PPiSeq: High-Throughput Protein-Protein Interaction Sequencing

项目概要/摘要 新出现的和地方性的病毒病原体是对人类健康、全球经济和国家准备的持续威胁。病毒蛋白与宿主蛋白相互作用以劫持宿主细胞并复制可传播的病毒颗粒。人-病毒和病毒-病毒蛋白质-蛋白质相互作用（PPI）已经针对有限的一组病毒进行了全面表征，鉴定了筛选的每种病毒的“PPI谱”。然而，这些努力只描述了一小部分已知病毒的特征。完整的病毒-人类PPI图谱将是一个宝贵的资源，可以分析相互作用的病毒蛋白质与常见的人类蛋白质靶点、细胞功能或途径聚集的频率，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图谱与人类群体遗传分析相结合，将能够表征现存和古老流行病的分子机制，以及这些PPI如何驱动人类的大部分适应。此外，人类病毒的PPI谱可用于帮助筛选动物宿主，以在人畜共患病溢出发生之前识别潜在威胁。尽管其具有很大的前景，但鉴于目前病毒-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。目前的高通量测定也缺乏定量输出，这意味着出现的人-病毒PPI图谱或病毒-病毒PPI图谱将难以用于重要的下游变体扫描或药物筛选应用。在此，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约2400万个病毒-人或病毒-病毒蛋白质对之间的PPl。我们将进一步将这项技术开发成一个大规模并行药物筛选平台，并使用它来筛选超过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社区的宝贵资源。此外，这项工作将建立新的大规模并行和定量PPI和药物PPI筛选技术，这些技术将随着基因合成，诱变和测序的进展而扩展，从而能够并行筛选现存，新兴和潜在的人畜共患病病毒的数万个基因和基因变体。

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.