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Principal Investigator: PHILIP J SANTANGELO
Organization: UNIVERSITY OF WISCONSIN-MADISON
Fiscal Year: 2019
Award: $385,366
Funding agency: National Institute of General Medical Sciences
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.
Terms: <0-11 years old><Address><Animals><Antiviral Agents><Antiviral Drugs><Antivirals><Area><Assay><Bioassay><Biochemical><Biologic Assays><Biological Assay><Bronchiolitis><Cell Body><Cell Membrane Lipid Rafts><Cell membrane><Cells><Cessation of life><Child><Child Youth><Children (0-21)><Clinical Treatment Moab><Codon><Codon Nucleotides><Complex><Cryo-electron Microscopy><Cryo-electron tomography><Cryoelectron Microscopy><Cryofixation><Cryopreservation><Cytoplasmic Membrane><Cytosine><Death><Development><Drug Screening><Elderly><Electron Cryomicroscopy><Electron Microscopy><Electrons><Event><Fluorescence><Fluorescence Light Microscopy><Fluorescence Microscopy><Fostering><Future><General Viruses><Glycoproteins><Goals><Gold><Hand><Human><Image><Immunocompromised><Immunocompromised Host><Immunocompromised Patient><Immunosuppressed Host><In Situ><Individual><Infant><Infection><Influenza Virus><Investigation><Knowledge><Label><Life Cycle><Life Cycle Stages><Light><Macromolecular Complexes><Macromolecular Protein Complexes><Mechanical ventilation><Membrane><Membrane Microdomains><Metallothionein><Methods><Modern Man><Monoclonal Antibodies><Multiprotein Complexes><Negative Beta Particle><Negatrons><Non-Polyadenylated RNA><Organization Charts><Passive Immunization><Photoradiation><Plasma Membrane><Prophylactic treatment><Prophylaxis><Proteins><Public Health><RNA><RNA Gene Products><Resolution><Respiratory Failure><Respiratory syncytial virus><Ribonucleic Acid><Ribonucleoproteins><Role><S-nitro-N-acetylpenicillamine><SNAP><Site><Sphingolipid Microdomains><Sphingolipid-Cholesterol Rafts><Structural Protein><Structure><Technology><Testing><Time><Translating><Vaccines><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Pneumonia><Viral Proteins><Virion><Virus><Virus Assembly><Virus Diseases><Virus Particle><Virus Replication><advanced age><anti-viral agents><anti-viral drugs><anti-virals><cell type><children><childrens'><cold preservation><cold storage><cryo-EM><cryo-EM tomography><cryoEM><cryoEM tomography><cryoelectron tomography><death in first year of life><developmental><elders><electron cryo-tomography><experiment><experimental research><experimental study><fluorescence imaging><fluorescent imaging><genomic RNA><geriatric><gold nano particle><gold nanoparticle><human model><human pathogen><imaging><imaging approach><imaging based approach><imaging study><immunosuppressed patient><improved><infant death><infant demise><infantile death><influenzavirus><inhibitor><inhibitor/antagonist><interest><late life><later life><life course><lipid raft><mAbs><macromolecule><mechanical respiratory assist><membrane structure><nano gold><nano meter><nano meter scale><nano meter sized><nano scale><nanoGold><nanometer><nanometer scale><nanometer sized><nanoscale><new drug target><new druggable target><new pharmacotherapy target><new therapeutic target><new therapy target><novel drug target><novel druggable target><novel pharmacotherapy target><novel therapeutic target><novel therapy target><older adult><older person><organizational structure><particle><plasmalemma><polymerization><protein complex><protein structure><public health relevance><pulmonary failure><recruit><respiratory insufficiency/failure><senior citizen><social role><success><therapeutic agent development><therapeutic development><viral RNA><viral assembly><viral infection><viral multiplication><viral replication><virus RNA><virus infection><virus multiplication><virus protein><virus-induced disease><youngster>