The study of molecular mechanism of hepatitis C virusproduction

利用丙型肝炎病毒JFH1株建立了丙型肝炎病毒复制子细胞和丙型肝炎病毒生产系统，使我们能够研究丙型肝炎病毒的整个生命周期和产生丙型肝炎病毒所必需的宿主因素。在这项研究中，我们旨在通过研究丙型肝炎病毒蛋白的结合伙伴来阐明丙型肝炎病毒产生的分子机制。我们分析了丙型肝炎病毒核心结合蛋白hnRNPH1/H2/F。加入丙型肝炎病毒RNA抑制了核心蛋白与hnRNPs的结合。核糖核酸酶恢复了这种抑制作用。这些结果表明，核心蛋白和hnRNPs之间的相互作用需要RNA。我们的结果表明，这些hnRNPH和hnRNPF协同调节丙型肝炎病毒复制。然后我们鉴定了丙型肝炎病毒NS5A结合蛋白、泛素连接酶p138、转录因子HNF-1a和组蛋白甲基转移酶SMYD3。P138蛋白与NS5A蛋白结合，但不与其他丙型肝炎病毒蛋白结合。我们的数据表明，p138蛋白促进了NS5A蛋白的泛素化，并增强了其蛋白酶体的降解，这表明p138蛋白调控着丙型肝炎病毒的复制。我们利用一系列缺失突变体分析了NS5A蛋白上的p138结合域。然后我们利用13个点突变对NS5A蛋白的泛素化位点进行了分析。这些研究将有助于更好地了解丙型肝炎病毒的生命周期以及病毒的致病机制。

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.