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 丝状病毒严重依赖于细胞蛋白来促进复制，并容易受到细胞的抑制 抗病毒系统。研究项目3(RP03)测试宿主蛋白复合体在病毒复制和 建立了一种评估大型数据集在病毒感染中的生物学重要性的方法。这项工作建立了 在我们的全基因组siRNA、CRISPR-Cas9、BioID、酵母双杂交(Y2H)和共亲和纯化加质量上 已确定在EBOV和MARV中起作用的100个宿主因素的光谱筛选数据 复制。传统上，对如此大的热门歌曲集的后续工作一直很缓慢，受到足够多的 吞吐量和信息量读出，以提供所需的优先顺序。此外，命中倾向于在 孤立，无视每一次打击的功能或细胞联系的关联性。在这里，我们应用先进的 计算机算法，获奖斯坦纳森林(PCSF)算法，通过已知关联HIT蛋白质， 高度可信，已出版的蛋白质-蛋白质相互作用(PPI)网络。重叠病毒蛋白相互作用 在Y2H和蛋白质组学工作中检测到的宿主蛋白质簇与一种常见的病毒蛋白相互作用。 此外，使用来自不同来源的基因本体论(GO)术语覆盖最高概率蛋白质功能 数据库揭示了通过可能的细胞功能与肌动蛋白调控和RNA相关的宿主蛋白簇 加工是最有代表性的，但也是与蛋白质修饰有关的途径，通过 泛素化、和素化或磷酸化明显(见RP01和RP02)。基于这些 新的发现，我们提出假设，宿主因子驻留在与普通病毒相关的子网络中 蛋白质、功能或两者在特定的病毒复制步骤中起着相同的作用。在这里，我们通过以下方式验证这一假设 应用一种新的、统计高功率的光学集合筛查平台，其表型评估 通过同时测量病毒蛋白和病毒RNA表达水平以及亚细胞水平的感染结局 通过已知宿主PPI将病毒功能相关性与相关性相关联的染色模式。这种方法 允许通过确定受影响步骤的机械分析来有效地确定评估因素组的优先顺序 在病毒感染中，然后确定宿主的区域和负责感染结果的病毒蛋白。 这项工作首先从代表肌动蛋白和RNA加工网络的高优先级验证线索开始，然后 评估我们现有的高要求网络，然后通过从以下方面反馈的新数据对其进行扩展和丰富 RP01和RP02。我们的团队由在表演和分析大型主持人方面有良好记录的专家组成 因子遗传和蛋白质组筛选，并进行病毒机制分析。通过广泛的互动 在每一组之间，我们期望获得对已识别的宿主因子蛋白的作用的机械性洞察 子网络，这将为针对这些目标的干预措施的设计提供基于知识的选择 相互作用，用于EBOV和MARV。