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HCV cDNA transfection to HepG2 cells

N Hiramatsu1, S Dash, M A Gerber

  • 1Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA.

Journal of Viral Hepatitis
|January 1, 1997
PubMed
Summary

Researchers established a new in vitro system for hepatitis C virus (HCV) propagation in HepG2 cells. This system enables prolonged study of HCV replication and protein expression, crucial for understanding viral pathogenicity.

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Area of Science:

  • Hepatology and Virology
  • Molecular Biology
  • Cell Culture Systems

Background:

  • Establishing an in vitro system for hepatitis C virus (HCV) propagation is crucial for studying viral replication, persistence, and pathogenicity.
  • HepG2 cells offer a potential model for HCV research, but efficient gene transfer methods are needed.

Purpose of the Study:

  • To establish a functional in vitro system for hepatitis C virus (HCV) replication in HepG2 cells.
  • To optimize gene transfer methods for introducing infectious HCV cDNA into HepG2 cells.
  • To characterize the duration and efficacy of HCV replication and protein expression in the established cell system.

Main Methods:

  • Evaluated various gene transfer methods including cationic liposomes (lipofectin, lipofectamine, DOTAP), DEAE-dextran, and replication-deficient adenovirus for transfecting HepG2 cells with a lacZ reporter gene.

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  • Utilized the most efficient method (lipofectamine and adenovirus combination) to transfect HepG2 cells with HCV cDNA.
  • Assessed HCV RNA (genomic and negative strands) presence using strand-specific reverse transcription (RT) followed by nested PCR.
  • Detected HCV proteins (core, NS3, NS4, NS5) via immunostaining with polyclonal antibodies.

    • Main Results:

    • The combination of lipofectamine and adenovirus achieved the highest transfection efficiency (20%) in HepG2 cells.
    • Positive-strand HCV RNA was detected for over 6 weeks post-transfection.
    • HCV replication, confirmed by the presence of negative-strand RNA, persisted for over 4 weeks.
    • HCV proteins (core, NS3, NS4, NS5) were successfully detected in the cytoplasm of transfected HepG2 cells.

    Conclusions:

    • Successful and prolonged replication and translation of hepatitis C virus (HCV) were achieved in HepG2 cells following HCV cDNA transfection.
    • This optimized in vitro system provides a valuable tool for extensive studies on HCV replication, persistence, and pathogenicity.
    • The findings pave the way for further research into antiviral therapies and mechanisms of HCV infection.