Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase.
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.
Arnold
Jamie J
JJ
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Vignuzzi
Marco
M
Stone
Jeffrey K
JK
Andino
Raul
R
Cameron
Craig E
CE
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D009711:Nucleotides / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics*; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000502:physiology*; D013329:Structure-Activity Relationship
D009711:Nucleotides; D012367:RNA, Viral; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D012321:DNA-Directed RNA Polymerases