Function and assembly of the Ebola virus nucleocapsid

 描述（由申请人提供）：埃博拉病毒是已知的最致命的病毒之一，并在2013-2015年在西非造成持续和持续的流行病。埃博拉病毒RNA转录、复制和病毒组装背后的奥秘在一定程度上仍然是个谜，因为还没有高分辨率的结构模型来解释这些过程。该项目的使命是确定埃博拉病毒RNA合成和病毒组装的结构和生化机制。病毒核衣壳是位于每个病毒体核心的多蛋白质-RNA复合物，其充当分子机器以合成和复制病毒RNA基因组的新拷贝。该机器中的病毒蛋白质对病毒RNA合成具有积极和消极的影响，并控制转录和复制的平衡。了解这种核衣壳机器的结构、蛋白质-蛋白质相互作用和组装是开发抗病毒药物的关键。然而，目前的低分辨率模型的核衣壳提出了相互矛盾的模型。在这里，我们提出了互补的晶体学分析，建立核衣壳（NP-RNA，NP-VP 35，NP-VP 24，NP-VP 30和NP-VP 40），结合组装的核衣壳的高分辨率电子显微镜重建的每个复合物。EM重建将使用最先进的仪器，创新技术和高质量的纯化样品来实现，结构结果将得到生化，生物物理和功能分析的支持。重要的是，几乎所有提出的复合物都有晶体在手，功能测定在实验室中可操作。由此产生的病毒核衣壳及其组件组件的结构将改变我们对埃博拉病毒和新兴丝状病毒的组装和复制的理解。此外，该提案的结果将为这些病毒的功能分析提供路线图，以及开发抗病毒药物以保护人类免受其害的策略。

 DESCRIPTION (provided by applicant): Ebola virus is among the deadliest viruses known and has caused on ongoing and sustained epidemic in Western Africa 2013-2015. The mysteries behind Ebola virus RNA transcription, replication and viral assembly remain mysteries in part, because no high- resolution structural models yet exist by which we may interpret these processes. The mission of this project is to define the structural and biochemical mechanisms governing Ebola virus RNA synthesis and viral assembly. The viral nucleocapsid is a multi-protein-RNA complex at the core of each virion that serves as a molecular machine to synthesize and encapsidate new copies of the viral RNA genome. The viral proteins in this machine have both positive and negative influences on viral RNA synthesis and control the balance of transcription and replication. Understanding the structures, protein-protein interactions, and assemblies of this nucleocapsid machine is key to development of antivirals. However, current low resolution models of the nucleocapsid present conflicting models. Here, we propose complementary crystallographic analysis of each complex that builds the nucleocapsid (NP-RNA, NP-VP35, NP-VP24, NP-VP30 and NP-VP40), combined with high-resolution electron microscopy reconstructions of assembled nucleocapsids. The EM reconstructions will be achieved using state-of-the-art instrumentation, innovative techniques, and high-quality purified samples, and the structural findings will be supported by biochemical, biophysical and functional analysis. Importantly, crystals are in hand for nearly all proposed complexes and functional assays are operational in the laboratory. The resulting structures of the viral nucleocapsid and its component assemblies will transform our understanding of the assembly and replication of Ebola and emerging filoviruses. Further, the results of this proposal will provide roadmaps for functional analysis of these viruses as well as strategies for development of antivirals to defend human populations against them.