Towards the Disruption of Viral RNA Replication

描述（由申请人提供）：由正义单链RNA病毒（包括脊髓灰质炎病毒、鼻病毒、丙型肝炎病毒和SARS）引起的疾病是全球范围内的重大健康问题。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靶点。 该提案重点关注脊髓灰质炎病毒，（i）为了利用脊髓灰质炎病毒结构和功能特性的广泛知识作为这些蛋白质-蛋白质相互作用的详细表征的基础，（ii）解决针对脊髓灰质炎病毒的新抗病毒策略的需求，2007年美国国家科学院小组断言将大大加强根除工作，并防止其作为生物武器对未接种疫苗的人群造成威胁。 脊髓灰质炎病毒RNA聚合酶的组装，复制复合物的功能中心，将进行分析的分子间相互作用，负责其稳定性和解释的基础上，高分辨率的晶体学数据和广泛的突变和生化数据。随着结构电子显微镜领域向常规亚纳米分辨率的发展，生物学问题（如本文所问的问题）继续推动技术创新。在具体目标1中，将开发用于常规使用冷冻电子显微镜来解析一侧上小至10个单位细胞的纳米晶体的结构的方法，并用于分析RNA聚合酶的纳米晶体的结构并表征其中的分子间相互作用。这将是远远超出聚合酶的拟议研究的价值，使结构分析的“淋浴”的蛋白质纳米晶体，经常出现在结晶托盘，到目前为止已被丢弃的失败的实验，最近已经吸引了极大的兴趣。在特定 目的2，用于稳定聚合酶-RNA复合物和支持RNA复制的机制，通过跨聚合酶界面的带正电荷的通道将通过包含野生型和突变聚合酶的复合物的结构和功能分析来测试。在特定目标3中，将病毒复制蛋白转染到哺乳动物细胞中将用于（i）确定细胞膜重组成双双层所需的最小病毒组分，以及（ii）表征稳定含聚合酶的寡聚体的蛋白质-蛋白质相互作用，因为这些界面代表病毒复制破坏和开发为潜在药物靶标的脆弱性位点。

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.