When viruses infect cells, they act like well-oiled machines, hijacking cellular resources and cranking out countless copies of their genetic material to help spread the infection. A new study published in the Journal of Biological Chemistry and led by researchers at the University of Wisconsin-Madison Institute for Molecular Virology and Center for Quantitative Cell Imaging offers a promising way to slow down the replication of RNA viruses like SARS-CoV-2 and poliovirus. Work like this can pave the way for new anti-viral treatments.
Assistant Professor Robert Kirchdoerfer and his team of researchers found that drugs designed to mimic the building blocks of the genetic codecan stall the replication of viral RNA. Viral RNA replication relies on an enzyme machine called an “RNA-dependent RNA polymerase”, which reads the viral genetic blueprint and builds a matching new strand of RNA. The polymerase grabs free floating building blocks called nucleotides and links them in the right order, building a new RNA chain with the nucleotides spelling out the viral genetic code.
What happens to RNA polymerase activity when some of these building blocks are misshapen? Kirchdoerfer and his colleagues tested two specific analogs, arabinose-CTP and arabinose-UTP (ara-CTP and ara-UTP), which are nearly identical to the natural nucleotide building blocks but with a single chemical group flipped to the wrong side of the molecule.
“When the polymerase accidentally picks up one of these nucleotide look-alikes, it incorporates the analog but then struggles for a long time to add the next building block in the chain.” explains Kirchdoerfer
Using a sophisticated technique called magnetic tweezers, the team was able to observe individual RNA polymerase molecules’ activity in real time. The team found that the polymerase has a strong preference for using normal nucleotides over the misshapen ones, but when the polymerase accidentally incorporates one of the analogs, the polymerase jams up for minutes.
“We found that when ara-CTP or ara-UTP was incorporated in the RNA molecule, the polymerase paused for an average of 200 seconds. This is a very lengthy pause as the entire RNA genome replication process is expected to only take a few minutes.” Said Kirchdoerfer.
The team then took a closer look at the jammed-up RNA polymerase, literally. They used a technique called cryo-electron microscopy (cryo-EM)to directly visualize the active site of the enzyme from SARS-CoV-2. Cryo-EM can produce incredibly detailed, freeze-frame images of moleculesat a near-atomic level. They saw that ara-CTP or ara-UTP fit right into the RNA polymerase and joined the growing RNA molecule. When the polymerase tried to add the next nucleotide, the arabinose building block didn’t want to link. The polymerase keeps trying to force the connection for a very long time before it finally makes the bond and the polymerase can carry on.
The discovery that RNA polymerases from both a coronavirus and a poliovirus jam up when exposed to ara-CTP or ara-UTP is a major win for antiviral research because it points the way toward developing drugs that work on new or multiple viruses.
“While there are a lot of differences between SARS-CoV-2 and poliovirus RNA polymerases, both paused for long periods of time when exposed to the arabinose analogs.” Said Kirchdoerfer. “These findings suggest that this mode of antiviral activity could be useful to explore for treatments to different viral diseases.”
Kirchdoerfer added, “To keep pace with emerging viruses we need antiviral treatments that work on a broad-spectrum of viruses. Arabinose analogs, like the ones that we used in our study, are a useful framework to start turning insights about molecular mechanisms into treatments that could someday be used for patients.”
In addition to Kirchdoerfer’s team at UW-Madison, this collaborative work made use of researcher expertise at Vrije Universiteit Amsterdam (Amsterdam, Netherlands) and the University of North Carolina-Chapel Hill.
Full citation:
Incorporation of arabinose-CTP and arabinose-UTP inhibits viral polymerases by inducing long pauses. Xiao Z, Das A, Jain A, Anderson TK, Cameron CE, Arnold JJ, Dulin D, Kirchdoerfer RN. J Biol Chem. (2026) 302:111027. doi: 10.1016/j.jbc.2025.111027.
The Institute for Molecular Virology (IMV) and the Center for Quantitative Cell Imaging (CQCI) are two research centers administered by the University of Wisconsin-Madison Office of the Vice Chancellor for Research. They are composed of faculty with tenure homes or other appointments homes in the Departments of Biochemistry, Biomedical Engineering, Biomolecular Chemistry, Botany, Cell & Regenerative Biology, Integrative Biology, Medical Microbiology & Immunology, Medical Physics, Oncology, and Plant Pathology.
The IMV is focused on uncovering how viruses replicate, evolve, and cause disease, developing strategies for prevention and treatment of viral infections, and harnesses viruses as tools to understand biological processes. Housed in the Robert M. Bock Laboratories, IMV anchors a much broader network of over 40 virology-focused researchers across UW-Madison who make up the Madison Virology Collective.
The CQCI emphasizes the development and application of cutting-edge imaging technologies to visualize and measure biological processes at the molecular, cellular, and tissue levels. In addition, CQCI manages the 3D Cell Electron Microscopy Core Facility and serves collaborative hub with the mission of sharing advanced imaging approaches across campus and with the broader scientific community. The CQCI aims to reveal the hidden mechanisms underlying cellular processes, health, and disease.