Determinants of RNA-Dependent RNA Polymerase (In)fidelity Revealed by Kinetic Analysis of the Polymerase Encoded by a Foot-and-Mouth Disease Virus Mutant with Reduced Sensitivity to Ribavirin

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Abstract
A mutant poliovirus (PV) encoding a change in its polymerase (3Dpol) at a site remote from the catalytic center (G64S) confers reduced sensitivity to ribavirin and forms a restricted quasispecies, because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouth disease virus (FMDV) mutant that encodes a change in the polymerase catalytic site (M296I) exhibits reduced sensitivity to ribavirin without restricting the viral quasispecies. In order to resolve this apparent paradox, we have established a minimal kinetic mechanism for nucleotide addition by wild-type (WT) FMDV 3Dpol that permits a direct comparison to PV 3Dpol as well as to FMDV 3Dpol derivatives. Rate constants for correct nucleotide addition were on par with those of PV 3Dpol, but apparent binding constants for correct nucleotides were higher than those observed for PV 3Dpol. The A-to-G transition frequency was calculated to be 1/20,000, which is quite similar to that calculated for PV 3Dpol. The analysis of FMDV M296I 3Dpol revealed a decrease in the calculated ribavirin incorporation frequency (1/8,000) relative to that (1/4,000) observed for the WT enzyme. Unexpectedly, the A-to-G transition frequency was higher (1/8,000) than that observed for the WT enzyme. Therefore, FMDV selected a polymerase that increases the frequency of the misincorporation of natural nucleotides while specifically decreasing the frequency of the incorporation of ribavirin nucleotide. These studies provide a mechanistic framework for understanding FMDV 3Dpol structure-function relationships, provide the first direct analysis of the fidelity of FMDV 3Dpol in vitro, identify the β9-α11 loop as a (in)fidelity determinant, and demonstrate that not all ribavirin-resistant mutants will encode high-fidelity polymerases.