Novel VP30-host Interactions that Negatively Regulate Ebola Virus Infection

摘要 丝状病毒，埃博拉和马尔堡病毒(EBOV和MARV)，是一种新出现的负链RNA病毒 与严重病毒性出血热暴发有关。这些病毒的毒性和新出现的性质 人畜共患病原体使其成为对人类健康的重大威胁，潜在的生物恐怖主义因素，以及 NIAID A类优先病原体。目前，还没有获得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。这个修订的R01应用程序的总体目标是描述EBOV VP30的特征 (EVP30)，一种促进病毒转录的关键病毒蛋白，及其与宿主因子的相互作用。我们的计划 建立在最近在结构和功能上表征eVP30如何与病毒相互作用方面的成功的基础上 调节EBOV RNA合成的核蛋白(NP)和联合(Amarasinghe、Basler和Krogan Groups)以EBOV蛋白为诱饵的无偏蛋白质组学筛选发现了193个高置信度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。 该复合体的晶体结构表明，RBBP6和病毒NP竞争相同的VP30结合 浮出水面。结合eVP30的NP和RBBP6多肽的比较显示了一个共同的PPxPxY基序，即 这是互动所必需的。而内源性RBBP6的敲除可刺激病毒转录和 EBOV感染力增加，RBBP6或其肽的过表达严重抑制EBOV转录和 感染。有趣的是，我们的数据集中至少还有两个eVP30交互作用子(hnRNP L和hnRNP UL1) 也有PPxPxY图案。基于这些发现，我们提出了一个多学科的方法来研究(1) 确定eVP30 N-末端的结构，并确定其与RNA和蛋白质配体的关系 NP的存在和缺失；(2)确定eVP30与蛋白RBBP6相互作用的机制， HnRNP L和hnRNP UL1调节eVP30功能和RNA合成；以及(3)检验以下假设 EVP30调控宿主因子RBBP6、hnRNP L和hnRNP UL1的功能。这些研究将 描述对EBOV复制进行负面调控的独特宿主交互作用，目的是定义 EBOV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.