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

ABSTRACT: 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.

SUBMITTER: Kempf BJ

PROVIDER: S-EPMC3648189 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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

Kempf Brian J BJ Kelly Michelle M MM Springer Courtney L CL Peersen Olve B OB Barton David J DJ

Journal of virology 20130306 10

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 ident ...[more]

PMID: 23468507

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Project description:Picornaviruses 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 pseudorevertants (L420V and L420I) were selected from all independent lineages of L420A, G64Fix L420A, and G64S L420A parental viruses. Ribavirin resistance G64S mutations were selected in two independent lineages, and novel ribavirin resistance mutations were selected in the polymerase in other lineages (M299I, M323I, M392V, and 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, K375R, and R376K). L420A revertants and pseudorevertants (L420V and L420I) restored efficient viral RNA recombination, confirming that ribavirin-induced error catastrophe coincides with defects in sexual RNA replication mechanisms. Viruses containing M392 mutations (M392A, M392L, and M392V) and primer grip mutations (K375R and 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, and R376, contributes to the fidelity of RNA synthesis and to efficient sexual RNA replication mechanisms.IMPORTANCE Picornaviruses 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 nonhomologous 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 nonhomologous RNA templates during sexual RNA replication.

Dataset Information

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

Publications

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

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