title: Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines.
abstract: A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann-Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.
authors.affiliation: Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8544, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Takeda
authors.affiliation: Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8544, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Ikeda
authors.affiliation: Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8544, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ariumi
authors.affiliation: Department of Virology II, National Institute of Infectious Disease, Tokyo 162-8640, Japan.
authors.forename: Takaji
authors.initials: T
authors.lastname: Wakita
authors.affiliation: Department of Tumor Virology, Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8544, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kato
mesh_terms: D000220:Adaptation, Biological; D019943:Amino Acid Substitution; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D016174:Hepacivirus / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D020125:Mutation, Missense; D012367:RNA, Viral / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014776:Virus Cultivation / Q000379:methods; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase