Function and assembly of the Ebola virus nucleocapsid

 描述(申请人提供)：埃博拉病毒是已知的最致命的病毒之一，并已在2013-2015年间在西非持续流行。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，因为目前还没有高分辨率的结构模型来解释这些过程。该项目的任务是确定控制埃博拉病毒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粒子核心的多蛋白质-RNA复合体，充当合成和封装病毒RNA基因组的新副本的分子机器。这台机器中的病毒蛋白对病毒RNA的合成既有积极的影响，也有消极的影响，控制着转录和复制的平衡。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，目前低分辨率的核衣壳模型呈现出相互矛盾的模型。在这里，我们建议对构建核衣壳的每个络合物(NP-RNA、NP-VP35、NP-VP24、NP-VP30和NP-VP40)进行互补结晶学分析，并结合组装的核衣壳的高分辨率电子显微镜重建。EM重建将使用最先进的仪器、创新的技术和高质量的纯化样本来实现，结构发现将得到生化、生物物理和功能分析的支持。重要的是，几乎所有拟议的络合物都有晶体可用，功能分析也可以在实验室进行。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出现的丝状病毒组件和复制的理解。此外，这项提案的结果将为这些病毒的功能分析提供路线图，并为开发抗病毒药物以保护人类免受它们的攻击提供战略。

 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.