The study of molecular mechanism of hepatitis C virusproduction

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Development of HCV replicon cells and HCV production system using HCV JFH1 strain enabled us to investigate the whole life cycle of HCV and the host factors essential for HCV production. In this study, we aimed to clarify molecular mechanism of HCV production by investigating binding partners for HCV proteins. We analyzed HCV core-binding proteins, hnRNPH1/H2/F. Addition of HCV RNA inhibited the binding between the core protein and hnRNPs. RNase recovered this inhibition. Theseresults suggest that RNA is required for the interaction between the core protein and hnRNPs. Our results suggest that these hnRNPH and hnRNPF cooperatively regulate HCV replication. We then identified HCV NS5A-binding proteins, ubiquitin ligase p138, transcription factor HNF-1a, and histone methyltransferase SMYD3. The p138 protein bound NS5A protein, but not other HCV proteins. Our data suggest that p138 protein promotes ubiquitylation of NS5A protein and enhances its proteasomal degradation, suggesting that p138 regulates HCV replication. We analyzed the p138-binding domain on the NS5A protein using a series of deletion mutants. Then weanalyzed the ubiquitylation site on the NS5A protein using 13 point-mutants. These studies would contribute to the better understanding of HCV life cycles as well as the viral pathogenesis.