Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases.

Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication.

Crystal structure of a soluble fragment of poliovirus 2CATPase.

Poliovirus (PV) 2CATPase is the most studied 2C protein in the Picornaviridae family. It is involved in RNA replication, encapsidation and uncoating and many inhibitors have been found that target PV 2CATPase. Despite numerous investigations to characterize its functions, a high-resolution structure of PV 2C has not yet been determined. We report here the crystal structure of a soluble fragment of PV 2CATPase to 2.55Å, containing an ATPase domain, a zinc finger and a C-terminal helical domain but missing the N-terminal domain. The ATPase domain shares the common structural features with EV71 2C and other Superfamily 3 helicases. The C-terminal cysteine-rich motif folds into a CCCC type zinc finger in which four cysteine ligands and several auxiliary residues assist in zinc binding. By comparing with the known zinc finger fold groups, we found the zinc finger of 2C proteins belong to a new fold group, which we denote the "Enterovirus 2C-like" group. The C-terminus of PV 2CATPase forms an amphipathic helix that occupies a hydrophobic pocket located on an adjacent PV 2CATPase in the crystal lattice. The C-terminus mediated PV 2C-2C interaction promotes self-oligomerization, most likely hexamerization, which is fundamental to the ATPase activity of 2C. The zinc finger is the most structurally diverse feature in 2C proteins. Available structural and virological data suggest that the zinc finger of 2C might confer the specificity of interaction with other proteins. We built a hexameric ring model of PV 2CATPase and visualized the previously identified functional motifs and drug-resistant sites, thus providing a structure framework for antiviral drug development.

Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein.

RNA viruses induce specialized membranous structures for use in genome replication. These structures are often referred to as replication organelles (ROs). ROs exhibit distinct lipid composition relative to other cellular membranes. In many picornaviruses, phosphatidylinositol-4-phosphate (PI4P) is a marker of the RO. Studies to date indicate that the viral 3A protein hijacks a PI4 kinase to induce PI4P by a mechanism unrelated to the cellular pathway, which requires Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1, GBF1, and ADP ribosylation factor 1, Arf1. Here we show that a picornaviral 3CD protein is sufficient to induce synthesis of not only PI4P but also phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). Synthesis of PI4P requires GBF1 and Arf1. We identified 3CD derivatives: 3CDm and 3CmD, that we used to show that distinct domains of 3CD function upstream of GBF1 and downstream of Arf1 activation. These same 3CD derivatives still supported induction of PIP2 and PC, suggesting that pathways and corresponding mechanisms used to induce these phospholipids are distinct. Phospholipid induction by 3CD is localized to the perinuclear region of the cell, the outcome of which is the proliferation of membranes in this area of the cell. We conclude that a single viral protein can serve as a master regulator of cellular phospholipid and membrane biogenesis, likely by commandeering normal cellular pathways.

Rational Control of Poliovirus RNA-Dependent RNA Polymerase Fidelity by Modulating Motif-D Loop Conformational Dynamics.

The conserved structural motif D is an important determinant of the speed and fidelity of viral RNA-dependent RNA polymerases (RdRps). Structural and computational studies have suggested that conformational changes in the motif-D loop that help to reposition the catalytic lysine represent critical steps in nucleotide selection and incorporation. Conformations of the motif-D loop in the poliovirus RdRp are likely controlled in part by noncovalent interactions involving the motif-D residue Glu364. This residue swivels between making interactions with Lys228 and Asn370 to stabilize the open and closed loop conformations, respectively. We show here that we can rationally control the motif-D loop conformation by breaking these interactions. The K228A variant favors a more active closed conformation, leading to increased nucleotide incorporation rates and decreased nucleotide selectivity, and the N370A variant favors a less active open conformation, leading to decreased nucleotide incorporation rates and increased nucleotide selectivity. Similar competing interactions likely control nucleotide incorporation rates and fidelity in other viral RdRps. Rational engineering of these interactions may be important in the generation of live, attenuated vaccine strains, considering the established relationships between RdRp function and viral pathogenesis.

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection.

Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cproin vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner.IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection. Although several host protein targets have been identified, the entire list of proteins that are targeted is not known. In this study, we used a novel unbiased proteomics approach to identify ∼100 novel host targets of the enterovirus 3C protease, thus providing further insights into the network of cellular pathways that are modulated to promote virus infection.

Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis.

