Towards the Disruption of Viral RNA Replication

致力于破坏病毒 RNA 复制

• 批准号: 8341918
• 负责人: ESTHER BULLITT
• 金额: $ 36.01万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8341918
• 关键词: Address; Antiviral Agents; Automobile Driving; Autophagosome; Avidity; Biochemical; Biological; Cell physiology; Cells; Cellular Membrane; Charge; Complex; Cryoelectron Microscopy; Crystallization; Cytoplasmic Vesicles; DNA-Directed RNA Polymerase; Data; Data Collection; Development; Disease; Disease Progression; Dose; Drug Delivery Systems; Drug Design; Electron Microscopy; Electrons; Encephalomyocarditis virus; Future; Genes; Genome; Health; Hepatitis C; Hepatitis C virus; Human poliovirus; Image; Individual; Intervention; Knowledge; Lipids; Location; Mammalian Cell; Membrane; Membrane Proteins; Methods; Molecular; Molecular Models; Molecular Target; Nature; Patients; Pharmaceutical Preparations; Polioviruses; Polymerase; Population; Preparation; Property; Proteins; Publishing; RNA; RNA Binding; RNA Viruses; RNA replication; Research; Resolution; Rhinovirus; Sampling; Severe Acute Respiratory Syndrome; Side; Site; Structure; Surface; System; Test Result; Testing; Therapeutic; Therapeutic Intervention; Transfection; Tube; United States National Academy of Sciences; Vesicle; Vesnarinone; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Work; base; computerized data processing; electron tomography; innovation; interest; intermolecular interaction; macromolecule; method development; molecular modeling; mutant; nanocrystal; nanometer; prevent; protein protein interaction; protein structure; reconstruction; research study; viral RNA

DESCRIPTION (provided by applicant): Diseases caused by positive-sense single-stranded RNA viruses, including poliovirus, rhinovirus, hepatitis C virus, and SARS, are critical health issues worldwide. Progression of disease requires replication of the virus genome by oligomeric complexes assembled on virally-induced membrane structures in the host cell. Design of drugs that interfere with protein-protein interactions has made considerable progress in recent years, making disruption of the viral replication complex an attractive target for antiviral intervention. This proposal focuses on poliovirus (i) in order to use the extensive knowledge of the structural and functional properties of poliovirus as a basis for detailed characterization of these protein-protein interactions, and (ii) to address the need for new antiviral strategies against poliovirus that a 2007 National Academy of Sciences panel asserted would significantly strengthen the eradication effort, and prevent its threat as a bioweapon against an unvaccinated population. Assemblies of poliovirus RNA polymerase, the functional centerpiece of the replication complex, will be analyzed in terms of the intermolecular interactions responsible for their stability and interpreted on the basis of high resolution crystallographic data and extensive mutational and biochemical data. As the field of structural electron microscopy advances toward routine sub-nanometer resolution, it is biological questions such as those asked here that continue to motivate technical innovations. In Specific Aim 1, methods for the routine use of cryo-electron microscopy to solve structures of nanocrystals as small as 10 unit cells on a side will be developed and used to analyze the structure of nanocrystals of RNA polymerase and characterize the intermolecular interactions therein. This will be of value far beyond the proposed studies of polymerase, enabling structural analyses of the 'shower' of protein nanocrystals that often appear in crystallization trays, that until now have been discarded as failed experiments, and that recently have been attracting greatly increased interest. In Specific Aim 2, a mechanism for stabilizing polymerase-RNA complexes and supporting RNA replication via a positively charged channel across the polymerase interface will be tested by structural and functional analyses of complexes comprising wild-type and mutant polymerases. In Specific Aim 3 transfection of viral replication proteins into mammalian cells will be used to (i) define th minimum viral component required for the membrane reorganization of cellular membrane into double-bilayers, and (ii) characterize the protein-protein interactions that stabilize polymerase-containing oligomers, as these interfaces represent sites of vulnerability for disruption of viral replication and for development as potential drug targets. PUBLIC HEALTH RELEVANCE: New antiviral medications against poliovirus and similar viruses, such as hepatitis C and SARS, are needed to treat patients with these diseases, and to help prevent the threat of their use as a bioweapon against an unvaccinated population. A promising approach is to target the proteins needed for the virus to make new copies of itself. The proposed research will look at assemblies formed by the proteins that are essential for copying the virus gene, and locate vulnerable regions for future drug design.

描述（由申请人提供）：由正链单链RNA病毒引起的疾病，包括脊髓灰质炎病毒、鼻病毒、丙型肝炎病毒和SARS，是世界范围内的严重健康问题。疾病的进展需要通过在宿主细胞中病毒诱导的膜结构上组装的寡聚复合物来复制病毒基因组。近年来，干扰蛋白质-蛋白质相互作用的药物设计取得了相当大的进展，使得破坏病毒复制复合物成为抗病毒干预的有吸引力的目标。 该提案重点关注脊髓灰质炎病毒（i），以便利用对脊髓灰质炎病毒结构和功能特性的广泛了解，作为详细表征这些蛋白质-蛋白质相互作用的基础，以及（ii）满足对针对脊髓灰质炎病毒的新抗病毒策略的需求，2007年美国国家科学院的一个小组声称，这将显着加强根除工作，并防止其作为针对脊髓灰质炎病毒的生物武器的威胁。 未接种疫苗的人群。 脊髓灰质炎病毒 RNA 聚合酶的组装是复制复合物的功能核心，将根据决定其稳定性的分子间相互作用进行分析，并根据高分辨率晶体学数据和广泛的突变和生化数据进行解释。随着结构电子显微镜领域向常规亚纳米分辨率发展，正是诸如此处提出的生物学问题继续推动技术创新。在具体目标1中，将开发常规使用冷冻电子显微镜来解析单侧小至10个单位细胞的纳米晶体结构的方法，并用于分析RNA聚合酶纳米晶体的结构并表征其中的分子间相互作用。这将远远超出所提出的聚合酶研究的价值，从而能够对经常出现在结晶托盘中的蛋白质纳米晶体“簇射”进行结构分析，迄今为止，这些蛋白质纳米晶体已被视为失败的实验而被丢弃，而最近却引起了极大的兴趣。具体来说 目标 2，通过对包含野生型和突变型聚合酶的复合物进行结构和功能分析，将测试通过聚合酶界面上的带正电通道稳定聚合酶-RNA 复合物并支持 RNA 复制的机制。在具体目标 3 中，将病毒复制蛋白转染到哺乳动物细胞中，用于 (i) 定义细胞膜重组为双层所需的最小病毒成分，以及 (ii) 表征稳定含聚合酶寡聚体的蛋白质-蛋白质相互作用，因为这些界面代表了病毒复制破坏和潜在发育的脆弱位点 药物靶点。 公共卫生相关性：需要针对脊髓灰质炎病毒和类似病毒（例如丙型肝炎和 SARS）的新型抗病毒药物来治疗患有这些疾病的患者，并帮助防止将其用作针对未接种疫苗的人群的生物武器的威胁。一种有前景的方法是针对病毒复制自身所需的蛋白质。拟议的研究将着眼于复制病毒基因所必需的蛋白质形成的组装体，并找到未来药物设计的脆弱区域。