title: Cell-based models of sustained, interferon-sensitive hepatitis C virus genotype 1 replication.
abstract: We have previously reported hepatitis C virus (HCV) replication using a novel binary expression system in which mammalian cells were transfected with a T7 polymerase-driven full-length genotype 1a HCV cDNA plasmid (pT7-flHCV-Rz) and infected with vaccinia-T7 polymerase. We hypothesized that the use of replication-defective adenoviral vectors expressing T7 (Ad-T7pol) or cell lines stably transfected with T7 (Huh-T7) would alleviate cell toxicity and allow for more sustained HCV replication. CV-1, Huh7, and Huh-T7 cells were transfected with pT7-flHCV-Rz and treated with Ad-T7pol (CV-1 and Huh7 only). Protein and RNA were harvested from cells on days 1, 2, 3, 5, 7, and 9 post-infection. No cytotoxicity was observed at 9 days post-infection in any cell type. HCV positive- and negative-strand RNA expression were strongest during days 1-3 post-infection; however, HCV RNA remained detectable throughout the 9-day observation period. Furthermore, transfection with a replication-incompetent plasmid suggested that efficient HCV replication is dependent upon NS5B gene expression. Finally, after 1-2 days of IFN treatment, HCV positive-strand levels decreased significantly compared to HCV-infected but untreated samples (p<0.05). In conclusion, these refined binary systems offer more durable and authentic models for identification of host cellular processes critical to HCV replication and will permit longer-term analysis of virus-host interactions critical to HCV pathogenesis and the treatment of genotype 1 infections.
authors.affiliation: Gastrointestinal Unit, Massachusetts General Hospital and Harvard Medical School, GRJ 816, 55 Fruit Street, Boston, MA 02114, USA.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Hiasa
authors.forename: Jason T
authors.initials: JT
authors.lastname: Blackard
authors.forename: Wenyu
authors.initials: W
authors.lastname: Lin
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kamegaya
authors.forename: Norio
authors.initials: N
authors.lastname: Horiike
authors.forename: Morikazu
authors.initials: M
authors.lastname: Onji
authors.forename: Emmett V
authors.initials: EV
authors.lastname: Schmidt
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Chung
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D012367:RNA, Viral / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication / Q000187:drug effects; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D007372:Interferons; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase