protein inhibited viral transcription. However, M protein mutants with weakened nuclear localization signals (NLS) and deficient nuclear interactions with the SYNV nucleocapsid protein were unable to suppress transcription. Moreover, SYNV with M NLS mutations exhibited compromised SIE against wild-type SYNV. From these data, we propose that M protein accumulating in nuclei with primary SYNV infections either coils or prevents uncoiling of nucleocapsids released by the superinfecting SYNV virions and suppresses transcription of superinfecting genomes, thereby preventing superinfection. Our model suggests that the rhabdovirus M protein regulates the transition from replication to virion assembly and renders the infected cells non-permissive for secondary infections.
IMPORTANCE Superinfection exclusion (SIE) is a widespread phenomenon in which an established virus infection prevents re-infection by closely related viruses. Understanding the mechanisms governing SIE will not only advance our basic knowledge of virus infection cycles, but may also lead to improved design of anti-viral measures. Despite its significance, our knowledge about viral SIE determinants and their modes of actions remain limited. In this study, we show that sonchus yellow net virus (SYNV) SIE is mediated by the viral matrix (M) protein. During primary infections, accumulation of M protein in infected nuclei results in coiling of genomic nucleocapsids and suppression of viral transcription. Consequently, nucleocapsids released by potential superinfectors are sequestered and are unable to initiate new infections. Our data suggest that SYNV SIE is caused by M protein-mediated transition from replication to virion assembly and that this process prevents secondary infections.
Proteoglycans function not only as structural components of the extracellular compartment but also as regulators of various cellular events, including cell migration, inflammation, and infection. Many microbial pathogens utilize proteoglycans to facilitate adhesion and invasion into host cells. Here, we report a secreted form of a novel heparan sulfate proteoglycan that functions against virus infection. The expression of SPOCK2/testican2 was significantly induced in virus-infected lungs or in IFN-treated alveolar lung epithelial cells. Overexpression from a SPOCK2 expression plasmid alone or the treatment of cells with recombinant SPOCK2 protein efficiently blocked influenza virus infection at the step of viral attachment to the host cell and entry. Moreover, mice treated with purified SPOCK2 were protected against virus infection. Sialylated glycans and heparan sulfate chains covalently attached to the SPOCK2 core protein were critical for its antiviral activity. Neuraminidase (NA) of influenza virus cleaves the sialylated moiety of SPOCK2, thereby blocking its binding to the virus. Our data suggest that IFN-induced SPOCK2 functions as a decoy receptor to bind and block influenza virus infection, thereby restricting entry of the infecting virus into neighboring cells.
IMPORTANCE Here, we report a novel proteoglycan protein, testican2/SPOCK2, that prevents influenza virus infection. Testican/SPOCK2 is a complex type of secreted proteoglycan with heparan sulfate GAG chains attached to the core protein. SPOCK2 expression is induced upon virus infection or by interferons and it is secreted to an extracellular compartment, where it acts directly to block virus-cell attachment and entry. Treatment with purified testican/SPOCK2 protein can efficiently block influenza virus infection in vitro and in vivo. We also identified the heparan sulfate moiety as a key regulatory module for this inhibitory effect. Based on its mode of action (cell-attachment/entry blocker) and site of action (extracellular compartment), we propose testican/SPOCK2 as a potential antiviral agent that can efficiently control influenza virus infection.
The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wildtype, replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by over-expression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not MLV infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication by altering the cellular milieu through targeting factors including TNPO3.
HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study we identified the interferon-inducible miR-128, as a novel antiviral mediator, which suppresses the expression of the host gene TNPO3 known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.
The adenovirus (Ad) E4orf4 protein was reported to contribute to inhibition of ATM- and ATR-regulated DNA damage signaling during Ad infection and following treatment with DNA damaging drugs. Inhibition of these pathways improved Ad replication and when expressed alone E4orf4 sensitized transformed cells to drug-induced toxicity. However, the mechanisms utilized were not identified. Here we show that E4orf4 associates with the DNA damage sensor Poly (ADP-ribose) polymerase 1 (PARP-1), and the association requires PARP activity. During Ad infection PARP is activated but its activity is not required for recruitment of either E4orf4 or PARP-1 to virus replication centers, suggesting that their association occurs following recruitment. Inhibition of PARP-1 assists E4orf4 in reducing DNA damage signaling during infection, and E4orf4 attenuates virus- and DNA damage-induced parylation. Furthermore, E4orf4 reduces PARP-1 phosphorylation on Serine residues, which likely contributes to PARP-1 inhibition, as phosphorylation of this enzyme was reported to enhance its activity. PARP-1 inhibition is important to Ad infection since treatment with a PARP inhibitor enhances replication efficiency. When E4orf4 is expressed alone it associates with Poly (ADP-ribose) (PAR) chains and is recruited to DNA damage sites in a PARP-1 dependent manner. This recruitment is required for inhibition of drug-induced ATR signaling by E4orf4 and for E4orf4-induced cancer cell death. Thus the results presented here demonstrate a novel mechanism by which E4orf4 targets and inhibits DNA damage signaling through an association with PARP-1 for the benefit of the virus and impacting E4orf4-induced cancer cell-death.
Replication intermediates and ends of viral DNA genomes can be recognized by the cellular DNA damage response network (DDR) as DNA damage whose repair may lead to inhibition of virus replication. Therefore, many viruses evolved mechanisms to inhibit the DDR. We have previously shown that the adenovirus (Ad) E4orf4 protein inhibits DDR signaling but the mechanisms were not identified. Here we describe an association of E4orf4 with the DNA damage sensor Poly (ADP-ribose) polymerase 1 (PARP-1). E4orf4 reduces phosphorylation of this enzyme and inhibits its activity. PARP-1 inhibition assists E4orf4 in reducing Ad-induced DDR signaling and improves the efficiency of virus replication. Furthermore, the ability of E4orf4, when expressed alone, to accumulate at DNA damage sites and to kill cancer cells is attenuated by chemical inhibition of PARP-1. Our results indicate that the E4orf4-PARP-1 interaction has an important role in Ad replication and in promotion of E4orf4-induced cancer-selective cell death.
Among the innate immune sentinels, the complement system is a formidable first line of defense against pathogens including viruses. Chandipura virus (CHPV), a neurotropic vesiculovirus of the family Rhabdoviridae is a deadly human pathogen known to cause fatal encephalitis especially among children. The nature of interaction and the effect of human complement on CHPV are unknown. Here, we report that CHPV is a potent activator of complement and thus is highly sensitive to complement proteins in normal human serum (NHS). Utilizing a panel of specific complement component depleted/reconstituted human serum we have demonstrated that CHPV neutralization is C3, C4 and C1q dependent and independent of factor B suggesting the importance of the classical pathway in limiting CHPV. Employing a range of biochemical approaches we could show a) direct association of C1q to CHPV b) deposition of complement proteins C3b, C4b and C1q on CHPV and c) virus aggregation. Depletion of C8, an important component of the pore forming complex of complement had no effect on CHPV further supporting the finding that aggregation and not virolysis is the mechanism of virus neutralization. With no approved vaccines or treatment modalities in place against CHPV, insights into such interactions can be exploited to develop potent vaccines or therapeutics targeting CHPV.
IMPORTANCE Chandipura virus is a clinically important human pathogen of the Indian subcontinent. The rapidity in death associated with CHPV infection in addition to the absence of an effective vaccine or therapeutics results in poor clinical prognosis. The biology of the virus and its interaction with the host immune system including the complement system is understudied. Our investigation reveals the susceptibility of CHPV to fluid phase complement and also dissects the pathway involved and the mechanism of virus neutralization. Direct binding of C1q, an important upstream component of the classical pathway of complement to CHPV and the strong dependency on C1q for virus neutralization highlights the significance of identifying such interactions to better understand CHPV pathogenesis and devise strategies to target this deadly pathogen.
As many tumour cells synthetize vascular endothelial growth factors (VEGF) that promote neo-vascularization and metastasis, front-line cancer therapies often administer aalpha;-VEGF antibodies. To target the oncolytic parvovirus minute virus of mice (MVM) to the tumour vasculature, we have studied the functional tolerance, evasion of neutralization, and induction of aalpha;-VEGF antibodies, of chimeric viruses in which the footprint of a neutralizing monoclonal antibody within the threefold capsid spike was replaced by VEGF blocking peptides: P6L (PQPRPL), and A7R (ATWLPPR). Both peptides allowed viral genome replication and nuclear translocation of chimeric capsid subunits. MVM-P6L efficiently propagated in culture exposing the heterologous peptide on the capsid surface, and evaded neutralization by the anti spike monoclonal antibody. In contrast, MVM-A7R yielded low infectious titers and was poorly recognized by an aalpha;-A7R monoclonal antibody. MVM-A7R showed a deficient assembly pattern, suggesting that A7R impaired a transitional configuration that the subunits must undergo in the threefold axis for closing-up the capsid shell. The MVM-A7R chimeric virus consistently evolved in culture to a mutant carrying the P6Q amino acid substitution within the A7R sequence, which restored normal capsid assembly and infectivity. Consistent with this finding, anti-native VEGF antibodies were induced in mice by a single injection of MVM-A7R empty capsids, but not by MVM-A7R virions. This fundamental study provides insights to endow immune anti-neovascularization and evasion capacities to an infectious parvovirus by replacing an antibody footprint in the capsid threefold axis with VEGF blocking peptides, and it also illustrates the evolutionary capacity of ssDNA viruses to overcome engineered capsid structural restrictions.
Targeting the VEGF signalling required for neovascularization by vaccination with chimeric capsids of oncolytic viruses may boost therapy for solid tumours. VEGF-blocking peptides (VEbp) engineered in the capsid threefold axis endowed infectious parvovirus MVM with the ability to induce aalpha;-VEGF antibodies without adjuvant, and to evade neutralization by MVM-specific antibodies. However, these properties may be compromised by structural restrains that the capsid imposes on peptide configuration, and by misassembly caused by the heterologous peptides. Significantly, chimeric MVM-VEbp was able to evolve resolving the structural restrictions by selecting mutations within the engineered peptides that restored efficient capsid assembly. This data shows the promise of anti-neovascularization vaccines using chimeric VEbp-icosahedral capsids of oncolytic viruses, but also raises safety concerns regarding the genetic stability of manipulated infectious parvoviruses in cancer and gene therapies.
A fusion protein expressed on the surface of enveloped viruses mediates fusion of the viral and cellular membranes to facilitate virus infection. Pre- and post-fusion structures of viral fusion proteins have been characterized, but conformational changes between them remain poorly understood. Here, we examined the intermediate conformation of the murine coronavirus fusion protein, called the spike protein, which must be cleaved by a cellular protease following receptor binding. Western blot analysis of protease digestion products revealed that two subunits (67 and 69 kDa) are produced from a single spike protein (180 kDa). These two subunits were considered to be by-products derived from conformational changes and were useful for probing the intermediate conformation of the spike protein. Interaction with a heptad repeat (HR) peptide revealed that these subunits adopt packed and unpacked conformations, respectively, and two-dimensional electrophoresis revealed a trimeric assembly. Based on biochemical observations, we propose an asymmetric trimer model for the intermediate structure of the spike protein. Receptor binding induces the membrane-binding potential of the trimer, in which at least one HR motif forms a packed-hairpin structure, while membrane fusion subunits are covered by the receptor-binding subunit, thereby preventing the spike protein from forming the typical homotrimeric pre-hairpin structure predicted by the current model of class I viral fusion protein. Subsequent proteolysis induces simultaneous packing of the remaining unpacked HRs upon assembly of three HRs at the central axis to generate a six-helix bundle. Our model proposes a key mechanism for membrane fusion of enveloped viruses.
