原钙黏蛋白β5介导丙型肝炎病毒入胞的作用及机制研究

Hepatitis C virus (HCV) infection represents a major public health threat with a world-wide 170 million peoples infected and no vaccine available for prevention. Viral entry represents a promising target for antiviral intervention. More recently, a cell culture system that supports the production and propagation of infectious HCV in cell culture (HCVcc) has been developed. Several host cell receptors have been proposed to be involved in the viral entry. .Our preliminary studies discovered protocadherin beta 5 (PCDHB5), a cellular adhesion molecule, as a cellular co-factor for HCV entry. In this proposal, we plan to further discover the role of PCDHB5 in HCV entry, and generate a neutralizing monoclonal antibody for future in vivo studies to address the feasibility of an antibody therapeutic..First, the functional domains of PCDHB5 involved in conferring HCV infectivity and E2 binding will be mapped. The specific interaction of PCDHB5 with HCV E2 may be part of the mechanism for its ability to confer infectivity. It would be important to identify specific regions of the respective proteins involved or the nature of the interactions, as well as to determine if such interaction can induce down-stream signaling that is important for virus infection. Different truncated PCDHB5 expression plasmids will be constructed and the method of co-immunoprecipitation will be performed. .Second, the potential interactions of PCDHB5 and other HCV co-receptors SRB1, CD81, CLDN1 and OCLD will be determined. There are several receptors identified mediating HCV viral entry. However, the exact roles of these receptors in different steps of viral entry pathway, as well as the contribution of complex receptor interactions in viral entry, are still unclear. It is believed that the interactions among different HCV co-receptors are involved in facilitating virus binding and internalization. Thus, we propose to investigate the potential interaction of PCDHB5 with the other well-known HCV co-receptors SRB1, CD81, CLDN1 and OCLD. The studies will be carried out in 293T or HepG2 cells with transiently transfected PCDHB5 and one of the known co-receptors and co-immunoprecipitation will be performed. .Last, the feasibility of potential antibody therapeutics for HCV will be tested. Monoclonal antibodies generation using mouse hybridoma will be made and the neutralizing activity of these antibodies on HCV viral infection will be tested using HCVcc and HCVpp..The identification of novel co-factor for HCV entry is useful to explore HCV life cycle and pathogenesis, and may provide a new target for anti-HCV infection.

全球HCV感染者约为1.7亿，目前尚无预防用疫苗,因此HCV感染是日益严重的公共卫生问题。HCV入胞相关宿主因子是潜在药物靶点。近年来随着HCV体外细胞培养体系的突破性进展，多个参与HCV入胞的宿主因子的发现使HCV入胞过程逐渐清晰，但尚未完全明了。本课题前期工作发现细胞黏附分子- - 原钙黏蛋白β5（PCDHB5）可介导HCV的入胞过程。本申请拟构建不同截短长度PCDHB5表达质粒，研究PCDHB5与HCV E2的结合功能域；分别共转染PCDHB5和已知HCV入胞相关分子CD81/SR-B1/CLDN1/OCLN的表达质粒，用免疫共沉淀研究PCDHB5与这些相关分子的相互作用；制备抗PCDHB5单克隆抗体，筛选具有抑制不同基因型HCV入胞功能的高亲和力的中和抗体。深入研究PCDHB5介导HCV入胞作用及机制，以进一步解析HCV生活周期，为全面阐明HCV致病机制和研制新的防治策略提供理论依据。

丙型肝炎病毒(HCV)是引起病毒性肝炎的主要病原体之一，全球约有1.7亿感染者。约80%感染者将发展为慢性丙型肝炎，并最终进展为肝硬化和肝细胞癌，且丙型肝炎尚无疫苗可供预防，因此已成为全球性的公共卫生问题。.HCV入胞相关的宿主因子可以作为防治HCV感染的药物靶点。近年来随着HCV体外细胞培养体系的突破性进展，越来越多的研究表明HCV入胞是一个缓慢而又复杂的多级过程。.本课题组发现不同基因型的HCV pseudoparticles (HCVpp) 可以感染非肝细胞来源的神经母细胞瘤细胞系SKNMC。用基因芯片分析了HCV受纳细胞和HCV非受纳细胞的基因表达情况，筛选到23个表达差异的细胞膜蛋白，包括原钙黏蛋白β5（PCDHB5）和CLDN1等分子。在一系列HCV非受纳细胞过表达PCDHB5可以使这些细胞系感染HCV；siRNA干扰技术沉默PCDHB5基因表达后HCV感染率下降；抗PCDHB5多克隆抗体具有拮抗HCV感染的效应；免疫共沉淀实验证实PCDHB5可特异性结合HCV E2区域。.本课题进一步利用体外表达的PCDHB5和HCV包膜蛋白研究了PCDHB5与HCV包膜蛋白的结合作用，以及HCVcc培养系统感染HCV后行co-IP，多角度证实了PCDHB5可结合HCV E2。.PCDHB5分子有5个胞外功能域（N端）和1个胞内区（C端），C-tail有可变区域和保守区域，我们构建了不同截短的PCDHB5表达质粒：（1）不同C端截短；（2）不同N端截短；（3）不同C-tail截短；（4）糖基化位点的5个突变体。结果显示，C端缺失和N端缺失的PCDHB5丧失了介导HCV入胞的能力；C-tail保守区域缺失的PCDHB5丧失了功能，而可变区域缺失则无明显影响；糖基化位点突变对PCDHB5功能无明显影响。.我们研究了PCDHB5和CD81、SR-B1和CLDN1等分子的相互作用。结果显示，PCDHB5可结合CLDN1，而不依赖CD81和SR-B1。.本课题用编码PCDHB5全序列的真核表达质粒作为免疫原，制备了PCDHB5的单克隆抗体，获得了5株单克隆抗体。HCVcc细胞模型筛选到其中一株单克隆抗体具有抑制HCV感染细胞的作用。.以上研究成果对深入研究PCDHB5介导HCV入胞作用及机制，进一步解析HCV生活周期以及为全面阐明HCV致病机制和研制新的防治策略具有重要意义。