Function of NS2 in Hepatitis C Virus Assembly and Release

描述（由申请人提供）：超过2%的世界人口感染丙型肝炎病毒（HCV），其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，已经开发了使用基因型2a JFH 1的完全允许的HCV细胞培养系统。预期该系统将通过使用复制子系统推进当前HCV特异性药物发现工作。随后，我们通过使用临床上更相关的基因型1a H77 S开发了HCV感染系统。通过利用这种新开发的HCV体外组织培养模型，我们建议阐明NS 2在调节感染性病毒生产过程中的机制。我们的假设，即“NS 2蛋白，这是已知的含有NS 2/NS 3蛋白酶的催化结构域，在HCV组装和释放中起着关键作用”是来自我们广泛的初步研究，使用两种不同的嵌合体编码H77结构蛋白的背景下的JFH 1。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，NS 2序列与其周围的结构和非结构序列的不相容性是病毒生产缺陷的主要原因，在NS 2以及其他区域，包括E1，p7和NS 3积累的补偿突变可以挽救感染性病毒生产。我们也有更直接的证据表明，NS 2参与病毒的生产和NS 2特异性的病毒装配缺陷可以反式互补。在这个提议中，我们将首先研究NS 2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，我们将把补偿突变介导的增强病毒产生的表型与涉及NS 2的蛋白质-蛋白质相互作用相关联。在我们的第二个目标，我们将调查基因型特异性决定因素（S）的组装和释放的C-末端区域内的NS 2通过使用最近可用的结构信息在这个区域内。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NS 2的特定性质和通过使用反式互补测定描绘参与这些过程的结构域来表征参与病毒组装和成熟的NS 2的功能结构域。从这个提议中产生的数据将推进我们对感染性病毒产生机制的认识。此外，如果NS 2参与病毒产生的特定作用通过该提议得到阐明，则将有可能开发针对HCV NS 2参与病毒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）感染通过引起严重的肝脏疾病而导致公共卫生问题。我们的初步数据表明，HCV NS 2蛋白，已知含有NS 2/NS 3蛋白酶的催化结构域，在HCV组装和释放中起着关键作用。从我们的提案中产生的数据将推进我们与HCV病毒生产相关的知识，并使开发针对HCV NS 2双重功能的特异性抑制剂成为可能，HCV NS 2参与病毒RNA复制和病毒颗粒形成。

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.