Molecular determinants of hepatitis C virus assembly

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是急性和慢性肝病的主要原因，并有助于肝细胞癌的发展。丙型肝炎病毒相关疾病是美国肝移植的主要原因。目前可用的丙型肝炎病毒治疗方法昂贵、耐受性差，并且只在一小部分患者中成功控制了病毒。为了制定更有效的抗病毒策略，对病毒生命周期的清楚了解是至关重要的。这个生命周期中最不被理解的方面是病毒组装，这是通过分泌途径中的病毒颗粒在细胞内萌发而发生的。病毒颗粒的形成与细胞的极低密度脂蛋白组装途径有着不同寻常的依赖关系，但这些过程之间的联系尚不清楚。此外，病毒非结构蛋白(NS)在病毒组装中发挥着重要但尚未明确的调节作用。到目前为止，丙型肝炎病毒颗粒组装的中间阶段还没有被严格定义。为了解决这些问题，我们正在确定丙型肝炎病毒NS2和NS3-4A蛋白对病毒组装的作用。在初步数据中，我们：1)开发了在细胞培养中生长丙型肝炎病毒的高效系统；2)利用有用的报告基因、亲和标签和荧光标记对感染性病毒变体进行了工程；3)对病毒NS2基因进行了突变研究，揭示了对病毒组装至关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)开发了在活细胞中对全功能的荧光标记的丙型肝炎病毒核心蛋白进行成像的方法。根据我们对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酶复合体相互作用来协调病毒组装的不同早期步骤。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，我们将通过从病毒产生细胞中专门捕获NS2及其相关蛋白来定义控制病毒组装的NS2蛋白-蛋白质相互作用。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，通过使用一组在病毒组装中存在缺陷的NS2突变体，我们将绘制它们的决定因素。在目标2中，我们将通过活细胞显微镜和固定细胞显微镜以及细胞分离来检查我们的NS2突变体的子集，从而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有重点的突变和其他相关条件上，我们将对丙型肝炎病毒组装途径有更详细的了解。在目标3中，我们将扩展我们的遗传和生物化学相结合的方法来定义病毒组装的NS3-4A决定因素。这些研究完成后，将有助于更清楚地了解丙型肝炎病毒的组装过程，并揭示可作为抗病毒设计目标的基本蛋白质-蛋白质相互作用。 公共卫生相关性：丙型肝炎病毒是一种非常成功的人类病原体，可导致持续性感染、慢性肝炎和肝细胞癌。我们的研究表征了病毒颗粒的组装过程，这将为靶向丙型肝炎病毒从而预防或治疗疾病提供新的途径。

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.