Molecular determinants of hepatitis C virus assembly

描述（由申请人提供）：丙型肝炎病毒（HCV）是急性和慢性肝脏疾病的主要原因，并有助于肝细胞癌的发展。在美国，hcv相关疾病是肝移植的主要原因。目前可用的HCV治疗方法价格昂贵，耐受性差，并且仅在一小部分患者中成功控制病毒。为了制定更有效的抗病毒策略，对病毒生命周期有一个清晰的认识是必不可少的。这个生命周期中最不为人所知的方面是病毒组装，这是通过分泌途径中病毒颗粒的细胞内出芽而发生的。病毒颗粒的形成不同寻常地依赖于细胞VLDL组装途径，但这些过程之间的联系尚不清楚。此外，病毒非结构（NS）蛋白在病毒组装过程中起着重要但不明确的调节作用。迄今为止，丙型肝炎病毒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)开发了一种高效的HCV细胞培养系统；2)具有有用的报告基因、亲和标签和荧光标记的工程感染病毒变体；3)对病毒NS2基因进行诱变研究，揭示了病毒组装过程中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)开发了在活细胞中成像全功能、荧光标记的HCV核心蛋白的方法。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，协调了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Aim 1中，我们将定义NS2蛋白-蛋白相互作用，通过特异性地从病毒产生细胞中捕获NS2及其相关蛋白来控制病毒组装。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，通过使用一组在病毒组装中有缺陷的NS2突变体，我们将绘制它们的决定因子。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离检查我们的NS2突变体的一个子集来识别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上，我们将对HCV组装途径有更详细的了解。在Aim 3中，我们将扩展我们的遗传和生化结合方法来定义病毒组装的NS3-4A决定因素。一旦完成，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基本蛋白质-蛋白质相互作用。

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.