Structural and Functional Characterization of the Ebola Virus Replication Complex

摘要：埃博拉病毒RNA合成结构与功能研究项目综述 埃博拉病毒(EBOV)是一种非节段性负链RNA病毒(NNSVs)，属于A类病毒 导致频繁致命性出血热的生物防御病原体，包括破坏性的和正在进行的 西非爆发疫情。缺乏获得批准的抗EBOV疗法。EBOV在中国高效复制 体内有效躲避宿主先天抗病毒免疫。不受限制的复制和关联 炎症会导致死亡。因此，对EBOV致病机理的完整理解需要 了解病毒RNA依赖的RNA聚合酶(RDRP)复合体如何与宿主因子相互作用。 本计划项目提案的首要目标是通过以下方式了解RDRP的作用机制 定义其组件之间的相互作用以及与宿主蛋白质组的相互作用，并通过定义 规范其功能。为了实现这一目标，项目1将确定影响EBOV的顺式和反式作用因素 RDRP活性。项目2将为EBOV RDRP复合体定义结构基础，确定监管 机制，并确定物种和菌株的特异性，项目3将使用全基因组RNAi和 确定调节RDRP功能的宿主因子的蛋白质组学策略。项目1提供了功能上下文 其中可以解释项目3的子集“命中”，而项目3可能会识别相关的宿主因素 项目1的调查结果。项目2将提供一个结构框架，用于整合调查结果 来自两个项目1和3。这些研究将提供对蛋白质-蛋白质如何 蛋白质-vRNA相互作用控制EBOV RDRP功能。研究项目由一个 管理核心，蛋白质生产和蛋白质相互作用的核心，关键是，生物安全级别4 (BSL4)核心。核心B将产生独特的试剂，包括克隆抗体和合成抗体。核心C (BSL4)将提供独特的能力，从野生型和突变型获得材料并进行测试 EBOV以及致病机制的评价。这项工作将由高生产率和协作性强的人员完成 在拟议研究的各个方面都有专业知识的调查人员，包括生物化学、EBOV 发病机制、高通量筛选和数据分析、免疫学、蛋白质组学、结构生物学和 病毒学。这些研究将对EBOV RDRP的组成和其 与宿主的相互作用以及影响RDRP复合体的物种和菌株差异。我们也 期望将特定的病毒和宿主因子相互作用确定为新的治疗靶点。

Abstract: Project Overview for Structural and Functional Studies of Ebola Virus RNA synthesis Ebolaviruses (EBOVs) are non-segmented negative-strand RNA viruses (NNSVs) and Category A Priority biodefense pathogens that cause frequent lethal hemorrhagic fever, including the devastating and ongoing outbreak in West Africa. Approved anti-EBOV therapeutics are lacking. EBOV replicates with high efficiency in vivo while effectively evading host innate antiviral immunity. The unrestrained replication and associated inflammation leads to death. A complete understanding of EBOV pathogenesis therefore requires understanding how the viral RNA dependent RNA polymerase (RDRP) complex interacts with host factors. The overarching goal of this program project proposal is to understand the mechanisms of RDRP function by defining the interactions among its components and with the host proteome and by defining signals that regulate its function. Toward this goal Project 1 will identify cis- and trans-acting factors that impact EBOV RDRP activity. Project 2 will define a structural basis for the EBOV RDRP complex, identify regulatory mechanisms, and determine species and strain specificities, and Project 3 will use genome-wide RNAi and proteomic strategies to identify host factors that modulate RDRP function. Project 1 provides functional context into which a subset of Project 3 “hits” can be interpreted, while Project 3 will likely identify host factors relevant to the findings of Project 1. Project 2 will provide a structural framework that will be used to integrate findings from both Projects 1 and 3. Together, these studies will provide mechanistic insights into how protein-protein and protein-vRNA interactions control EBOV RDRP function. Research projects are supported by an Administrative Core, a Protein Production and Protein Interaction Core, and critically, a Biosafety Level 4 (BSL4) Core. Core B will generate unique reagents, including monoclonal and synthetic antibodies. Core C (BSL4) will provide unique capabilities to obtain materials from and perform tests using wild type and mutant EBOVs as well as pathogenesis evaluation. The work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, EBOV pathogenesis, high throughput screening and data analysis, immunology, proteomics, structural biology, and virology. These studies will provide unprecedented insights into the components of the EBOV RDRP and in its interaction with the host as well as species and strain differences that affect the RDRP complex. We also expect to identify specific viral and host factor interactions as novel therapeutic targets.