Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication

• Published in: Journal of virological methods 2010-11, Vol.169 (2), p.380-384
• Main Authors: Yu, Lijuan, Aoki, Chie, Shimizu, Yohko, Shimizu, Kazufumi, Hou, Wei, Yagyu, Fumihiro, Wen, Xianzi, Oshima, Masamichi, Iwamoto, Aikichi, Gao, Bin, Liu, Wenjun, Gao, George Fu, Kitamura, Yoshihiro
• Format: Article
• Language: English
• Subjects: Adaptation; Adaptation, Biological; Antiviral Agents - pharmacology; Biological and medical sciences; Cell Line; DNA; Drug Evaluation, Preclinical - methods; Drug screening; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Hepacivirus - drug effects; Hepacivirus - physiology; Hepatitis C virus; Hepatocytes - virology; Humans; Interferon-alpha - pharmacology; Luciferases, Renilla - genetics; Luciferases, Renilla - metabolism; Microbial Sensitivity Tests - methods; Microbiology; Renilla luciferase; Replication; Reporter; RNA, Viral - genetics; Sequence Analysis, DNA; Serial Passage; Staining and Labeling - methods; Techniques used in virology; Virology; Virus Cultivation - methods; Virus Replication - drug effects
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2010.08.009

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.