Recent studies using single-particle cryo-electron microscopy (cryoEM) revealed the mechanism underlying activation of viral fusion protein at the priming stage. However, characterizing the subsequent triggering stage underpinning transition from pre- to post-fusion structures is difficult because single-particle cryoEM excludes unstable structures that appear as heterogeneous shapes. Therefore, population-based biochemical analysis is needed to capture features of unstable proteins. Here, we analyzed protease digestion products of a coronavirus fusion protein during activation; their sizes appear to be affected directly by the conformational state. We propose a model for the viral fusion protein in the intermediate state, which involves a compact structure and conformational changes that overcome steric hindrance within the three fusion protein subunits.
The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine-substituted mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at aalpha;2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the aalpha;2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine-substituted mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localizations of the flaviviral capsid protein and the formation of infectious viral particles.
This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants, and found that the capsid protein localizes at the nucleolus in a different manner compared to its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of anti-flavivirus small molecules that can target the flavivirus capsid protein.
Human T-cell Leukemia Virus type 1 (HTLV-1) causes multiple pathological effects, ranging from a form of leukemia to a spectrum of inflammatory-mediated diseases. These diseases arise from one or several infected CD4+ T-cells among thousands acquiring proliferation and survival advantages and ultimately becoming pathogenic. Given the low incidence of HTLV-1-associated diseases among carriers, such cellular evolutionary processes appear to occur rarely. Therefore, infectious spread of HTLV-1 within the T-cell population may be one underlying factor influencing disease development. Free HTLV-1 virions are poorly infectious, so infection of T-cells relies on direct contact between infected and target cells. Following contact, virions pass to target cells through a virological synapse or cellular conduits, or are transferred to target cells within an extracellular matrix. LFA-1 on the surface of the target cell engaging with its ligand, ICAM-1, on the surface of the infected cell (effector cell) initiates and stabilizes cell-cell contact for infection. We found that stable expression of the HTLV-1 accessory protein, HTLV-1 bZIP factor (HBZ), in Jurkat T-cells increases homotypic aggregation. This phenotype was attributed to elevated ICAM-1 expression in the presence of HBZ. Using a single-cycle replication-dependent luciferase assay, we found that HBZ expression in Jurkat cells (used as effector cells) increases HTLV-1 infection. Despite this effect, HBZ could not replace the critical infection-related functions of the HTLV-1 regulatory protein, Tax. However, in HTLV-1-infected T-cells, knockdown of HBZ expression did lead to a decrease in infection efficiency. These overall results suggest that HBZ contributes to HTLV-1 infectivity.
Human T-cell Leukemia Virus type 1 (HTLV-1) causes a variety of diseases, ranging from a fatal form of leukemia to immune-mediated inflammatory diseases. These diseases occur rarely, arising from one or a small subset of virally-infected cells infrequently evolving into a pathogenic state. Thus, the process of HTLV-1 cell-to-cell transmission within the host helps influence the probability of disease-development. HTLV-1 primarily infects T-cells, and initially spreads within this cell population when virally-infected T-cells dock to uninfected target T-cells and then transfer HTLV-1 virus particles to the target cells. Here we found that the viral protein HTLV-1 bZIP factor (HBZ) promotes infectivity. HBZ accomplishes this task by increasing the surface abundance of a cellular adhesion protein known as ICAM-1, which helps initiate and stabilize contact (docking) between infected and target T-cells. These results define a novel and unexpected function of HBZ, diverging from its defined functions in cellular survival and proliferation.
Major histocompatibility complex E (MHC-E) is a highly conserved non-classical MHC-Ib molecule that tightly binds peptides derived from leader sequences of classical MHC-Ia molecules for presentation to natural killer cells. However, MHC-E also binds diverse foreign and neoplastic self-peptide antigens for presentation to CD8+ T cells. Although the determinants of MHC-E-restricted T cell priming remain unknown, these cells are induced in humans infected with pathogens containing genes that inhibit the transporter associated with antigen processing (TAP). Indeed, mice vaccinated with TAP-inhibited autologous dendritic cells develop T cells restricted by the murine MHC-E homologue, Qa-1b. Here, we tested whether rhesus macaques (RM) vaccinated with viral constructs expressing a TAP inhibitor would develop insert-specific MHC-E-restricted CD8+ T cells. We generated viral constructs co-expressing SIVmac239 Gag in addition to one of three TAP inhibitors: herpes simplex virus-2 ICP47, bovine herpes virus-1 UL49.5, or rhesus cytomegalovirus Rh185. Each TAP inhibitor reduced surface expression of MHC-Ia molecules, but did not reduce surface MHC-E expression. In agreement with modulation of surface MHC-Ia levels, TAP inhibition diminished presentation of MHC-Ia-restricted CD8+ T cell epitopes without impacting presentation of peptide antigen bound by MHC-E. Vaccination of macaques with vectors dually expressing SIVmac239 Gag with ICP47, UL49.5, or Rh185 generated Gag-specific CD8+ T cells classically restricted by MHC-Ia, but not MHC-E. These data demonstrate that, in contrast to mice, TAP inhibition alone is insufficient for priming of MHC-E-restricted T cell responses in primates, and suggest that additional unknown mechanisms govern the induction of CD8+ T cells recognizing MHC-E-bound antigen.
Importance: Due to the near monomorphic nature of MHC-E in the human population and inability of many pathogens to inhibit MHC-E-mediated peptide presentation, MHC-E-restricted T cells have become an attractive vaccine target. However, little is known concerning how these cells are induced. Understanding the underlying mechanisms that induce these T cells would provide a powerful new vaccine strategy to an array of neoplasms and viral and bacterial pathogens. Recent studies have indicated a link between TAP-inhibition and induction of MHC-E-restricted T cells. The significance of our research is in demonstrating that TAP inhibition alone does not prime MHC-E-restricted T cell generation and suggests that other, currently unknown mechanisms regulate their induction.
Human T cell leukemia virus type-1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose function in humans is to counteract innate and adaptive responses and support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by the mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO. In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both HTLV-1WT and HTLV-1p12KO. We found that NOD/SCID/C-/- c-kit+ mice engrafted with human tissues one day from birth, designated NSG-1d, are highly susceptible to infection by HTLV-1WT and a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+CD25+ T cells to vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo. Infection by HTLV-1p12KO in a Bone Marrow-Liver-Thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks of exposure and was associated with high HTLV-1 DNA in blood and the expansion of CD4+CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with higher fitness.
IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 nndash; 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.
CD69 is highly expressed on the leukocyte surface upon viral infection, and its regulatory role in the vaccinia virus (VACV) immune response has been recently demonstrated using CD69-/- mice. Here, we show augmented control of VACV infection using the anti-human CD69 monoclonal antibody (mAb) 2.8 as both preventive and therapeutic treatment for mice expressing human CD69. This control was related to increased natural killer (NK) cell reactivity and increased numbers of cytokine-producing T and NK cells in the periphery. Moreover, similarly increased immunity and protection against VACV were reproduced over both long and short periods in anti-mouse CD69 mAb 2.2-treated immunocompetent wild-type (WT) mice and immunodeficient Rag2-/- CD69+/+ mice. This result was not due to synergy between infection and anti-CD69 treatment since, in the absence of infection, anti-human CD69 targeting induced immune activation, which was characterized by mobilization, proliferation, and enhanced survival of immune cells as well as marked production of several innate proinflammatory cytokines by immune cells. Additionally, we showed that the rapid leukocyte effect induced by anti-CD69 mAb treatment was dependent on mTOR signaling. These properties suggest the potential of CD69-targeted therapy as an antiviral adjuvant to prevent derived infections.
In this study, we demonstrate the influence of human and mouse anti-CD69 therapies on the immune response to VACV infection. We report that targeting CD69 increases the leukocyte numbers in the secondary lymphoid organs during infection and improves the capacity to clear the viral infection. Targeting CD69 increases the numbers of gamma interferon (IFN)- and tumor necrosis factor alpha (TNFaalpha;)-producing NK and T cells. In mice expressing human CD69, treatment with an anti-CD69 mAb produces increases in cytokine production, survival and proliferation mediated in part by mTOR signaling. These results, together with the fact that we have mainly worked with a human-CD69 transgenic model, reveal CD69 as a treatment target to enhance vaccine protectiveness.
Initiation of RNA synthesis by the Hepatitis C virus (HCV) RNA-dependent-RNA-polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5' UTR, which was anticipated to support non-canonical initiation. Indeed, a shorter RNA species was detected by northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that the capability for internal initiation it is a common and conserved feature of viral RdRps.
Many aspects of viral RNA replication of Hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA-polymerase NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study we present a replicon model, which allows the intracellular assessment of non-canonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive strand RNA viruses, which might contain the intrinsic ability to initiate internally.
Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, especially due to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR) and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow for the production of infectious virus. NanoLuc allowed to quantitatively monitor HEV infection and replication by luciferase assay. HA-tagged replicons and full-length genomes allowed to localize putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlap with ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow to further investigate the structure and composition of the viral replication complex.
Hepatitis E virus (HEV) is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. Especially, low expression of the ORF1 protein and/or limited sensitivity of currently available antibodies limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow to further characterize the HEV replication complex and to improve our understanding of the viral life cycle.
Increased frequencies of immunosuppressive regulatory T-cells (Tregs) are associated with gut lymphoid tissue fibrosis and dysfunction which, in turn, contribute to disease progression in chronic SIV/HIV infection. Mesenteric lymph nodes (MLNs), which drain the large and small intestine, are critical sites for the induction and maintenance of gut mucosal immunity. However, the dynamics of Tregs in MLNs are not well understood due to the lack of accessibility to these tissues in HIV-infected individuals. Herein, dynamics of Tregs in blood and MLNs were assessed in SIV-infected rhesus macaques following early anti-retroviral (ARV) initiation. Early ARV initiation reduced T-cell immune activation, as assessed by HLA-DR/CD39 expression, and prevented the depletion of memory CCR6+Th17 cells in both blood and MLNs. Untreated animals showed higher frequencies of Treg, CD39+Tregs, thymic Tregs and new memory CD4 populations sharing similarity with Tregs as CTLA4+PD1- and CTLA4+PD1-FoxP3+ T-cells. Despite early ARV, the frequencies of these Treg sub-sets remained unchanged within the MLNs and, in contrast to blood normalization, the Th17/Treg ratio remained distorted in MLNs. Furthermore, our results highlighted that the expressions of IDO-1, TGFbbeta;1 and collagen-1 mRNA remained unchanged in MLN of ARV-treated RMs. ARV interruption didn't affect T-cell immune activation and Th17/Treg ratios in MLN. Altogether, our data demonstrated that early ARV initiation within the first few days of SIV infection is unable to reduce the frequencies and homing of various subsets of Tregs within the MLNs which, in turn, may result in tissue fibrosis, impairment in MLN function and HIV persistence.
Tregs contribute to SIV/HIV disease progression by inhibition of anti-viral specific responses and effector T-cells proliferation. Tregs also cause tissue fibrosis via TGF-bbeta;1 production and collagen deposition, which are associated with microbial translocation and generalized immune activation. Early ARV initiation upon viral exposure is recommended globally and results in improved immune function recovery and reduced viral persistence. Here, using an acute SIV infection model of rhesus macaques, we demonstrated for the first time that despite clear improvements in mucosal CD4 T-cells, in contrast to blood, Tregs frequencies in MLNs remained elevated despite early ARV initiation. The particular Th17/Treg balance observed in MLNs can contribute, in part, to maintenance of mucosal fibrosis during suppressive ARV. Our results provide a better understanding of gut mucosal immune dynamics following early ARV initiation. These findings suggest that Tregs-based treatments could serve as a novel immunotherapeutic approach to decrease gut mucosal damage during SIV/HIV infections.
