Viral and Cellular Determinants of HCV Assembly

描述（由申请人提供）：丙型肝炎病毒（HCV）感染仍然是公共卫生的主要负担，影响全球约1.7亿人和3-4百万美国人。hcv相关终末期肝病是肝移植的主要指标。目前的标准治疗是聚乙二醇干扰素？与利巴韦林联合使用仅能达到有限（<50%）的抗病毒反应，并引起严重的副作用。目前临床试验中的丙型肝炎病毒蛋白酶和聚合酶特异性抑制剂很有希望，但由于丙型肝炎病毒耐药突变体的迅速出现而受到损害。未来丙型肝炎的抗病毒治疗可能需要针对丙型肝炎病毒生命周期不同阶段的几种药物的联合治疗。缺乏对HCV生命周期的分子细节的了解严重阻碍了针对HCV感染的抗病毒药物的发现和开发。更全面地了解病毒和细胞蛋白在HCV生命周期中的作用将为抗HCV药物的发现提供额外的新靶点。我们最近的研究表明，人类载脂蛋白E （apoE）是HCV感染和组装结果的重要决定因素。在我们的初步研究中也发现ApoE与HCV NS5A相互作用。在Specific Aim 1中，我们将剖析apoE结构域和在HCV感染和组装中具有双重功能的氨基酸残基。我们还将分别确定apoE及其与NS5A相互作用在HCV感染和组装中的重要性和潜在的分子机制。在初步研究中，我们已经证明HCV非结构（NS）蛋白的适应性突变极大地增强了HCV的产生和细胞致病性。我们还发现HCV NS蛋白与病毒结构蛋白相互作用，通过哺乳动物双杂交系统和共免疫沉淀实验证实了这一点。与我们的发现一致，最近由其他人完成的遗传学研究表明，HCV NS蛋白在HCV组装和/或生产中起重要作用。然而，HCV NS蛋白在HCV生命周期中的潜在分子机制尚未明确。在特异性目标2中，我们将解读细胞培养适应性突变在HCV组装和诱导细胞致病性中的作用和作用机制。在特异性目标3中，我们将确定病毒NS蛋白的重要性及其与HCV组装中的结构蛋白的相互作用。我们将利用我们实验室最近开发的强大的HCV反向遗传系统，结合诱变研究和生化、免疫学和细胞生物学方法来实现这些特定目标。从这些研究中获得的新知识将导致关于病毒NS蛋白在HCV组装中的作用的病毒学范式转变。我们的研究也将为发现有效治疗丙型肝炎的抗病毒药物提供新的靶点。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection continues to be a major burden on public health, affecting approximately 170 million people worldwide and 3-4 million Americans. HCV-associated end-stage liver disease is the leading indicator of liver transplantation. Current standard therapy with pegylated interferon-? in combination with ribavirin only achieves limited (<50%) antiviral response and causes severe side effects. The HCV protease- and polymerase-specific inhibitors currently in clinical trials are promising but are undermined by rapid emergence of drug-resistant HCV mutants. Future antiviral therapy for hepatitis C likely requires a combination of several drugs targeting different steps of the HCV life cycle. The lack of knowledge about the molecular details of the HCV life cycle has significantly impeded the discovery and development of antiviral drugs against HCV infection. A more complete understanding of the roles of viral and cellular proteins in the HCV life cycle will provide additional novel targets for anti-HCV drug discovery. Our recent studies have demonstrated that human apolipoprotein E (apoE) is an important determinant for the outcomes of HCV infection and assembly. ApoE was also found to interact with the HCV NS5A in our preliminary studies. In Specific Aim 1, we will dissect apoE domains and amino acid residues important for its dual functions in HCV infection and assembly. We will also determine the importance and the underlying molecular mechanisms of apoE and its interaction with NS5A in HCV infection and assembly, respectively. In preliminary studies, we have demonstrated that adaptive mutations in the HCV non-structural (NS) proteins greatly enhanced HCV production and cytopathogenicity in cell culture. We have also found that HCV NS proteins interact with viral structural proteins, as demonstrated by experiments with the mammalian two-hybrid system and co-immunoprecipitation assay. Consistent with our findings, recent genetic studies done by others suggest that HCV NS proteins play important roles in HCV assembly and/or production. However, the underlying molecular mechanisms of the HCV NS proteins in the HCV life cycle have not been defined. In Specific Aim 2, we will decipher the roles and mechanisms of action of cell culture adaptive mutations in HCV assembly and induction of cytopathogenicity. In Specific Aim 3, we will determine the importance of viral NS proteins and their interactions with structural proteins in HCV assembly. We will accomplish these specific aims using a robust HCV reverse genetics system recently developed in our lab in conjunction with mutagenesis studies and the use of biochemical, immunological, and cell biological approaches. New knowledge derived from these studies will lead to a paradigm shift in virology with respect to the roles of viral NS proteins in HCV assembly. Our studies will also provide novel targets for discovery of antiviral drugs to effectively treat hepatitis C.