RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platform to monitor the elongation dynamics of a prototypical RdRp over thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRp-RNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides. Dulin et al. find that a prototypical RNA-dependent RNA polymerase (RdRp) visits several states during nucleotide synthesis, of which only one incorporates nucleotide analogs with therapeutic potential. Different analogs exhibit distinct kinetic signatures, with an analog thought to induce chain termination actually promoting RdRp backtracking.@en

Enteroviruses are well known for their ability to cause neurological damage and paralysis. The model enterovirus is poliovirus (PV), the causative agent of poliomyelitis, a condition characterized by acute flaccid paralysis. A related virus, enterovirus 71 (EV-A71), causes similar clinical outcomes in recurrent outbreaks throughout Asia. Retrospective phylogenetic analysis has shown that recombination between circulating strains of EV-A71 produces the outbreak-associated strains which exhibit increased virulence and/or transmissibility. While studies on the mechanism(s) of recombination in PV are ongoing in several laboratories, little is known about factors that influence recombination in EV-A71. We have developed a cell-based assay to study recombination of EV-A71 based upon previously reported assays for poliovirus recombination. Our results show that (i) EV-A71 strain type and RNA sequence diversity impacts recombination frequency in a predictable manner that mimics the observations found in nature; (ii) recombination is primarily a replicative process mediated by the RNA-dependent RNA polymerase; (iii) a mutation shown to reduce recombination in PV (L420A) similarly reduces EV-A71 recombination, suggesting conservation in mechanism(s); and (iv) sequencing of intraserotypic recombinant genomes indicates that template switching occurs by a mechanism that may require some sequence homology at the recombination junction and that the triggers for template switching may be sequence independent. The development of this recombination assay will permit further investigation on the interplay between replication, recombination and disease.IMPORTANCE Recombination is a mechanism that contributes to genetic diversity. We describe the first assay to study EV-A71 recombination. Results from this assay mimic what is observed in nature and can be used by others to predict future recombination events within the enterovirus species A group. In addition, our results highlight the central role played by the viral RNA-dependent RNA polymerase (RdRp) in the recombination process. Further, our results show that changes to a conserved residue in the RdRp from different species groups have a similar impact on viable recombinant virus yields, which is indicative of conservation in mechanism.@en

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.@en

Enterovirus A71 (EV-A71) is an important nonpolio enterovirus that causes severe neurological complications. In 1998, Taiwan experienced an EV-A71 outbreak that caused 78 deaths. Since then, periodic epidemics of EV-A71 associated with newly emerging strains have occurred. Several of these strains are known to be recombinant; however, how these strains arose within such a short period of time remains unknown. Here, we sequenced 64 full-length genomes from clinical isolates collected from 2005 to 2016 and incorporated all 91 Taiwanese genomes downloaded from the Virus Pathogen Resource to extensively analyze EV-A71 recombination in Taiwan. We found that the B3 subgenotype was a potential recombinant parent of the EV-A71 C2-like and C4 strains by intratypic recombination. Such B3-similar regions were also found in many cocirculating coxsackieviruses belonging to Enterovirus A species (EV-A) through a series of intertypic recombinations. Therefore, locally enriched outbreaks of cocirculating viruses from different genotypes/serotypes may facilitate recombination. Most recombination breakpoints we found had nonrandom distributions and were located within the region spanning from the boundary of P1 (structural gene) and P2 (nonstructural) to the cis-Acting replication element at P2, indicating that specific genome reassembly of structural and nonstructural genes may be subject to natural selection. Through intensive recombination, 11 EV-A71-like signatures (including one in 3A, two in 3C, and eight in 3D) were found to be present in a variety of recently cocirculating EV-A viruses worldwide, suggesting that these viruses may be targets for wide-spectrum antiviral development.@en