Human coronavirus NL63 (HCoV-NL63) is a common respiratory virus that causes moderately severe infections. We have previously shown that the virus uses heparan sulfate proteoglycans (HSPGs) as the initial attachment factors, facilitating viral entry into the cell. In the present study, we show that the membrane protein (M) of the HCoV-NL63 mediates this attachment. Using virus-like particles lacking the spike protein (S) we demonstrated that the binding to the cell is not S protein-dependent. Furthermore, we mapped the M protein site responsible for the interaction with HSPG and confirmed its relevance using a viable virus. Importantly, in silico analysis of the region responsible for HSPG binding in different clinical isolates and the Amsterdam I strain did not exhibit any signs of cell culture adaptation.
IMPORTANCE It is generally accepted that the coronaviral S protein is responsible for viral interaction with a cellular receptor. Here we showed that the M protein is also an important player during early stages of HCoV-NL63 infection and that a concerted action of the two proteins (M and S) is a prerequisite for effective infection. We believe that this study broadens the understanding of HCoV-NL63 biology and may also alter the way in which we perceive the first steps of cell infection with the virus. The data presented herein may also be important for future research into vaccine or drug development.
Autosomal dominant STAT1 mutations in humans have been associated with chronic mucocutaneous candidiasis (CMC), as well as increased susceptibility to herpesvirus infections. Prior studies have focused on mucosal and Th17-mediated immunity against Candida, but mechanisms of impaired antiviral immunity have not previously been examined. To begin to explore mechanisms of STAT1-associated immunodeficiency against herpesviruses, we generated heterozygous STAT1 R274W knockin mice that have a frequently reported STAT1 mutation associated with susceptibility to herpesvirus infections in humans. In primary macrophages and fibroblasts, we found that STAT1 R274W had no appreciable effect on cell-intrinsic immunity against herpes simplex virus-1 or gammaherpesvirus-68 (HV68) infection. However, intraperitoneal inoculation of mice with HV68 was associated with impaired control of infection at day 14 in STAT1 R274W mice compared with WT littermate control animals. Infection of STAT1 R274W mice was associated with paradoxically decreased expression of IFN-stimulated genes (ISGs) and interferon- (IFN-), likely secondary to defective CD4+ and CD8+ T cell responses, including diminished numbers of antigen-specific CD8+ T cells. Viral pathogenesis studies in WT and STAT1 R274W mixed bone marrow chimeric mice revealed that the presence of WT leukocytes was sufficient to limit infection, and that antigen-specific STAT1 R274W CD8+ T cell responses were impaired even in the presence of WT leukocytes. Thus, in addition to regulating Th17 responses against Candida, a STAT1 gain-of-function mutant impedes antigen-specific T cell responses against a common gammaherpesvirus in mice.
Mechanisms of immunodeficiency related to STAT1 gain-of-function have not previously studied in an animal model of viral pathogenesis. Using virological and immunological techniques, we examined the immune response to HV68 in heterozygous mice that have an autosomal dominant mutation in the STAT1 coiled-coil domain (STAT1 R274W). We observed impaired control of infection, which was associated with diminished production of IFN-, fewer effector CD4+ and CD8+ T cells, and a reduction in the number of antigen-specific CD8+ T cells. These findings indicate that a STAT1 gain-of-function mutation limits production of antiviral T cells, likely contributing to immunodeficiency against herpesviruses.
Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler's murine encephalomyelitis virus (TMEV), Saffold virus (SAFV) and Encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2aalpha; phosphorylation, thus acting downstream of eIF2aalpha;; on the other hand, it can inhibit double-stranded RNA-activated protein kinase (PKR) activation and the consequent PKR-mediated eIF2aalpha; phosphorylation. Interestingly, co-immunostaining experiments revealed that PKR co-localizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR co-precipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I:C). No interaction was however observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA.
IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler's virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of double-stranded RNA-activated protein kinase (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism, by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins such as influenza virus NS1, the leader protein appears to interact neither with PKR nor with double stranded RNA, suggesting that it acts indirectly, to trigger the inhibition of the kinase.
Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs) including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets including transitional, mature naïve, memory, and plasma cells were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablasts, plasma, transitional, and mature naïve B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively.
IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon re-infection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.
Transforming Growth Factor bbeta; (TGFbbeta;) has been shown to play a role in immunity towards different pathogens in vitro, and in vivo against parasites. However, its role in viral infections in vivo is incompletely understood. Using a neonatal mouse model of heterologous Rhesus Rotavirus vaccination, we showed that the vaccine induced Rotavirus-specific CD4 T cells, the majority of which lacked expression of KLRG1 or CD127, and a few regulatory Rotavirus-specific CD4 T cells that expressed surface Latency Associated Peptide (LAP)-TGFbbeta;. In these mice, inhibiting TGFbbeta;, both with a neutralizing antibody or with an inhibitor of TGFbbeta; receptor signaling (ALK5i), did not change the development or intensity of the mild diarrhea induced by the vaccine, the Rotavirus-specific T cell response, or the protection to a subsequent challenge with a murine EC-Rotavirus. However, mice treated with anti-LAP antibodies had improved protection after a homologous EC-Rotavirus challenge, when compared with control Rhesus Rotavirus-immunized mice. Thus, oral vaccination with a heterologous Rotavirus stimulates regulatory RV-specific CD4 LAP+ T cells, and depletion of LAP+ cells increases vaccine induced protection.
IMPORTANCE Despite the introduction of several live attenuated animal and human rotaviruses as efficient oral vaccines, rotaviruses continue to be the leading etiological agent for diarrhea mortality among children under 5 years of age worldwide. Improvement of these vaccines has been partially delayed because immunity to rotaviruses is incompletely understood. In the intestine (were rotavirus replicates) regulatory T cells that express Latency Associated peptide (LAP) play a prominent role, which has been explored for many diseases but not specifically for infectious agents. In this paper we show that neonatal mice given a live oral rotavirus vaccine develop rotavirus specific LAP+ T cells and that depletion of these cells improves the efficiency of the vaccine. These findings may proof useful to design strategies to improve rotavirus vaccines.
Animal hepaciviruses represent promising surrogate models for hepatitis C virus (HCV), for which there are no efficient immunocompetent animal models. Experimental infection of laboratory rats with rodent hepacivirus isolated from feral Rattus norvegicus (RHV-rn1) mirrors key aspects of HCV infection in humans, including chronicity, hepatitis and steatosis. Moreover, RHV has been adapted to infect immunocompetent laboratory mice. RHV in vitro systems have not been developed, but would enable detailed studies of the viral life-cycle crucial for designing animal experiments to model HCV infection.
Here, we established efficient RHV-rn1 selectable subgenomic replicons with and without reporter genes. Rat and mouse liver-derived cells did not readily support the complete RHV life-cycle, but replicon-containing cell clones could be selected with and without acquired mutations. Replication was significantly enhanced by mutations in NS4B and NS5A and in cell clones cured of replicon RNA. These mutations increased RHV replication of both mono- and bi-cistronic constructs, and CpG/UpA-dinucleotide optimization of reporter genes allowed replication. Using the replicon system, we show that the RHV-rn1 NS3-4A protease cleaves a human mitochondrial antiviral-signaling protein reporter providing a sensitive readout for virus replication. RHV-rn1 replication was inhibited by the HCV polymerase inhibitor sofosbuvir and high concentrations of HCV NS5A antivirals, but not by NS3 protease inhibitors. The microRNA-122 antagonist, miravirsen, inhibited RHV-rn1 replication, demonstrating importance of this HCV host-factor also for RHV. These novel RHV in vitro systems will be useful for studies of tropism, molecular virology and characterization of virus-host interactions, thereby providing important complements to in vivo systems.
IMPORTANCE A vaccine against hepatitis C virus (HCV) is crucial for global control of this important pathogen inducing fatal human liver diseases. Vaccine development has been hampered by the lack of immunocompetent animal models. Discovery of rodent hepacivirus (RHV) enabled establishment of novel surrogate animal models. These allow robust infection, reverse genetic and immunization studies of laboratory animals, which develop HCV-like chronicity. Currently, there are no RHV in vitro systems available to study tropism and molecular virology.
Here, we established the first culture systems for RHV, recapitulating the intracellular phase of the viral life-cycle in vitro. These replicon systems enabled identification of replication-enhancing mutations and selection of cells highly permissive to RHV replication, which allow study of virus-host interactions. HCV antivirals targeting NS5A, NS5B and microRNA-122 efficiently inhibited RHV replication. Hence, several important aspects of HCV replication are shared by the rodent virus system, reinforcing its utility as an HCV model.
For members of the Flaviviridae it is known that beside the structural proteins also nonstructural (NS) proteins play a critical role in virion formation. Pestiviruses such as bovine viral diarrhea virus (BVDV) rely on uncleaved NS2-3 for virion formation while its cleavage product NS3 is selectively active in RNA replication. This dogma was recently challenged by the selection of gain-of-function mutations in NS2 and NS3 which allowed virion formation in absence of uncleaved NS2-3 in BVDV-1 variants encoding either an ubiquitin (NS2-Ubi-NS3) or an IRES (NS2-IRES-NS3) between NS2 and NS3. To determine whether the ability to adapt to NS2-3-independent virion morphogenesisis is conserved among pestiviruses, we studied the corresponding NS2 and NS3 mutations (2/T444-V and 3/M132-A) in classical swine fever virus (CSFV). We observed that these mutations were only capable of restoring low level NS2-3-independent virion formation for CSFV NS2-Ubi-NS3. Interestingly, a second NS2 mutation (V439-D) identified by selection was essential for high titer virion production. Similar to previous findings for BVDV-1, these mutations in NS2 and NS3 allowed only for low titer virion production in CSFV NS2-IRES-NS3. For efficient virion morphogenesis additional exchanges in NS4A (A48-T) and NS5B (D280-G) were required, indicating that these proteins cooperate in NS2-3-independent virion formation. Interestingly, both NS5B mutations, selected independently for NS2-IRES-NS3 variants of BVDV-1 and CSFV, are located in the fingertip region of the viral RdRp, classifying this structural element as a novel determinant for pestiviral NS2-3-independent virion formation. Together, these findings will stimulate further mechanistic studies on genome packaging of pestiviruses.
IMPORTANCE For Flaviviridae members the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain-of-function. However, so far the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant have not been clarified. To unravel the course of genome packaging, the obtained additional sets of mutations for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.
A/H1N1 2009 pandemic influenza virus (A/H1N1/pdm09) was first identified as a novel pandemic influenza A virus (IAV) in 2009. Previously, we reported that many viral antigens were detected in type II alveolar epithelial cells (AEC-IIs) within autopsied lung tissue from a patient with A/H1N1/pdm09 pneumonia. It is important to identify the association between the virus and host cells to elucidate the pathogenesis of IAV pneumonia. To investigate the distribution of virus particles and morphological changes in host cells, the autopsied lung specimens from this patient were examined using transmission electron microscopy (TEM) and a novel scanning electron microscopy (SEM) method. We focused on AEC-IIs as viral antigen-positive cells, and on monocytes/macrophages (Ms/Ms) and neutrophils (Neus) as innate immune cells. We identified virus particles and intranuclear dense tubules, which are associated with matrix 1 (M1) proteins from IAV. Large-scale two-dimensional observation was enabled by digitally llsquo;stitchingrrsquo; together contiguous SEM images. A single whole cell analysis using a serial section array (SSA)-SEM identified virus particles in vesicles within the cytoplasm and/or around the cell surface of AEC-IIs, Ms/Ms, and Neus; however, intranuclear dense tubules were found only in AEC-IIs. Computer-assisted processing of SSA-SEM images from each cell type enabled 3D modeling of the distribution of virus particles within an ACE-II, a M/M, and a Neu.
