title: Inducible system in human hepatoma cell lines for hepatitis C virus production.
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 nai;ve Huh7 and stable Huh7-human CD81 cells.
authors.affiliation: Collaborative Anti-viral Research Laboratory, Institute of Molecular and Cell Biology, 30 Medical Drive, 117609, Singapore. mcblimsp@imcb.nus.edu.sg
authors.forename: Siew Pheng
authors.initials: SP
authors.lastname: Lim
authors.forename: Hui Meng
authors.initials: HM
authors.lastname: Soo
authors.forename: Yin Hwee
authors.initials: YH
authors.lastname: Tan
authors.forename: Sydney
authors.initials: S
authors.lastname: Brenner
authors.forename: Heinrich
authors.initials: H
authors.lastname: Horstmann
authors.forename: Jason M
authors.initials: JM
authors.lastname: MacKenzie
authors.forename: Mah Lee
authors.initials: ML
authors.lastname: Ng
authors.forename: Seng Gee
authors.initials: SG
authors.lastname: Lim
authors.forename: Wan jin
authors.initials: Wj
authors.lastname: Hong
mesh_terms: D006528:Carcinoma, Hepatocellular; D002499:Centrifugation, Density Gradient; D003470:Culture Media; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D018936:Hepatitis C Antigens / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D012367:RNA, Viral / Q000032:analysis / Q000096:biosynthesis; D013752:Tetracycline; D014162:Transfection; D014407:Tumor Cells, Cultured; D014758:Viral Core Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism; D014771:Virion / Q000302:isolation & purification / Q000648:ultrastructure; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D003470:Culture Media; D018936:Hepatitis C Antigens; D012367:RNA, Viral; D013752:Tetracycline; D014758:Viral Core Proteins; D014759:Viral Envelope Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase