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

J Biol Chem. 2000 Aug 18;275(33):25523-32. doi: 10.1074/jbc.M002671200.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Monophosphate / metabolism
  • Amino Acid Motifs
  • DNA Primers
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • HIV Reverse Transcriptase / chemistry
  • HeLa Cells
  • Humans
  • Kinetics
  • Ligands
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Phenotype
  • Poliovirus / enzymology*
  • Protein Structure, Tertiary
  • RNA Replicase / chemistry*
  • RNA, Viral / biosynthesis
  • Temperature
  • Time Factors
  • Transfection

Substances

  • DNA Primers
  • DNA, Complementary
  • Ligands
  • RNA, Viral
  • Adenosine Monophosphate
  • RNA Replicase
  • HIV Reverse Transcriptase