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

基本信息

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
项目类别：
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

项目摘要

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 项目总结/摘要丝状病毒严重依赖细胞蛋白来促进复制，并且容易受到细胞蛋白的抑制抗病毒系统。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。工作建立关于我们的全基因组 siRNA、CRISPR-Cas9、BioID、酵母双杂交 (Y2H) 和共亲和纯化加质量光谱筛选数据已识别出数百种在 EBOV 和 MARV 中发挥作用的宿主因子复制。传统上，对如此大的命中集的后续行动一直很缓慢，受到足够的分析的阻碍。吞吐量和信息读出，以提供所需的优先级。此外，点击往往会解决隔离，忽略功能或细胞关联的每个命中的相关性。在这里，我们应用了先进的计算机算法，收集斯坦纳森林（PCSF）算法，通过已知的方式关联命中蛋白质，高可信度、已发表的蛋白质-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宿主蛋白簇与常见病毒蛋白相互作用。此外，使用来自不同国家的基因本体论（GO）术语覆盖最高概率的蛋白质功能数据库揭示了与可能的细胞功能、肌动蛋白调节和 RNA 相关的宿主蛋白簇加工是最常见的，但也是与蛋白质修饰相关的途径泛素化、苏酰化或磷酸化很明显（如 RP01 和 RP02 中所示）。基于这些新的发现，我们提出了这样的假设：宿主因素驻留在与常见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发挥相同的作用。在这里，我们通过以下方式检验这个假设应用一种新颖的、统计上高功率的光学联合筛选平台来进行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来确定感染结果将病毒功能相关性与已知宿主 PPI 的相关性关联起来的染色模式。这种做法允许对因素组进行有效的优先级排序，以便通过识别受影响步骤的机械分析进行评估病毒感染，然后定义负责感染结果的宿主区域和病毒蛋白。这项工作从代表肌动蛋白和 RNA 处理网络的高优先级验证线索开始，然后评估我们现有的高严格网络，然后通过来自的新数据进行扩展和丰富 RP01 和 RP02。我们的团队由在执行和分析大型主机方面拥有丰富经验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制洞察子网络，将为设计针对这些问题的干预措施提供基于知识的选择 EBOV 和 MARV 的相互作用。