Remote Site Control of an Active Site Fidelity Checkpoint in a ViralRNA-dependent RNAPolymerase

Jamie J Arnold,Marco Vignuzzi,Jeffrey K Stone,Raul Andino,Craig E Cameron

doi:10.1074/jbc.m503444200

Abstract

The kinetic, thermodynamic, and structural basis for fidelity of nucleic acid polymerases remains controversial. An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness. Here we show that a conformational change step preceding phosphoryl transfer is a key fidelity checkpoint for the poliovirus RdRp (3Dpol). We provide evidence that this conformational change step is orientation of the triphosphate into a conformation suitable for catalysis, suggesting a kinetic and structural model for RdRp fidelity that can be extrapolated to other classes of nucleic acid polymerases. Finally, we show that a site remote from the catalytic center can control this checkpoint, which occurs at the active site. Importantly, similar connections between a remote site and the active site exist in a wide variety of viral RdRps. The capacity for sites remote from the catalytic center to alter fidelity suggests new possibilities for targeting the viral RdRp for antiviral drug development.

Highlights

During each cycle of nucleotide incorporation, nucleic acid polymerases are presented with the challenge of having to select a nucleotide with the correct sugar configuration and the correct base
An understanding of viral RNA-dependent RNA polymerase (RdRp) fidelity has become a topic of considerable interest as a result of recent experiments that show that a 2-fold increase in fidelity attenuates viral pathogenesis and a 2-fold decrease in fidelity reduces viral fitness
Studies with PV have shown that the mechanism of action of ribavirin, a clinically useful antiviral nucleoside, is lethal mutagenesis [24]

Summary

The abbreviations used are

RdRp, RNA-dependent RNA polymerase; PV, poliovirus; WT, wild-type; RTP, ribavirin triphosphate; T7 Rp, bacteriophage T7 RNA polymerase; NV, Norwalk virus; HCV, hepatitis C virus; nt, nucleotide(s); AMP␣S, adenosine 5Ј-O-(thiomonophosphate); RMP, ribavirin monophosphate. The active site, the backbone of this residue is connected via hydrogen bonding to the conserved structural motif A that functions, in part, to hold the triphosphate of the incoming nucleotide in a conformation appropriate for catalysis. Similar connections between remote sites and motif A exist in polymerases from a wide variety of RNA viruses. Together, these data provide genetic evidence for a role of the first conformational change step in polymerase fidelity, provide a link between the conformational change and the orientation of the triphosphate of the incoming nucleotide, and define sites remote from the polymerase active site as targets for development of antiviral agents capable of functioning by modulating nucleotide incorporation fidelity. The conclusions of this study regarding fidelity mechanisms employed by the RdRp are germane to all classes of nucleic acid polymerases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION