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Journal of Virology, December 2002, p. 13001-13014, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.13001-13014.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Highly Permissive Cell Lines for Subgenomic and Genomic Hepatitis C Virus RNA Replication

Keril J. Blight,* Jane A. McKeating, and Charles M. Rice*

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021

Received 16 April 2002/ Accepted 9 September 2002

Hepatitis C virus (HCV) replication appears to be restricted to the human hepatoma cell line Huh-7, indicating that a favorable cellular environment exists within these cells. Although adaptive mutations in the HCV nonstructural proteins typically enhance the replicative capacity of subgenomic replicons in Huh-7 cells, replication can only be detected in a subpopulation of these cells. Here we show that self-replicating subgenomic RNA could be eliminated from Huh-7 clones by prolonged treatment with alpha interferon (IFN-{alpha}) and that a higher frequency of cured cells could support both subgenomic and full-length HCV replication. The increased permissiveness of one of the cured cell lines allowed us to readily detect HCV RNA and antigens early after RNA transfection, eliminating the need for selection of replication-positive cells. We also demonstrate that a single amino acid substitution in NS5A is sufficient for establishing HCV replication in a majority of cured cells and that the major phosphate acceptor site of subtype 1b NS5A is not essential for HCV replication.

* Corresponding author. Present address for Keril J. Blight: Department of Molecular Microbiology, Center for Infectious Disease Research, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8230, St. Louis, MO 63110. Phone: (314) 286-0065. Fax: (314) 362-1232. E-mail: blight{at}borcim.wustl.edu. Mailing address for Charles M. Rice: Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

Journal of Virology, December 2002, p. 13001-13014, Vol. 76, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.24.13001-13014.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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