The goals of this proposal are to markedly improve the methodology for imaging, reconstructing, and interpreting the three-dimensional structures of icosahedral viruses and to apply this methodology to a number of significant questions related to viral structure and function in collaboration with program-project colleagues at Parti. The primary tools of investigation are cryo-electron microscopy and image reconstruction. These methods currently allow three-dimensional structural information to be obtained at the 20-30 angstroms resolution level for macromolecular specimens maintained in an aqueous environment that closely mimics 'native' (physiological) conditions. Addition of a high resolution slow- scan, charge-coupled device (CCD) camera and associated peripherals and computer equipment to a transmission electron microscope and the development of computer software to capture and analyze digital, low- irradiation images of frozen-hydrated specimens with spot scan procedures will greatly improve the ability to study viruses and virus complexes at resolutions exceeding 20 angstroms. The structures of several viruses and also virus-antibody and virus- receptor complexes will be studied with the new technology. These studies include examinations of: alphaviruses (to investigate i) conformational changes in the virus envelope induced by various physical treatments, ii) interactions with monoclonal antibodies and cellular receptors, and iii) reconstituted core particles); chimeras of cowpea mosaic virus which include polypeptide loops, 15-22 residues in length, from human rhinovirus serotype 14, and loops from proteins gp41 and gp120 of human immunodeficiency virus; complexes of human rhino virus and neutralizing antibodies and the intercellular adhesion molecule, ICAM-1; complexes of canine and human parvoviruses with monoclonal antibodies; and proheads of homologous bacteriophages phiX174, G4, and alpha3.
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