IMPORTANCE Generally, it is difficult to observe IAV particles in post-mortem samples from patients with seasonal influenza. In fact, only a few viral antigens are detected in bronchial epithelial cells from autopsied lung sections. Previously, we detected many viral antigens in AEC-IIs from the lung. This was because the majority of A/H1N1/pdm09 in the lung tissue harbored an aspartic acid to glycine substitution at position 222 (D222G) of the hemagglutinin protein. A/H1N1/pdm09 harboring the D222G substitution has a receptor-binding preference for aalpha;-2,3-linked sialic acids expressed on human AECs and infects them in the same way as H5N1 and H7N9 avian IAVs. Here, we report the first successful observation of virus particles not only in AEC-IIs, but also in Ms/Ms and Neus, using electron microscopy. The finding of a M/M harboring numerous virus particles within vesicles and at the cell surface suggests that Ms/Ms are involved in the pathogenesis of IAV primary pneumonia.
Crimean-Congo hemorrhagic fever virus is a cause of severe hemorrhagic fever. Its tick reservoir and vector is widely distributed throughout Africa, Southern and Eastern Europe, the Middle East and Asia. Serological evidence suggests that CCHFV can productively infect a wide variety of species but only humans develop severe sometimes fatal disease. The role of the host adaptive immunity in control or contribution to the severe pathology seen in CCHF cases is largely unknown. Study of adaptive immune responses to CCHFV have been limited due to lack of suitable small animal models. Wild-type mice are resistant to CCHFV-infection and type I interferon deficient mice typically develop a rapid onset fatal disease, prior to development of adaptive immune responses. We report here a mouse model in which type I interferon deficient mice infected with a clinical isolate of CCHFV develop a severe inflammatory disease but ultimately recover. Recovery was co-incident with development of CCHFV-specific B and T-cell responses that were sustained for weeks post-infection. We also found that recovery from a primary CCHFV infection could protect against disease following homologous or heterologous re-infection. Together this model enables study of multiple aspects of CCHFV pathogenesis, including convalescence, an important aspect of CCHF disease that existing mouse models have been unsuitable for studying.
Importance. The role of antibody or virus-specific T-cell responses in control of acute Crimean-Congo hemorrhagic fever virus infection is largely unclear. This is a critical gap in our understanding of CCHF and investigation of convalescence following severe acute CCHF has been limited by a lack of suitable small animal models. We report here a mouse model of CCHF in which infected mice develop severe disease but ultimately most recover. Although mice developed an inflammatory immune response along with severe liver and spleen pathology, these mice also developed CCHFV-specific B and T-cell responses and were protected from re-infection. This model provides a valuable tool to investigate how host immune responses control acute CCHFV infection, how these responses may contribute to the severe disease seen in CCHFV-infected humans and develop therapeutic interventions that promote protective immune responses.
Human papillomaviruses (HPVs) infect squamous epithelia and cause several important cancers. Immune evasion is critical for viral persistence. Fibroblasts in the stromal microenvironment provide growth signals and cytokines that are required for proper epithelial differentiation, maintenance, and immune responses, and are critical in the development of many cancers. In this study, we examined the role of epithelial-stromal interactions in the HPV16 life cycle using organotypic (raft) cultures as a model. Rafts were created using uninfected human foreskin keratinocytes (HFKs), and HFKs containing either wild type HPV16 or HPV16 with a stop mutation to prevent the expression of the viral oncogene E5. Microarray analysis revealed significant changes in gene expression patterns in the stroma in response to HPV16, some of which were E5 dependent. Suppression of interferon (IFN) stimulated genes (ISGs) and extracellular matrix remodeling genes were the most prominent pathways affected. STAT1, IFNAR1, IRF3 and IRF7 were knocked down in stromal fibroblasts using lentiviral shRNA transduction. HPV late gene expression and viral copy number in the epithelium were increased when the stromal IFN pathway was disrupted, indicating that the stroma helps control the late phase of the HPV life cycle in the epithelium. Increased late gene expression correlated increased late keratinocyte differentiation but not decreased in IFN signaling in the epithelium. These studies show HPV16 has a paracrine effect on stromal innate immunity, reveal a new role for E5 as a stromal innate immune suppressor, and suggest that stromal IFN signaling may influence keratinocyte differentiation.
IMPORTANCE Persistence of high-risk human papillomavirus (HPV) infections is the key risk factor for developing HPV-associated cancers. The ability of HPV to evade host immunity is a critical component of its ability to persist. The environment surrounding a tumor is increasingly understood to be critical in cancer development, including immune evasion. Our studies show that HPV can suppress the expression of immune-related genes in neighboring fibroblasts in a 3D model of human epithelium. This finding is significant because it indicates that HPV can control innate immunity not only in the infected cell but also in the microenvironment. In addition, the ability of HPV to regulate stromal gene expression depends in part on the viral oncogene E5, revealing a new function for this protein as an immune evasion factor.
Rho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation and apoptosis. Here, we demonstrate that ROCK has antiviral properties and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection ROCK1 translocates to the nucleus and concentrates in the nucleolus were it co-localizes with the stress related chaperone, heat shock cognate 71 kDa protein (Hsc70) . Gene expression measurements showed that inhibition of ROCK activity does not affect the cellular stress response. We further demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the anti-viral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus.
ROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation and apoptosis. Here we reveal a novel anti-viral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus where it mainly localizes to the nucleolus. Our findings suggest that ROCK's anti-viral activity may be related to the activation of the actomyosin network and inhibition of capsid egress out of the nucleus.
Human metapneumovirus (hMPV) is a leading cause of viral lower respiratory tract infection in children. The sole target of neutralizing antibodies targeting hMPV is the fusion (F) protein, a class I viral fusion protein mediating virus-cell membrane fusion. There have been several monoclonal antibodies (mAbs) isolated that neutralize hMPV, however, determining the antigenic sites on the hMPV F protein mediating such neutralizing antibody generation would assist efforts for effective vaccine design. In this report, the isolation and characterization of four new human mAbs is described, termed MPV196, MPV201, MPV314, and MPV364. Among the four mAbs, MPV364 was found to be the most potent neutralizing mAb in vitro. Binding studies to monomeric and trimeric hMPV F revealed that MPV364 had the weakest binding affinity to monomeric hMPV F as compared to the other three mAbs, yet binding experiments to trimeric hMPV F showed limited difference in binding affinity, suggesting MPV364 targets an antigenic site incorporating two protomers. Epitope binning studies showed that MPV364 targets antigenic site III on the hMPV F protein and competes for binding with previously discovered mAbs MPE8 and 25P13, which both cross-react with the respiratory syncytial virus (RSV) F protein. However, MPV364 does not cross-react with the RSV F protein, and the competition profile suggests it binds to the hMPV F protein in a binding pose slightly shifted from mAbs MPE8 and 25P13. MPV364 was further assessed in vivo and was shown to substantially reduce viral replication in the lungs of BALB/c mice. Overall, these data reveal a new binding region near antigenic site III of the hMPV F protein that elicits potent neutralizing hMPV F-specific mAbs, and provide a new panel of neutralizing mAbs that are candidates for therapeutic development.
IMPORTANCE Recent progress in understanding the human immune response to respiratory syncytial virus has paved the way for new vaccine antigens and therapeutics to prevent and treat disease. Progress toward understanding the immune response to human metapneumovirus (hMPV) has lagged behind, although hMPV is a leading cause of lower respiratory tract infection in children. In this report, we advanced the field by isolating a panel of human mAbs to the hMPV F protein. One potent neutralizing mAb, MPV364, targets antigenic site III on the hMPV F protein and incorporates two protomers into its epitope, yet is unique from previously discovered site III-mAbs as it does not cross-react with the RSV F protein. We further examined MPV364 in vivo, and found it limits viral replication in BALB/c mice. Altogether, these data provide new mAb candidates for therapeutic development, and provide insights into hMPV vaccine development.
Hepatitis C virus (HCV) is a significant contributor to the global disease burden and development of an effective vaccine is required to eliminate HCV infections worldwide. CD4+ and CD8+ T cell immunity correlate with viral clearance in primary HCV infection, and intrahepatic CD8+ tissue-resident memory T (TRM) cells provide lifelong and rapid protection against hepatotropic pathogens. Consequently, we aimed to develop a vaccine to elicit HCV-specific CD4+ and CD8+ T cells, including CD8+ TRM cells in the liver given that HCV primarily infects hepatocytes. To achieve this we vaccinated wild-type BALB/c mice with a highly immunogenic cytolytic DNA vaccine encoding a model HCV (genotype 3a) non-structural protein 5B (NS5B) and a mutant perforin (pVAX-NS5B-PRF) as well as a recombinant adeno-associated virus (AAV) encoding NS5B (rAAV-NS5B). A novel fluorescent target array (FTA) was used to map immunodominant CD4+ T helper (TH) cell and cytotoxic CD8+ T cell epitopes of NS5B in vivo which were subsequently used to design a KdNS5B451-459 tetramer and analyse NS5B-specific T cell responses in vaccinated mice in vivo. The data showed that intradermal prime/boost vaccination with pVAX-NS5B-PRF was effective in eliciting TH and cytotoxic CD8+ T cell responses, and intrahepatic CD8+ TRM cells, but a single intravenous dose of hepatotropic rAAV-NS5B was significantly more effective. As a T-cell based vaccine against HCV should ideally result in localised T cell responses in the liver this study describes primary observations in the context of HCV vaccination which can be used to achieve this goal.
IMPORTANCE There are currently at least 71 million individuals with chronic HCV worldwide, and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offer highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. A HCV vaccine which elicits T cell immunity in the liver will be highly protective for the following reasons: 1) T cell responses against non-structural proteins of the virus are associated with clearance of primary infection, and 2) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.
Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference, or inhibiting its activity by treatment with Brefeldin A (BFA) or Golgicide A (GCA) significantly reduce the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly since in the presence of either BFA or GCA the assembly of infectious mature triple-layered virions was significantly prevented and only doubled layered-particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface VP7, a step required for its incorporation into virus particles. Although a post-translational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking-down its expression reduces VP7 trimerizarion. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when co-transfected with NSP4.
Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite the significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for the COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4, and the assembly of the virus surface proteins VP7 and VP4.
HIV-exposed seronegative KIR3DS1 homozygotes have a reduced risk of HIV infection. HLA-F is the ligand for the activating NK cell receptor (NKR) KIR3DS1. HLA-F is expressed on HIV-infected CD4 T cells. Co-culture of sorted, HIV-infected CD4- (siCD4-) T cells with NK cells activated a higher frequency of KIR3DS1+ than KIR3DS1- NK cells from KIR3DS1 homozygotes to elicit anti-HIV functions such as CCL4, IFN- and CD107a expression. This was the case whether KIR3DS1+/- NK cells were analyzed inclusively or exclusively by gating out NK cells co-expressing the NKRs, KIR2DL1/L2/L3, 3DL2, KIR2DS1/S2/S3/S5, NKG2A and ILT2. Blocking the interaction of HLA-F on siCD4- cells with KIR3DS1 on exclusively gated KIR3DS1+ NK cells with KIR3DS1-Fc chimeric protein or an HLA-F specific monoclonal antibody, reduced the frequency of activated KIR3DS1+ cells compared to control conditions. KIR3DS1+ NK cell activation by HIV-infected CD4 cells may underlie the reduced risk of KIR3DS1 homozygotes to HIV infection.
IMPORTANCE This manuscript investigates a mechanism that may underly epidemiological studies showing that carriage of the KIR3DS1 homozygous genotype is more frequent among HIV exposed seronegative subjects than among HIV susceptible individuals. Carriage of this genotype is associated with a reduced risk of HIV infection. The protective mechanism involves the interaction of HLA-F on CD4 cells infected with replication competent HIV with the activating NK receptor, KIR3DS1. This interaction leads to the activation of KIR3DS1+ NK cells for secretion of cytokines and chemokines with anti-HIV activity. Among these is the secretion of CCL4, which binds and blocks CCR5, the co-receptor for HIV entry of HIV into new target cells. In the setting of an exposure to HIV, incoming HIV-infected cells expressing HLA-F rapidly activate KIR3DS1+ NK cells to elicit anti-HIV activity. Exclusive gating strategies and blocking experiments support the notion that the HLA-F/KIR3DS1 interaction is sufficient to activate NK cell functions.
