title: A protocol for analyzing hepatitis C virus replication.
abstract: Hepatitis C Virus (HCV) affects 3% of the world's population and causes serious liver ailments including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.
authors.affiliation: Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center.
authors.forename: Songyang
authors.initials: S
authors.lastname: Ren
authors.affiliation: Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center.
authors.forename: Deisy
authors.initials: D
authors.lastname: Contreras
authors.affiliation: Liver Program at Regenerative Medicine Institute, Department of Biomedical Sciences, Department of Surgery, Cedars-Sinai Medical Center; Department of Surgery, David Geffen School of Medicine at UCLA; arumugaswami@cshs.org.
authors.forename: Vaithilingaraja
authors.initials: V
authors.lastname: Arumugaswami
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000821:virology; D045744:Cell Line, Tumor; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology; D014773:Virology / Q000379:methods*; D014779:Virus Replication / Q000502:physiology*