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Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies 1 . Viperin, a member of the radical S -adenosyl- l -methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus 2 and HIV 3 , 4 . Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins 3 , 4 . Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes. Viperin inhibits the replication of various viruses by catalysing the conversion of CTP to ddhCTP, which is a unique nucleotide that functions as replication-chain terminator of RNA-dependent RNA polymerases.

Mutation rates can evolve through genetic drift, indirect selection due to genetic hitchhiking, or direct selection on the physicochemical cost of high fidelity. However, for many systems, it has been difficult to disentangle the relative impact of these forces empirically. In RNA viruses, an observed correlation between mutation rate and virulence has led many to argue that their extremely high mutation rates are advantageous because they may allow for increased adaptability. This argument has profound implications because it suggests that pathogenesis in many viral infections depends on rare or de novo mutations. Here, we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. We find that a poliovirus antimutator, 3DG64S, has a significant replication defect and that wild-type (WT) and 3DG64S populations have similar adaptability in 2 distinct cellular environments. Experimental evolution of 3DG64S under selection for replicative speed led to reversion and compensation of the fidelity phenotype. Mice infected with 3DG64S exhibited delayed morbidity at doses well above the lethal level, consistent with attenuation by slower growth as opposed to reduced mutational supply. Furthermore, compensation of the 3DG64S growth defect restored virulence, while compensation of the fidelity phenotype did not. Our data are consistent with the kinetic proofreading model for biosynthetic reactions and suggest that speed is more important than accuracy. In contrast with what has been suggested for many RNA viruses, we find that within-host spread is associated with viral replicative speed and not standing genetic diversity.

RNA recombination in positive-strand RNA viruses is a molecular-genetic process, which permits the greatest evolution of the genome and may be essential to stabilizing the genome from the deleterious consequences of accumulated mutations. Enteroviruses represent a useful system to elucidate the details of this process. On the biochemical level, it is known that RNA recombination is catalyzed by the viral RNA-dependent RNA polymerase using a template-switching mechanism. For this mechanism to function in cells, the recombining genomes must be located in the same subcellular compartment. How a viral genome is trafficked to the site of genome replication and recombination, which is membrane associated and isolated from the cytoplasm, is not known. We hypothesized that genome translation was essential for colocalization of genomes for recombination. We show that complete inactivation of internal ribosome entry site (IRES)-mediated translation of a donor enteroviral genome enhanced recombination instead of impairing it. Recombination did not occur by a nonreplicative mechanism. Rather, sufficient translation of the nonstructural region of the genome occurred to support subsequent steps required for recombination. The noncanonical translation initiation factors, eIF2A and eIF2D, were required for IRES-independent translation. Our results support an eIF2A/eIF2D-dependent mechanism under conditions in which the eIF2-dependent mechanism is inactive. Detection of an IRES-independent mechanism for translation of the enterovirus genome provides an explanation for a variety of debated observations, including nonreplicative recombination and persistence of enteroviral RNA lacking an IRES. The existence of an eIF2A/eIF2D-dependent mechanism in enteroviruses predicts the existence of similar mechanisms in other viruses.

Viruses, where wrong is right The replication of RNA viruses is associated with a higher mutation rate than is seen in organisms using DNA as their genetic material. This can produce nonviable individuals but also, it has been suggested, some useful variation that could enhance the fitness of virus populations by allowing them to adapt to changing environments encountered during infection. Until now there has been no experimental support for this suggestion, known as the ‘quasispecies’ hypothesis. But now a search for viruses that copy their genome too accurately has provided support for this idea. Poliovirus isolates carrying a ‘super accurate’ RNA polymerase are less varied and less infectious than normal viruses. These results could have implications for the development of antiviral drugs. An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies 1 , 2 , 3 , 4 . Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication 5 , 6 , 7 , 8 . Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a ‘cloud’ of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection 4 , 9 , 10 , 11 . Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population 4 , 12 . According to this view, viral populations, rather than individual variants, are the target of evolutionary selection 4 , 12 . Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

