胞质型磷脂酶PLA2G4C对丙型肝炎病毒复制的影响及其机制

The genome replication of Hepatitis C Virus (HCV) takes place in a remodeled intracellular membranous structure known as the "membranous web (MW)" induced by NS4B. Though many host proteins have been identified as critical factors required for this process, the detailed mechanism of MW formation remains obscure, as well as how HCV replication complex transfers to the lipid droplet from MW to start viron assembly. Previous study showed that PLA2G4C, a kind of phospholipase A2 with the membrane remodeling function, is upregulated by HCV infection. Silencing PLA2G4C expression by siRNA inhibits virus replication in HCV subgenomic replicon cells. PLA2G4C was found to localize around the lipid droplet which is an important organelle for virus assembly. According to the preliminary results, the following hypothesis was made. PLA2G4C participates in the formation of membranous web induced by HCV, and may be involved in the assembly process. This project is designed to test this hypothesis. HCVpp system, subgenomic system, pIRES reporter plasmid and HCVcc system will be used to study the role of PLA2G4C in different steps of virus life cycle such as virus enty, RNA replication, protein translation, assembly and release. The questions of whether and how PLA2G4C participates in the formation of membranous web will be answered. The involvement and function of PLA2G4C in replication complex and viron assembly will be investigated. The interaction between PLA2G4C and HCV proteins will be studied by co-immunoprecipitation to explore the mechanism of PLA2G4C functionality. This project will explore the role of PLA2G4C in HCV replication and its mechanism, and may provide a new target for antiviral drug screening.

HCV诱导的膜网结构是病毒基因组复制位点，NS4B及多种细胞因子参与该过程，但其形成机制尚未完全揭示；病毒复制复合体如何转移到脂滴周围完成病毒粒子组装的机制也未阐明。前期研究发现，具有调控膜结构功能的胞质型磷脂酶A2－PLA2G4C在HCV感染后表达上调，而干扰该基因下调HCV复制；发现该蛋白定位于脂滴周围。提示PLA2G4C可能参与HCV诱导的膜网结构形成，脂滴定位可能与病毒包装相关。本项目围绕这一假说利用HCV假病毒、亚复制子体系、pIRES报告质粒、HCVcc体系等研究PLA2G4C对病毒进入、RNA复制、蛋白翻译和包装释放过程的影响；回答其是否参与病毒诱导的膜网结构的形成及其机制；明确其是否病毒复制复合体的组分及在病毒粒子包装中的功能；分析其与HCV蛋白是否有相互作用以揭示其发挥功能的可能机制。该研究将揭示PLA2G4C在HCV复制中的功能及机制，可望为抗病毒药物筛选提供新的靶点。

丙型肝炎病毒（HCV）是一种单股正链RNA病毒，其生活周期的完成需要宿主因子的参与。本项目的研究鉴定并分析了宿主细胞胞质型磷脂酶PLA2G4C参与HCV生活周期的机制。研究发现HCV感染导致宿主细胞PLA2G4C的表达水平上调，其上调是由于多种HCV蛋白的协同作用导致的，其中core蛋白发挥重要作用。利用RNA干扰降低PLA2G4C的表达后发现HCV感染受到明显影响，通过HCV假病毒、pHCV-IRES报告质粒、HCV亚复制子及HCVcc等体系对病毒进入、RNA复制、蛋白翻译和包装释放过程进行研究发现PLA2G4C对HCV病毒的影响发生在RNA复制及病毒粒子的包装步骤。磷脂酶水解甘油磷脂的重要活性产物花生四烯酸对HCV病毒的复制无明显影响，提示PLA2G4C对病毒复制的影响不是通过其水解产物发生的；而电镜及免疫荧光结果显示在感染细胞或亚复制子细胞中降低PLA2G4C的表达可以显著抑制细胞内膜网结构的形成。进一步通过免疫荧光及免疫共沉淀实验发现在病毒感染的细胞中PLA2G4C蛋白与HCV复制复合体成分NS5A、NS4B及双链RNA存在共定位情况，并且可以与NS4B发生蛋白-蛋白相互作用。实验中还发现干扰PLA2G4C表达会导致细胞内脂滴结构减少，而回补PLA2G4C的表达可以回复细胞内脂滴结构同时回复HCV病毒的包装效率。综上，本项目的结果表明PLA2G4C参与HCV病毒生活周期，通过影响病毒诱导膜网结构的形成参与HCV基因组RNA复制，此外可能通过影响脂滴的生成影响HCV病毒粒子的包装。