PMID:17400557

Citation	Korneeva, VS and Cameron, CE (2007) 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. J. Biol. Chem. 282:16135-45
Abstract	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.
Links	PubMed PMC2116994 Online version:10.1074/jbc.M610090200
Keywords	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/enzymology; Poliovirus/genetics; RNA Replicase/chemistry; RNA Replicase/genetics; RNA Replicase/metabolism; Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism; Virus Replication/genetics
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
POL1M:POLG	involved_in	GO:0039690: positive stranded viral RNA replication	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

References

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