High Resolution of Structural Studies of Insect Viruses

昆虫病毒的高分辨率结构研究

基本信息

批准号：
7210482
负责人：
John Emil Johnson
金额：
$ 46.6万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-12-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7210482
关键词：
3-Dimensional Acetone Address Adenoviruses Affinity Amino Acids Animal Viruses Antibodies Area Artificial Membranes Aspartic Acid Award Baculovirus Expression System Baculoviruses Base Sequence Binding Biochemical Biochemistry Biological Biological Assay Biological Models Bluetongue virus Capsid Capsid Proteins Cell Adhesion Molecules Cell Nucleus Cell Survival Cell membrane Cell surface Cells Cellular Membrane Cellular biology Centrifugation Chemicals Chimeric Proteins Cleaved cell Cloning Collaborations Color Computers Coupled Cryoelectron Microscopy Crystallography Cytolysis Cytoplasmic Protein Cytoskeleton DNA DNA-Directed RNA Polymerase Data Collection Deposition Detergents Development Drosophila genus Drosophila inturned protein Drosophila lachesin protein Durcupan Dyes Electron Microscopy Electrons Electrospray Ionization Employee Strikes Endopeptidases Endosomes Enzymes Ethanol Event Evolution Family Picornaviridae Fishes Fluorescence Fluorescence Microscopy Fluorescent Probes Freeze Substitution Freezing Funding Gel Genbank Gene Proteins Genes Genome Genomics Genotype Glutaral Goals Grant Green Fluorescent Proteins Helix (Snails)Hour Housing Human Human poliovirus Hypotonic Solutions Image Imagery Imaging Techniques Immunoglobulins In Situ In Vitro Infection Insect Viruses Insecta Institution Intercellular Junctions Investigation Label Lead Leadership Left Length Letters Life Life Cycle Stages Light Lipid Bilayers Localized Location Malawi Measurement Measures Membrane Membrane Proteins Methods Microscope Microscopy Microtomy Mind Mitochondria Modeling Molecular Molecular Conformation Molecular Genetics Molecular Weight Mus Mutation Necrosis Negative Staining None or Not Applicable Nonidet P-40 Nucleic Acids Numbers Object Attachment Organelles Oryctolagus cuniculus Osmium Tetroxide Pathway interactions Peptide Hydrolases Peptides Peroxidase Peroxidases Persons Phenotype Plant Resins Plants Plasmids Polioviruses Polymerase Polymers Population Positioning Attribute Precipitation Principal Investigator Procedures Process Production Progress Reports Property Protein Binding Protein Biosynthesis Proteins Publications Pyroxylin R-factor RNA RNA I RNA Interference RNA replication RNA-Directed RNA Polymerase Range Reaction Reagent Recombinants Recycling Reovirus Research Research Personnel Resolution Resources Sampling Sepharose Site Staining method Stains Standards of Weights and Measures Structure Students Surface System Talents Techniques Technology Thick Time Tomogram Transcript Transfection Translations Trypan Blue Two-Dimensional Gel Electrophoresis United States National Institutes of Health Viral Viral Cytopathogenic Effect Viral Genome Viral Proteins Virion Virus Virus Assembly Virus Diseases Virus Receptors Virus Replication Virus-like particle Water Western Blotting Work X-Ray Crystallography Yeasts base cell fixing cricket paralysis virus cytotoxic cytotoxicity day ear helix electron tomography endosome membrane experience follow-up handbook human poliovirus receptor image reconstruction improved in vivo insight interest lachesine link protein mitochondrial membrane mutant nano-electrospray non-genomic oxidation parsnip yellow fleck virus particle polypeptide polypeptide C pressure prevent professor programs protein aminoacid sequence receptor receptor mediated endocytosis replicase research study resistance factors sample fixation size skills statistics structural biology tandem mass spectrometry tomography uranyl acetate virology virus development

