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。这些病毒的毒性和新出现的性质 人畜共患病病原体使其成为人类健康的重大威胁，是生物恐怖主义的潜在因素， NIAID A类优先病原体。目前，没有批准的抗丝状病毒治疗剂可用。 重要的是，我们对宿主因素在疾病关键阶段的作用的理解存在重大差距。 病毒复制周期此次修订的R 01申请的总体目标是表征EBOV VP 30 （eVP 30），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eVP 30如何与病毒相互作用的成功基础上， 核蛋白（NP）调节EBOV RNA合成和关节（Amarasinghe，Basler，and Krogan 使用EBOV蛋白作为诱饵的无偏蛋白质组学筛选，发现了193个高置信度的EBOV- 人蛋白质-蛋白质相互作用（PPI），包括eVP 30和宿主泛素连接酶RBBP 6之间的相互作用。 该复合物的晶体结构显示RBBP 6和病毒NP竞争相同的VP 30结合 面结合eVP 30的NP和RBBP 6肽的比较揭示了一个共同的PPxPxY基序， 互动所必需的。而内源性RBBP 6的敲低刺激了病毒的转录， EBOV感染性增加，RBBP 6或其肽的过表达严重抑制EBOV转录， 感染有趣的是，来自我们数据集的至少两个额外的eVP 30相互作用物（hnRNP L和hnRNP UL 1） 也具有PPxPxY基序。基于这些发现，我们提出了一个多学科的方法（1） 确定eVP 30 N-末端的结构，并确定其与RNA和蛋白质配体的结合， NP的存在和不存在;（2）确定eVP 30相互作用蛋白RBBP 6， hnRNP L和hnRNP UL 1调节eVP 30功能和RNA合成;和（3）检验以下假设： eVP 30调节宿主因子RBBP 6、hnRNP L和hnRNP UL 1的功能。这些研究将 表征负调节EBOV复制的独特宿主相互作用，目的是定义如何 EBOV操纵宿主途径并鉴定新的治疗靶点。

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.