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Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the “backtracked” state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity., Accepted Author Manuscript, BN/Cees Dekker Lab, BN/Nynke Dekker Lab

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., BN/Nynke Dekker Lab

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., BN/Nynke Dekker Lab