Perturbing virus replication with interfering peptides

用干扰肽扰乱病毒复制

• 批准号: 10354399
• 负责人: Douglas J. LaCount
• 金额: $ 25.27万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-25 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354399
• 关键词: 2019-nCoV; Address; Affect; Affinity; Antiviral Response; Binding; Binding Proteins; Binding Sites; Bioinformatics; C-terminal; COVID-19 pandemic; Cell Communication; Cell Line; Cells; Cessation of life; Data; Defect; Disease Outbreaks; Ebola; Ebola virus; Europe; Follow-Up Studies; Frequencies; Generations; Goals; Health; Human; Infection; Lead; Libraries; Link; Mammalian Cell; Maps; Mediating; N-terminal; Nucleoproteins; Pathogenesis; Peptide Fragments; Peptide Library; Peptide Mapping; Peptides; Phosphoric Monoester Hydrolases; Population; Positioning Attribute; Proline; Protein Dephosphorylation; Protein Fragment; Protein Interaction Mapping; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Publishing; Reporting; Research; Resistance; Resolution; Survivors; Testing; United States; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; West Nile virus; ZIKA; base; cell killing; chikungunya; deep sequencing; drug discovery; emerging pathogen; experience; experimental study; follow-up; genome-wide; global health; improved; innovation; method development; new therapeutic target; obligate intracellular parasite; pathogen; protein protein interaction; tool

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.

新出现的病毒对人类健康构成了重大挑战，当前的新冠病毒大流行就是一个例子。然而，除了 SARS-CoV-2 之外，每年都会出现新的病毒，并且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，该病毒持续引发零星疫情。就在 2021 年 2 月，据报道又有两人死亡。与所有病毒一样，埃博拉病毒是一种专性细胞内寄生虫，其复制依赖于细胞蛋白，并且容易受到细胞抗病毒反应的抑制。为了促进自身复制并克服细胞防御，病毒蛋白与病毒蛋白和细胞蛋白相互作用。在过去的十年中，数十项已发表的研究报告了数千种病毒蛋白-蛋白相互作用（PPI）。然而，这些相互作用中的绝大多数尚未被表征，并且选择优先进行有针对性的、假设驱动的后续实验的相互作用通常是一项具有挑战性的任务。需要更好的工具来识别关键病毒 PPI。为了解决这一差距，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鉴定可以破坏病毒 PPI 并抑制病毒复制的病毒肽。我们的假设是，关键的病毒-病毒和病毒-宿主细胞 PPI 由短线性相互作用域介导，当在宿主细胞中表达时，该相互作用域可以显着干扰病毒复制。已发表的埃博拉病毒和其他几种病毒的报告为这一假设提供了支持。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。在哺乳动物细胞中将这些肽表达为 GFP 融合物会破坏 NP 和 VP30 之间的相互作用并抑制 EBOV 复制。在这里，我们建议使用包含所有可能的 30 聚体 EBOV 肽的平铺肽库来系统地识别相似的肽。在目标 1 中，我们将这些肽库生成为 GFP 的 N 端和 C 端融合体，并在对 EBOV 诱导的细胞病变效应敏感的细胞中表达它们。表达抑制肽的细胞将抑制埃博拉病毒复制并更有可能存活，从而增加其在群体中的出现频率。这些肽将通过对感染前和感染后群体进行深度测序来识别，以找到丰度增加的肽。在目标 2 中，我们验证了这些肽的抑制作用，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破坏的 EBOV 蛋白相互作用域的高分辨率、全基因组图谱，以及快速表征新出现病毒的改进工具。该项目的数据将增强我们对埃博拉病毒复制的理解，并可能引导新药物靶点的识别。