Perturbing virus replication with interfering peptides

用干扰肽扰乱病毒复制

• 批准号: 10613481
• 负责人: Douglas J. LaCount
• 金额: $ 19.87万
• 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 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10613481
• 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; Cell Survival; 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; Nucleoproteins; Pathogenesis; Peptide Fragments; Peptide Library; Peptide Mapping; Peptides; Phosphoric Monoester Hydrolases; Population; Positioning Attribute; Predisposition; 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; 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重大挑战，当前的COVID 19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年爆发最大规模疫情以来，该病毒继续引起零星疫情。就在2021年2月，又报告了两例死亡病例。与所有病毒一样，EBOV是一种专性细胞内寄生虫，其复制依赖于细胞蛋白质，并且易受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质-蛋白质相互作用（PPI）。然而，这些相互作用中的绝大多数尚未被表征，并且选择哪些相互作用优先用于重点的、假设驱动的后续实验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的病毒PPI。为了解决这一差距，在本项目中，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是由短的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复制。EBOV和其他几种病毒的已发表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RBBP 6的短的富含脯氨酸的序列与EBOV VP 30结合。在哺乳动物细胞中表达这些肽作为与GFP的融合物破坏NP和VP 30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-mer EBOV肽的平铺肽库来系统地鉴定类似的肽。在目标1中，我们将这些肽文库作为与GFP的N-和C-末端融合物产生，并在对EBOV诱导的细胞病变效应敏感的细胞中表达它们。表达抑制性肽的细胞将抑制EBOV复制并且更可能存活，从而在群体中的频率增加。这些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一个高分辨率的EBOV蛋白质相互作用结构域的全基因组图谱，这些结构域最容易被破坏，以及快速表征新出现的病毒的改进工具。该项目的数据将增强我们对EBOV复制的理解，并可能导致新药物靶点的确定。