STRUCTURAL STUDIES OF VIRUSES

University Of Chicago, Chicago IL

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We study the structure of viruses and their components. We are interested in viral assembly pathways viral interaction with the host and entry mechanisms. We make extensive use of electron microscopy to study viruses or fragments of viruses that cannot be crystallized. These relatively low resolution maps can be augmented by crystallographic studies of the protein components. 1. Flaviviruses [1]: (West Nile virus [2 3 4 5 6] and Dengue virus [7 8 2 9 10 11 12 13 14] The analysis of West Nile virus crystals that diffract to about 16Angstrom resolution is in progress. The virions in these crystals appear to have undergone fusion with neighboring particles in the crystal lattice. Structure of components of flaviviruses such as envelope protein (E) the pre maturation membrane protein (prM) and PrM-E complex are being studies. Also being studied are crystal structures of various Fab fragments in complex with E. In addition we are studying various Fab fragments complexed with the whole virus by cryoEM. Combining these crystallographic and EM studies has given us insight into the mechanisms of neutralization by the antibodies as well as the fusion process that occurs during virus entry into cells. 2. Alphaviruses [15 16 17 18 7 19 13 14]: Electron microscopy studies of alpha viruses (Sindbis virus Ross River virus Chikungunya virus) are being combined with crystal studies of the E1 and E2 surface glycoprotein. The crystal structure of an E1-E2 fusion protein (with the trans-membrane regions omitted) shows a trimeric spike which has a structure the same as found in the EM studies of the whole virus. This structure still requires phase improvement (using SeMet SAD data) in order to completely establish the the previousily unknown structure of E2. We have recently obtained an excellent cryoEM structure of Chikungunya virus(CHIKV) like particles (VLPs). CHIKV is an emerging pathogen in SE Asia with lethal implications. We have initiated started to work on the crystal structures of a number of Fab fragments of neutralizing antibodies. Combining these structures with the cryoEM structures of their complexes with virus will be informative as to the mode of neutralization the antigenic sites on the virus and other essential information for the design of VLP vaccines. We are also working on Rubella virus (the causative agent of German Measels) by combining cryo electron microscopy with the structure determination of its E1 and E2 glycoprotein components and its capsid protein. We are using the same cloning expression and crystalization techniques as we used for the study of dengue virus 13 and Sindbis virus17. 3. Parvoviridae: Capsid structures of several parvoviruses [20 21] including a human parvovirus (B19) [22 23] have been determined. The crystal structures of a shrimp pavovirus and a silk worm pavovirus are being determined. Attempts are being made to crystallize the PLA2 unique region of the capsid protein of B19. 4. Structural components of T4 phage: We have solved about 15 out of about 40 proteins so far [24 25 26 27 28 31]. We have recently determined the structure of T4 soc and crystals of T4 hoc and soc the two minor head accessory proteins. 5. Structural components of other phages: We are have crystals of fragments of the H pilot protein of phiX174 29and have crystalized various proteins of phiKZ 30 gp13 gp9 cell lysing enzymes of phi29 [32 33]. 6. Mimivirus structural proteins We are studying the giant Mimivirus using cryoEM and cryo tomography. We are currently identifying various structural proteins for crystallization of recombinant proteins including the major capsid protein. References are attached as a separate file due to space limitation.