Macrophages are susceptible to HIV infection and play an important role in viral dissemination through cell-cell contacts with T cells. However, our current understanding of macrophage-to-T cell HIV transmission are derived from studies that do not consider the robust migration and cell-cell interaction dynamics between these cells. Here, we performed live-cell imaging studies in 3-dimensional (3D) collagen that allows CD4+ T cells to migrate, locate and engage HIV-infected macrophages, modeling the dynamic aspects of the in situ environment in which these contacts frequently occur. We show that HIV+ macrophages form stable contacts with CD4+ T cells that are facilitated by both gp120:CD4 and LFA-1:ICAM-1 interactions, and that prolonged contacts are a pre-requisite for efficient viral spread. LFA-1:ICAM-1 adhesive contacts function to restrain highly motile T cells, since their blockade substantially de-stabilized macrophage:T cell contacts, resulting in abnormal tethering events that reduced cell-cell viral spread. HIV-infected macrophages displayed strikingly elongated podosomal extensions that were dependent on Nef expression, but were dispensable for stable, cell-cell contact formations. Finally, we observed persistent T cell infection during dynamic MDM:T cell co-cultures in the presence of single high antiretroviral drug concentrations, but achieved complete inhibition with combination therapy. Together, our data implicate macrophages as drivers of T cell infection by altering physiological MDM:T cell contact dynamics to access and restrain large numbers of susceptible, motile T cells within lymphoid tissues.
Once HIV enters the lymphoid organs, exponential viral replication in T cells ensues. Given the densely-packed nature of these tissues, where infected and uninfected cells are in near-constant contact with one another, efficient HIV spread is thought to occur through cell-cell contacts in vivo. However, this has not been formally demonstrated. In this study, we performed live-cell imaging studies within a 3-dimensional space to recapitulate the dynamic aspects of the lymphoid microenvironment, and asked whether HIV can alter the morphology, migration capacity and cell-cell contact behaviors between macrophages and T cells. We show that HIV-infected macrophages can engage T cells into stable contacts through binding of virus- and host-derived adhesive molecules, and that stable macrophage:T cell contacts were required for high viral spread. Thus, HIV alters physiological macrophage:T cell interactions in order to access and restrain large numbers of susceptible, motile T cells, thereby playing an important role in HIV progression.
Chikungunya virus (CHIKV) is a re-emerged arbovirus, member of the Togaviridae family. It circulates through mosquito vectors mainly of the Aedes family and a mammalian host. CHIKV causes chikungunya fever, a mild to severe disease characterized by arthralgia, with some fatal outcomes described. In the past years, several outbreaks mainly caused by enhanced adaptation of the virus to the vector and ineffective control of the contacts between infected mosquito populations and the human host have been reported. Vaccines represent the best solution for the control of insect-borne viruses, including CHIKV, but are often unavailable. We designed live attenuated CHIKV by applying a rational genomic design based on multiple replacements of synonymous codons. In doing so, the virus mutational robustness (capacity to maintain phenotype despite introduction of mutations to genotype) is decreased, driving the viral population towards deleterious evolutionary trajectories. When tested in the insect and mammalian hosts, we observed overall strong attenuation in both and greatly diminished signs of disease. Moreover, we found the vaccine candidates elicited protective immunity, related to the production of neutralizing antibodies after a single dose. During an experimental transmission cycle between mosquitoes and naïve mice, vaccine candidates could be transmitted by mosquito bite leading to asymptomatic infection in mice with compromised dissemination. Using deep sequencing technology we observed an increase in detrimental (stop) codons, which confirmed the effectiveness of this genomic design. Because the approach involves hundreds of synonymous modifications to the genome, the reversion risk is significantly reduced, rendering them promising vaccine candidates.
IMPORTANCE Chikungunya fever is a debilitating disease that causes severe pain to the joints, which can compromise the patient's lifestyle for several months and even in some grave cases lead to death. The etiological agent is chikungunya virus, an alphavirus transmitted by mosquito bite. Currently there are no approved vaccines or treatments against the disease. In our research, we developed novel live attenuated vaccine candidates against chikungunya virus by applying an innovative genomic design. When tested in the insect and mammalian host, the vaccine candidates did not cause disease, elicited strong protection against further infection and had low risk of reversion to pathogenic phenotypes.
The Zika virus (ZIKV) life cycle involves multiple steps and requires interactions with host factors. However, the inability to systematically identify host regulatory factors for ZIKV has hampered antiviral development and our understanding of pathogenicity. Here, using a bioactive compound library with 2659 small molecules, we applied a high-throughput and imaging-based screen to identify host factors that modulate ZIKV infection. The screen yielded hundreds of hits that markedly inhibited or potentiated ZIKV infection in SNB-19 glioblastoma cells. Among the hits, URMC-099, a mixed lineage kinase 3 (MLK3) inhibitor, significantly facilitated ZIKV replication in both SNB-19 cells and the neonatal mouse brain. Using gene silencing and overexpression, we further confirmed that MLK3 was a host restriction factor against ZIKV. Mechanistically, MLK3 negatively regulated ZIKV replication through inducing the inflammatory cytokines IL-6, IL-8, TNF-aalpha; and MCP-1 but did not modulate host interferon related pathways. Importantly, ZIKV activated the MLK3/MKK7/JNK pathway in both SNB-19 cells and neonatal mouse brain. Together, these findings reveal a critical role for MLK3 in regulating ZIKV infection and facilitate the development of anti-ZIKV therapeutics by providing a number of screening hits.
IMPORTANCE Zika fever, an infectious disease caused by the Zika virus (ZIKV), normally results in mild symptoms. Severe infection can cause Guillainnndash;Barreeacute; syndrome in adults and birth defects, including microcephaly, in newborns. Although ZIKV was first identified in Uganda in 1947 in rhesus monkeys, a widespread epidemic of ZIKV infection in South and Central America in 2015-2016 raised major concerns. To date, there is no vaccine or specific medicine for ZIKV. The significance of our research is the systematic discovery of small molecule candidates that modulate ZIKV infection, which will allow the development of antiviral therapeutics. In addition, we identified MLK3, a key mediator of host signaling pathways that can be activated during ZIKV infection and limits virus replication by inducing multiple inflammatory cytokines. These findings broaden our understanding of ZIKV pathogenesis.
The potential avian influenza pandemic remains a threat to public health, as the avian-originated influenza A(H7N9) virus has caused more than 1560 laboratory-confirmed human infections since 2013, with nearly 40% mortality. Development of low pathogenic candidate vaccine viruses (CVVs) for vaccine production is essential for pandemic preparedness. However, the suboptimal growth of CVVs in mammalian cells and chicken eggs is often a challenge. By introducing a single adaptive substitution, G218E, into the hemagglutinin (HA), we generated reassortant A(H7N9)-G218E CVVs that were characterized by significantly enhanced growth in both cells and eggs. These G218E CVVs retained the original antigenicity as determined by hemagglutination inhibition assay, and effectively protected ferrets from lethal challenge with the highly pathogenic parental virus. We found that the suboptimal replication of the parental H7 CVVs was associated with impeded progeny virus release as a result of strong HA receptor binding relative to weak neuraminidase (NA) cleavage of receptors. In contrast, the G218E-mediated growth improvement was attributed to relatively balanced HA and NA functions, resulted from reduced HA binding to both human- and avian-type receptors, and thus facilitated NA-mediated virus release. Our findings revealed that a single amino acid mutation at residue 218 of the HA improved the growth of A(H7N9) influenza virus by balancing HA and NA functions, shedding light on an alternative approach for optimizing certain influenza CVVs.
The circulating avian influenza A(H7N9) has caused recurrent epidemic waves with high mortality in China since 2013, in which the alarming fifth wave crossing 2016 and 2017 was highlighted by large number of human infections and emergence of highly pathogenic avian influenza (HPAI) A(H7N9) strains in human cases. We generated low pathogenic reassortant CVVs derived from the emerging A(H7N9) with improved virus replication and protein yield in both MDCK cells and eggs by introducing a single substitution, G218E, into HA, which was associated with reducing HA receptor binding and subsequently balancing HA-NA functions. The in vitro and in vivo experiments demonstrated comparable antigenicity of the G218E CVVs with that of their WT counterparts, and both the WT and G218E CVVs fully protected ferrets from parental HPAI virus challenge. With high yield traits and the anticipated antigenicity, the G218E CVVs should benefit the pandemic preparedness against the A(H7N9) influenza threat.
Gastrointestinal immune system competency is dependent upon interactions with commensal microbiota, which can be influenced by wide-ranging pharmacologic interventions. In SIV-infected Asian macaque models of HIV infection, we previously noted that initiation of antiretroviral therapy (ART) is associated with a specific imbalance (dysbiosis) of the composition of the intestinal bacteriome. To determine if ART itself might contribute to dysbiosis or immune dysfunction, we treated healthy rhesus macaques with protease-, integrase- or reverse transcriptase inhibitors for 1-2 or 5-6 weeks and evaluated intestinal immune function and the composition of the fecal bacterial microbiome. We observed that individual antiretrovirals (ARVs) modestly altered intestinal T-cell pro-inflammatory responses without disturbing total or activated T-cell frequencies. Moreover, we observed transient disruptions in bacterial diversity coupled with perturbations in the relative frequencies of bacterial communities. Shifts in specific bacterial frequencies were not persistent post-treatment, however, with individual taxa showing only isolated associations with T-cell pro-inflammatory responses. Our findings suggest that intestinal bacterial instability and modest immunological alterations can result from ART itself. These data could lead to therapeutic interventions which stabilize the microbiome in individuals prescribed ART.
IMPORTANCE Dysbiosis of the fecal microbiome is a common feature observed in ARV-treated people living with HIV. The degree to which HIV infection itself causes this dysbiosis remains unclear. Here we demonstrate that medications used to treat HIV infection can influence the composition of the GI tract immune responses and its microbiome in the non-human primate SIV model.
Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral N (nucleoprotein), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric rVHSV by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout-virulence phenotype of either parental strain. However, reciprocal exchanges of the viral nucleoprotein (N) gene resulted in a partial gain-of-function in the chimeric trout-avirulent strain (22% mortality), and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and phosphoprotein (P) genes together resulted in complete gain-of-function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout-virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout.
IMPORTANCE Rainbow trout farming is a major food-source industry world-wide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout-virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout-virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) proteins.
Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral-signaling protein (MAVS) and subsequent cellular interferon response. On the other hand, viruses explore multiple strategies to resist the host immunity. In this study, we found that NDV infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-bbeta; production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with the wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-bbeta; production at the late stage of infection. Several other paramyxovirus V proteins showed similar activities of degrading MAVS and blocking IFN production as NDV V protein did. The present study revealed a novel role of NDV V protein on targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit.
Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5, and subsequently initiates downstream signaling through interaction with mitochondrial antiviral-signaling protein (MAVS). On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). The MAVS degradation leads to the inhibition of the downstream IFN-bbeta; pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.
Genomic material from many neurotropic RNA viruses (e.g., measles virus (MV), West Nile virus (WNV), Sindbis virus (SV), rabies virus (RV), and influenza A virus (IAV)) remains detectable in the mouse brain parenchyma long after resolution of the acute infection. The presence of these RNAs in the absence of overt central nervous system (CNS) disease has led to the suggestion that they are viral remnants, with little or no potential to reactivate. Here, we show that MV RNA remains detectable in permissive mouse neurons long after challenge with MV, and moreover, that immune suppression can cause RNA and protein synthesis to rebound, triggering neuropathogenesis months after acute viral control. Robust recrudescence of viral transcription and protein synthesis occur after experimental depletion of cells of the adaptive immune response, and is associated with a loss of T resident memory (Trm) lymphocytes within the brain. The disease associated with loss of immune control is distinct from that seen during the acute infection: immune cell-depleted, long-term infected mice display severe gait and motor problems, as compared to the wasting and lethal disease that occurs during acute infection of immunodeficient hosts. These results illuminate the potential consequences of non-cytolytic, immune-mediated viral control in the CNS, and demonstrate that, what were once considered "resolved" RNA viral infections may, in fact, induce diseases later in life that are distinct from those caused by acute infection.
