Towards the Disruption of Viral RNA Replication

致力于破坏病毒 RNA 复制

基本信息

批准号：
8882459
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
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2017-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8882459
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 Design Drug Targeting Electron Microscopy Electrons Encephalomyocarditis virus Future Genes 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 Genome 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

项目摘要

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.

描述（由申请人提供）：由poliovirus，Rhinovirus，rhpatitis c Virus和SARS在内的阳性单链RNA病毒引起的疾病是全球关键的健康问题。疾病的进展需要在宿主细胞中病毒诱导的膜结构上组装的寡聚复合物复制病毒基因组。近年来，干扰蛋白质蛋白质相互作用的药物的设计已取得了长足的进步，使病毒复制复合物的破坏成为抗病毒干预的有吸引力的靶标。 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未接种的人群。将根据负责其稳定性的分子间相互作用来分析脊髓灰质炎病毒RNA聚合酶的组装，这是复制复制复合物的功能性核心，并根据高分辨率晶体学数据以及广泛的突变和生物化学数据来解释。随着结构电子显微镜的领域朝着常规的子纳米分辨率迈进，这是生物学问题，例如这里被问到的问题继续激励技术创新。在特定的目标1中，将开发并使用冷冻电子显微镜的常规使用方法来求解侧面小至10个单位细胞的结构，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。这将远远超出了对聚合酶的拟议研究的价值，从而实现了经常出现在结晶托盘中的蛋白质纳米晶体“淋浴”的结构分析，直到现在，这些蛋白质纳米晶体已被丢弃为失败的实验，并且最近吸引了人们的兴趣大大增加。具体 AIM 2，一种稳定聚合酶-RNA复合物并通过跨聚合酶界面的带正电通道的RNA复制的机制将通过组成野生型和突变聚合酶的结构和功能分析进行测试。在特定目标中3将病毒复制蛋白转染到哺乳动物细胞中将用于（i）定义将细胞膜重组为双层膜的最小病毒成分，（ii）表征蛋白质 - 蛋白质蛋白相互作用，以稳定这些脉动脉络膜的范围，以致于稳定范围的差异范围，以致是脉动的差异。药物靶标。