An Extended Primer Grip of Picornavirus Polymerase Facilitates Sexual RNA Replication Mechanisms

Abstract

ABSTRACTPicornaviruses have both asexual and sexual RNA replication mechanisms. Asexual RNA replication mechanisms involve one parental template whereas sexual RNA replication mechanisms involve two or more parental templates. Because sexual RNA replication mechanisms counteract ribavirin-induced error catastrophe, we selected for ribavirin-resistant poliovirus to identify polymerase residues that facilitate sexual RNA replication mechanisms. We used serial passage in ribavirin, beginning with a variety of ribavirin-sensitive and ribavirin-resistant parental viruses. Ribavirin-sensitive virus contained an L420A polymerase mutation while ribavirin-resistant virus contained a G64S polymerase mutation. A G64 codon mutation (G64Fix) was used to inhibit emergence of G64S-mediated ribavirin resistance. Revertants (L420) or pseudo-revertants (L420V, L420I) were selected from all independent lineages of L420A, G64FixL420A and G64S L420A parental viruses. Ribavirin-resistant G64S mutations were selected in two independent lineages and novel ribavirin-resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, T353I). The structural orientation of M392, immediately adjacent to L420 and the polymerase primer grip region, led us to engineer additional polymerase mutations into poliovirus (M392A, M392L & M392V and K375R & R376K). L420A revertants and pseudorevertants (L420V, L420I) restored efficient sexual RNA replication mechanisms, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L & M392V) and primer grip mutations (K375R & R376K) exhibited divergent RNA recombination, ribavirin sensitivity and biochemical phenotypes, consistent with changes in the fidelity of RNA synthesis. We conclude that an extended primer grip of the polymerase, including L420, M392, K375 & R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.IMPORTANCEPicornaviruses have both asexual and sexual RNA replication mechanisms. Sexual RNA replication shapes picornavirus species groups, contributes to the emergence of vaccine-derived polioviruses and counteracts error catastrophe. Can viruses distinguish between homologous and non-homologous partners during sexual RNA replication? We implicate an extended primer grip of the viral polymerase in sexual RNA replication mechanisms. By sensing RNA sequence complementarity near the active site, the extended primer grip of the polymerase has the potential to distinguish between homologous and non-homologous RNA templates during sexual RNA replication.