Studies of large and small-scale virus capsid dynamics associated with four virus systems will be extended by crystallography, electron cryomicroscopy (cryo-EM), image reconstruction, molecular genetics and a variety of other biophysical methods. (I) Two classes of insect tetraviruses (omega-like and beta-like T=4 capsids), will be studied to extend and refine models describing the driving force of large-scale (20-30Angstroms subunit translations and 20 degree rotations), pH dependent, reorganization of capsid quaternary structure and the chemical principles that determine the trajectory of subunit motions. Crystallographic studies of omega-like virus-like, particles (VLPs) at low pH (410Angstroms diameter) and in an expanded form (450Angstroms diameter) will be performed, as will studies of a beta-like authentic virus. Cryo-EM structures of intermediates titrated between the omega-like expanded (pH 7.0) and compact form (pH 5.0) will be modeled with the high resolution coordinates from the 2.8Angstroms structure of Nudaurelia capensis omega virus (NomegaV). Mutagenesis studies, high resolution titrations, derivitization and spectroscopy will be performed to confirm the helix (pH 7.0)-coil (pH 5.0) transition believed to drive the transition and to probe the surfaces of interaction during the transition. (II) Experimental studies on salt-stable mutants and the swollen-form of CCMV and computational analysis of swelling will be finished. High resolution cryo-EM and crystallography will be performed on a 120 subunit particle formed when it is expressed in yeast or assembled in vitro under conditions favoring pentamer formation. (III) The novel dsRNA virus L-A (400Angstroms diameter capsid formed by 60 homo dimers) that infects yeast will be studied by structure-based mutagenesis to determine the mechanism of a unique removal of cellular mRNA 7 methyl guanidine caps that is encoded in the capsid protein. Dynamics of the capsid associated with cap removal, substrate entry and RNA exit will be investigated with controlled proteolysis and mass spectrometry. (IV) The structure of yellow stone virus (YSV), a dsDNA, 520Angstrom diameter particle with 200Angstrom pentameric turrets, infecting the archea sulfolobus, will be studied by cryo-EM and crystallography.
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