IMPORTANCE Viral infections of neurons are often not cytopathic; thus, once-infected neurons survive, and viral RNAs can be detected long after apparent viral control. These RNAs are generally considered viral fossils, unlikely to contribute to central nervous system (CNS) disease. Using a mouse model of measles virus (MV) neuronal infection, we show that MV RNA is maintained in the CNS of infected mice long after acute control and in the absence of overt disease. Viral replication is suppressed by the adaptive immune response; when these immune cells are depleted, viral protein synthesis recurs, inducing a CNS disease that is distinct from that observed during acute infection. The studies presented in this manuscript provide the basis for understanding how persistent RNA infections in the CNS are controlled by the host immune response, as well as the pathogenic consequences of non-cytolytic viral control.
The New World (NW) arenaviruses are a diverse group of zoonotic viruses including several causative agents of severe hemorrhagic fevers in humans. All known human pathogenic NW arenaviruses belong to clade B, where they group into sub-lineages with phylogenetically closely related non-pathogenic viruses, e.g. the highly pathogenic Junin (JUNV) and Machupo viruses with the non-pathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of non-pathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cells innate anti-viral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune response against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in presence or absence of an IFN-I response. However, compared to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2aalpha;. Confocal microscopy studies revealed similar sub-cellular co-localization of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV, but promoted TCRV multiplication, providing first evidence for differential innate recognition and control of pathogenic and non-pathogenic NW arenaviruses by PKR.
New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of non-pathogenic NW arenaviruses with their highly pathogenic cousins suggest that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune response against the highly pathogenic Junin virus (JUNV) and the related non-pathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivity to IFN-I treatment. However, the dsRNA-dependent protein kinase R (PKR) contributed to control of TCRV, but not JUNV, providing first evidence for differential innate recognition and control of JUNV and TCRV.
Marek's disease virus (MDV), which causes T cell lymphomas in chickens, is economically important and has contributed to knowledge of herpesvirus-associated oncogenicity. The DNA-sensing pathway induces innate immune responses against DNA virus infection, and nuclear factor B (NF-B) signaling is critical for the establishment of innate immunity. Here, we report that RLORF4, an MDV-specific protein directly involved in viral attenuation, is an inhibitor of the DNA-sensing pathway. The results showed that ectopically expressed RLORF4 blocked IFN-bbeta; promoter activation induced by cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). RLORF4 selectively inhibited the activation of NF-B but not IFN-regulatory factor 7. RLORF4 was found to bind the endogenous NF-B subunits p65 and p50, and it also bound to the Rel homology domains of these subunits. Furthermore, RLORF4 suppressed the nuclear translocation of p65 and p50 mediated by tumor necrosis factor alpha and interferon stimulatory DNA. Finally, deletion of RLORF4 from the MDV genome promoted IFN-bbeta; and IL-6 production in vitro and in vivo. In the absence of RLORF4, the host cellular immunity was significantly increased, and reduced viral titers were observed during infection of chickens. Our results suggest that the RLORF4-mediated suppression of the host antiviral innate immunity might play an important role in MDV pathogenesis.
IMPORTANCE Marek's disease virus (MDV) RLORF4 has been shown to be directly involved in the attenuation of MDV upon serial passages in vitro, however, the exact function of this protein during viral infection was not well characterized. This study demonstrated that RLORF4 significantly inhibits cGAS-STING-mediated NF-B activation by binding to the Rel homology domains of the NF-B subunits p65 and p50, interrupting their translocation to the nuclei and thereby inhibiting IFN-bbeta; production. Furthermore, RLORF4 deficiency promoted the induction of IFN-bbeta; and downstream IFN-stimulated genes during MDV infection in chickens. Our results suggest that the contribution of RLORF4 to MDV virulence may stem from its inhibition of viral DNA-triggered IFN-bbeta; responses.
Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both the G6P WC3 and mono-reassortant G4P strains recognized the aalpha;Gal HBGA. The infectivity of both P-bearing strains was significantly reduced in aalpha;Gal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by co-incubation with the aalpha;2,6-linked SA-specific Sambucus nigra lectin, but not by the aalpha;2,3-linked specific sialidase or by Maackia amurensis lectin. Free NeuAc and the aalpha;Gal trisaccharide also prevented the infectivity of both strains. This indicated that both P-bearing strains utilize aalpha;2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack aalpha;2,6-linked SA or aalpha;Gal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight junction proteins bound directly to the VP5* domain can be used by the P-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P-bearing strains have potential for cross-species transmission.
IMPORTANCE Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P vaccine strains recognize both aalpha;2,6-linked SA and aalpha;Gal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P-bearing strains. However, we observed that the P-bearing WC3 and G4P RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.
Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma-retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ~10-fold less and KoRV-B expression being ~100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas compared to koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.
IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared vulnerable' by the Australian Government and officially listing it as a threatened species. KoRV is clearly contributing to the overall health status of koalas and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new, more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.
Hepatitis C virus (HCV) infection promotes metabolic disorders, and the severity of lipogenic disease depends upon the infecting virus genotype. Here, we have examined HCV genotype 1, 2, or 3 specific regulation of lipid metabolism, involving TGF-bbeta; regulated phospho-Akt and PPARaalpha; axes. Since HCV core protein is one of the key players in metabolic regulation, we also examined its contribution in lipid metabolic pathways. The regulatory molecules, TGF-bbeta;1/2, phospho-Akt (Ser473), PPARaalpha;, SREBP-1, FASN, hormone-sensitive lipase (HSL), and acyl dehydrogenases expression were analyzed in virus infected hepatocytes. Interestingly, HCV genotype 3a exhibited much higher activation of TGF-bbeta; and p-Akt, with a concurreent decrease in PPARaalpha; expression and fatty acid oxidation. A significant and similar decrease in HSL, unlike HCV genotype 1a, was observed with both genotype 2a and 3a. Similar observations were made from ectopic expression of the core genomic region from each genotype. The key role of TGF-bbeta; was further verified using specific siRNA. Together, our results highlighted a significant difference in TGF-bbeta; induced activity for HCV genotype 2a or 3a induced lipogenic pathway, exhibiting higher triglyceride synthesis and a decreased lipolytic mechanism. These results may help in therapeutic modalities for early treatment of HCV genotype associated lipid metabolic disorders.
IMPORTANCE Hepatic steatosis is a frequent complication associated with chronic HCV infection and is a key prognostic indicator for progression to fibrosis and cirrhosis. Several mechanisms are proposed for the development of steatosis, especially with HCV genotype 3a. Our observations suggest that TGF-bbeta; and PPARaalpha; associated mechanistic pathway differ in HCV genotypes 2a and 3a infected hepatocytes from genotype 1a. The results suggested a targeted therapeutic approach for enhanced PPARaalpha; and lipolysis may reduce HCV genotype associated lipid metabolic disorder in liver disease.
Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (~27 kb) and has a complex replication strategy that includes the production of a nested set of sub-genomic mRNAs (sgmRNAs). Here, we used RNA sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-CK strains at sub-codon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-CK strain, one of which would generate a sgmRNA encoding a ribosomally occupied ORF (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33nndash;40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (4b and 4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global up-regulation of immune-related mRNA transcripts following infection, and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.
IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV, and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intra-species variation, with single nucleotide polymorphisms associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection.
Aedes aegypti mosquitoes are the main vectors of arthropod-borne viruses (arboviruses) of public health significance such as the flaviviruses causing dengue (DENV) and Zika (ZIKV). Mosquitoes are also the natural hosts of a wide range of viruses that are insect-specific, raising the question of their influence on arbovirus transmission in nature. Cell-fusing agent virus (CFAV) was the first described insect-specific flavivirus, initially discovered in an Ae. aegypti cell line and subsequently detected in natural Ae. aegypti populations. It was recently shown that DENV and the CFAV strain isolated from the Ae. aegypti cell line have mutually beneficial interactions in mosquito cells in culture. However, whether natural strains of CFAV and DENV interact in live mosquitoes is unknown. Using a wild-type CFAV isolate recently derived from Thai Ae. aegypti mosquitoes, we found that CFAV negatively interferes with both DENV type 1 and ZIKV, in vitro and in vivo. For both arboviruses, prior infection by CFAV reduced the dissemination titer in mosquito head tissues. Our results indicate that interactions observed between arboviruses and the CFAV strain derived from the cell line might not be a relevant model of the viral interference that we observed in vivo. Overall, our study supports the hypothesis that insect-specific flaviviruses may contribute to reduce transmission of human-pathogenic flaviviruses.
IMPORTANCE The mosquito Aedes aegypti carries several arthropod-borne viruses (arboviruses) that are pathogenic to humans including dengue and Zika viruses. Interestingly, Ae. aegypti is also naturally infected with insect-only viruses such as cell-fusing agent virus. Although interactions between cell-fusing agent virus and dengue virus have been documented in mosquito cells in culture, whether wild strains of cell-fusing agent virus interfere with arbovirus transmission by live mosquitoes was unknown. We used an experimental approach to demonstrate that cell-fusing agent virus infection reduces the propagation of dengue and Zika viruses in Ae. aegypti. These results support the idea that insect-only viruses in nature can modulate the ability of mosquitoes to carry arboviruses of medical significance, and that they could possibly be manipulated to reduce arbovirus transmission.
Over 2 million people are infected with HIV-1 annually. Approximately half of these new infections occur in women residing in low-income countries, where their access to and control over HIV-1 preventative measures are often limited, indicating that female-controlled prevention options for HIV-1 are urgently needed. Microbicides that can be topically applied to the vaginal tract in advance of sexual activity represent a promising female-controlled prevention option for HIV-1. We have previously described the development of an HIV-1 specific microbicide using the Surface or S-layer recombinant protein display capabilities of the non-pathogenic, freshwater bacterium Caulobacter crescentus. Recombinant C. crescentus were created that displayed proteins that interfere with the HIV-1 attachment and entry process, and were able to provide significant protection of TZM-bl cells from infection with HIV-1 pseudovirus. These studies have been expanded to investigate if these recombinant C. crescentus are able to maintain efficacy with replication competent HIV-1 and both TZM-bl cells and human peripheral blood mononuclear cells (PBMCs). In addition we utilized the humanized bone marrow-liver-thymus (BLT) mouse model to determine if vaginal application of recombinant C. crescentus at the time of HIV-1JR-CSF infection could provide protection from HIV-1 infection. Recombinant C. crescentus expressing Griffithsin, GB virus C E2 protein, elafin, aalpha;-1-antitrypsin, indolicidin and the fusion inhibitor T-1249 were able to protect 40-75% of the BLT mice from vaginal infection with HIV-1JR-CSF, with Cc-Griffithsin being the most effective. Taken together this data suggests that a C. crescentus based microbicide could be a safe and effective method for HIV-1 prevention.
Importance: Human immunodeficiency virus (HIV) disproportionally infects young women in sub-Saharan Africa. Current HIV-1 prevention options have had limited success among women, suggesting that alternative, female-controlled prevention options need to be developed. Microbicides that can be applied to the vaginal tract are a promising prevention option. In this study we describe testing of fifteen potential candidates to inhibit HIV-1 infection in a humanized mouse model of HIV-1. Four of these candidates were able to provide significant protection from vaginal infection with HIV-1, with the most successful candidate protecting 75% of the mice from infection. This study describes preclinical testing of a new strategy that could be a safe and effective option for HIV-1 prevention in women.
