title: An in vitro model of hepatitis C virion production.
abstract: The hepatitis C virus (HCV) is a major cause of liver disease worldwide. The understanding of the viral life cycle has been hampered by the lack of a satisfactory cell culture system. The development of the HCV replicon system has been a major advance, but the system does not produce virions. In this study, we constructed an infectious HCV genotype 1b cDNA between two ribozymes that are designed to generate the exact 5' and 3' ends of HCV. A second construct with a mutation in the active site of the viral RNA-dependent RNA polymerase (RdRp) was generated as a control. The HCV-ribozyme expression construct was transfected into Huh7 cells. Both HCV structural and nonstructural proteins were detected by immunofluorescence and Western blot. RNase protection assays showed positive- and negative-strand HCV RNA. Sequence analysis of the 5' and 3' ends provided further evidence of viral replication. Sucrose density gradient centrifugation of the culture medium revealed colocalization of HCV RNA and structural proteins in a fraction with the density of 1.16 g/ml, the putative density of HCV virions. Electron microscopy showed viral particles of approximately 50 nm in diameter. The level of HCV RNA in the culture medium was as high as 10 million copies per milliliter. The HCV-ribozyme construct with the inactivating mutation in the RdRp did not show evidence of viral replication, assembly, and release. This system supports the production and secretion of high-level HCV virions and extends the repertoire of tools available for the study of HCV biology.
authors.affiliation: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Theo
authors.initials: T
authors.lastname: Heller
authors.forename: Satoru
authors.initials: S
authors.lastname: Saito
authors.forename: Jonathan
authors.initials: J
authors.lastname: Auerbach
authors.forename: Tarice
authors.initials: T
authors.lastname: Williams
authors.forename: Tzivia Rachel
authors.initials: TR
authors.lastname: Moreen
authors.forename: Allison
authors.initials: A
authors.lastname: Jazwinski
authors.forename: Brian
authors.initials: B
authors.lastname: Cruz
authors.forename: Neha
authors.initials: N
authors.lastname: Jeurkar
authors.forename: Ronda
authors.initials: R
authors.lastname: Sapp
authors.forename: Guangxiang
authors.initials: G
authors.lastname: Luo
authors.forename: T Jake
authors.initials: TJ
authors.lastname: Liang
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics; D014771:Virion / Q000235:genetics / Q000502:physiology / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D016337:RNA, Catalytic; D012367:RNA, Viral; D014764:Viral Proteins