Function of NS2 in Hepatitis C Virus Assembly and Release

描述（由申请人提供）：世界上超过2%的人口感染丙型肝炎病毒（HCV），导致严重的肝脏疾病，包括脂肪变性、进行性纤维化、肝硬化和肝细胞癌。最近，利用基因型2a JFH1开发了一种完全允许的HCV细胞培养系统。预计该系统将通过使用复制子系统来推进当前的HCV特异性药物发现工作。随后，我们利用临床更相关的基因型1a H77S开发了HCV感染系统。利用新建立的HCV体外组织培养模型，我们拟阐明NS2调控感染性病毒产生过程的机制。我们的假设是“已知含有NS2/NS3蛋白酶催化结构域的NS2蛋白在HCV的组装和释放中起关键作用”，这是我们在JFH1背景下使用两种不同的嵌合体编码H77结构蛋白进行的广泛的初步研究得出的。这些嵌合体在导致病毒产生的途径中显示出明显的缺陷。我们的研究结果表明，NS2序列与其周围结构和非结构序列的不亲和性是病毒产生缺陷的主要原因，在NS2以及其他区域（包括E1、p7和NS3）积累的补偿性突变可以挽救感染性病毒的产生。我们也有更直接的证据表明NS2参与了病毒的产生，并且NS2特异性的病毒组装缺陷可能是反式互补的。在本文中，我们将首先研究NS2是否与结构蛋白以及其他非结构蛋白相互作用，如果是这样的话，我们将把补偿性突变介导的增强病毒产生的表型与涉及NS2的蛋白-蛋白相互作用联系起来。在我们的第二个目标中，我们将利用最近获得的NS2 c端区域的结构信息，研究该区域内组装和释放的基因型特异性决定因素。在我们的第三个目标中，我们将通过分析与病毒产生相关的NS2的特定特性，并通过使用反式互补试验描绘这些过程中涉及的区域，来表征参与病毒组装和成熟的NS2的功能域。从这一建议中产生的数据将促进我们对传染性病毒产生机制的认识。此外，如果NS2参与病毒产生的具体作用被阐明，将有可能开发出针对HCV NS2参与病毒RNA复制和病毒颗粒形成的双重功能的特异性抑制剂。公共卫生相关性：丙型肝炎病毒（HCV）感染会引起严重的肝脏疾病，从而导致公共卫生问题。我们的初步数据表明，已知含有NS2/NS3蛋白酶催化结构域的HCV NS2蛋白在HCV的组装和释放中起关键作用。从我们的提案中产生的数据将推进我们对HCV病毒产生的相关知识，并使开发针对HCV NS2参与病毒RNA复制和病毒颗粒形成的双重功能的特异性抑制剂成为可能。

DESCRIPTION (provided by applicant): Over 2% of the world's population is infected with hepatitis C virus (HCV), which causes severe liver diseases, including steatosis, progressive fibrosis, cirrhosis and hepatocellular carcinoma. Recently, a fully permissive HCV cell culture system using genotype 2a JFH1 has been developed. It is expected that this system will advance, by using a replicon system, the current HCV specific drug discovery effort. Subsequently, we developed an HCV infection system by using the clinically more relevant genotype 1a H77S. By taking advantage of this newly developed in vitro tissue culture model of HCV, we propose to elucidate the mechanism of NS2 in regulating the infectious virus production process. Our hypothesis that "NS2 protein, which was known to contain a catalytic domain of NS2/NS3 protease, plays a critical role in HCV assembly and release" is derived from our extensive preliminary studies using two different chimeras encoding H77 structural proteins within the background of JFH1. These chimeras showed distinct defects in the pathway leading to virus production. Our results suggest that the incompatibility of the NS2 sequence with its surrounding structural and nonstructural sequences is the major reason there is a defect in virus production, and the compensatory mutations accumulated at NS2 as well as other regions, including E1, p7 and NS3, could rescue infectious virus production. We also have more direct evidence that NS2 is involved in virus production and that NS2-specific viral assembly defect could be trans-complemented. In this proposal, we will first investigate if NS2 interacts with structural, as well as other nonstructural proteins, and, if that is the case, we will correlate the compensatory mutation-mediated phenotype of enhanced virus production with protein-protein interactions involving NS2. In our second aim, we will investigate the genotype-specific determinant(s) of assembly and release within the C-terminal region of NS2 by using recently available structural information within this region. In our third aim, we will characterize functional domain(s) of NS2 involved in viral assembly and maturation by analyzing specific properties of NS2 related to virus production and by delineation of the domain(s) involved in these processes using a trans-complementation assay. The data generated from this proposal will advance our knowledge of the mechanism of infectious virus production. Moreover, if the specific role of NS2 involved in virus production is elucidated through this proposal, it will make possible the development of specific inhibitors against the dual functions of HCV NS2 involved in both viral RNA replication and viral particle formation. PUBLIC HEALTH RELEVANCE: Hepatitis C Virus (HCV) infection leads to public health problems by causing severe liver diseases. Our preliminary data indicate that HCV NS2 protein, known to contain a catalytic domain of NS2/NS3 protease, plays a critical role in HCV assembly and release. The data generated from our proposal will advance our knowledge related to HCV virus production and make possible the development of specific inhibitors against the dual functions of HCV NS2 involved in both viral RNA replication and viral particle formation.