Structure-function relationships of the viral RNA-dependent RNA polymerase: fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket

• Published in: The Journal of biological chemistry 2007-06, Vol.282 (22), p.16135-16145
• Main Authors: Korneeva, Victoria S, Cameron, Craig E
• Format: Article
• Language: English
• Subjects: Animals; Asparagine - chemistry; Asparagine - genetics; Asparagine - metabolism; Bacteriophages - enzymology; Bacteriophages - genetics; Binding Sites - genetics; Glutamic Acid - chemistry; Glutamic Acid - genetics; Glutamic Acid - metabolism; HeLa Cells; Humans; Kinetics; Mutation; Nucleotides - chemistry; Nucleotides - metabolism; Poliovirus; Poliovirus - enzymology; Poliovirus - genetics; RNA-Dependent RNA Polymerase - chemistry; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M610090200

Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2'-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg2+, the apparent affinity of Glu-297 3Dpol for 2'-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2'-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4- to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.