Defining the Role of Host Factors in Ebola Virus RNA Synthesis

定义宿主因子在埃博拉病毒 RNA 合成中的作用

• 批准号: 9312744
• 负责人: Douglas J. LaCount
• 金额: $ 74.16万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9312744
• 关键词: Address; Affect; Affinity; Affinity Chromatography; Binding; Binding Proteins; Biochemical; Bioinformatics; Biological Assay; Biophysics; Cell Communication; Cells; Cellular biology; Cessation of life; Clinical; Clinical Trials; Communities; Complex; Data; Data Set; Democratic Republic of the Congo; Dengue Virus; Dependence; Development; Disease Outbreaks; Drug effect disorder; Ebola virus; Filoviridae; Filovirus; Genetic Transcription; Genomic Library; Genotype; Goals; Guinea; Health; Hepatitis C; Human; Individual; Integration Host Factors; Libraries; Manuscripts; Maps; Mass Spectrum Analysis; Methods; Mission; Molecular; Mus; Mutation; National Security; Network-based; Pathway interactions; Pharmaceutical Preparations; Polymerase; Preparation; Principal Investigator; Proteins; Proteomics; RNA Interference; RNA chemical synthesis; RNA interference screen; RNA replication; Reporting; Research; Risk; Role; Small Interfering RNA; Systems Biology; Techniques; Technology; Testing; Two-Hybrid System Techniques; Viral; Viral Proteins; Virus; Virus Diseases; Virus Replication; Western Africa; Work; Yeasts; Zaire Ebola virus; cellular imaging; data sharing; experience; experimental study; follow-up; gammaherpesvirus; genome-wide; genome-wide analysis; high throughput screening; improved; insight; killings; novel; novel strategies; pathogen; programs; protein protein interaction; screening; statistics; transcriptome sequencing; vaccine candidate; virus host interaction; web site

Program Director/Principal Investigator (Last, First, Middle): The overall goal of this Project is to identify cellular proteins that are required for Ebola virus replication. Ebola viruses pose significant risks to human health and national security. The 2014 outbreak in Western Africa infected more than 28,000 people and caused more than 11,000 deaths (per WHO statistics). Although vaccine candidates are showing promise in clinical trials, no clinically approved drugs are available and basic information about Ebola virus replication is lacking. Like all viruses, Ebola virus depends on cellular proteins for its replication. This dependence creates a potential “Achilles heel” that may be exploited to develop new approaches to treat Ebola virus infections. By studying Ebola virus-host cell interactions, we gain insight into the mechanisms of action of viral proteins and the strategies that the virus uses to interface with its host. However, relatively few host factors of Ebola virus replication have been reported and no systematic, genome- wide analyses have been performed. To address this gap, we will apply complementary technologies to develop high quality maps of Ebola virus-host cell interactions. In Aim 1 we perform a high content, genome- wide RNAi screen under BSL4 conditions with wildtype Ebola virus to identify novel host factors required for Ebola virus replication. The capacity to perform such a screen is unique to the research team. Since the host factors identified in the siRNA screen could act directly or indirectly to affect Ebola virus replication, in Aim 2 we propose to identify cellular proteins that bind to Ebola virus replication proteins. Complementary protein- protein interaction discovery technologies (yeast two-hybrid assay and co-affinity purification plus mass spectrometry) are used to develop a more comprehensive Ebola virus-host cell protein interaction network than could be achieved with any single approach. Since the sequences of Ebola virus strains differ between outbreaks and the impact of these genetic changes on host cell interactions is not known, we systematically compare host cell binding partners of a 2014 outbreak strain to those of Ebola Zaire (1976). Host cell factors from Aims 1 and 2 are integrated with existing large-scale data sets in Aim 3 to develop strain-specific Ebola virus-host cell protein interaction networks. To create a more complete understanding of regulatory effects of the virus-host interactions, we will enrich the network in indirect transcriptional interactions using high- throughput RNA-seq profiling of a subset of host factors selected from Aims 1 and 2. Our research team is well qualified to accomplish the goals of this Project, having expertise in high-throughput screening of BSL4 viruses, virus-host cell protein interactions, RNA-Seq profiling, and network-based bioinformatic analyses. Host factors will be tested for their effects on virus infection in Projects 1 and 3, and characterized by biophysical and structural methods in Core B and Project 2. Together these studies will develop the most comprehensive analysis of Ebola virus-host cell interactions to date. The data is integral to the mission of this Program and is expected to stimulate hypothesis-driven experiments in broader Ebola virus research community. 0925-0001/0002 (Rev. 08/12) Page Continuation Format Page

项目负责人/主要研究者（最后一名、第一名、中间名）： 该项目的总体目标是确定埃博拉病毒复制所需的细胞蛋白。埃博拉 病毒对人类健康和国家安全构成重大威胁。2014年西非疫情 感染了28，000多人，造成11，000多人死亡（根据世卫组织统计）。虽然 候选疫苗在临床试验中显示出希望，没有临床批准的药物可用， 关于埃博拉病毒复制的信息缺乏。像所有病毒一样，埃博拉病毒依赖于细胞蛋白 为了它的复制。这种依赖性造成了一个潜在的“阿喀琉斯之踵”，可能会被利用来开发新的 治疗埃博拉病毒感染的方法。通过研究埃博拉病毒与宿主细胞的相互作用， 病毒蛋白的作用机制以及病毒与宿主相互作用的策略。 然而，埃博拉病毒复制的宿主因素相对较少，没有系统的，基因组- 进行了广泛的分析。为了弥补这一差距，我们将应用互补技术， 开发埃博拉病毒-宿主细胞相互作用的高质量图谱。在目标1中，我们执行高含量的基因组- 在BSL 4条件下用野生型埃博拉病毒进行广泛RNAi筛选，以鉴定 埃博拉病毒复制。进行这种筛选的能力是研究小组所独有的。由于主机 在Aim 2中，在siRNA筛选中鉴定的因子可以直接或间接影响埃博拉病毒的复制， 我们打算鉴定与埃博拉病毒复制蛋白结合的细胞蛋白。互补蛋白- 蛋白质相互作用发现技术（酵母双杂交测定和共亲和纯化加质谱 光谱法）被用来开发一个更全面的埃博拉病毒-宿主细胞蛋白相互作用网络， 可以通过任何一种方法来实现。由于埃博拉病毒株的序列在 爆发和这些遗传变化对宿主细胞相互作用的影响尚不清楚，我们系统地 将2014年爆发菌株的宿主细胞结合伴侣与扎伊尔埃博拉病毒（1976年）的宿主细胞结合伴侣进行比较。宿主细胞因子 将目标1和目标2中的数据与目标3中现有的大规模数据集相结合，以开发埃博拉病毒株特异性 病毒-宿主细胞蛋白质相互作用网络。为了更全面地了解 病毒-宿主相互作用，我们将丰富网络的间接转录相互作用，使用高， 本发明的目的是提供选自目的1和2的宿主因子子集的通量RNA-seq分析。我们的研究团队 有资格完成本项目的目标，具有高通量筛选BSL 4的专业知识 病毒、病毒-宿主细胞蛋白质相互作用、RNA-Seq分析和基于网络的生物信息学分析。主机 在项目1和3中，将测试因子对病毒感染的影响，并通过生物物理特性 核心B和项目2中的结构方法。这些研究将共同开发最全面的 分析埃博拉病毒与宿主细胞的相互作用。这些数据是本计划使命不可或缺的一部分， 预计将在更广泛的埃博拉病毒研究界激发假设驱动的实验。 0925-0001/0002（2012年8月修订版）续页格式页