Role of HCV p7 and NS2 in infectious virus production

HCV p7 和 NS2 在感染性病毒产生中的作用

• 批准号: 7464662
• 负责人: Charles M Rice
• 金额: $ 42万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7464662
• 关键词: Active Sites; Affect; Alanine; Animal Model; Antiviral Agents; Antiviral Therapy; Area; Biochemical; Biochemical Genetics; Biological; Biological Assay; Capsid Proteins; Catalytic Domain; Cell Count; Cell Culture System; Cells; Cellular biology; Chemicals; Chimera organism; Chromosome Mapping; Chronic; Chronic Hepatitis C; Cirrhosis; Class; Clinical; Complement; Confocal Microscopy; Crystallization; Cultured Cells; Cysteine Protease; Data; Detergents; Development; Dimerization; Dissection; Elements; Endopeptidases; Fluorescence Resonance Energy Transfer; Genetic; Genome; Genotype; Glycoproteins; Goals; Gold; Hepatitis B; Hepatitis C virus; Hepatitis C virus p7 protein; Homo; Individual; Infection; Integration Host Factors; Ion Channel; Ions; Knowledge; Length; Liver Failure; Liver diseases; Malignant Neoplasms; Maps; Mediating; Membrane; Methods; Molecular; Molecular Cloning; Mutagenesis; Mutation; N-terminal; NS2-3 protease; Nonstructural Protein; Parents; Pathogenesis; Peptide Hydrolases; Physiologic pulse; Polymerase; Polyproteins; Primary carcinoma of the liver cells; Process; Production; Property; Protease Domain; Protein Fragment; Proteins; Proteomics; Public Health; Pulse taking; RNA; RNA Interference; RNA Viruses; RNA replication; Reporter; Research; Resistance; Resolution; Risk; Role; Scanning; Scheme; Serine Protease; Site; Structure; Suppressor Mutations; Surface; System; Therapeutic Intervention; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Western Blotting; base; crosslink; design; genetic analysis; helicase; inhibitor/antagonist; liver transplantation; mutant; protein protein interaction; research study; viral RNA

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.

描述（由申请人提供）：丙型肝炎病毒（HCV）是肠道外传播的非甲、非乙型病毒性肝炎的病原体。慢性感染使个体处于肝硬化、肝细胞癌和肝功能衰竭的发展风险中，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前景，但快速出现的耐药性表明需要额外的病毒靶点和抗病毒药物的组合来进行有效控制。两种HCV蛋白，p7和NS 2的功能研究有限。虽然这两种蛋白质都是必需的，但除了NS 2 -3蛋白酶介导的切割以释放NS 3丝氨酸蛋白酶/解旋酶的N-末端之外，HCV RNA复制都不需要。随着最近允许HCV RNA复制和病毒体产生（HCVRNA）的细胞培养系统的发展，探测p7和NS 2功能的能力发生了显著变化。利用单顺反子和双顺反子HCV基因组的初步实验表明，p7和NS 2都是感染性病毒生产所绝对需要的。NS 2在病毒生产中的作用与其自身蛋白水解活性无关; p7的作用是否与其推定的离子通道活性或其他机制有关仍有待确定。使用遗传学，生物化学和结构的方法相结合，我们建议剖析HCV p7和NS 2在病毒组装和释放中的作用。遗传分析将针对保守的功能，包括表面暴露的残基的NS 2 autoprotease域和残基假定参与其寡聚化，根据我们最近的X射线结构。基因间嵌合体，最初受损的病毒生产，将用于选择和映射第二个位点的抑制突变，以产生p7和NS 2相互作用的遗传图谱。这些遗传学研究将通过生物化学和细胞生物学方法进行补充，以检查有害和补偿突变对同源寡聚化、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（第7页）。最后，我们建议继续我们的结构研究，开始与后切割形式的NS 2自身蛋白酶（揭示了一个域交换的同源二聚体与两个复合半胱氨酸蛋白酶活性位点），包括全长NS 2。通过这些研究，我们希望开始阐明这些未充分研究的病毒蛋白在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：丙型肝炎病毒是导致包括癌症在内的肝脏疾病的主要原因。该提案旨在研究两种未充分研究的病毒蛋白质的功能，这两种蛋白质对感染性病毒的产生至关重要。研究结果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。