Underlying Mechanisms of ApoE in HCV Infection and Assembly

描述(申请人提供)：本申请的总体目标是确定细胞蛋白载脂蛋白E(ApoE)在丙型肝炎病毒(丙型肝炎病毒)感染和体内生产中的重要性，并确定载脂蛋白E在丙型肝炎病毒感染和组装中的潜在分子机制。丙型肝炎病毒慢性感染全球约1.7亿人。在美国，丙型肝炎病毒感染是肝移植最常见的适应症。丙型肝炎病毒也是导致肝细胞癌的主要原因。肝细胞癌是美国增长最快的癌症，每年有超过14,000人死亡。聚乙二醇化干扰素-a联合利巴韦林是治疗丙型肝炎的唯一选择。然而，超过50%感染丙型肝炎病毒的患者对干扰素和利巴韦林治疗没有反应。丙型肝炎病毒1型是感染的主要病毒，占感染的70%。因此，迫切需要开发更有效的抗丙型肝炎病毒药物。识别新的靶点是发现新型抗病毒药物的关键。我们已经证明apoE是丙型肝炎病毒的一个结构成分，在丙型肝炎病毒感染和病毒粒子组装中发挥重要作用。我们还证明了载脂蛋白E与丙型肝炎病毒NS5A相互作用，并且载脂蛋白E-NS5A相互作用对丙型肝炎病毒组装是重要的。更重要的是，我们的初步研究发现，载脂蛋白E而不是丙型肝炎病毒E2介导了丙型肝炎病毒的附着。然而，这些发现来自于对2a型丙型肝炎病毒(JFH1)细胞培养的研究。载脂蛋白E在体内丙型肝炎病毒生命周期中的意义尚未得到实验检验。在特定的目标1中，我们将确定载脂蛋白E在体内丙型肝炎病毒感染和产生中的重要性。在人源化的丙型肝炎小鼠模型中，将使用临床分离的1型丙型肝炎病毒，评估apoE特异性单抗的中和活性和apoE沉默shRNA的治疗效果。在特定的目标2，我们将确定apoE在丙型肝炎病毒感染中的潜在分子机制。对于受体结合至关重要的载脂蛋白E的特定结构域和关键残基将通过突变研究来确定。此外，我们还将确定介导载脂蛋白E结合和丙型肝炎病毒附着的特定细胞表面受体(S)。在具体目标3中，我们将阐明apoE在丙型肝炎病毒组装中的作用机制。具体地说，我们将确定apoE-NS5A相互作用的分子基础，并确定apoE介导的丙型肝炎病毒组装的细胞机制和/或途径。这些具体目标将使用多学科方法成功实现，包括强大的丙型肝炎病毒反向遗传学系统、定点突变、RNA沉默、细胞生物学、免疫学和生化方法。丙型肝炎病毒使用细胞蛋白(ApoE)进行细胞附着是史无前例的，挑战了目前病毒包膜蛋白通过与细胞表面受体结合来介导病毒附着的教条。来自这些研究的新知识将导致关于病毒和细胞蛋白在病毒感染中的作用的范式改变。本申请中描述的研究也将为抗丙型肝炎药物的发现提供新的靶点。

DESCRIPTION (provided by applicant): The overall goal of this application is to determine the importance of the cellular protein apolipoprotein E (apoE) in hepatitis C virus (HCV) infection and production in vivo and to define the underlying molecular mechanism of apoE in HCV infection and assembly. HCV chronically infects approximately 170 million people worldwide. HCV infection is the most common indication for liver transplantation in the United States. HCV is also the major cause of hepatocellular carcinoma (HCC), the most rapidly increasing cancer with more than 14,000 deaths each year in the U.S. Pegylated interferon-a in combination with ribavirin is the only option for treatment of hepatitis C. However, more than 50% patients infected with HCV genotype 1, the dominant virus accounting for ~70% infections, do not respond to IFN and ribavirin therapy. Therefore, there is an urgent need to develop more efficacious anti-HCV drugs. Identification of novel targets is key to discovery of new classes of antiviral drugs. We have demonstrated that apoE is a structural component of HCV and plays important roles in HCV infection and virion assembly. We have also demonstrated that apoE interacts with HCV NS5A and that the apoE-NS5A interaction is important for HCV assembly. More importantly, our preliminary studies found that apoE but not HCV E2 mediates HCV attachment. However, these findings were derived from the studies with a cell culture grown HCV of genotype 2a (JFH1). The significance of apoE in the HCV life cycle in vivo has not been experimentally examined. In specific aim 1, we will determine the importance of apoE in HCV infection and production in vivo. The HCV-neutralizing activity of an apoE-specific monoclonal antibody and the therapeutic efficacy of an apoE-silencing shRNA will be evaluated in a humanized HCV mouse model using clinical HCV isolates of genotype 1. In specific aim 2, we will define the underlying molecular mechanism of apoE in HCV infection. Specific domain and critical residues of apoE important for receptor-binding will be determined by mutagenesis studies. Additionally, we will identify specific cell surface receptor(s) that mediate apoE-binding and HCV attachment. In specific Aim 3, we will illustrate the mechanism of action of apoE in HCV assembly. Specifically, we will determine the molecular basis underlying the apoE-NS5A interaction and identify cellular mechanism and/or pathways responsible for the apoE-mediated HCV assembly. These specific aims will be successfully accomplished using multidisciplinary approaches, including a robust HCV reverse genetics system, site-directed mutagenesis, RNA silencing, cell biological, immunological, and biochemical methodologies. That HCV uses a cellular protein (apoE) for cell attachment is unprecedented and challenges the current dogma that viral envelope proteins mediate virus attachment by binding to cell surface receptors. New knowledge originating from these studies will result in a paradigm change regarding the roles of viral and cellular proteins in virus infection. The studies described in this application will also provide novel targets for anti-HCV drug discovery.