Role of HCV p7 and NS2 in infectious virus production

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是由肠道外传播的非甲非乙型病毒性肝炎的病原体。慢性感染使个人面临发展为肝硬变、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适应症。虽然丙型肝炎病毒特异的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，将需要更多的病毒靶点和抗病毒药物的组合来有效控制。对两种丙型肝炎病毒蛋白p7和ns2的功能研究一直很有限。虽然这两种蛋白质都是必需的，但除了NS2-3蛋白酶介导的裂解NS3丝氨酸蛋白酶/解旋酶的N末端外，这两种蛋白质都不是丙型肝炎病毒RNA复制所必需的。随着最近允许丙型肝炎病毒RNA复制和病毒产生(HCVcc)的细胞培养系统的发展，探测p7和NS2功能的能力发生了巨大变化。利用单顺反子和双顺反子HCVcc基因组进行的初步实验表明，p7和NS2都是产生传染性病毒所必需的。NS2的S(S)在病毒产生中的作用不依赖于其自身的蛋白分解活性；p7‘S的作用是否与其可能的离子通道活性或其他机制有关仍有待确定。结合遗传学、生物化学和结构学方法，我们建议剖析丙型肝炎病毒p7和NS2在病毒组装和释放中的作用。根据我们最近的X射线结构，遗传分析将针对保守的特征，包括NS2自蛋白酶结构域上暴露的表面残基和推测参与其寡聚的残基。最初不利于病毒产生的基因间嵌合体将被用于选择和定位第二个位置抑制突变，以产生p7和NS2相互作用的遗传图谱。这些遗传学研究将得到生化和细胞生物学方法的补充，以检查有害和补偿性突变对同源寡聚、亚细胞定位、与病毒和细胞伙伴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自身蛋白酶的裂解后形式开始(揭示了一个与两个复合半胱氨酸蛋白酶活性位点的区域交换的同源二聚体)，以包括全长NS2。通过这些研究，我们希望开始阐明这些未被充分研究的病毒蛋白在丙型肝炎病毒生命周期中的作用，并发现更多治疗干预的途径。公共卫生相关性：丙型肝炎病毒是包括癌症在内的肝病的主要原因。这项建议旨在研究两种未被研究的病毒蛋白的功能，这两种蛋白对传染性病毒的产生至关重要。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.