The Ebola vaccine based on Ad26.ZEBOV/MVA-BN-Filo prime-boost regimens is being evaluated in multiple clinical trials. The long-term immune response to the vaccine is unknown, including factors associated with the response and variability around the response. We analyzed data from three phase I trials performed by the EBOVAC1 consortium in four countries - UK, Kenya, Tanzania and Uganda. Participants were randomized in four groups based on the interval between prime and boost immunization (28 or 56 days) and the sequence in which Ad26.ZEBOV and MVA-BN-Filo were administered. Consecutive ELISA measurements of the IgG binding antibody concentrations against the Kikwit glycoprotein (GP) were available in 177 participants to assess the humoral immune response up to 1 year post prime. Using a mathematical model for the dynamics of the humoral response, from 7 days after the boost immunization up to 1 year after the prime immunization, we estimated the durability of the antibody response and the influence of different factors on the dynamics of the humoral response. Ordinary differential equations (ODEs) described the dynamics of antibody response and two populations of antibody-secreting cells (ASC), short-lived (SL) and long-lived (LL). Parameters of the ODEs were estimated using a population approach. We estimated that half of the LL ASCs could persist at least five years. The vaccine regimen significantly affected the SL ASCs and the antibody peak but not the long-term response. The LL ASCs compartment dynamics differed significantly by geographic regions analyzed, with a higher long-term antibody persistence in European subjects. These differences could not be explained by the observed differences in cellular immune response.
IMPORTANCE With no available licensed vaccines or therapies, the West African Ebola virus disease epidemic of 2014-2016 caused 11,310 deaths. Following this outbreak, the development of vaccines has been accelerated. Combining different vector-based vaccines as heterologous regimens could induce a durable immune response, assessed through antibody concentrations. Based on data from phase 1 trials in East Africa and Europe, the dynamics of the humoral immune response from 7 days after the boost immunization onwards were modeled to estimate the durability of the response and understand its variability. Antibody production is maintained by a population of long-lived cells. Estimation suggests that half of these cells can persist at least five years in humans. Differences in prime-boost vaccine regimens affect only the short-term immune response. Geographical differences in long-lived cell dynamics were inferred, with higher long-term antibody concentrations induced in European participants.
Neutralization by antibodies and complement limits the effective dose and thus the therapeutic efficacy of oncolytic viruses after systemic application. We and others previously showed that pseudotyping of oncolytic rhabdoviruses like the maraba virus and the vesicular stomatitis virus (VSV) with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) results in only a weak induction of neutralizing antibodies. Moreover, LCMV-GP-pseudotyped VSV (VSV-GP) was significantly more stable in normal human serum (NHS) than VSV. Here, we demonstrate that depending on the cell line used for virus production, VSV-GP showed different complement sensitivities in non-immune NHS. The NHS-mediated titer reduction of VSV-GP was dependent on the activation of the classical complement pathway mainly by natural IgM antibodies against xenoantigens like galactose-aalpha;-(1,3)-galactose (aalpha;-Gal) or N-glycolylneuraminic acid (Neu5Gc) expressed on non-human production cell lines. VSV-GP produced on human cell lines was stable in NHS. However, VSV-GP generated in transduced human cells expressing aalpha;-Gal became sensitive for NHS. Furthermore, GP-specific antibodies induced complement-mediated neutralization of VSV-GP independent of the producer cell line, suggesting that complement regulatory proteins potentially acquired by the virus during the budding process are not sufficient to rescue the virus from antibody-dependent complement-mediated lysis. Thus, our study points to the importance of a careful selection of cell lines for viral vector production for clinical use.
IMPORTANCE Systemic application aims to deliver oncolytic viruses to tumors as well as to metastatic lesions. However, we found that xenoantigens incorporated onto the viral surface from non-human producing cell lines are recognized by natural antibodies in human serum and that the virus is thereby inactivated by complement lysis. Hence, to maximize the effective dose, careful selection of cell lines for virus production is crucial.
Genomics analysis of a historically-intriguing and predicted emergent human adenovirus (HAdV) pathogen, which caused pneumonia and death, provides insight into a novel molecular evolution pathway involving "ping-pong" zoonosis and anthroponosis. The genome of this promiscuous pathogen is embedded with evidence of unprecedented multiple, multidirectional, stable, and reciprocal cross-species infections of hosts from three species (human, chimpanzee, and bonobo). This recombinant genome, typed as HAdV-B76, is identical to two recently reported simian AdV (SAdV) genomes isolated from chimpanzees and bonobos. Additionally, the presence of a critical adenoviral replication element found in HAdV genomes, in addition to genes that are highly similar with counterparts in other HAdVs, reinforce its potential as a human pathogen. Reservoirs in nonhuman hosts may explain periods of apparent absence and then re-emergence of human adenoviral pathogens, as well as present pathways for the genesis of those thought to be newly emergent. The nature of the HAdV-D76 genome has implications for the use of SAdVs as gene delivery vectors in human gene therapy and vaccines, selected to avoid preexisting and potentially fatal host immune responses to HAdV.
Importance: An emergent adenoviral human pathogen, HAdV-B76, associated with a fatality in 1965, shows a remarkable degree of genome identity with two recently isolated simian adenoviruses that contain cross-species genome recombination events from three hosts: human, chimpanzee, and bonobo. Zoonosis (nonhuman to human transmission) and anthroponosis (human to nonhuman transmission) may play significant roles in the emergence of human adenoviral pathogens.
Our previous study showed that pentagalloylglucose (PGG), a naturally-occurring hydrolysable phenolic tannin, possessed significant anti-rabies viral (RABV) activity. In BHK-21 cells, RABV induced over-activation of STAT3 (signal transducer and activator of transcription 3) by suppressing the expression of SOCS3 (suppressor of cytokine signaling 3). Inhibition of STAT3 by niclosamide, or siRNA, or exogenous expression of SOCS3, all significantly suppressed the replication of RABV. Additionally, RABV-induced upregulation of miR-455-5p downregulated SOCS3 by directly binding to the 3'-UTR of SOCS3. Importantly, PGG effectively reversed the expression of miR-455-5p and its following SOCS3/STAT3 signaling pathway. Finally, activated STAT3 elicited the expression of interleukin (IL)-6, thereby contributing to RABV-associated encephalomyelitis; however, PGG restored the level of IL-6 in vitro and in vivo in a SOCS3/STAT3-dependent manner. Altogether, these data identify a new miR-455-5p/SOCS3/STAT3 signaling pathway that contributes to viral replication and IL-6 production in RABV-infected cells, with PGG exerting its anti-viral effect by inhibiting production of miR-455-5p and activation of STAT3.
Importance: Rabies virus causes lethal encephalitis in mammals and poses a serious public health threat in many parts of the world. Numerous strategies have been explored to combat rabies; however, their efficacy has always been unsatisfactory. We previously reported a new drug, PGG, which possesses a potent inhibitory activity on RABV replication. Herein, we describe the underlying mechanisms by which PGG exerts its anti-RABV activity. Our results show that RABV induces over-activation of STAT3 in BHK -21 cells, which facilitates viral replication. Importantly, PGG effectively inhibits the activity of STAT3 by disrupting the expression of miR-455-5p and increases the level of SOCS3 by directly targeting 3'-UTR of SOCS3. Furthermore, the downregulated STAT3 inhibits the production of IL-6, thereby contributing to a reduction in the inflammatory response in vivo. Our study indicates that PGG effectively inhibits the replication of RABV by the miR-455-5p/SOCS3/STAT3/IL-6 dependent pathway.
Current combination antiretroviral therapies (cART) are unable to eradicate HIV-1 virus from infected individuals, because of the establishment of proviral latency in long-lived cellular reservoirs. The "shock and kill" approach aims to reactivate viral replication from the latent state ("shock") using latency reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells ("kill") by specific therapeutics. The NF-B RelA/p50 heterodimer has been characterized as an essential component of reactivation of the latent HIV-1 long terminal repeat. Nevertheless, prolonged NF-B activation contributes to the development of various autoimmune, inflammatory and malignant disorders. In the present study we established a cellular model of HIV-1 latency in JLat CD4+ T cells that stably expressed the NF-B super-repressor IB-aalpha; 2N4, and demonstrate that conventional treatments, with Bryostatin-1 and Hexamethylenebisacetammide (HMBA) or Ionomycin, synergistically reactivated HIV-1 from latency, even under conditions where NF-B activation was repressed. Using specific calcineurin phosphatase, p38, and MEK1/MEK2 kinase inhibitors, or specific shRNAs, c-Jun was identified as an essential factor binding to the LTR enhancer kB sites and mediating the combined synergistic reactivation effect. Furthermore, acetyl salicylic acid (ASA), a potent inhibitor of the NF-B activator kinase IKK-bbeta;, did not significantly diminish reactivation in a primary CD4+ T central memory (T
The "shock and kill" approach consists in the reactivation of HIV-1 replication from latency using latency reversing agents (LRAs), followed by the elimination of reactivated virus-producing cells. The cellular transcription factor NF-B is considered a master mediator of HIV-1 escape from latency induced by LRAs. Nevertheless, a systemic activation of NF-B in HIV-1 patients, resulting from the combined administration of different LRAs, could represent a potential risk especially in the case of a prolonged treatment. We here demonstrate that conventional treatments with Bryostatin-1 and Hexamethylenebisacetammide (HMBA) or Ionomycin synergistically reactivated HIV-1 from latency, even under conditions where NF-B activation was repressed. Our study provides a molecular proof of concept for the use of anti-inflammatory drugs, like aspirin, capable of inhibiting NF-B in patients under cART during the "shock and kill" approach, to avoid potential autoimmune and inflammatory disorders that can be elicited by combinations of LRAs.
Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales. Here we aimed to identify arterivirus proteins involved in the mode-of-action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to a ~2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA-resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely-related drug ALV CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.
Currently, no approved treatments are available to combat infections with nidoviruses, a group of plus-stranded RNA viruses including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporin A (CsA) and Alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and may thus represent a class of pan-nidovirus inhibitors. Here, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two trans-membrane subunits of the viral replication complex, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and other RNA viruses inhibited by these compounds.
Flavivirus is a positive single stranded RNA viral genus, with members causing severe diseases in humans such as tick-borne encephalitis (TBE), yellow fever and dengue fever. Flaviviruses are known to cause remodeling of intracellular membranes into small cavities where replication of the viral RNA takes place. Non-structural (NS) proteins are not part of the virus coat and are believed to participate in the formation of these viral replication compartments (RCs). Here, we used tick-borne encephalitis virus (TBEV) as a model for flaviviruses and developed a stable human cell line where the expression of NS proteins can be induced without viral RNA replication. The described model system provides a novel and benign tool for studies of the viral components under controlled expression levels. We show that the expression of six NS proteins is sufficient to induce infection-like dilation of the endoplasmic reticulum (ER) and the formation of RC-like membrane invaginations. The NS proteins form a membrane associated complex in the ER and electron tomography reveals that the dilated areas of the ER are closely associated with lipid droplets and mitochondria. We propose that the NS proteins drive the remodeling of ER membranes and that the viral RNA, RNA replication, the viral polymerase and the TBEV structural proteins are not required.
Tick-borne encephalitis virus (TBEV) infection causes a broad spectrum of symptoms ranging from mild fever to severe encephalitis. Similar to other flaviviruses, TBEV exploits intracellular membranes to build replication compartments (RCs) for viral replication. The viral non-structural (NS) proteins have been suggested to be involved in this process, however, the mechanism for how RCs are formed and the role of individual NS proteins remain unclear. To study how TBEV induces membrane remodeling, we developed an inducible stable cell system expressing the TBEV NS polyprotein in the absence of viral RNA replication. Using this system, we were able to reproduce RC-like vesicles which resemble the RCs formed in flavivirus-infected cells in terms of morphology and size. This cell system is a robust tool to facilitate the studies of flavivirus RCs formation and an ideal model for the screening of antiviral agents at lower biosafety level.
