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Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

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Abstract

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08–1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 107 copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.

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Acknowledgments

We are grateful to Dr. Simon Rayner for a critical review of the manuscript. The authors are grateful to professor Ralf Bartenschlager (University of Heidelberg) for providing the Huh7-lunet cell line. The work was supported by National Basic Research Program (2005CB522901).

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Authors and Affiliations

  1. State Key Lab of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, People’s Republic of China

    Xiangjie Yao, Qingxia Han, Jianhua Song, Changyong Liang, Rongge Yang & Xinwen Chen

  2. Graduate University of Chinese Academy of Sciences, Beijing, 10039, People’s Republic of China

    Xiangjie Yao & Qingxia Han

  3. Department of Virology II, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan

    Takaji Wakita

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  1. Xiangjie Yao
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  2. Qingxia Han
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Correspondence to Xinwen Chen.

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Yao, X., Han, Q., Song, J. et al. Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase. Mol Biotechnol 40, 186–194 (2008). https://doi.org/10.1007/s12033-008-9075-2

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