The poliovirus eradication initiative has spawned global immunization infrastructure and dramatically decreased the prevalence of the disease, yet the original virus eradication goal has not been met. The suboptimal properties of the existing vaccines are among the major reasons why the program has repeatedly missed eradication deadlines. Oral live poliovirus vaccine (OPV), while affordable and effective, occasionally causes the disease in the primary recipients, and the attenuated viruses rapidly regain virulence and can cause poliomyelitis outbreaks. Inactivated poliovirus vaccine (IPV) is safe but expensive and does not induce the mucosal immunity necessary to interrupt virus transmission. While the need for a better vaccine is widely recognized, current efforts are focused largely on improvements to the OPV or IPV, which are still beset by the fundamental drawbacks of the original products. Here we demonstrate a different design of an antipoliovirus vaccine based on in situ production of virus-like particles (VLPs). The poliovirus capsid protein precursor, together with a protease required for its processing, are expressed from a Newcastle disease virus (NDV) vector, a negative-strand RNA virus with mucosal tropism. In this system, poliovirus VLPs are produced in the cells of vaccine recipients and are presented to their immune systems in the context of active replication of NDV, which serves as a natural adjuvant. Intranasal administration of the vectored vaccine to guinea pigs induced strong neutralizing systemic and mucosal antibody responses. Thus, the vectored poliovirus vaccine combines the affordability and efficiency of a live vaccine with absolute safety, since no full-length poliovirus genome is present at any stage of the vaccine life cycle.IMPORTANCE A new, safe, and effective vaccine against poliovirus is urgently needed not only to complete the eradication of the virus but also to be used in the future to prevent possible virus reemergence in a postpolio world. Currently, new formulations of the oral vaccine, as well as improvements to the inactivated vaccine, are being explored. In this study, we designed a viral vector with mucosal tropism that expresses poliovirus capsid proteins. Thus, poliovirus VLPs are produced in vivo, in the cells of a vaccine recipient, and are presented to the immune system in the context of vector virus replication, stimulating the development of systemic and mucosal immune responses. Such an approach allows the development of an affordable and safe vaccine that does not rely on the full-length poliovirus genome at any stage.

Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases.

The nucleotide incorporation fidelity of the viral RNA-dependent RNA polymerase (RdRp) is important for maintaining functional genetic information but, at the same time, is also important for generating sufficient genetic diversity to escape the bottlenecks of the host's antiviral response. We have previously shown that the structural dynamics of the motif D loop are closely related to nucleotide discrimination. Previous studies have also suggested that there is a reorientation of the triphosphate of the incoming nucleotide, which is essential before nucleophilic attack from the primer RNA 3'-hydroxyl. Here, we have used 31P NMR with poliovirus RdRp to show that the binding environment of the triphosphate is different when correct versus incorrect nucleotide binds. We also show that amino acid substitutions at residues known to interact with the triphosphate can alter the binding orientation/environment of the nucleotide, sometimes lead to protein conformational changes, and lead to substantial changes in RdRp fidelity. The analyses of other fidelity variants also show that changes in the triphosphate binding environment are not always accompanied by changes in the structural dynamics of the motif D loop or other regions known to be important for RdRp fidelity, including motif B. Altogether, our studies suggest that the conformational changes in motifs B and D, and the nucleoside triphosphate reorientation represent separable, "tunable" fidelity checkpoints.

A novel point mutation (L70P) inactivates poliovirus 3C protease.

Poliovirus (PV) contains a single-stranded positive-sense RNA genome, which is translated into a single polyprotein. Viral proteases process this polyprotein to produce several individual as well as fused proteins. The major viral protease 3C cleaves at nine of the eleven cleavage sites. During the process of expressing PV 3ABC protein in Escherichia coli, we identified a 3C mutant (L70P), which lost its protease activity. This loss of function was confirmed by generating recombinant adenoviruses expressing mutant and wild-type 3C. Further, infectious PV could not be recovered from PV full-length cDNA containing the L70P mutation. However, 3C L70P mutant cDNA could complement a PV cDNA containing a 1AB deletion, producing a viable virus population containing defective complementing genomes. Structural analysis of the mutant protein indicated that the L70P mutation resulted in the loss of a hydrogen bond between two residues located within a loop between two ß-sheets, potentially leading to strain on the catalytic site. We conclude that L70P inactivates 3C protease because of its close proximity to the 3C catalytic site.

A Single Amino Acid Substitution in Poliovirus Nonstructural Protein 2CATPase Causes Conditional Defects in Encapsidation and Uncoating.

