title: Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication.
abstract: Replication of infectious hepatitis C virus in Huh7 cells, a human hepatocyte cell line, has become possible due to the unique properties of the JFH1 isolate. Developing reporter virus systems for a simple titration has been attempted by integrating heterologous reporter genes into the JFH1 genome, resulting in a big infectivity reduction that limits the usefulness of such reporter systems. To overcome this problem, JFH1-infected Huh7 cells were cultured continuously for 2 years to obtain Huh7-adapted JFH1 variants capable of yielding up to 1000-fold higher titers. Sequence analysis of variant genome RNA suggested that this adapted population consisted mainly of two variants. By joining the 5'-half of the obtained representative viral complementary DNA (cDNA) fragments of the variants with the 3'-half of the wild-type's, two prototype clones, A/WT and B/WT, were constructed. Replication of A/WT and B/WT viruses in Huh7 cells showed up to 100-1000-fold higher titers than the wild-type. A Renilla luciferase cDNA was inserted into the Nonstructural Protein 5A region of the A/WT and B/WT cDNA to generate A/WT-Rluc and B/WT-Rluc, respectively. Transfection of Huh7 cells with in vitro-transcribed A/WT-Rluc and B/WT-Rluc RNA resulted in production of infectious viruses with approximately 15- and 25-fold higher titers, respectively, than the wild-type RNA. The replication of A/WT-Rluc and B/WT-Rluc viruses was more vigorous than the wild-type even with insertion of the luciferase cDNA showing a good correlation of luciferase activities with infectious titers. Furthermore, interferon-alpha inhibited the replication of A/WT-Rluc and B/WT-Rluc viruses in a dose-dependent manner as determined by a luciferase assay. These results imply that our system is potentially a tool useful for screening anti-hepatitis C virus drugs in a simple and time/cost-saving manner.
authors: China-Japan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology, Chinese Academy of Sciences (CAS), Beijing 100101, China.
authors: Lijuan
authors: L
authors: Yu
authors: Chie
authors: C
authors: Aoki
authors: Yohko
authors: Y
authors: Shimizu
authors: Kazufumi
authors: K
authors: Shimizu
authors: Wei
authors: W
authors: Hou
authors: Fumihiro
authors: F
authors: Yagyu
authors: Xianzi
authors: X
authors: Wen
authors: Masamichi
authors: M
authors: Oshima
authors: Aikichi
authors: A
authors: Iwamoto
authors: Bin
authors: B
authors: Gao
authors: Wenjun
authors: W
authors: Liu
authors: George Fu
authors: GF
authors: Gao
authors: Yoshihiro
authors: Y
authors: Kitamura
mesh_terms: D000220:Adaptation, Biological; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D017930:Genes, Reporter; D016174:Hepacivirus / Q000187:drug effects* / Q000502:physiology*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D017422:Sequence Analysis, DNA; D012692:Serial Passage; D013194:Staining and Labeling / Q000379:methods; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D012367:RNA, Viral; D049410:Luciferases, Renilla