Structure-Function Relationships of the Viral RNA-dependent RNA Polymerase

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 Mg 2؉ , 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.RNA viruses cause a variety of acute and chronic diseases in humans: common cold, summer flu, hepatitis, severe acute respiratory syndrome, and liver cancer, to name a few (1-5). The genomes of these viruses are transcribed and replicated by a virus-encoded RNA-dependent RNA polymerase (RdRp) 2 (4, 6, 7). Like other viral polymerases, the RdRp represents an important target for antiviral drug development (8 -11). The RdRp from poliovirus (PV), 3Dpol, has emerged as an important model for understanding the structure, function, and mechanism of this class of nucleic acid polymerases (12-15).PV 3Dpol will incorporate a ribonucleotide (rNMP) with an incorrect base at a frequency of ϳ10 Ϫ7 to 10 Ϫ4 (16, 17). However, this enzyme is much more tolerant of nucleotides with an incorrect sugar configuration. Both 2Ј-and 3Ј-deoxynucleotides (dNMPs) are incorporated at a frequency of ϳ10 Ϫ2 (15). It is known that incorporation of more than one incorrect ribonucleotide per PV genome decreases the specific infectivity of the RNA (16, 18). Whether or not 2Ј-dNMP incorporation has an effect on viral RNA infectivity is not known.Several factors likely limit 2Ј-dNMP incorporation into the genomes of RNA viruses of eukaryotes. First, dNTP pools in cells are thought to be low, in the 5-30 M range (19). Low dNTP levels are maintained by regulating the activity and localizat...

Picornavirus Genome Replication

“…Importantly, this model explains and predicts biological phenotypes (2,4,5,20). However, VPg uridylylation occurs 10 -50-fold slower in vitro than necessary to support the rate constant (ϳ0.1/s) for initiation calculated from biological data (see Ref.…”

“…Processive uridylylation is likely essential as VPg-pU does not chase into VPg-pUpU in vitro (20). In addition, a highly active 3Dpol derivative that produces much more 4 I. G. Goodfellow, unpublished results.…”

“…Uridylylation of PV VPg occurs processively to form VPgpUpU (20). Processive uridylylation is likely essential as VPg-pU does not chase into VPg-pUpU in vitro (20).…”

“…Both uridylate residues are templated by a single adenylate residue in the oriI loop by using a slideback mechanism (13). VPg-pUpU must occur processively as VPg-pU appears to be catalytically incompetent (20).…”

Changes in Protein Domains outside the Catalytic Site of the Bacteriophage Qβ Replicase Reduce the Mutagenic Effect of 5-Azacytidine

“…Therefore, it is striking that these two substitutions, located in different domains of the Q␤ replicase and outside the catalytic site, gave rise to such similar phenotypes with respect to AZC tolerance, revealing a redundancy that has not been observed in other viral systems. Previous work has demonstrated that the fidelity of RNA virus replicases can be modulated by protein domains located outside the catalytic site (66)(67)(68). For instance, a conformational change consisting of the reorientation of the triphosphate moiety of the incoming nucleotide is a key fidelity checkpoint for the poliovirus replicase (68).…”