Please use this identifier to cite or link to this item: https://doi.org/10.1006/viro.2002.1687
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dc.titleInducible system in human hepatoma cell lines for hepatitis C virus production
dc.contributor.authorLim, S.P.
dc.contributor.authorSoo, H.M.
dc.contributor.authorTan, Y.H.
dc.contributor.authorBrenner, S.
dc.contributor.authorHorstmann, H.
dc.contributor.authorMacKenzie, J.M.
dc.contributor.authorNg, M.L.
dc.contributor.authorLim, S.G.
dc.contributor.authorHong, W.-J.
dc.date.accessioned2016-11-28T10:20:34Z
dc.date.available2016-11-28T10:20:34Z
dc.date.issued2002
dc.identifier.citationLim, 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
dc.identifier.issn00426822
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/131465
dc.description.abstractWe 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).
dc.description.urihttp://libproxy1.nus.edu.sg/login?url=http://dx.doi.org/10.1006/viro.2002.1687
dc.sourceScopus
dc.subjectHepatitis C virus
dc.subjectHepatoma cell lines
dc.subjectTetracycline-inducible
dc.typeArticle
dc.contributor.departmentBIOLOGICAL SCIENCES
dc.contributor.departmentDEAN'S OFFICE (MEDICINE)
dc.contributor.departmentMICROBIOLOGY
dc.contributor.departmentMEDICINE
dc.contributor.departmentBIOCHEMISTRY
dc.description.doi10.1006/viro.2002.1687
dc.description.sourcetitleVirology
dc.description.volume303
dc.description.issue1
dc.description.page79-99
dc.description.codenVIRLA
dc.identifier.isiut000179828800008
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