项目摘要

Flock House Virus (FHV) is a simple RNA insect virus that infects drosophila line 1 (DL1) cells. The capsid contains 180 copies of a single protein type arranged with T=3 quasi symmetry. The virus genome is split into two RNA molecules; RNA1 encodes a RNA directed RNA polymerase and a small protein that inhibits the cellular RNAi activity against FHV replication; RNA2 encodes the 407 amino acid capsid protein alpha that assembles into capsids and then undergoes auto-catalytic clevage into beta (residues 1-363) and gamma (364-407), an event required for infectivity. FHV is the simplest non enveloped animal virus, but it shares entry properties with reovirus, blue tongue virus poliovirus and adenovirus. The relationship between genotype and phenotype is extremely well defined. During the first four years of support we have assembled reagents and techniques that allow us to study events associated with virus entry in vivo and in vitro. This provides an exceptional opportunity to understand non enveloped virus during the second half of this MERIT award. Specific Aims: (1) Image FHV (tetra-cys containing) VLP entry into live DL1 cells with the fluorescent probes FIAsH and ReAsH. Confocal imaging will allow the determination of the precise time course of entry without infection, as VLPs do not contain the viral genome. This will define the time points for study with high pressure freezing and electron tomography. (2) Time course studies of high pressure frozen, thick sections will be performed with electron tomography to follow the entry pathway of virus particles, the location of uncoating and the translocation of RNA across endosomal membranes. (3) Studies with VLPs depositing tetra-cys modifies gamma peptides, but not infecting cells, will be investigated to determine exactly where gamma peptides are deposited and what membranes are effected. The studies will be performed with ReAsH, allowing fluorescence and high resolution EM studies of the DL1 cells. (4) A cryoEM reconstructionis being performed on the eluted particles of FHV, corresponding to a disassembly intermediate. (5) Features of the gamma peptide required for membrane alteration and infection will be determined with the gamma in trans procedure discovered during the last period of support. (6) The putative lachesin receptor for FHV will be used as a reagent to understand virus particle destabilization in vitro. (7) Crystallographic studies will be performed with the RNA polymerase to establish a structural basis for its multi-functionality.

羊群病毒（FHV）是一种简单的RNA昆虫病毒，它感染了果蝇1（DL1）细胞。这 Capsid包含180种具有T = 3准对称性的单蛋白类型的副本。病毒基因组分为两个RNA分子。 RNA1编码RNA定向RNA聚合酶和A 抑制细胞RNAi活性对FHV复制的小蛋白质； RNA2编码407 氨基酸衣壳蛋白alpha聚集成衣壳，然后进行自动催化 Clevage进入β（残基1-363）和伽马（364-407），这是感染性所需的事件。 FHV是最简单的非包围动物病毒，但它与蓝肠病毒，蓝色舌头共享入口属性病毒脊髓灰质炎病毒和腺病毒。基因型和表型之间的关系非常好定义。在支持的前四年，我们组装了试剂和技术，允许美国研究与体内和体外病毒进入有关的事件。这提供了一个特殊的在该优点奖的下半年中了解非包裹病毒的机会。具体的目的：（1）图像FHV（包含四个Cys）VLP进入Live DL1细胞，并带有荧光探针 fiash and reash。共聚焦成像将允许确定进入的精确时间课程没有感染，因为VLP不含病毒基因组。这将定义学习时间点高压冻结和电子断层扫描。（2）高压时间研究冻结的厚部分将使用电子断层扫描进行遵循病毒的进入途径颗粒，脱涂的位置和RNA跨内体膜的易位。（3）通过沉积四边形的VLP的研究会修饰γ肽，但不会感染细胞，将是进行了研究以确定伽马肽沉积的位置以及哪些膜是实现。研究将通过REASH进行，允许荧光和高分辨率EM DL1细胞的研究。（4）在洗脱的颗粒上进行冷冻重建 FHV，对应于拆卸中间体。（5）所需的伽马肽的特征膜的改变和感染将在发现的反式过程中用伽马确定在支持的最后一个阶段。（6）推定的FHV的Lachesin受体将用作试剂了解体外病毒颗粒不稳定。（7）将使用晶体学研究 RNA聚合酶为其多功能性建立结构基础。