CXCR2 is a chemokine receptor expressed on oligodendroglia that has been implicated in the pathogenesis of neuroinflammatory demyelinating diseases as well as enhancing the migration, proliferation, and myelin production by oligodendroglia. Using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system, we were able to assess how timed ablation of Cxcr2 in oligodendroglia affected disease following intracranial infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Generation of Plp-Cre-ER(T) ::Cxcr2fl/fl transgenic mice (termed Cxcr2-CKO mice) allows for Cxcr2 to be silenced in oligodendrocytes in adult mice following treatment with tamoxifen. Ablation of oligodendroglia Cxcr2 did not influence clinical severity in response to intracranial infection with JHMV. Infiltration of activated T cells or myeloid cells into the central nervous system (CNS) was not affected nor was the ability to control viral infection. In addition, the severity of demyelination was similar between tamoxifen-treated mice compared to vehicle-treated controls. Notably, deletion of Cxcr2 resulted in increased remyelination as assessed by g-ratio calculation when compared to vehicle-treated control mice. Collectively, our findings argue that CXCR2 signaling in oligodendroglia is dispensable with regards to contributing to neuroinflammation but its deletion enhances remyelination in a pre-clinical model of the human demyelinating disease multiple sclerosis (MS).
IMPORTANCE: Signaling through the chemokine receptor CXCR2 in oligodendroglia is important for developmental myelination in rodents, while chemical inhibition or non-specific genetic deletion of CXCR2 appears to augment myelin repair in animal models of the human demyelinating disease multiple sclerosis (MS). To better understand the biology of CXCR2 signaling on oligodendroglia, we generated transgenic mice in which Cxcr2 is selectively ablated in oligodendroglia upon treatment with tamoxifen. Using a viral model of neuroinflammation and demyelination, we demonstrate that genetic silencing of CXCR2 on oligodendroglia does not affect clinical disease, neuroinflammation, nor demyelination yet there was increased remyelination. These findings support and extend previous findings suggesting that targeting CXCR2 may offer a therapeutic avenue for enhancing remyelination in patients with demyelinating diseases.
Chronic viruses like herpes simplex virus type 1 (HSV-1) evade the hosts' immune system, by inducing exhaustion of antiviral T cells. In the present study, we found that exhausted HSV-specific CD8+ T cells, with elevated expression of programmed death ligand-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) receptors were frequent in symptomatic (SYMP) patients, with a history of numerous episodes of recurrent corneal herpetic disease, compared to asymptomatic (ASYMP) patients who never had corneal herpetic disease. Subsequently, using a rabbit model of recurrent ocular herpes, we found that the combined blockade of PD-1 and LAG-3 pathways with antagonist antibodies significantly restored the function of tissue-resident antiviral CD8+ TRM cells in both cornea and TG. An increased number of functional tissue-resident HSV-specific CD8+ TRM cells in latently infected rabbits was associated with protection against recurrent herpes infection and disease. Compared to the PD-1 or LAG-3 blockade alone, the combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional Ki-67+, IFN-+, CD107+ CD8+ T cells. Moreover, using the human leukocyte antigen (HLA) transgenic rabbit model, we found that dual blockade of PD-1 and LAG-3 reinforced the effect of a multiepitope vaccine, in boosting the frequency of HSV-1 specific CD8+ TRM cells and reducing disease severity. Thus, both the PD-1 and LAG-3 exhaustion pathways play a fundamental role in ocular herpes T cell immunopathology and provide important immune checkpoint targets to combat ocular herpes.
IMPORTANCE HSV-specific tissue-resident memory CD8+ TRM cells play a critical role in preventing virus reactivation from latently infected TG and subsequent virus shedding in tears that trigger the recurrent corneal herpetic disease. In this report, we determined how the dual blockade of PD-1 and LAG-3 immune checkpoints combined with vaccination improved the function of CD8+ TRM cells associated with a significant reduction in recurrent ocular herpes in HLA Tg rabbit model. The combined blockade of PD-1 and LAG-3 appeared to have a synergistic effect in generating frequent polyfunctional CD8+ TRM cells that infiltrated both the cornea and TG. The pre-clinical findings using the established HLA Tg rabbit model of recurrent herpes highlight that blocking immune checkpoints combined with a T cell-based vaccine would provide an important strategy to combat recurrent ocular herpes in the clinic.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive sense RNA genome that also serves as the mRNA for four non-structural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed trans-replication systems, where production of replication competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce non-capped RNA templates, which are poorly translated relative to CHIKV replicase generated capped RNAs. It was found that, in human cells, constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast, RNA polymerase I promoters from Aedes mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells, novel trans-replicase assays had exceptional sensitivity, with up to 105-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyse CHIKV nsP1 functionality, several mutations that severely reduced, but did not completely block, CHIKV replicase activity were identified: (i) tagging the N-terminus of nsP1 with eGFP; (ii) mutations D63A and Y248A blocking the RNA capping; (iii) mutation R252E affecting nsP1 membrane anchoring. In contrast, a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirms that this novel system provides a valuable tool to study CHIKV replicase, RNA replication and virus-host interactions.
IMPORTANCE Chikungunya virus (CHIKV) is a medically important pathogen responsible for recent large-scale epidemics. The development of efficient therapies against CHIKV has been hampered by gaps in our understanding of how non-structural proteins (nsPs) function to form the viral replicase and replicate virus RNA. Here we describe an extremely sensitive assay to analyse the effects of mutations on virus RNA synthesis machinery in both cells of mammalian (host) and mosquito (vector) origin. Using this system several lethal mutations in CHIKV nsP1 were shown to reduce but not completely block the ability of its replicase to synthesize viral RNAs. However, in contrast to related alphaviruses, CHIKV replicase was completely inactivated by mutations preventing palmitoylation of nsP1. These data can be used to develop novel, virus-specific antiviral treatments.
Simian-human immunodeficiency viruses (SHIVs) have been utilized to test vaccine efficacy and characterize mechanisms of viral transmission and pathogenesis. However, the majority of SHIVs currently available have significant limitations in that they were developed using sequences from chronically HIV-infected individuals or uncommon HIV subtypes or were optimized for the macaque model by serially passaging the engineered virus in vitro or in vivo. Recently, a newly developed SHIV, SHIV.C.CH505.375H.dCT (SHIV.CH505) that incorporates vpu-env (gp140) sequences from a transmitted/founder HIV-1 subtype C strain was shown to retain attributes of primary HIV-1 strains. However, a comprehensive analysis of the immunopathology that results from infection with this virus, especially in critical tissue compartments like the intestinal mucosa, has not been completed. In this study, we evaluated the viral dynamics and immunopathology of SHIV.CH505 in rhesus macaques. In line with previous findings, we found that SHIV.CH505 is capable of infecting and replicating efficiently in rhesus macaques, resulting in peripheral viral kinetics similar to that seen in pathogenic SIV and HIV infection. Furthermore, we observed significant and persistent depletions of CCR5+ and CCR6+ CD4+ T cells in mucosal tissues, decreases in CD4+ T cells producing Th17 cell-associated cytokines, CD8+ T cell dysfunction, and alterations of B cell and innate immune cell function, indicating that SHIV.CH505 elicits intestinal immunopathology typical of SIV/HIV infection. These findings suggest that SHIV.CH505 recapitulates the early viral replication dynamics and immunopathogenesis of HIV-1 infection of humans and thus can serve as a new model for HIV-1 pathogenesis, treatment, and prevention research.
IMPORTANCE The development of chimeric SHIVs has been instrumental in advancing our understanding of HIV-host interactions and allowing for in vivo testing of novel treatments. However, many of the currently available SHIVs have distinct drawbacks and are unable to fully reflect the features characteristic of primary SIV and HIV strains. Here, we utilize rhesus macaques to define the immunopathogenesis of the recently developed SHIV.CH505, which was designed without many of the limitations of previous SHIVs. We observed that infection with SHIV.CH505 leads to peripheral viral kinetics and mucosal immunopathogenesis comparable with that caused by pathogenic SIV and HIV. Overall, these data provide evidence of the value of SHIV.CH505 as an effective model of SIV/HIV infection and an important tool that can be used in future studies, including preclinical testing of new therapies or prevention strategies.
Epstein-Barr virus is associated with several human malignancies including nasopharyngeal carcinoma, gastric cancer and lymphoma. Latently infected cells carry a circularized EBV episome where the origin of replication (OriP) is comprised of two elements: the family of repeats (FR) and dyad symmetry (DS). The viral protein Epstein-Barr Nuclear Antigen-1 (EBNA1) binds to FR and DS to promote EBV episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 binding to the DS constitutes a minimal origin of DNA replication. Here, we report the crystal structure of two EBNA1 DNA-binding domain dimers bound to a DS half site. This structure shows that the DNA is smoothly bent allowing for stabilizing interactions between the dimers. The dimer-dimer interface requires an intricate hydrogen bonding network involving residues R491 and D581. When this interface is disrupted, we note loss of stable dimer-dimer complex formation on the DNA, compromised OriP-containing plasmid replication in cells and impaired recruitment of the MCM3 complex to the OriP. Surface conservation analysis reveals that these residues are part of a larger conserved surface that may be critical for recruitment of replication machinery to the OriP. Our results reveal a new region of EBNA1 critical for its activity and one that may be exploited by targeted small molecules to treat EBV-associated disease.
Epstein-Barr Virus (EBV) is a causative agent of various malignancies and may also contribute to autoimmune disease. The latent and episomal form of the virus is known to drive EBV-associated oncogenesis. Persistence of the viral episome in proliferating tumor cells requires the interaction of the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) with the viral origin of plasmid replication OriP. The Dyad Symmetry (DS) element in OriP is the essential minimal replicator of OriP. Here, we report the X-ray crystal structure of EBNA1 bound to DS. The structure reveals a previous unrecognized interface formed between dimers of EBNA1 necessary for cooperative DNA-binding, recruitment of cellular replication machinery, and replication function. These findings provide new insights into the mechanism of EBNA1 function at the replication origin and new opportunities to inhibit EBV latent infection and pathogenesis.
Given the unprecedented scale of the recent Ebola and Zika viral epidemics, it is crucial to understand the biology of host factors with broad antiviral action in order to develop novel therapeutic approaches. Here, we look into one such factor; zinc-finger antiviral protein (ZAP) inhibits a variety of RNA and DNA viruses. Alternative splicing results in two isoforms that differ at their C-termini; ZAPL (long), encodes a poly(ADP-ribose) polymerase (PARP)-like domain that is missing in ZAPS (short). Previously it has been shown that ZAPL is more antiviral than ZAPS while the latter is more induced by interferon (IFN). In this study, we discovered and confirmed the expression of two additional splice variants of human ZAP mmdash; ZAPXL (extra-long) and ZAPM (medium). We also found two haplotypes of human ZAP. Since ZAPL and ZAPS have differential activities, we hypothesize that all four ZAP isoforms have evolved to mediate distinct antiviral and/or cellular functions. By taking a gene knockout and reconstitution approach, we have characterized the antiviral, translational inhibition, and IFN activation activities of individual ZAP isoforms. Our work demonstrates that ZAPL and ZAPXL are more active against alphaviruses and hepatitis B virus (HBV) than ZAPS and ZAPM and elucidates the effects of splice variants on the action of a broad spectrum antiviral factor.
ZAP is an IFN-induced host factor that can inhibit a wide range of viruses and there is great interest in fully characterizing its antiviral mechanism. This is the first study that defines the antiviral capacity of individual ZAP isoforms in the absence of endogenous ZAP expression and hence crosstalk with other isoforms. Our data demonstrate that ZAP is expressed as four different forms mmdash; ZAPS, ZAPM, ZAPL and ZAPXL. The longer ZAP isoforms better inhibit alphaviruses and HBV while all isofoms equally inhibit Ebola virus transcription and replication. In addition, there is no difference in the ability of ZAP isoforms to enhance the induction of type I IFN expression. Our results show that the full spectrum of ZAP activities can change depending on the virus target and the relative levels of basal expression and induction by IFN or infection.