Structural Investigations Of Macromolecular Complexes Critical To hRSV Life Cycle

对 hRSV 生命周期至关重要的大分子复合物的结构研究

• 批准号: 9195113
• 负责人: PHILIP J SANTANGELO
• 金额: $ 38.7万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195113
• 关键词: Address; Animals; Antiviral Agents; Area; Biochemical; Biological Assay; Bronchiolitis; Cell membrane; Cells; Cessation of life; Child; Codon Nucleotides; Complex; Cryoelectron Microscopy; Cytosine; Development; Elderly; Electron Microscopy; Electrons; Event; Fluorescence; Fluorescence Microscopy; Fostering; Future; Glycoproteins; Goals; Gold; Hand; Human; Image; Immunocompromised Host; In Situ; Individual; Infant; Infection; Investigation; Knowledge; Label; Life Cycle Stages; Light; Macromolecular Complexes; Mechanical ventilation; Membrane; Membrane Microdomains; Metallothionein; Methods; Modeling; Monoclonal Antibodies; Multiprotein Complexes; Passive Immunization; Preclinical Drug Evaluation; Prophylactic treatment; Proteins; Public Health; RNA; Recruitment Activity; Resolution; Respiratory Failure; Respiratory syncytial virus; Ribonucleoproteins; Role; S-nitro-N-acetylpenicillamine; Site; Structural Protein; Structure; Technology; Testing; Time; Translating; Vaccines; Viral; Viral Pneumonia; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; cell type; electron tomography; experimental study; fluorescence imaging; genomic RNA; imaging approach; imaging study; improved; infant death; influenzavirus; inhibitor/antagonist; interest; macromolecule; nanoGold; nanometer; nanoscale; new therapeutic target; particle; pathogen; permissiveness; polymerization; protein complex; public health relevance; success; therapeutic development; vaccine development; viral RNA

 DESCRIPTION (provided by applicant): Human respiratory syncytial virus (hRSV) is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, mechanical ventilation, and viral death in infants in the USA and worldwide. There are no effective vaccines for hRSV disease. In order to identify new targets for drug screening, it is imperative that we gain more structural information regarding critical RNA and protein complexes in the virus life cycle, specifically during assembly of the infectious virion. Here we will investigate: 1) the structural and functional implications of cellular and viral membrane structure and composition for ordering hRSV assembly and scission of the virus from the cell membrane; 2) the in situ structures of complexes formed between F, G, M, M2-1, and the genomic RNA during hRSV assembly; and 3) develop labeling strategies specifically for cryo-EM/cryo-ET technologies to address the ultrastructural analyses of hRSV and many other viruses. To answer these questions, we are using the A2 strain of hRSV to infect permissive, model human-derived cells for correlative fluorescence microscopy and cryo-electron microscopy experiments. The three areas to be investigated are: 1. Determine the optimal strategy for labeling hRSV viral complexes assembling into viral particles in live cells. In this aim, we will develop, test, and use several strategies to label viral proteins in live cells in order to study virus assembly through correlatie fluorescence light microscopy (FLM) and cryo-electron tomography (cryo-ET) approaches. Alternative labeling strategies will include metallothionein - fluorescent protein conjugates, SNAP, and CLIP tags along with PEG-nanogold-benzylguanine or cytosine conjugates delivered to live cells. Efficient incorporation of the labeled protein into viral structures and vral titers similar to wild-type infections will be criterions for success. 2. Define the coordinated roe of the membrane and viral glycoproteins for fostering virus assembly and budding events. Experiments will determine if F and G reside within specific plasma membrane microdomains and if this facilitates recruitment of M, M2-1, and RNP complexes. Experiments will employ multiple cell types to examine and define any potential relationships, differences, or commonalities between cell type and virus replication. 3. Determine the structure of complexes formed between M, M2-1 and the ribonucleoprotein (RNP) complex during hRSV assembly. Experiments will determine the structures of the complexes formed between hRSV structural proteins, M and M2-1, and the components of the RNP complex, the genomic RNA, N, P, and L, during replication and assembly through correlative imaging strategies. Cryo-immuno-EM approaches and hRSV RNA-specific probes will be used to further define the organization of the structural proteins, M and M2-1, and the RNP complex during cryo-ET analysis of cryo-preserved virions and virus- infected cells.

 描述（由适用提供）：人类呼吸道合胞病毒（HRSV）是病毒性肺炎，支气管炎，呼吸衰竭，机械通气和美国和全世界婴儿的病毒死亡的主要原因。没有有效的HRSV疾病疫苗。为了确定药物筛查的新靶标，我们必须在病毒生命周期中获得有关关键RNA和蛋白质复合物的更多结构信息，在这里我们将研究：1）细胞和病毒膜结构和组成的结构和功能含义，用于订购HRSV的HRSV组装和科学，并从细胞膜中订购病毒的科学； 2）在HRSV组装过程中F，G，M，M2-1和基因组RNA之间形成的配合物的原位结构； 3）制定专门针对Cryo-EM/Cryo-ET技术的标签策略，以解决HRSV和许多其他病毒的超微结构分析。为了回答这些问题，我们正在使用HRSV的A2菌株来感染允许的，衍生的人类衍生细胞，以进行矫正荧光显微镜和冷冻电子显微镜实验。要研究的三个区域是：1。确定将HRSV病毒复合物标记为活细胞中病毒颗粒的最佳策略。在此目标中，我们将开发，测试和使用几种策略在活细胞中标记病毒蛋白，以通过相关荧光光学显微镜（FLM）和冷冻电子层析成像（Cryo-ET）方法研究病毒组装。替代标记策略将包括金属硫氨酸 - 荧光蛋白结合物，快照和夹标签以及PEG-Nanogold-BenzylGuanine或胞嘧啶结合物传递到活细胞。标记的蛋白质的有效纳入病毒结构和类似于野生型感染的Vral滴度将是成功的标准。 2。定义膜和病毒糖蛋白的协调ROE，以促进病毒组装和发芽事件。实验将确定F和G是否驻留在特定的质膜微区内，以及这是否有助于M，M2-1和RNP复合物的募集。实验将采用多种细胞类型来检查和定义细胞类型和病毒复制之间的任何潜在关系，差异或共同点。 3。确定HRSV组装过程中M，M2-1和核糖核蛋白（RNP）配合物之间形成的复合物的结构。实验将确定通过相关成像策略在复制和组装过程中，HRSV结构蛋白M和M2-1之间形成的复合物以及RNP复合物，基因组RNA，N，P和L的成分。冷冻免疫 - EM方法和HRSV RNA特异性问题将用于进一步定义结构蛋白，M和M2-1的组织，以及在冷冻保存的病毒和感染病毒感染的细胞的冷冻分析过程中的Cryo-ET分析期间的RNP复合物。