Poliovirus RNA-dependent RNA polymerase (3D(pol)) - Structural, biochemical, and biological analysis of conserved structural motifs A and B

We have constructed a structural model for poliovirus RNA-dependent RNA pol ymerase (3D(pol)) in complex with a primer-template (sym/sub) and ATP. Resi dues found in conserved structural motifs A (Asp-238) and B (Asn-297) are i nvolved in nucleotide selection. Asp-238 appears to couple binding of nucle otides with the correct sugar configuration to catalytic efficiency at the active site of the enzyme. Asn-297 is involved in selection of ribonucleosi de 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 ribonucleo side triphosphate. Substitutions at position 238 or 297 of 3D(pol) produced derivatives exhibiting a range of catalytic efficiencies when assayed in v itro for poly(rU) polymerase activity or sym/sub elongation activity. A dir ect correlation existed between activity on sym/sub and biological phenotyp es; 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 th e biological relevance of the sym/sub system, and provide additional eviden ce for kinetic coupling between RNA synthesis and subsequent steps in the v irus life cycle.