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(申请人提供)：人类呼吸道合胞病毒(HRSV)是导致美国和世界各地婴儿病毒性肺炎、毛细支气管炎、呼吸衰竭、机械通气和病毒性死亡的主要原因。目前还没有针对HRSV病的有效疫苗。为了确定药物筛选的新靶点，我们必须获得更多关于病毒生命周期中关键RNA和蛋白质复合体的结构信息，特别是在传染性病毒粒子的组装过程中。在这里，我们将调查：1)细胞和病毒膜结构和组成对HRSV组装和病毒从细胞膜上分离的有序的结构和功能的影响；2)在HRSV组装过程中F、G、M、M2-1和基因组RNA形成的复合体的原位结构；以及3)开发专门用于冷冻-EM/冷冻-ET技术的标记策略，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 A2株感染允许的、模型人类来源的细胞，进行相关的荧光显微镜和冷冻电子显微镜实验。需要研究的三个方面是：1.确定在活细胞中标记组装成病毒颗粒的HRSV病毒复合体的最佳策略。为此，我们将开发、测试和使用几种方法来标记活细胞中的病毒蛋白，以便通过相关的荧光显微镜(FLM)和低温电子断层扫描(CRYO-ET)方法来研究病毒组装。可供选择的标记策略包括金属硫蛋白-荧光蛋白结合物、SNAP和片段标签以及传递给活细胞的聚乙二醇纳米金-苄基鸟嘌呤或胞嘧啶结合物。有效地将标记蛋白整合到病毒结构和类似野生型感染的病毒滴度将是成功的标准。2.确定用于促进病毒组装和萌发事件的膜和病毒糖蛋白的协调RoE。实验将确定F和G是否位于特定的质膜微域内，以及这是否有助于M、M2-1和RNP复合体的招募。实验将使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、差异或共性。3.确定M、M2-1与核糖核蛋白(RNP)复合体在HRSV组装过程中形成的复合体的结构。实验将通过相关成像策略确定HRSV结构蛋白M和M2-1在复制和组装过程中形成的复合体的结构，以及RNP复合体的组成成分基因组RNA、N、P和L。在冷冻保存的病毒粒子和病毒感染细胞的冷冻分析过程中，将使用冷冻免疫EM方法和HRSV RNA特异性探针来进一步确定结构蛋白M和M2-1以及RNP复合体的组织。