Alphaviruses pose a growing global health threat, with Chikungunya virus (CHIKV) epidemics ongoing. Although several CHIKV vaccine candidates have progressed to late-stage clinical evaluation, none have yet achieved licensure or widespread availability. The CHIKV nonstructural proteins nsP2 and nsP4 encode essential enzymatic activities that represent key targets for antiviral development, yet the biochemical basis of nsP4 RNA-dependent RNA polymerase (RdRp) activity remains poorly understood. Here, we identify a minimal, functional precursor form of nsP4 derived from the nsP3-nsP4 polyprotein (P34) that is active in a cell-based RNA replicon system. Using synthetic, capped mRNAs, we show that cleavage of P34 by the nsP2 protease is required for robust reporter expression, and that a truncated form retaining only the C-terminal 50 residues of nsP3 (CT50-P34) supports near-wild-type replication. Unexpectedly, ubiquitin-nsP4 fusions failed to substitute for P34, likely reflecting the transient expression supported by our RNA-based system. We propose that precursor forms of nsP4 interact with the nsP1 dodecamer at the site of genome replication, where cleavage activates the RdRp and localization within the nsP1 dodecamer maintains nsP4 in its active conformation. Dissociation from the nsP1 dodecamer triggers a conformational switch to an inactive state. Together, these findings establish a tractable framework for interrogation of the assembly, activation, and regulation of the alphavirus polymerase.

The absence of ‘shovel-ready’ anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. To multiply and spread from cell to cell, the virus responsible for COVID-19 (also known as SARS-CoV-2) must first replicate its genetic information. This process involves a ‘polymerase’ protein complex making a faithful copy by assembling a precise sequence of building blocks, or nucleotides. The only drug approved against SARS-CoV-2 by the US Food and Drug Administration (FDA), remdesivir, consists of a nucleotide analog, a molecule whose structure is similar to the actual building blocks needed for replication. If the polymerase recognizes and integrates these analogs into the growing genetic sequence, the replication mechanism is disrupted, and the virus cannot multiply. Most approaches to study this process seem to indicate that remdesivir works by stopping the polymerase and terminating replication altogether. Yet, exactly how remdesivir and other analogs impair the synthesis of new copies of the virus remains uncertain. To explore this question, Seifert, Bera et al. employed an approach called magnetic tweezers which uses a magnetic field to manipulate micro-particles with great precision. Unlike other methods, this technique allows analogs to be integrated under conditions similar to those found in cells, and to be examined at the level of a single molecule. The results show that contrary to previous assumptions, remdesivir does not terminate replication; instead, it causes the polymerase to pause and backtrack (which may appear as termination in other techniques). The same approach was then applied to other nucleotide analogs, some of which were also found to target the SARS-CoV-2 polymerase. However, these analogs are incorporated differently to remdesivir and with less efficiency. They also obstruct the polymerase in distinct ways. Taken together, the results by Seifert, Bera et al. suggest that magnetic tweezers can be a powerful approach to reveal how analogs interfere with replication. This information could be used to improve currently available analogs as well as develop new antiviral drugs that are more effective against SARS-CoV-2. This knowledge will be key at a time when treatments against COVID-19 are still lacking, and may be needed to protect against new variants and future outbreaks.

Virus spread at the single-cell level is largely uncharacterized. We have designed and constructed a microfluidic device in which each nanowell contains a single, infected cell (donor) and a single, uninfected cell (recipient). Using a GFP-expressing poliovirus as our model, we observed both lytic and non-lytic spread. Donor cells supporting lytic spread established infection earlier than those supporting non-lytic spread. However, non-lytic spread established infections in recipient cells substantially faster than lytic spread and yielded higher rates of genome replication. While lytic spread was sensitive to the presence of capsid entry/uncoating inhibitors, non-lytic spread was not. Consistent with emerging models for non-lytic spread of enteroviruses using autophagy, reduction in LC3 levels in cells impaired non-lytic spread and elevated the fraction of virus in donor cells spreading lytically. The ability to distinguish lytic and non-lytic spread unambiguously will enable discovery of viral and host factors and host pathways used for non-lytic spread of enteroviruses and other viruses as well.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.