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|Title:||Inducible system in human hepatoma cell lines for hepatitis C virus production||Authors:||Lim, S.P.
|Keywords:||Hepatitis C virus
Hepatoma cell lines
|Issue Date:||2002||Citation:||Lim, S.P., Soo, H.M., Tan, Y.H., Brenner, S., Horstmann, H., MacKenzie, J.M., Ng, M.L., Lim, S.G., Hong, W.-J. (2002). Inducible system in human hepatoma cell lines for hepatitis C virus production. Virology 303 (1) : 79-99. ScholarBank@NUS Repository. https://doi.org/10.1006/viro.2002.1687||Abstract:||We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naïve Huh7 and stable Huh7-human CD81 cells. © 2002 Elsevier Science (USA).||Source Title:||Virology||URI:||http://scholarbank.nus.edu.sg/handle/10635/131465||ISSN:||00426822||DOI:||10.1006/viro.2002.1687|
|Appears in Collections:||Staff Publications|
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