title: Establishment of hepatitis C virus RNA-replicating cell lines possessing ribavirin-resistant phenotype.
abstract: BACKGROUND
  Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells.
  
  
  RESULTS
  By repetitive RBV (100 μM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 μM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor.
  
  
  CONCLUSIONS
  These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Satoh
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Mori
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Youki
authors.initials: Y
authors.lastname: Ueda
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Hiroe
authors.initials: H
authors.lastname: Sejima
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Dansako
authors.affiliation: Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, 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, 2-5-1 Shikata-cho, Okayama 700-8558, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kato
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral; D005814:Genes, Viral; D016174:Hepacivirus / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D010641:Phenotype; D012254:Ribavirin / Q000494:pharmacology*; D014779:Virus Replication
chemical_list: D000998:Antiviral Agents; D012254:Ribavirin