Novel VP30-host Interactions that Negatively Regulate Ebola Virus Infection

摘要 丝状病毒、埃博拉病毒和马尔堡病毒（EBOV 和 MARV）是新兴的负链 RNA 病毒 与严重病毒性出血热的爆发有关。这些病毒的毒力和新出现的性质 人畜共患病病原体对人类健康构成重大威胁，是生物恐怖主义的潜在媒介， NIAID A 类优先病原体。目前，还没有批准的抗丝状病毒疗法。 重要的是，我们对宿主因素在关键阶段的作用的理解存在重大差距。 病毒复制周期。此修订版 R01 应用程序的总体目标是表征 EBOV VP30 （eVP30），一种促进病毒转录及其与宿主因子相互作用的关键病毒蛋白。我们的计划 建立在最近在结构和功能表征 eVP30 如何与病毒相互作用方面取得的成功的基础上 核蛋白 (NP) 调节 EBOV RNA 合成并作用于关节（Amarasinghe、Basler 和 Krogan） 使用 EBOV 蛋白作为诱饵进行无偏见的蛋白质组学筛选，发现了 193 个高可信度的 EBOV- 人类蛋白质-蛋白质相互作用 (PPI)，包括 eVP30 和宿主泛素连接酶 RBBP6 之间的相互作用。 该复合物的晶体结构揭示了 RBBP6 和病毒 NP 竞争相同的 VP30 结合 表面。结合 eVP30 的 NP 和 RBBP6 肽的比较揭示了一个共同的 PPxPxY 基序，即 交互所必需的。而内源性 RBBP6 的敲除会刺激病毒转录并 EBOV 感染性增加，RBBP6 或其肽的过度表达严重抑制 EBOV 转录， 感染。有趣的是，我们的数据集中至少有两个额外的 eVP30 交互器（hnRNP L 和 hnRNP UL1） 还具有 PPxPxY 基序。基于这些发现，我们提出了一种多学科方法：(1) 确定 eVP30 N 末端的结构并定义其与 RNA 和蛋白质配体的关联 NP 的缺失和存在； (2) 确定 eVP30 相互作用蛋白 RBBP6、 hnRNP L 和 hnRNP UL1 调节 eVP30 功能和 RNA 合成； (3) 检验假设 eVP30 调节宿主因子 RBBP6、hnRNP L 和 hnRNP UL1 的功能。这些研究将 描述对 EBOV 复制产生负面调节的独特宿主相互作用，其目标是定义如何 埃博拉病毒操纵宿主途径并识别新的治疗靶点。

ABSTRACT The filoviruses, Ebola and Marburg viruses (EBOV and MARV), are emerging, negative-strand RNA viruses associated with outbreaks of severe viral hemorrhagic fever. The virulence and emerging nature of these zoonotic pathogens makes them a significant threat to human health, potential agents of bioterrorism, and NIAID category A priority pathogens. Currently, no approved anti-filovirus therapeutics are available. Importantly, there is a major gap in our understanding with regard to the role of host factors at critical stages in the viral replication cycle. The overall goal of this revised R01 application is to characterize EBOV VP30 (eVP30), a key viral protein that facilitates viral transcription, and its interactions with host factors. Our plan builds on recent successes in structurally and functionally characterizing how eVP30 interacts with the viral nucleoprotein (NP) to modulate EBOV RNA synthesis and on a joint (Amarasinghe, Basler, and Krogan groups) unbiased proteomics screen using EBOV proteins as bait that uncovered 193 high-confidence EBOV- human protein-protein interactions (PPIs), including one between eVP30 and the host ubiquitin ligase RBBP6. A crystal structure of this complex revealed that RBBP6 and the viral NP compete for the same VP30 binding surface. Comparison of NP and RBBP6 peptides that bind eVP30 revealed a common PPxPxY motif that is necessary for the interaction. Whereas knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV infectivity, overexpression of RBBP6 or its peptide severely inhibited EBOV transcription and infection. Interestingly, at least two additional eVP30 interactors from our dataset (hnRNP L and hnRNP UL1) also possess PPxPxY motifs. Based on these findings, we propose a multidisciplinary approach to (1) Determine the structure of eVP30 N-terminus and define its association with RNA and protein ligands in the absence and presence of NP; (2) Determine the mechanisms by which eVP30-interacting proteins RBBP6, hnRNP L, and hnRNP UL1 modulate eVP30 function and RNA synthesis; and (3) Test the hypothesis that eVP30 modulates the function of host factors RBBP6, hnRNP L, and hnRNP UL1. These studies will characterize unique host interactions that negatively regulate EBOV replication with the goals of defining how EBOV manipulates host pathways and identifying novel therapeutic targets.