Cryo-reconstructions of Helminthosporium victoriae virus 190S, Alternaria alternata virus-1, and bacteriophage P22
Structural studies of viruses in this thesis demonstrate how the determination of three dimensional (3D) structures can reveal insights concerning assembly, flexibility, and organization of the capsid protein and viral capsid shells. The generation of 3D density maps of viral capsids can provide information to what is already established for the viral life cycle, capsid organization, and subunit interactions. This thesis has involved the investigation of the structures of three different viruses using the methods of cryo-electron microscopy and 3D image reconstruction.
Chapter II discusses Helminthosporium victoriae virus 190S (HvV190S), a virus that infects the filamentous fungus Helminthosporium victoriae. Structures of the virion, and two virus like particles of Hv190S were all determined at ~7-8Å resolution. The capsid was found to have a relatively thin and featureless structure. The asymmetric unit of the capsid is a dimmer comprised of two, chemically identical subunits organized in a so called "T=2" lattice. The individual capsid protein was found to have a fold that is unique compared to the fold of the well characterized, Saccharomyces cerevisiae virus -L-A, and other members of the family Totiviridae. The cryo-reconstruction of HvV190S, represents the first ever structure determination for a virion that belongs to this genus Victorivirus.
In Chapter III, the structures of the virion and empty capsids of Alternaria alternata virus-1 (AaV-1) are reported. Currently, AaV-1 is an unclassified virus and a 3D reconstruction of the capsid will aid in establishing its proper classification. Cryo-reconstructions were computed from images of full and empty particles at 14Å and 25Å, respectively. The capsid has large protrusions at the 5-fold axes of symmetry and displays other features that are similar to the mycovirus, Penicillium chrysogenum virus.
Chapter IV presents a study where difference mapping was used to identify flexibility changes as a result of single amino acid residue substitutions to the coat protein of bacteriophage P22. The residue F170 is located within a flexible region of the coat protein, termed the "beta-hinge," which has been shown to play a critical role in proper monomer assembly. Procapsid reconstructions of three coat protein variants of the F170 residue, F170L, F170K, and F170A were generated at 13.5Å, 10.4Å, and 10.3Å resolution, respectively.