Perturbing virus replication with interfering peptides

新出现的病毒对人类健康构成重大挑战，当前的COVID19大流行就是例证。然而，除了SARS-CoV-2之外，每年都会出现新的病毒，最近出现的病毒继续构成威胁。该项目的重点是埃博拉病毒(EBOV)，自2014年最大规模疫情爆发以来，埃博拉病毒继续造成零星疫情。就在2021年2月，据报道又有两人死亡。与所有病毒一样，EBOV是一种专性的细胞内寄生虫，既依赖细胞蛋白进行复制，又容易受到细胞抗病毒反应的抑制。为了促进自身复制和克服细胞防御，病毒蛋白与病毒和细胞蛋白相互作用。在过去的十年里，数十项已发表的研究报告了数以千计的病毒蛋白质-蛋白质相互作用(PPI)。然而，这些交互作用中的绝大多数还没有被描述出来，选择哪些交互作用优先进行有重点的、假设驱动的后续实验往往是一项具有挑战性的任务。需要更好的工具来识别关键的病毒PPI。为了弥补这一差距，在这个项目中，我们开发了一种创新的、普遍适用的方法来全面识别能够干扰病毒PPI并抑制病毒复制的病毒多肽。我们的假设是，关键的病毒-病毒和病毒-宿主细胞PPI是由短线性相互作用结构域介导的，当在宿主细胞中表达时，这些结构域可以主要干扰病毒的复制。EBOV和其他几种病毒发布的报告支持了这一假设。例如，来自EBOV核蛋白NP或来自细胞蛋白如RBBP6的富含脯氨酸的短序列与EBOV VP30结合。在哺乳动物细胞中表达这些肽作为与GFP的融合破坏了NP和VP30之间的相互作用，并抑制了EBOV的复制。在这里，我们建议使用包含所有可能的30聚体EBOV多肽的平铺多肽文库来系统地鉴定相似的多肽。在目标1中，我们以N端和C端与GFP融合的形式构建了这些肽库，并在对EBOV诱导的细胞病变效应敏感的细胞中表达它们。表达抑制性多肽的细胞会抑制EBOV的复制，更有可能存活，从而增加种群中的频率。这些多肽将通过对感染前和感染后的人群进行深度测序来识别，以找到丰度增加的人群。在目标2中，我们验证了这些多肽的抑制作用，并评估了它们对与病毒和细胞蛋白相互作用的影响。该项目的成功完成将产生最容易受到干扰的高分辨率全基因组EBOV蛋白质相互作用域图谱，以及改进的工具，以快速表征新出现的病毒。来自该项目的数据将增强我们对EBOV复制的理解，并可能引导识别新的药物靶点。

Emerging viruses pose significant challenges to human health, as exemplified by the current COVID19 pandemic. However, in addition to SARS-CoV-2, new viruses emerge each year and recently emerged viruses continue to pose threats. The focus of this project, the Ebola virus (EBOV), has continued to cause sporadic outbreaks since the largest outbreak in 2014. As recently as February 2021, two additional deaths were reported. EBOV, as with all viruses, is an obligate intracellular parasites that is both dependent upon cellular proteins for its replication and susceptible to inhibition by cellular antiviral responses. To promote their own replication and to overcome the cellular defenses, virus proteins interact with both viral and cellular proteins. Over the last decade, dozens of published studies have reported thousands of virus protein-protein interactions (PPIs). However, the vast majority of these interactions have not been characterized and choosing which interactions to prioritize for focused, hypothesis driven follow up experiments is often a challenging task. Better tools are needed to identify critical virus PPIs. To address this gap, in this project we develop an innovative, generally applicable approach to comprehensively identify virus peptides that can disrupt virus PPIs and inhibit virus replication. Our hypothesis is that critical virus-virus and virus-host cell PPIs are mediated by short linear interaction domains that can dominantly interfere with virus replication when expressed in host cells. Published reports from EBOV and several other viruses provide support for this hypothesis. For example, short proline-rich sequences from the EBOV nucleoprotein NP or from cellular proteins such as RBBP6 bind to EBOV VP30. Expressing these peptides in mammalian cells as fusions to GFP disrupts the interaction between NP and VP30 and inhibits EBOV replication. Here, we propose to systematically identify similar peptides using tiled peptide libraries containing all possible 30-mer EBOV peptides. In aim 1, we generate these peptide libraries as N- and C-terminal fusions to GFP and express them in cells that are sensitive to EBOV-induced cytopathic effect. Cells expressing inhibitory peptides will suppress EBOV replication and be more likely to survive, thus increasing in frequency in the population. These peptides will be identified by deep sequencing the pre- and post-infection populations to find those whose abundance increases. In aim 2, we verify the inhibitory effect of these peptides and evaluate their impact on interactions with viral and cellular proteins. Successful completion of this project will yield a high-resolution, genome-wide map of EBOV protein interaction domains that are most susceptible to disruption, as well as improved tools to rapidly characterize newly emerged viruses. The data from this project will enhance our understanding of EBOV replication and may lead the identification of new drug targets.