Abstract
Hepatitis C virus (HCV) RNA-dependent RNA polymerase replicates the viral genomic RNA and is a primary drug target for antiviral therapy. Previously, we described the purification of an active and stable polymerase-primer-template elongation complex. Here, we show that, unexpectedly, the polymerase elongation complex can use NTPs to excise the terminal nucleotide in nascent RNA. Mismatched ATP, UTP, or CTP could mediate excision of 3'-terminal CMP to generate the dinucleoside tetraphosphate products Ap(4)C, Up(4)C, and Cp(4)C, respectively. Pre-steady-state kinetic studies showed that the efficiency of NTP-mediated excision was highest with ATP. A chain-terminating inhibitor, 3'deoxy-CMP, could also be excised through this mechanism, suggesting important implications for nucleoside drug potency and resistance. The nucleotide excision reaction catalyzed by recombinant hepatitis C virus polymerase was 100-fold more efficient than the corresponding reaction observed with HIV reverse transcriptase.
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