Roles of host factor protein subnetworks in regulating steps of filovirus infection

宿主因子蛋白亚网在丝状病毒感染调节步骤中的作用

• 批准号: 10555057
• 负责人: ROBERT A DAVEY
• 金额: $ 62.76万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-07 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555057
• 关键词: Actins; Address; Affect; Affinity Chromatography; Algorithms; Antibodies; Behavior; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Classification; Clustered Regularly Interspaced Short Palindromic Repeats; Computational algorithm; Computer Models; Data; Data Set; Databases; Disease; Ebola virus; Evaluation; Filoviridae Infections; Filovirus; Fluorescence; Genes; Genetic; Genetic Transcription; Inclusion Bodies; Infection; Integration Host Factors; Liver; Mass Spectrum Analysis; Measures; Microscopy; Modeling; Molecular; Network-based; Ontology; Optics; Outcome; Outcome Measure; Pathway Analysis; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Post-Translational Protein Processing; Predisposition; Prize; Probability; Production; Proteins; Proteomics; Publishing; RNA; RNA Processing; RNA chemical synthesis; Records; Regulation; Research Project Grants; Resolution; Role; Scheme; Set protein; Small Interfering RNA; Stains; Sumoylation Pathway; System; Systems Biology; Techniques; Testing; Translations; Ubiquitin; Viral; Viral Proteins; Virus; Virus Assembly; Virus Diseases; Virus Replication; Work; Yeasts; cell type; complex data; domain mapping; follow-up; forest; genome-wide; insight; knock-down; knockout gene; knowledge base; large datasets; link protein; monocyte; mutant; network models; novel; overexpression; protein complex; protein expression; protein function; protein protein interaction; risk mitigation; screening; synergism; therapy design; tool; viral RNA; yeast two hybrid system

RP03 Project Summary/Abstract Filoviruses critically depend on cellular proteins to facilitate replication and are susceptible to inhibition by cellular antiviral systems. Research Project 3 (RP03) tests the roles of host protein complexes in virus replication and establishes an approach to evaluate large datasets for biological importance in virus infection. The work builds on our genome-wide siRNA, CRISPR-Cas9, BioID, yeast two-hybrid (Y2H), and co-affinity purification plus mass spectrometry screening data that has identified 100s of host factors that play roles in EBOV and MARV replication. Traditionally, follow up on such large hit sets has been slow, hampered by assays of sufficient throughput and informative read-out to provide needed prioritization. Additionally, hits tend to addressed in isolation, disregarding the relatedness of each hit by function or cellular association. Here, we apply an advanced computer algorithm, the Prize Collecting Steiner Forest (PCSF) algorithm to associate hit proteins by known, high confidence, published, protein-protein interaction (PPI) networks. Overlaying virus protein interactions detected in Y2H and proteomics work revealed clusters of host proteins that interact with a common virus protein. Furthermore, overlaying highest probability protein function using Gene Ontology (GO) terms from different databases, revealed clusters of host proteins related by likely cellular function, with actin regulation and RNA processing being the most over-represented but also pathways related to protein modifications through ubiquitinylation, sumoylation or phosphorylation being evident (as seen in RP01 and RP02). Based on these novel findings, we propose the hypothesis that the host factors residing in subnetworks related by common virus proteins, function or both play the same role in a specific virus replication step. Here, we test this hypothesis by applying a novel, statistically high powered optical pooled screening platform that phenotypically evaluates infection outcome by measuring both virus protein and virus RNA expression levels together with subcellular staining patterns to associate viral functional relatedness to relatedness by known host PPI. This approach allows efficient prioritization of groups of factors for evaluation by mechanistic assays that identify affected steps in virus infection and then defining regions of the host and virus proteins responsible for the infection outcome. The work starts with high priority validated leads representing the actin and RNA processing networks, then evaluates our existing high stringency network, which is then expanded and enriched through new data fed from RP01 and RP02. Our team consists of experts with strong track records in performing and analyzing large host factor genetic and proteomic screens, and performing virus mechanistic analysis. Through extensive interaction between each group, we expect to gain mechanistic insight into roles for identified host factor protein subnetworks, which will provide knowledge-based choices for design of interventions targeting these interactions, for both EBOV and MARV.

RP03项目摘要/摘要 丝状病毒严重依赖细胞蛋白来促进复制，并且容易受到细胞的抑制作用 抗病毒系统。研究项目3（RP03）测试宿主蛋白复合物在病毒复制中的作用和 建立一种评估大型数据集在病毒感染中生物学重要性的方法。工作建立 在我们全基因组的siRNA，CRISPR-CAS9，Bioid，酵母两杂交（Y2H）和共亲密纯化加质量上 光谱筛选数据已经确定了在EBOV和MARV中起作用的100个主机因子 复制。传统上，跟进如此大的命中量一直很慢，受到足够的测定的阻碍 吞吐量和信息丰富的读数，以提供所需的优先级。此外，命中倾向于解决 隔离，无视每个受功能或细胞关联命中的相关性。在这里，我们应用了高级 计算机算法，收集Steiner Forest（PCSF）算法的奖品，以相关的命中蛋白 高度置信，已发表，蛋白质 - 蛋白质相互作用（PPI）网络。覆盖病毒蛋白相互作用 在Y2H和蛋白质组学工作中检测到​​的揭示了与常见病毒蛋白相互作用的宿主蛋白簇。 此外，使用来自不同的基因本体学（GO）术语覆盖最高概率蛋白函数 数据库，揭示了与可能细胞功能相关的宿主蛋白的簇，肌动蛋白调节和RNA 处理是最有代表性的，但也是与蛋白质修饰有关的途径 明显的泛素化，sumoylation或磷酸化（如RP01和RP02所示）。基于这些 新发现，我们提出了以下假设：驻留在子网中的宿主因子与普通病毒相关的宿主因子 蛋白质，功能或两者都在特定病毒复制步骤中起相同的作用。在这里，我们通过 应用一个新颖的，统计上高功率的光学合并筛选平台，以表型评估 通过测量病毒蛋白和病毒RNA表达水平以及亚细胞的感染结果 通过已知宿主PPI将病毒功能相关性与相关性相关的染色模式。这种方法 允许通过识别受影响步骤的机械测定法进行有效的因素优先级以进行评估 在病毒感染中，然后定义宿主和病毒蛋白的区域，负责感染结果。 这项工作从代表肌动蛋白和RNA处理网络的高优先级验证的潜在客户开始，然后 评估我们现有的高严格网络，然后通过馈送的新数据扩展并丰富了该网络 RP01和RP02。我们的团队由专家组成，在表演和分析大型主持的方面具有很强的记录 因子遗传和蛋白质组学筛选，并进行病毒机理分析。通过广泛的互动 在每组之间，我们期望获得对已识别宿主因子蛋白的作用的机械洞察力 子网络将提供基于知识的选择，以设计针对这些的干预措施 Ebov和Marv的互动。