High Resolution of Structural Studies of Insect Viruses

昆虫病毒的高分辨率结构研究

• 批准号: 7931506
• 负责人: John Emil Johnson
• 金额: $ 22.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931506
• 关键词: Affect; Antibodies; Area; Baculovirus Expression System; Baculoviruses; Biochemistry; Biological; Biophysics; Capsid Proteins; Cells; Cellular biology; Chemistry; Chimeric Proteins; Cloning; Collaborations; Computers; Cryoelectron Microscopy; Crystallography; DNA; Data Collection; Drosophila genus; Electron Microscopy; Enzymes; Evolution; Family Picornaviridae; Fishes; Fluorescence; Fluorescence Microscopy; Gene Proteins; Genes; Genome; Goals; Green Fluorescent Proteins; Housing; Image; In Vitro; Insect Viruses; Insecta; Investigation; Label; Lead; Life Cycle Stages; Light; Location; Mammalian Cell; Membrane; Methods; Microscopy; Modeling; Molecular; Molecular Virology; Necrosis; Negative Staining; Organism; Peptides; Phenotype; Plants; Polymerase; Protein Biosynthesis; Proteins; RNA; RNA I; RNA replication; RNA-Directed RNA Polymerase; Research; Resolution; Role; Site; Site-Directed Mutagenesis; Structure; Time; Translations; Viral; Viral Cytopathogenic Effect; Virus; Virus Assembly; Virus Diseases; Virus Receptors; Virus-like particle; Yeasts; base; cricket paralysis virus; image reconstruction; improved; in vivo; mutant; novel; parsnip yellow fleck virus; particle; polypeptide; professor; receptor; research study; viral RNA

Crystallographic, biophysical,and molecular biological studies were performed by our group and collaborators on insect nodaviruses and picorna-likc viruses. A number of observations were made that apparently relate to specific functions of the particle in the virus life cycle. This renewal application describes experiments in the area of cell biology, as well as crystallography,biophysics and molecular virology,as efforts are made to define viral phenotypes on the basis of chemistry discerned from atomic resolution and in vitro studies. The essence of the proposal is to span the range from atoms to cells for simple, yet elegant, organisms, the non enveloped, ssRNA viruses of insects and plants. The variationsobserved in structure and hypothesized function lead naturally to an investigation of the evolution of these particles. Specifically we seek to: (1) Employ fluoresencc and electron microscopy to determine the mode of entry of flock house virus (FHV) and, to employ biochemistry and biophysics to determine the role of the receptor in RNA release. (2) Employ in vivo expression of GFP fusion proteins in mammalian and drosophila cells to establish the role of a membrane active peptide derived from the capsid protein of FHV by a maturationcleavage. (3) Determine the structure of the RNA directed RNA polymerasc of FHV and establish its novel features that allow it to replicate and CAP RNA molecules in insect, plant, yeast and mammalian cells. (4) Investigate, by crystallography, cryoEM, baculovirus expression and site directed mutagenesis, factors that affect assembly, maturation and genome organization of insect and fish nodaviruses. (5) Determine the sites of FHV RNA replication, capsid protein synthesis and particle assembly by fluorcsence and electron microscopy. (6) Establish evolutionaryrelationships among insect and fish nodaviruses and among insect and plant picorna and picorna-like viruses.

晶体学、生物物理学和分子生物学研究由我们的小组和合作者进行， 昆虫诺达病毒和小核糖核酸样病毒。提出了一些意见，显然与具体的 在病毒生命周期中的作用。此更新应用程序描述了细胞领域的实验 生物学，以及晶体学，生物物理学和分子病毒学，因为努力定义病毒表型 基于从原子分辨率和体外研究中辨别的化学。该提案的实质是跨越 从原子到细胞的简单而优雅的生物体，昆虫的无包膜ssRNA病毒， 植物观察到的结构和假设功能的变化自然导致了对 这些粒子的演化。具体而言，我们寻求：（1）采用荧光和电子显微镜，以确定 进入方式的鸡舍病毒（FHV），并采用生物化学和生物物理学，以确定的作用， RNA释放的受体。(2)利用GFP融合蛋白在哺乳动物和果蝇细胞中的体内表达， 通过成熟裂解，确定了来源于FHV衣壳蛋白的膜活性肽的作用。（三） 确定FHV的RNA指导的RNA聚合酶的结构，并建立其新的特征，使其能够 在昆虫、植物、酵母和哺乳动物细胞中复制和CAP RNA分子。(4)通过晶体学研究， cryoEM、杆状病毒表达和定点突变、影响组装、成熟和基因组的因素 昆虫和鱼类诺达病毒的组织。(5)确定FHV RNA复制、衣壳蛋白合成的位点 荧光显微镜和电子显微镜观察颗粒的组装。(6)建立昆虫之间的进化关系 和鱼类诺达病毒以及昆虫和植物小核糖核酸和小核糖核酸样病毒。