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: 7995498
• 负责人: Charles M Rice
• 金额: $ 41.41万
• 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/7995498
• 关键词: Active Sites; Alanine; Animal Model; Antiviral Agents; Antiviral Therapy; Area; Biochemical; Biological; Biological Assay; Capsid Proteins; Catalytic Domain; Cell Count; Cell Culture System; Cell Culture Techniques; Cells; Cellular biology; Chemicals; Chimera organism; Chromosome Mapping; Chronic; Chronic Hepatitis C; Cirrhosis; Clinical; Complement; Confocal Microscopy; Cysteine Protease; Development; Dimerization; Dissection; Elements; Fluorescence Resonance Energy Transfer; Genetic; Genome; Genotype; Glycoproteins; Goals; Gold; Health; Hepatitis C virus; Hepatitis C virus p7 protein; Homo; Individual; Infection; Integration Host Factors; Ion Channel; Length; Liver Failure; Liver diseases; Malignant Neoplasms; Maps; Mediating; Membrane; 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; RNA; RNA Viruses; RNA replication; Reporter; Research; Research Personnel; Resistance; Resolution; Rice; Risk; Role; Scanning; Scheme; Serine Protease; Site; Structural Protein; Structure; Suppressor Mutations; Surface; System; Therapeutic Intervention; Viral; Viral Proteins; Viral hepatitis; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; Western Blotting; base; crosslink; effective therapy; genetic analysis; helicase; inhibitor/antagonist; liver transplantation; programs; 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）是肠胃外传播的非甲、非乙型病毒性肝炎的病原体。慢性感染使个体面临肝硬化、肝细胞癌和肝衰竭的风险，使慢性丙型肝炎成为肝移植的主要适应症。虽然 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标和抗病毒药物组合才能有效控制。对两种 HCV 蛋白 p7 和 NS2 的功能研究有限。虽然这两种蛋白质都是必需的，但除了 NS2-3 蛋白酶介导的裂解以释放 NS3 丝氨酸蛋白酶/解旋酶的 N 末端之外，HCV RNA 复制都不需要这两种蛋白质。随着最近细胞培养系统的发展，允许 HCV RNA 复制和病毒颗粒产生 (HCVcc)，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 基因组的初步实验表明，p7 和 NS2 都是感染性病毒产生所必需的。 NS2 在病毒产生中的作用与其自身蛋白水解活性无关； p7 的作用是否与其假定的离子通道活性或其他机制有关仍有待确定。我们建议结合遗传、生化和结构方法来剖析 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 自蛋白酶结构域上表面暴露的残基以及根据我们最近的 X 射线结构推测参与其寡聚化的残基。基因间嵌合体最初因病毒产生而受损，将用于选择和绘制第二位点抑制突变图谱，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，以检查有害突变和补偿突变对同源寡聚化、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 自蛋白酶的切割后形式（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）开始，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 HCV 生命周期中的作用，并揭示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病的主要原因。该提案旨在研究两种正在研究的病毒蛋白的功能，这两种蛋白对于感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。