PPiSeq: High-Throughput Protein-Protein Interaction Sequencing

PPiSeq：高通量蛋白质-蛋白质相互作用测序

• 批准号: 10449402
• 负责人: SASHA F LEVY
• 金额: $ 41.89万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-16 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10449402
• 关键词: Animals; Antiviral Agents; Bar Codes; Biological Assay; Cell Culture Techniques; Cell physiology; Cells; Communities; Complement; Cowpox; DHFR gene; Data; Data Set; Dihydrofolate Reductase; Drug Screening; Drug Targeting; Epidemic; Funding; Grant; Health; Hepatitis E; Herpesviridae; Human; Infection; Influenza B Virus; Influenza C Virus; Learning; Libraries; Maps; Measures; Molecular; Mutagenesis; Natural regeneration; Open Reading Frames; Output; Pathway interactions; Pharmaceutical Preparations; Process; Protein Fragment; Protein-Protein Interaction Map; Proteins; Rabies; Readiness; Research; Resources; Scanning; System; Technology; Testing; Variant; Viral; Viral Proteins; Virulence; Virus; Work; Yeasts; Zoonoses; base; dimer; drug testing; gene synthesis; genetic analysis; genetic variant; genome-wide; high throughput screening; human coronavirus; human population genetics; particle; pathogenic virus; protein protein interaction; screening; sequencing platform; small molecule; small molecule libraries; zoonotic spillover

Project Summary/Abstract Emerging and endemic viral pathogens are a persistent threat to human health, the global economy, and national readiness. Viral proteins interact with host proteins to hijack host cells and replicate transmissible viral particles. Human-viral and viral-viral protein-protein interactions (PPls) have been comprehensively characterized for a limited set of viruses, identifying a "PPI profile" for each virus screened. However, these efforts have characterized only a small fraction of the known viruses. A complete viral-human PPI map would be an invaluable resource, enabling analyses of how often interacting viral proteins converge on common human protein targets, cellular functions, or pathways, and which of these interactions are associated with transmissibility or virulence. Combining the viral-human PPI map with human population genetic analyses will enable characterization of the molecular mechanisms underlying extant and ancient epidemics and how these PPIs drive much of human adaptation. In addition, PPI profiles of human viruses could be used to aid screening of animal reservoirs to identify potential threats before a zoonotic spillover occurs. Despite its great promise, characterization of the viral-human PPI map remains a challenge given the throughput of current viral-human PPI screening assays. Current high-throughput assays also lack a quantitative output, meaning that the emergent human-viral PPI map, or viral-viral PPI maps, would be difficult to exploit for important downstream variant scanning or drug screening applications. Here we will use a quantitative sequencing-based protein-protein interaction assay platform to screen for PPls between -24 million viral-human or viral-viral protein pairs. We will further develop this technology into a massively parallel drug screening platform and use it to screen >3 million drug-PPI combinations for small-molecule compounds that promote or antagonize a PPI. The viral-human PPI and drug-PPI maps developed here will be an invaluable resource for a broad research community. In addition, this work will establish new massively parallel and quantitative PPI and drug-PPI screening technologies that will scale with advances in gene synthesis, mutagenesis and sequencing, enabling parallel screening of tens of thousands of gene and gene variants of extant, emerging, and potentially zoonotic viruses.

项目概要/摘要 新出现的地方性病毒病原体对人类健康、全球经济和国家准备状况构成持续威胁。病毒蛋白与宿主蛋白相互作用，劫持宿主细胞并复制可传播的病毒颗粒。人类-病毒和病毒-病毒蛋白-蛋白相互作用 (PPls) 已针对一组有限的病毒进行了全面表征，为每种筛选的病毒确定了“PPI 图谱”。然而，这些努力仅描述了一小部分已知病毒的特征。完整的病毒-人类 PPI 图谱将是一种宝贵的资源，可以分析相互作用的病毒蛋白在常见的人类蛋白靶点、细胞功能或途径上聚合的频率，以及这些相互作用中哪些与传播性或毒力相关。将病毒-人类 PPI 图谱与人群遗传分析相结合，将能够表征现存和古代流行病的分子机制，以及这些 PPI 如何推动人类适应。此外，人类病毒的 PPI 谱可用于帮助筛查动物宿主，以便在人畜共患病蔓延发生之前识别潜在威胁。尽管前景广阔，但考虑到当前病毒-人 PPI 筛选测定的通量，病毒-人 PPI 图谱的表征仍然是一个挑战。目前的高通量检测还缺乏定量输出，这意味着新兴的人类-病毒 PPI 图谱或病毒-病毒 PPI 图谱将难以用于重要的下游变异扫描或药物筛选应用。在这里，我们将使用基于定量测序的蛋白质-蛋白质相互作用测定平台来筛选-2400万个病毒-人类或病毒-病毒蛋白质对之间的PPls。我们将进一步将该技术开发成大规模并行药物筛选平台，并用它来筛选超过300万种药物-PPI组合，以寻找促进或拮抗PPI的小分子化合物。这里开发的病毒-人类 PPI 和药物-PPI 图谱将成为广泛研究界的宝贵资源。此外，这项工作将建立新的大规模并行定量 PPI 和药物 PPI 筛选技术，这些技术将随着基因合成、诱变和测序的进步而扩展，从而能够并行筛选现有、新兴和潜在人畜共患病毒的数万个基因和基因变体。