Viral and Cellular Determinants of HCV Assembly

HCV 组装的病毒和细胞决定因素

• 批准号: 8465174
• 负责人: GUANGXIANG George LUO
• 金额: $ 34.51万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465174
• 关键词: Address; Adverse effects; Affect; American; Amino Acids; Antiviral Agents; Antiviral Response; Antiviral Therapy; Apolipoprotein E; Binding; Biochemical; Biological; Biological Assay; Cell Culture System; Cell Culture Techniques; Cells; Cessation of life; Cirrhosis; Clinical Trials; Co-Immunoprecipitations; Combined Modality Therapy; Development; Drug Targeting; Drug resistance; Ectopic Expression; Future; Genetic; Genome; Genotype; Goals; HCV Vaccine; Hepatitis; Hepatitis C; Hepatitis C Therapy; Hepatitis C virus; Human; Infectious hepatitides; Interferons; Knowledge; Lead; Life Cycle Stages; Liver diseases; Molecular; Mutagenesis; Mutation; Outcome; Peptide Hydrolases; Play; Polymerase; Primary carcinoma of the liver cells; Production; Protein Isoforms; Proteins; Public Health; RNA Sequences; RNA replication; Research; Research Design; Ribavirin; Role; Sequence Analysis; Staging; Structural Protein; Structure; System; United States; Variant; Viral; Viral Nonstructural Proteins; Viral Proteins; Viral Structural Proteins; Virion; Virus; Virus Assembly; anti-hepatitis C; base; drug development; drug discovery; inhibitor/antagonist; liver transplantation; mutant; novel; positional cloning; research study; viral RNA; virology; yeast two hybrid system

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.

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相关的终末期肝病是肝移植的主要指标。目前的标准治疗与聚乙二醇干扰素-？与利巴韦林组合仅实现有限的（<50%）抗病毒应答并引起严重的副作用。目前在临床试验中的HCV蛋白酶和聚合酶特异性抑制剂是有希望的，但由于HCV耐药突变体的迅速出现而受到破坏。丙型肝炎的未来抗病毒治疗可能需要针对HCV生命周期不同阶段的几种药物的组合。缺乏对HCV生命周期分子细节的了解，严重阻碍了抗HCV感染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，人载脂蛋白E（apoE）是一个重要的决定因素，丙型肝炎病毒感染和组装的结果。在我们的初步研究中还发现ApoE与HCV NS 5A相互作用。在具体目标1，我们将剖析载脂蛋白E结构域和氨基酸残基的重要性，其双重功能，在丙型肝炎病毒感染和组装。我们还将确定的重要性和潜在的分子机制，载脂蛋白E及其相互作用与NS 5A在HCV感染和组装，分别。在初步研究中，我们已经证明，适应性突变的HCV非结构（NS）蛋白大大提高了HCV的生产和细胞培养中的致细胞病变性。我们还发现HCV NS蛋白与病毒结构蛋白相互作用，如哺乳动物双杂交系统和免疫共沉淀试验所示。与我们的研究结果一致，最近的遗传学研究表明，HCV NS蛋白在HCV组装和/或生产中发挥重要作用。然而，HCV NS蛋白在HCV生命周期中的潜在分子机制尚未确定。在具体目标2中，我们将破译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。在具体目标3中，我们将确定病毒NS蛋白及其与HCV组装中结构蛋白的相互作用的重要性。我们将使用我们实验室最近开发的一个强大的HCV反向遗传学系统，结合诱变研究和生物化学，免疫学和细胞生物学方法的使用来实现这些特定的目标。从这些研究中获得的新知识将导致病毒学中关于病毒NS蛋白在HCV组装中的作用的范式转变。我们的研究还将为发现有效治疗丙型肝炎的抗病毒药物提供新的靶点。