Hepatitis C Virus RNA Synthesis in a Cell-Free System Isolated from Replicon-Containing Hepatoma Cells

A number of hepatitis C virus (HCV) proteins, including NS5B, the RNA-dependent RNA polymerase, were detected in membrane fractions from Huh7 cells containing autonomously replicating HCV RNA replicons. These membrane fractions were used in a cell-free system for the analysis of HCV RNA replication. Initial characterization revealed a reaction in which the production of replicon RNA increased over time at temperatures ranging from 25 to 40°C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn(2+) and Mg(2+) cations could be used in the reaction; however, concentrations of Mn(2+) greater than 1 mM were inhibitory. Compounds shown to inhibit recombinant NS3 and NS5B activity in vitro were found to inhibit RNA synthesis in the cell-free system. This system should be useful for biochemical analysis of HCV RNA synthesis by a multisubunit membrane-associated replicase and for evaluating potential antiviral agents identified in biochemical or cell-based screens.