Emerging virus-host cell protein interaction networks

 描述（由申请方提供）：本提案的总体目标是鉴定和表征与病毒性出血热病毒结合的细胞蛋白。具体来说，我们专注于 对来自四个科的RNA病毒：丝状病毒埃博拉病毒和马尔堡病毒;沙粒病毒朱宁病毒和拉沙热病毒;以及布尼亚病毒克里米亚-刚果出血热病毒。这些新出现的病毒对人类健康和国家安全构成重大风险。虽然每种编码的蛋白质类型不同，但它们具有相似的疾病结果，导致严重的，通常是致命的人类感染;所有这些都被归类为NIAID A类优先病原体。由于需要对其中许多病毒进行高水平的遏制，我们对这些病毒的理解存在重大差距，目前没有具体的批准治疗方法。像所有病毒一样，这些新兴病毒依赖于宿主细胞蛋白来复制。这种依赖性代表了病毒的一个潜在的“致命弱点” 生命周期，可以利用开发新的方法来治疗病毒感染。然而，本研究中仅报告了少量病毒-宿主细胞蛋白相互作用，未对新出现的病毒-宿主细胞蛋白相互作用进行系统分析。在这个项目中，我们开发和评估新出现的病毒-人类蛋白质相互作用的高质量图谱。在目标1中，我们使用互补酵母双杂交筛选和共亲和纯化加质谱法来鉴定与来自同一家族的新出现的病毒和密切相关的非致病性病毒结合的细胞蛋白。在目标2中，我们确定了新兴病毒的宿主细胞相互作用网络的共享和病毒特异性特征。我们使用直向同源蛋白质-蛋白质相互作用测定来鉴定每个病毒家族内的共享和病毒特异性相互作用。为了获得这些结果的置信度，将每个检测试剂盒与人阳性和随机参考集进行基准比较。然后，我们采用信息学方法来发现新兴病毒优先靶向的途径，开发组织特异性病毒-宿主细胞蛋白相互作用网络，并确定预测抑制病毒复制的小分子激动剂或拮抗剂。在目标3中，我们测试了通过生物信息学分析产生的预测。一个由多种病毒靶向的高兴趣细胞蛋白的子集将被实验性地询问它们对使用RNA干扰的感染的贡献。然后采用最先进的分子技术来确定它们促进病毒复制的机制。评价病毒和组织特异性相互作用对病毒复制的贡献及其对细胞增殖的影响。 信号通路最后，在我们的网络模型中鉴定的小分子抑制剂被评估其对细胞培养物中的病毒复制的影响。该项目产生的数据将提高我们对单个病毒蛋白功能的理解，深入了解病毒性出血热病毒与其宿主细胞相互作用的总体策略，并可能导致发现治疗新兴病毒的新靶点。

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to identify and characterize cellular proteins that bind to viral hemorrhagic fever viruses. Specifically, we focus on RNA viruses from four families: the filoviruses Ebolavirus and Marburg virus; the Arenaviruses Junin virus and Lassa Fever virus; and the Bunyavirus Crimean-Congo hemorrhagic fever virus. These emerging viruses pose significant risks to human health and national security. While each differs in the types of proteins encoded, they share similar disease outcomes, causing severe, often fatal infections in humans; all are classified as NIAID Category A Priority Pathogens. Due to the high level containment needed to work with many of them, there is a significant gap in our understanding of these viruses and no specific, approved therapies are currently available. Like all viruses, these emerging viruses rely on host cell proteins in order to replicate. This dependence represents a potential "Achilles heel" in the virus life cycle that may be exploited to develop new approaches to treat viral infections. However, only a small number of virus-host cell protein interactions have been reported for the viruses in this study, and no systematic analysis of emerging virus-host cell protein interactions has been performed. In this project we develop and evaluate high quality maps of emerging virus-human protein interactions. In Aim 1 we use complementary yeast two-hybrid screening and co-affinity purification plus mass spectrometry approaches to identify cellular proteins that bind to emerging viruses and closely related non-pathogenic viruses from the same family. In aim 2 we identify shared and virus-specific features of the host cell interaction networks of emerging viruses. We use orthologonal protein-protein interaction assays to identify shared and virus specific interactions within each virus family. To gain confidence in these results, each assay is benchmarked against the human positive and random reference sets. We then employ informatic approaches to discover pathways that targeted preferentially by emerging viruses, develop tissue-specific virus-host cell protein interaction networks, and identify small molecule agonists or antagonists predicted to inhibit virus replication. In aim 3, we test the predictions generated through the bioinformatic analyses. A subset of high-interest cellular proteins targeted by multiple viruses will be experimentally interrogated for their contribution to infectio using RNA interference. State-of-the-art molecular techniques are then employed to determine the mechanisms by which they contribute to virus replication. Virus- and tissue-specific interactions are evaluated for their contribution to virus replication and their effect on cellular signaling pathways. Finally, small molecule inhibitors identified in our network models are evaluated for their effect on virus replication in cell culture. The data generated from this projet will improve our understanding of the functions of individual viral proteins, provide insight into the overall strategies used by viral hemorrhagic fever virus to interface with their host cells, an may lead to the discovery of new targets for treatments of emerging viruses.