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Journal of Virology, November 2005, p. 13963-13973, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.13963-13973.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Robust Production of Infectious Hepatitis C Virus (HCV) from Stably HCV cDNA-Transfected Human Hepatoma Cells

Zhaohui Cai,1 Chen Zhang,1 Kyung-Soo Chang,1 Jieyun Jiang,1 Byung-Chul Ahn,1 Takaji Wakita,2 T. Jake Liang,3 and Guangxiang Luo1*

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, Kentucky 40536-0298,1 Department of Microbiology and Immunology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan,2 Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 208923

Received 3 June 2005/ Accepted 22 August 2005

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5' end and a hepatitis delta virus ribozyme at the 3' end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 108 copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, 800 Rose Street, Lexington, KY 40536-0298. Phone: (859) 257-5577. Fax: (859) 257-8994. E-mail: gluo0{at}uky.edu.

Journal of Virology, November 2005, p. 13963-13973, Vol. 79, No. 22
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.22.13963-13973.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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