Molecular determinants of hepatitis C virus assembly

丙型肝炎病毒组装的分子决定因素

• 批准号: 7861755
• 负责人: Brett D. Lindenbach
• 金额: $ 41.38万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7861755
• 关键词: Acute; Address; Affinity; Antiviral Agents; Antiviral Therapy; Biochemical; Biological; Cell Culture Techniques; Cells; Chronic; Chronic Hepatitis; Complementary DNA; Complex; Core Protein; Data; Defect; Dependence; Development; Disease; Engineering; Flaviviridae; Fractionation; Genes; Genetic; Glycoproteins; Goals; Hepatitis C virus; Hepatocyte; Human; Image; Infection; Infectious hepatitides; Label; Life; Life Cycle Stages; Link; Lipids; Lipoproteins; Liver Failure; Liver diseases; Maps; Mass Spectrum Analysis; Methods; Microscopy; Modeling; Molecular; Multienzyme Complexes; Mutagenesis; Nonstructural Protein; Nucleocapsid; Pathway interactions; Patients; Phenotype; Primary carcinoma of the liver cells; Process; Property; Proteins; RNA Binding; RNA Helicase; RNA Viruses; Reporter Genes; Research Personnel; Role; Serine Protease; Serum; Site; Staging; Sterols; Structure; Suppressor Mutations; System; Testing; Ultracentrifugation; United States; Variant; Very low density lipoprotein; Viral; Viral Core Proteins; Viral Nonstructural Proteins; Viral Proteins; Viral Structural Proteins; Virion; Virus; Virus Assembly; base; cell fixing; cellular imaging; design; genetic analysis; hepatitis C virus nucleocapsid protein; inhibitor/antagonist; liver transplantation; member; mutant; particle; pathogen; positional cloning; prevent; protein function; protein protein interaction; protein structure function; public health relevance; skills; tool; trafficking; virus culture

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a major cause of acute and chronic liver disease, and contributes to the development of hepatocellular carcinoma. HCV-associated disease is the leading cause of liver transplantation in the United States. Currently available HCV therapies are expensive, poorly tolerated, and successfully control the virus in only a fraction of patients. To develop more effective antiviral strategies, a clear understanding of the viral life cycle is essential. The least understood aspect of this life cycle is virus assembly, which occurs by intracellular budding of virus particles within the secretory pathway. Formation of virus particles has an unusual dependence on the cellular VLDL assembly pathway, but the connection between these processes is unclear. Furthermore, the viral nonstructural (NS) proteins contribute important but poorly defined regulatory roles in virus assembly. And to date, intermediate stages in HCV particle assembly have not been rigorously defined. To address these issues, we are determining how the HCV NS2 and NS3-4A proteins contribute to virus assembly. In Preliminary Data, we: 1) developed a highly efficient system for growing HCV in cell culture; 2) engineered infectious virus variants with useful reporter genes, affinity tags, and fluorescent markers; 3) conducted a mutagenesis study on the viral NS2 gene, revealing functional interactions that are important for virus assembly; 4) developed the biochemical tools necessary to study NS protein structure-function, and 5) developed methods to image fully functional, fluorescently-labeled HCV core protein in live cells. Based on our preliminary genetic, biochemical, and cell biological analysis of NS2, we hypothesize that NS2 coordinates distinct, early steps in virus assembly through interactions with the E1-E2 glycoprotein and NS3-4A enzyme complexes. We will test this hypothesis by completing three Specific Aims. In Aim 1, we will define NS2 protein-protein interactions that control virus assembly by specifically capturing NS2 and its associated proteins from virus-producing cells. We will examine interactions between NS2 and other viral and cellular proteins; then, by using a panel of NS2 mutants that are defective in virus assembly, we will map their determinants. In Aim 2, we will identify and characterize intermediate stages of virus assembly by examining a subset of our NS2 mutants with live- and fixed-cell microscopy and cellular fractionation. By integrating these analyses over a focused set of mutants and other relevant conditions, we will gain a more detailed understanding of the HCV assembly pathway. In Aim 3, we will extend our combined genetic and biochemical approach to define NS3-4A determinants of virus assembly. When completed, these studies will contribute to a clearer understanding of the HCV assembly process and reveal essential protein-protein interactions that can serve as targets for antiviral design. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a highly successful human pathogen that causes persistent infection, chronic hepatitis, and hepatocellular carcinoma. Our studies characterize the virus particle assembly process, which will provide new avenues to target HCV and thereby prevent or treat disease.

描述（由申请人提供）：丙型肝炎病毒（HCV）是急性和慢性肝病的主要原因，并有助于肝细胞癌的发展。HCV相关疾病是美国肝移植的主要原因。目前可用的HCV疗法昂贵，耐受性差，并且仅在一小部分患者中成功控制病毒。为了开发更有效的抗病毒策略，对病毒生命周期的清晰理解至关重要。这个生命周期最不为人所知的方面是病毒装配，它是通过分泌途径内病毒颗粒的细胞内出芽发生的。病毒颗粒的形成对细胞VLDL组装途径有着不寻常的依赖性，但这些过程之间的联系尚不清楚。此外，病毒非结构（NS）蛋白在病毒组装中发挥重要但定义不清的调节作用。到目前为止，HCV颗粒组装的中间阶段还没有严格定义。为了解决这些问题，我们正在确定HCV NS 2和NS 3 -4A蛋白如何有助于病毒组装。在初步数据中，我们：1）开发了在细胞培养物中生长HCV的高效系统; 2）用有用的报告基因、亲和标签和荧光标记物工程化感染性病毒变体; 3）对病毒NS 2基因进行诱变研究，揭示了对病毒组装重要的功能相互作用; 4）开发了研究NS蛋白结构-功能所必需的生物化学工具，以及5）开发了在活细胞中成像全功能的荧光标记的HCV核心蛋白的方法。基于我们对NS 2的初步遗传、生物化学和细胞生物学分析，我们假设NS 2通过与E1-E2糖蛋白和NS 3 -4A酶复合物的相互作用协调病毒组装中不同的早期步骤。我们将通过完成三个具体目标来验证这一假设。在目标1中，我们将定义NS 2蛋白质-蛋白质相互作用，通过从病毒产生细胞中特异性捕获NS 2及其相关蛋白质来控制病毒组装。我们将研究NS 2与其他病毒和细胞蛋白之间的相互作用，然后，通过使用一组在病毒组装中有缺陷的NS 2突变体，我们将绘制它们的决定簇。在目标2中，我们将通过使用活细胞和固定细胞显微镜和细胞分级法检查我们的NS 2突变体的子集来鉴定和表征病毒组装的中间阶段。通过将这些分析集中在一组突变体和其他相关条件下，我们将获得更详细的了解HCV组装途径。在目标3中，我们将扩展我们的遗传和生化相结合的方法来定义病毒组装的NS 3 -4A决定簇。完成后，这些研究将有助于更清楚地了解HCV组装过程，并揭示可作为抗病毒设计靶点的重要蛋白质-蛋白质相互作用。 公共卫生相关性：丙型肝炎病毒（HCV）是一种非常成功的人类病原体，可引起持续感染、慢性肝炎和肝细胞癌。我们的研究表征了病毒颗粒组装过程，这将为靶向HCV提供新的途径，从而预防或治疗疾病。