HCV cDNA transfection to HepG2 cells.
The establishment of an in vitro system for hepatitis C virus (HCV) propagation is essential to characterize virus replication, virus persistence and viral pathogenicity. The aim was to establish HCV replication in HepG2 cells by gene transfer of infectious HCV cDNA. First, several gene transfer methods were evaluated that employed cationic liposomes (lipofectin, lipofectamine. DOTAP), DEAE-dextran and replication-deficient adenovirus for transfection to HepG2 cells using a lacZ reporter gene. Highest transfection efficiency (20%) of cultured HepG2 cells was obtained by the combined use of lipofectamine and adenovirus. This method was used for transfection of HepG2 cells with HCV cDNA in a mammalian expression plasmid (pRC/CMV). The success and efficacy of HCV transfection to HepG2 cells was evaluated by testing for the presence of genomic and replicative (negative) strands of HCV RNA by strand-specific reverse transcription (RT) followed by nested PCR. Expression of structural and non-structural proteins of hepatitis C virus was detected using polyclonal antibodies to core, NS3, NS4 and NS5. Positive-strand HCV RNA was detected by RT-PCR for over 6 weeks in the HCV cDNA-transfected HepG2 cells. Presence of HCV replication in these cells was confirmed by detecting HCV negative-strand RNA by strand-specific RT-PCR and was observed to continue for over 4 weeks. HCV proteins (core, NS3, NS4 and NS5) were detected in the cytoplasm of the transfected cells by immunostaining. In summary, these findings suggest that replication and translation of HCV were achieved for a prolonged time in HepG2 cells after transfection with HCV cDNA and may provide an in vitro system for HCV studies.
Hiramatsu
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Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, LA 70112, USA.
Dash
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Gerber
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MA
D018076:DNA, Complementary; D004279:DNA, Viral; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D014162:Transfection; D014407:Tumor Cells, Cultured; D014758:Viral Core Proteins / Q000032:analysis; D017361:Viral Nonstructural Proteins / Q000032:analysis; D001616:beta-Galactosidase / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D018076:DNA, Complementary; D004279:DNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D001616:beta-Galactosidase; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C069130:nucleocapsid protein, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase