Assembly and Pre‐steady‐state Kinetic Analysis of the Hepatitis C Virus RNA Polymerase Elongation Complex

Abstract

The synthesis of RNA molecules by HCV RNA polymerase (NS5b) proceeds through two major phases: de novo initiation and elongation. To date, mechanistic studies of HCV polymerase at its elongation phase have been limited by the apparent difficulty to obtain preparations of the polymerase/primer/template elongation complex. The goal of this study was to assemble a functional HCV polymerase elongation complex and characterize the kinetics of nucleotide incorporation catalyzed by the polymerase in its elongation mode. NS5b(Δ21) was used in this study. After initiation from a GG primer and a 20‐nucleotide RNA template, the majority of NS5b(Δ21) was trapped into an elongation complex containing NS5b(Δ21) bound to the 20‐mer template and 9‐mer primer (P9/T20). Active site titration at various protein concentrations showed that more than 80% NS5b was trapped in the elongation complex, which was different from previous reports on HCV polymerase suggesting that only a small proportion of NS5b protein was active. The NS5b(Δ21)/P9/T20 complex was characterized using pre‐steady‐state kinetic methods. Challenging the elongation complex with enzyme trap (heparin or RNA) for up to 21 hr caused little dissociation of the complex, suggesting an extremely slow dissociation rate of the elongation complex. The kinetics of incorporation of CTP opposite GTP and misincorporation of A, U, G opposite GTP catalyzed by NS5b(Δ21) in the elongation complex were measured. The HCV elongation complex will be a valuable tool to study the mechanism of action of NS5b inhibitors.