UNLABELLED: The specificity of encapsidation of C-cluster enteroviruses depends on an interaction between capsid proteins and nonstructural protein 2C(ATPase) In particular, residue N252 of poliovirus 2C(ATPase) interacts with VP3 of coxsackievirus A20, in the context of a chimeric virus. Poliovirus 2C(ATPase) has important roles both in RNA replication and encapsidation. In this study, we searched for additional sites in 2C(ATPase), near N252, that are required for encapsidation. Accordingly, segments adjacent to N252 were analyzed by combining triple and single alanine mutations to identify residues required for function. Two triple alanine mutants exhibited defects in RNA replication. The remaining two mutations, located in secondary structures in a predicted three-dimensional model of 2C(ATPase), caused lethal growth phenotypes. Most single alanine mutants, derived from the lethal variants, were either quasi-infectious and yielded variants with wild-type (wt) or temperature-sensitive (ts) growth phenotypes or had a lethal growth phenotype due to defective RNA replication. The K259A mutation, mapping to an α helix in the predicted structure of 2C(ATPase), resulted in a cold-sensitive virus. In vivo protein synthesis and virus production were strikingly delayed at 33°C relative to the wt, suggesting a defect in uncoating. Studies with a reporter virus indicated that this mutant is also defective in encapsidation at 33°C. Cell imaging confirmed a much-reduced production of K259A mature virus at 33°C relative to the wt. In conclusion, we have for the first time linked a cold-sensitive encapsidation defect in 2C(ATPase) (K259A) to a subsequent delay in uncoating of the virus particle at 33°C during the next cycle of infection. IMPORTANCE: Enterovirus morphogenesis, which involves the encapsidation of newly made virion RNA, is a process still poorly understood. Elucidation of this process is important for future drug development for a large variety of diseases caused by these agents. We have previously shown that the specificity of encapsidation of poliovirus and of C-cluster coxsackieviruses, which are prototypes of enteroviruses, is dependent on an interaction of capsid proteins with the multifunctional nonstructural protein 2C(ATPase) In this study, we have searched for residues in poliovirus 2C(ATPase), near a presumed capsid-interacting site, important for encapsidation. An unusual cold-sensitive mutant of 2C(ATPase) possessed a defect in encapsidation at 37°C and subsequently in uncoating during the next cycle of infection at 33°C. These studies not only reveal a new site in 2C(ATPase) that is involved in encapsidation but also identify a link between encapsidation and uncoating.

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance.

UNLABELLED: RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3D(pol), as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3D(pol) may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3D(pol)-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3D(pol) decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3D(pol) increased the frequency of recombination. The 3D(pol) Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3D(pol) Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE: Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication.

The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

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.

Heterogeneous Nuclear Ribonucleoprotein M Facilitates Enterovirus Infection.

UNLABELLED: Picornavirus infection involves a dynamic interplay of host and viral protein interactions that modulates cellular processes to facilitate virus infection and evade host antiviral defenses. Here, using a proteomics-based approach known as TAILS to identify protease-generated neo-N-terminal peptides, we identify a novel target of the poliovirus 3C proteinase, the heterogeneous nuclear ribonucleoproteinM(hnRNP M), a nucleocytoplasmic shuttling RNA-binding protein that is primarily known for its role in pre-mRNA splicing. hnRNPMis cleaved in vitro by poliovirus and coxsackievirus B3 (CVB3) 3C proteinases and is targeted in poliovirus- and CVB3-infected HeLa cells and in the hearts of CVB3-infected mice. hnRNPMrelocalizes from the nucleus to the cytoplasm during poliovirus infection. Finally, depletion of hnRNPMusing small interfering RNA knockdown approaches decreases poliovirus and CVB3 infections in HeLa cells and does not affect poliovirus internal ribosome entry site translation and viral RNA stability. We propose that cleavage of and subverting the function of hnRNPMis a general strategy utilized by picornaviruses to facilitate viral infection. IMPORTANCE: Enteroviruses, a member of the picornavirus family, are RNA viruses that cause a range of diseases, including respiratory ailments, dilated cardiomyopathy, and paralysis. Although enteroviruses have been studied for several decades, the molecular basis of infection and the pathogenic mechanisms leading to disease are still poorly understood. Here, we identify hnRNPMas a novel target of a viral proteinase. We demonstrate that the virus subverts the function of hnRNPMand redirects it to a step in the viral life cycle. We propose that cleavage of hnRNPMis a general strategy that picornaviruses use to facilitate infection.

[Determination of the Minimal Fragment of the Poliovirus IRES Necessary for the Formation of a Specific Complex with the Human Glycyl-tRNA Synthetase].

