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Journal of Virology, February 2003, p. 2029-2037, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.2029-2037.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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

Richard W. Hardy,1,{dagger} Joseph Marcotrigiano,2 Keril J. Blight,2 John E. Majors,1 and Charles M. Rice2*

Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110,1 Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, New York 100212

Received 17 June 2002/ Accepted 6 November 2002

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 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.

* Corresponding author. Mailing address: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

{dagger} Present address: Department of Biology, Indiana University, Bloomington, IN 47405.

Journal of Virology, February 2003, p. 2029-2037, Vol. 77, No. 3
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.3.2029-2037.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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