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: 8448734
• 负责人: Brett D. Lindenbach
• 金额: $ 38.5万
• 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/8448734
• 关键词: Acute; Address; Affinity; Antiviral Agents; Antiviral Therapy; Biochemical; Biological; Cell Culture Techniques; Cells; 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; 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; chronic liver disease; 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.

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，并促进肝细胞癌的发展。 HCV 相关疾病是美国肝移植的主要原因。目前可用的 HCV 疗法价格昂贵、耐受性差，并且仅能在一小部分患者中成功控制病毒。为了制定更有效的抗病毒策略，清楚地了解病毒的生命周期至关重要。这个生命周期最不为人所知的方面是病毒组装，它是通过病毒颗粒在分泌途径内的细胞内出芽而发生的。病毒颗粒的形成对细胞 VLDL 组装途径具有不同寻常的依赖性，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着重要但不明确的调节作用。迄今为止，HCV 颗粒组装的中间阶段尚未得到严格定义。为了解决这些问题，我们正在确定 HCV NS2 和 NS3-4A 蛋白如何促进病毒组装。在初步数据中，我们：1) 开发了一种在细胞培养中培养 HCV 的高效系统； 2）具有有用的报告基因、亲和标签和荧光标记的工程感染性病毒变体； 3）对病毒NS2基因进行了诱变研究，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-功能所需的生化工具，5) 开发了对活细胞中全功能、荧光标记的 HCV 核心蛋白进行成像的方法。基于我们对 NS2 的初步遗传、生化和细胞生物学分析，我们假设 NS2 通过与 E1-E2 糖蛋白和 NS3-4A 酶复合物的相互作用来协调病毒组装中独特的早期步骤。我们将通过完成三个具体目标来检验这一假设。在目标 1 中，我们将定义 NS2 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 NS2 及其相关蛋白质来控制病毒组装。我们将研究 NS2 与其他病毒和细胞蛋白之间的相互作用；然后，通过使用一组病毒组装有缺陷的 NS2 突变体，我们将绘制它们的决定因素。在目标 2 中，我们将通过活细胞和固定细胞显微镜以及细胞分级检查 NS2 突变体的子集来识别和表征病毒组装的中间阶段。通过将这些分析整合到一组重点突变体和其他相关条件上，我们将对 HCV 组装途径有更详细的了解。在目标 3 中，我们将扩展遗传和生化相结合的方法来定义病毒组装的 NS3-4A 决定因素。完成后，这些研究将有助于更清楚地了解 HCV 组装过程，并揭示重要的蛋白质-蛋白质相互作用，可作为抗病毒设计的目标。