Towards the Disruption of Viral RNA Replication

描述(申请人提供)：由阳性单链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，是世界范围内的关键健康问题。疾病的发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体来复制病毒基因组。近年来，干扰蛋白质-蛋白质相互作用的药物设计取得了长足的进步，使破坏病毒复制复合体成为抗病毒干预的一个有吸引力的目标。 这项提案的重点是脊髓灰质炎病毒：(I)利用对脊髓灰质炎病毒结构和功能特性的广泛了解，作为详细描述这些蛋白质-蛋白质相互作用的基础；(Ii)解决针对脊髓灰质炎病毒的新的抗病毒战略的需要，2007年美国国家科学院的一个小组断言，这将大大加强根除努力，并防止其作为对未接种疫苗人群的生物武器的威胁。脊髓灰质炎病毒RNA聚合酶组件是复制复合体的功能核心，将根据导致其稳定性的分子间相互作用进行分析，并根据高分辨率结晶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的亚纳米分辨率发展，像这里提出的这样的生物学问题继续推动技术创新。在具体目标1中，将发展常规使用冷冻电子显微镜来解析纳米晶体结构的方法，该方法可用于分析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。这将比拟议的聚合酶研究更有价值，使人们能够对经常出现在结晶盘中的蛋白质纳米晶体的结构进行分析，到目前为止，这些纳米晶体一直被认为是失败的实验而被丢弃，最近引起了人们极大的兴趣。具体而言 目的2，通过对包含野生型和突变型聚合酶的复合体的结构和功能的分析，测试通过跨越聚合酶界面的正电荷通道稳定聚合酶-RNA复合体和支持RNA复制的机制。在特定目的3中，将病毒复制蛋白导入哺乳动物细胞将被用来(I)确定细胞膜重组为双层所需的最低病毒成分，以及(Ii)表征稳定含聚合酶的低聚物的蛋白质-蛋白质相互作用，因为这些界面代表着破坏病毒复制和作为潜在药物靶点开发的脆弱部位。 公共卫生相关性：需要针对脊髓灰质炎病毒和类似病毒(如丙型肝炎和SARS)的新的抗病毒药物来治疗这些疾病的患者，并帮助防止将其用作生物武器对未接种疫苗的人口构成威胁。一种有希望的方法是针对病毒制造新副本所需的蛋白质。这项拟议的研究将着眼于复制病毒基因所必需的蛋白质组成的组装，并为未来的药物设计定位脆弱区域。

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.