Triphosphate Re‐orientation of the Incoming Nucleotide as A Fidelity Checkpoint in Viral RNA‐dependent RNA Polymerases

Abstract

The fidelity of the virus‐encoded RNA‐dependent RNA polymerase (RdRp) plays an essential role in generating sufficient genetic diversity to escape the bottlenecks of the host's anti‐viral response. For many nucleic acid polymerases, a pre‐chemistry conformational change is at least partially rate‐limiting and represents a key fidelity checkpoint. There has been disagreement in the polymerase field about the structural rearrangements important in this step. One suggestion has been that this step represents a rearrangement of the triphosphate of the incoming nucleotide that will facilitate nucleophilic attack by the primer 3′‐hydroxyl group. We have now gained insight into the triphosphate re‐orientation in the poliovirus (PV) RdRp via 31P NMR. Modeling studies suggest that Lys167 and Arg174 on structural motif F make important interactions with the triphosphate. 31P NMR results demonstrate that WT RdRp interacts with “correct” nucleotide in a way different from “incorrect” nucleotide; R174K RdRp is unable to rearrange correct nucleotide like WT enzyme and instead interacts with correct nucleotide in a similar fashion as incorrect nucleotide; The R174K substitution also substantially reduces enzyme efficiency and leads to very high fidelity. The K167R variant has similar catalytic efficiency as WT when correct nucleotide is incorporated, however, incorrect nucleotide is more readily incorporated. Altogether these results suggest that triphosphate re‐orientation is an essential fidelity checkpoint and interactions with motif F residues are important for this alignment.The research is supported from Penn State start‐up funds and NIH R01 AI104878 to DDB.