Hepatitis C virus RNA synthesis in a cell-free system isolated from replicon-containing hepatoma cells. Academic Article uri icon

Overview

abstract

  • 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 degrees C. Heparin sensitivity and nucleotide starvation experiments suggested that de novo initiation was occurring in this system. Both Mn2+ and Mg2+ cations could be used in the reaction; however, concentrations of Mn2+ 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.

publication date

  • February 1, 2003

Research

keywords

  • Hepacivirus
  • RNA, Viral
  • Replicon

Identity

PubMed Central ID

  • PMC140877

Scopus Document Identifier

  • 0037303327

Digital Object Identifier (DOI)

  • 10.1128/jvi.77.3.2029-2037.2003

PubMed ID

  • 12525637

Additional Document Info

volume

  • 77

issue

  • 3