Poliovirus RNA-dependent RNA polymerase (3Dpol): structural, biochemical, and biological analysis of conserved structural motifs A and B

Gohara DW, Crotty S, Arnold JJ, Yoder JD, Andino R, Cameron CE

J. Biol. Chem. 2000 Aug;275(33):25523-32

PMID: 10827187


We have constructed a structural model for poliovirus RNA-dependent RNA polymerase (3D(pol)) in complex with a primer-template (sym/sub) and ATP. Residues found in conserved structural motifs A (Asp-238) and B (Asn-297) are involved in nucleotide selection. Asp-238 appears to couple binding of nucleotides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleoside triphosphates over 2′-dNTPs, a role mediated most likely via a hydrogen bond between the side chain of this residue and the 2′-OH of the ribonucleoside triphosphate. Substitutions at position 238 or 297 of 3D(pol) produced derivatives exhibiting a range of catalytic efficiencies when assayed in vitro for poly(rU) polymerase activity or sym/sub elongation activity. A direct correlation existed between activity on sym/sub and biological phenotypes; a 2.5-fold reduction in polymerase elongation rate produced virus with a temperature-sensitive growth phenotype. These data permit us to propose a detailed, structural model for nucleotide selection by 3D(pol), confirm the biological relevance of the sym/sub system, and provide additional evidence for kinetic coupling between RNA synthesis and subsequent steps in the virus life cycle.

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