Please use this identifier to cite or link to this item:
|Title:||Inducible system in human hepatoma cell lines for hepatitis C virus production|
|Keywords:||Hepatitis C virus|
Hepatoma cell lines
|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).|
|Appears in Collections:||Staff Publications|
Show full item record
Files in This Item:
There are no files associated with this item.
checked on Jan 12, 2019
WEB OF SCIENCETM
checked on Jan 2, 2019
checked on Oct 26, 2018
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.