Rate-limiting pyrophosphate release by hepatitis C virus polymerase NS5B improves fidelity

The hepatitis C virus RNA-dependent RNA polymerase NS5B is responsible for the replication of the viral genome. Previous studies have uncovered NTP-mediated excision mechanisms that may be responsible for aiding in maintaining fidelity (the frequency of incorrect incorporation events relative to correct), but little is known about the fidelity of NS5B. In this study, we used transient-state kinetics to examine the mechanistic basis for polymerase fidelity. We observe a wide range of efficiency for incorporation of various mismatched base pairs and have uncovered a mechanism in which the rate constant for pyrophosphate release is slowed for certain misincorporation events. This results in an increase in fidelity against these specific misincorporations. Furthermore, we discover that some mismatches are highly unfavorable and cannot be observed under the conditions used here. The calculated fidelity of NS5B ranges between 10−4–10−9 for different mismatches.