Cryo-EM studies of adenovirus cell entry, antibody neutralization, and the molecular architecture of vaults

The use of cryo-electron microscopy (cryo-EM) to observe unfixed and unstained single particles, along with three-dimensional (3-D) image reconstruction, has been successful in the analysis of large macromolecular complexes. The cryo-EM studies presented in this thesis address the role of the adenovirus fiber in virus cell entry, the mechanism of viral neutralization by a monoclonal antibody, and the molecular architecture of the vault.;The observation that adenovirus can efficiently infect a wide range of tissue types has made it a promising gene therapy vector. Cryo-EM imaging and gene delivery experiments have demonstrated that both the length and flexibility of the fiber are important for cell entry. A molecular explanation is proposed for the use of alternative receptors by various adenovirus subgroups.;In a separate study, a cryo-EM reconstruction of adenovirus complexed with an anti-hexon monoclonal antibody helped to elucidate the mechanism of neutralization. Complementary cryo-EM and confocal imaging experiments indicate that this antibody blocks entry of the viral genome into the nucleus by either preventing uncoating of the viral capsid or by blocking required interactions with elements of the nuclear import machinery.;Cryo-EM reconstructions of recombinant and tissue derived vaults with varying protein compositions are also presented. Vaults are intriguing particles as they have been associated with multi-drug resistance in cancer cells. Wild-type mammalian vaults are composed of three proteins, MVP, VPARP, and TEP1, as well as a small untranslated RNA, vRNA. A reconstruction of a recombinant vault composed of MVP and a chimeric protein, referred to Luciferase-INT, indicates that the MVP interaction (INT) domain of VPARP binds on the inner surface of the vault barrel. Difference maps also identify possible locations for the VPARP BRCT and PARP catalytic domains. Fold models for these domains, as well as for the VPARP VWA domain, were identified and aided the interpretation of the difference maps. Positioning of the fold models within the cryo-EM density implies that a maximum of sixteen copies of VPARP might bind inside one vault.