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

Abstract

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 which 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.

Pyrophosphate release acts as a kinetic checkpoint during high-fidelity DNA replication by the Staphylococcus aureus replicative polymerase PolC

“…HIV-RT demonstrates slow PP i release during both nucleotide misincorporation and processive synthesis in the presence of all four cognate nucleotides when utilizing an RNA template ( 37 ). HCV NS5B has demonstrated reversible chemistry and slow PP i release during nucleotide misincorporation ( 47 ). Recently, T7 DNA polymerase was shown to be partially limited by slow PP i release at low temperature ( 7 ).…”

“…Additionally, reversibility of chemistry due to the rate-limiting release of PP i , would enhance discrimination of incorrectly paired or non-cognate base pairs at the polymerase active site. This has been demonstrated as a mechanism for increasing fidelity by HIV-RT and HCV NS5B ( 37 , 47 ). Moreover, the polymerase could sense DNA damage before committing to replicate the damaged DNA if the damaged base fails to pair with the next incoming nucleotide.…”

Hepatitis C Viral Replication Complex

“…First among them is the mutation rate of NS5B polymerase. As mentioned earlier, the calculated fidelity of NS5B is between 10 −4 and 10 −9 [ 113 ]. The second is the replication rate of the virus.…”

“…However, NS5B has also been reported to have a nucleotide excision mechanism, which may allow limited error correction [ 112 ]. Indeed, a recent report showed that the calculated fidelity of NS5B ranges between 10 −4 and 10 −9 for different mismatches [ 113 ].…”

Trapping a non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase

“…93 Motif F can be linked to fidelity control as well since it guides the nucleotide into the tunnel as well as directs the pyrophosphate out of the tunnel after catalysis, which can be rate limiting in NS5B to improve the fidelity. 95 In the ESI, † Table S5, we provide a summary of motifs A-G and sequence alignments for various RdRps in comparison (from SARS-CoV-2, PV, EV, and HCV).…”