Aminoacyl-tRNA synthetases are an ancient enzyme family that specifically charge a tRNA molecule with a cognate amino acid required for protein synthesis. Glycyl-tRNA synthetase is one of the most interesting aminoacyl-tRNA synthetases due to its structure variability and functional features in the different organisms. It was shown recently that human glycyl-tRNA synthetase is a regulator of translational initiation of poliovirus mRNA. Details of this process and its mechanism still remain unknown. While exploring this stage of poliovirus functioning we have studied the interaction of the cytoplasmic form of human glycyl-tRNA synthetase and its domains with the fragments of the poliovirus IRES element. As a result, we have identified the minimal fragment of viral mRNA with which glycyl-tRNA synthetase fully interacts and estimated the contribution of some domains to the interaction of glycyl-tRNA synthetase with RNA.

Structural dynamics as a contributor to error-prone replication by an RNA-dependent RNA polymerase.

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity.

RNA virus population diversity, an optimum for maximal fitness and virulence.

The ability of an RNA virus to exist as a population of genetically distinct variants permits the virus to overcome events during infections that would otherwise limit virus multiplication or drive the population to extinction. Viral genetic diversity is created by the ribonucleotide misincorporation frequency of the viral RNA-dependent RNA polymerase (RdRp). We have identified a poliovirus (PV) RdRp derivative (H273R) possessing a mutator phenotype. GMP misincorporation efficiency for H273R RdRp in vitro was increased by 2-3-fold that manifested in a 2-3-fold increase in the diversity of the H273R PV population in cells. Circular sequencing analysis indicated that some mutations were RdRp-independent. Consistent with the population genetics theory, H273R PV was driven to extinction more easily than WT in cell culture. Furthermore, we observed a substantial reduction in H273R PV virulence, measured as the ability to cause paralysis in the cPVR mouse model. Reduced virulence correlated with the inability of H273R PV to sustain replication in tissues/organs in which WT persists. Despite the attenuated phenotype, H273R PV was capable of replicating in mice to levels sufficient to induce a protective immune response, even when the infecting dose used was insufficient to elicit any visual signs of infection. We conclude that optimal RdRp fidelity is a virulence determinant that can be targeted for viral attenuation or antiviral therapies, and we suggest that the RdRp may not be the only source of mutations in a RNA virus genome.

Inhibition of poliovirus-induced cleavage of cellular protein PCBP2 reduces the levels of viral RNA replication.

UNLABELLED: Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE: Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition.

Vaccine-derived mutation in motif D of poliovirus RNA-dependent RNA polymerase lowers nucleotide incorporation fidelity.

All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.

Polyprotein context regulates the activity of poliovirus 2CATPase bound to bilayer nanodiscs.

Positive-strand RNA viruses generally replicate in large membrane-associated complexes. For poliovirus, these replication complexes are anchored to the membrane via the viral 2B, 2C, and 3A proteins. 2C is an AAA+ family ATPase that plays a key role in host cell membrane rearrangement, is a putative helicase, and is implicated in virion assembly and packaging. However, the membrane-binding characteristics of all of these viral proteins have made it difficult to elucidate their exact roles in virus replication. We show here that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C, 2BC, and 2BC3AB polyproteins in a membrane-bound form. ATPase assays on these proteins show that the activity of the core 2C domain is stimulated ~0-fold compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. The proteins are active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Our data lead to a model wherein the replication complex can be assembled with a minimally active form of 2C that then becomes fully activated by proteolytic cleavage from the adjacent 2B viroporin domain.

Structural features of a picornavirus polymerase involved in the polyadenylation of viral RNA.

Picornaviruses have 3' polyadenylated RNA genomes, but the mechanisms by which these genomes are polyadenylated during viral replication remain obscure. Based on prior studies, we proposed a model wherein the poliovirus RNA-dependent RNA polymerase (3D(pol)) uses a reiterative transcription mechanism while replicating the poly(A) and poly(U) portions of viral RNA templates. To further test this model, we examined whether mutations in 3D(pol) influenced the polyadenylation of virion RNA. We identified nine alanine substitution mutations in 3D(pol) that resulted in shorter or longer 3' poly(A) tails in virion RNA. These mutations could disrupt structural features of 3D(pol) required for the recruitment of a cellular poly(A) polymerase; however, the structural orientation of these residues suggests a direct role of 3D(pol) in the polyadenylation of RNA genomes. Reaction mixtures containing purified 3D(pol) and a template RNA with a defined poly(U) sequence provided data consistent with a template-dependent reiterative transcription mechanism for polyadenylation. The phylogenetically conserved structural features of 3D(pol) involved in the polyadenylation of virion RNA include a thumb domain alpha helix that is positioned in the minor groove of the double-stranded RNA product and lysine and arginine residues that interact with the phosphates of both the RNA template and product strands.