Role of HCV p7 and NS2 in infectious virus production

描述（由申请人提供）：丙型肝炎病毒（HCV）是肠道传播的非甲、非乙型病毒性肝炎的病原。慢性感染使个体面临肝硬化、肝细胞癌和肝功能衰竭的风险，使慢性丙型肝炎成为肝移植的主要指征。虽然hcv特异性蛋白酶和聚合酶抑制剂在早期临床开发中显示出希望，但耐药性的迅速出现表明，需要额外的病毒靶点和抗病毒药物组合来进行有效控制。两种HCV蛋白p7和NS2的功能研究受到限制。尽管这两种蛋白都是必需的，但除了NS2-3蛋白酶介导的裂解释放NS3丝氨酸蛋白酶/解旋酶的n端外，HCV RNA复制都不需要这两种蛋白。随着近年来细胞培养系统的发展，检测p7和NS2功能的能力发生了巨大的变化，这些培养系统允许HCV RNA复制和病毒粒子产生（HCVcc）。利用单顺反子和双顺反子HCVcc基因组进行的初步实验表明，感染性病毒的产生绝对需要p7和NS2。NS2在病毒产生中的作用独立于其自身蛋白水解活性；p7的作用是否与其假设的离子通道活性或其他机制有关仍有待确定。利用遗传、生化和结构方法的结合，我们建议剖析HCV p7和NS2在病毒组装和释放中的作用。遗传分析将针对保守特征，包括NS2自蛋白酶结构域的表面暴露残基和基于我们最近的x射线结构假定参与其寡聚化的残基。基因间嵌合体，最初对病毒产生不利，将用于选择和绘制第二位点抑制突变，以产生p7和NS2相互作用的遗传图谱。这些遗传研究将通过生化和细胞生物学方法加以补充，以检查有害和补偿突变对同质寡聚化、亚细胞定位、与病毒和细胞伴侣的相互作用以及离子通道活性的影响（p7）。最后，我们建议继续我们的结构研究，从NS2自蛋白酶的切割后形式开始（揭示具有两个复合半胱氨酸蛋白酶活性位点的结构域交换同二聚体），包括全长NS2。通过这些研究，我们希望开始澄清这些未被研究的病毒蛋白在HCV生命周期中的作用，并发现治疗干预的其他途径。公共卫生相关性：丙型肝炎病毒是肝脏疾病包括癌症的主要原因。本研究旨在研究感染性病毒产生所必需的两种未被充分研究的病毒蛋白的功能。由此产生的发现应该有助于开发更有效的治疗方法，旨在根除这种致命的病毒性疾病。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. Chronic infection puts individuals at risk for the development of cirrhosis, hepatocellular carcinoma, and liver failure, making chronic hepatitis C the leading indication for liver transplantation. While HCV-specific protease and polymerase inhibitors are showing promise in early clinical development, rapid emergence of resistance indicates that additional viral targets and combinations of antivirals will be needed for effective control. Functional studies on two HCV proteins, p7 and NS2, have been limited. Although both proteins are essential, neither is required for HCV RNA replication other than the NS2-3 protease-mediated cleavage to liberate the N-terminus of the NS3 serine protease/helicase. The ability to probe p7 and NS2 functions has changed dramatically with recent development of cell culture systems allowing both HCV RNA replication and virion production (HCVcc). Preliminary experiments utilizing monocistronic and bicistronic HCVcc genomes indicate that both p7 and NS2 are absolutely required for infectious virus production. NS2's role(s) in virus production is independent of its autoproteolytic activity; whether p7's role is related to its putative ion channel activity or other mechanisms remains to be determined. Using a combination of genetic, biochemical and structural approaches, we propose to dissect the roles of HCV p7 and NS2 in virus assembly and release. Genetic analyses will target conserved features including surface exposed residues on the NS2 autoprotease domain and residues postulated to be involved in its oligomerization, based on our recent x-ray structure. Intergenic chimeras, initially impaired for virus production, will be used to select and map second site suppressor mutations to yield a genetic map of p7 and NS2 interactions. These genetic studies will be complemented by biochemical and cell biological approaches to examine the effect of deleterious and compensating mutations on homo-oligomerization, subcellular localization, interactions with viral and cellular partners, and ion channel activity (p7). Finally, we propose to continue our structural studies that began with the post-cleavage form of the NS2 autoprotease (revealing a domain exchanged homodimer with two composite cysteine protease active sites) to include full-length NS2. Through these studies, we hope to begin to clarify the role of these understudied viral proteins in the HCV lifecycle and uncover additional avenues for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Hepatitis C virus is a leading cause of liver disease including cancer. This proposal aims to study the function of two understudied viral proteins that are essential for infectious virus production. The resulting findings should aid development of more effective treatments aimed at eradicating this deadly viral disease.