Function of NS2 in Hepatitis C Virus Assembly and Release

描述(申请人提供)：全球超过2%的人口感染丙型肝炎病毒，它会导致严重的肝脏疾病，包括脂肪变性、进行性纤维化、肝硬变和肝细胞癌。最近，一种完全允许使用2aJFH1基因的丙型肝炎病毒细胞培养系统已经被开发出来。预计该系统将通过使用复制子系统来推进目前的丙型肝炎病毒特异性药物发现工作。随后，我们利用临床上更相关的基因1a H77S开发了一种丙型肝炎病毒感染系统。利用新建立的丙型肝炎病毒体外组织培养模型，我们建议阐明NS2在调控感染性病毒产生过程中的作用机制。我们的假设是“NS2蛋白在丙型肝炎病毒的组装和释放中起关键作用，它含有NS2/NS3酶的催化结构域”，这一假说是在JFH1背景下使用两种不同的编码H77结构蛋白的嵌合体进行的初步研究得出的。这些嵌合体在导致病毒产生的途径上显示出明显的缺陷。我们的结果表明，NS2序列与其周围的结构和非结构序列的不相容是导致病毒产生缺陷的主要原因，NS2以及其他区域(包括e1、p7和NS3)积累的补偿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，NS2特异性的病毒组装缺陷可能是反式互补的。在这个建议中，我们将首先研究NS2是否与结构蛋白和其他非结构蛋白相互作用，如果是这样的话，我们将把代偿突变介导的增强病毒产生的表型与涉及NS2的蛋白质-蛋白质相互作用联系起来。在我们的第二个目标中，我们将利用最近在NS2 C-末端区域获得的结构信息来研究该区域内组装和释放的基因型特异性决定因素(S)。在我们的第三个目标中，我们将通过分析与病毒生产相关的NS2的特定性质，并通过反式互补分析描述参与这些过程的结构域(S)，从而表征参与病毒组装和成熟的NS2的功能结构域(S)。这一提议产生的数据将促进我们对传染性病毒产生机制的了解。此外，如果通过这一建议阐明NS2在病毒产生中的具体作用，将有可能开发针对病毒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(丙型肝炎病毒)感染会导致严重的肝病，从而导致公共卫生问题。我们的初步数据表明，已知的含有NS2/NS3酶催化结构域的丙型肝炎病毒NS2蛋白在丙型肝炎病毒的组装和释放中起着关键作用。从我们的建议中产生的数据将促进我们对丙型肝炎病毒生产的相关知识，并使针对涉及病毒RNA复制和病毒颗粒形成的丙型肝炎病毒NS2的双重功能的特异性抑制剂的开发成为可能。

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.