Underlying Mechanisms of ApoE in HCV Infection and Assembly

ApoE 在 HCV 感染和组装中的潜在机制

• 批准号: 9011987
• 负责人: GUANGXIANG George LUO
• 金额: $ 36.75万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9011987
• 关键词: Accounting; Antibodies; Antiviral Agents; Apolipoprotein E; Binding; Biochemical; Biological; Biology; Cell Culture Techniques; Cell Surface Receptors; Cell-Matrix Junction; Cells; Cessation of life; Clinical; Clinical Trials; Combined Modality Therapy; Confocal Microscopy; Coupled; Drug resistance; Elements; Genes; Genotype; Glycoproteins; Goals; Hepatitis C; Hepatitis C virus; Hepatocyte; Human; Infection; Interferons; Knowledge; Life Cycle Stages; Liver diseases; Malignant Neoplasms; Mediating; Methodology; Molecular; Monoclonal Antibodies; Mutagenesis; Mutation; N-terminal; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Primary carcinoma of the liver cells; Production; Proteins; RNA; RNA Interference; Research; Ribavirin; Role; SCID Mice; Site-Directed Mutagenesis; Small Interfering RNA; System; Testing; Transplantation; Treatment Efficacy; United States; Viral; Viral Envelope Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; anti-hepatitis C; base; cell attachment protein; clinical efficacy; design; drug discovery; glycosylation; hepatitis C virus envelope 2 protein; in vivo; inhibitor/antagonist; interdisciplinary approach; liver transplantation; mouse model; novel; receptor binding; reverse genetics; small hairpin RNA; synthetic peptide; virology; virus envelope

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.

描述（由申请人提供）：本申请的总体目标是确定细胞蛋白载脂蛋白E（apoE）在体内丙型肝炎病毒（HCV）感染和产生中的重要性，并确定apoE在HCV感染和组装中的潜在分子机制。HCV慢性感染全球约1.7亿人。HCV感染是美国肝移植最常见的适应症。HCV也是肝细胞癌（HCC）的主要原因，肝细胞癌是增长最快的癌症，在美国每年有超过14，000人死亡。聚乙二醇化干扰素-a与利巴韦林组合是治疗丙型肝炎的唯一选择。然而，超过50%的HCV基因1型感染患者（占约70%感染的优势病毒）对IFN和利巴韦林治疗无反应。因此，迫切需要开发更有效的抗HCV药物。新靶点的识别是发现新类别抗病毒药物的关键。我们已经证明，载脂蛋白E是丙型肝炎病毒的结构组成部分，并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apoE与HCV NS 5A相互作用，并且apoE-NS 5A相互作用对于HCV组装是重要的。更重要的是，我们的初步研究发现，apoE而不是HCV E2介导HCV附着。然而，这些发现来自于对基因型2a（JFH 1）的细胞培养生长的HCV的研究。载脂蛋白E在体内HCV生命周期中的重要性尚未得到实验研究。在具体目标1中，我们将确定apoE在体内HCV感染和产生中的重要性。使用基因型1的临床HCV分离株，在人源化HCV小鼠模型中评价apoE特异性单克隆抗体的HCV中和活性和apoE沉默shRNA的治疗功效。在具体目标2中，我们将明确载脂蛋白E在HCV感染中的潜在分子机制。将通过诱变研究确定apoE对受体结合重要的特定结构域和关键残基。此外，我们还将鉴定介导apoE结合和HCV附着的特异性细胞表面受体。在具体目标3中，我们将阐明apoE在HCV组装中的作用机制。具体而言，我们将确定潜在的apoE-NS 5A相互作用的分子基础，并确定负责apoE介导的HCV组装的细胞机制和/或途径。这些具体目标将成功地实现使用多学科的方法，包括一个强大的HCV反向遗传学系统，定点诱变，RNA沉默，细胞生物学，免疫学和生物化学方法。HCV使用细胞蛋白（apoE）进行细胞附着是前所未有的，并挑战了目前的教条，即病毒包膜蛋白通过结合细胞表面受体介导病毒附着。从这些研究中产生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还将为抗HCV药物发现提供新的靶点。