CRYO-EM STRUCTURAL STUDIES OF FELINE, MURINE, AVIAN AND SARS CORONA VIRUSES

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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. Little is known about structure and function-associated conformational changes in coronavirus virion proteins. In addition to providing the framework for the virion, the four conserved structural proteins S, E, M and N are essential in assembly, RNA packaging, egress and ingress of the virion. We propose to characterize the two- and three- dimensional structure of the coronavirus structural protein complexes within the virion. Changes that occur during fusion activation will also be studied. The coronavirus that causes severe acute resporatory syndrome outbreak of 2002-2003 was marked by high mortality and morbidity. The reservoir of this virus has not been established. Understanding of how the viral proteins function within the virion and during interaction with the host would prove very useful in rational antiviral drug design. We have reconstructed the structural protein complexes of three arenaviruses, which are pleomorphic enveloped particles 40-500 nm in diameter, using electron cryomicroscopy. Preliminary two- and three-dimensional images of the SARS structural proteins are in refinement. The NRAMM component of this project would consist of imaging particles from the three coronavirus families: feline coronavirus (group I), murine coronavirus (group II) and avian infectious bronchitis virus (group III) for comparison with SARS coronavirus (distantly related to group II or perhaps assigned as the only member of group IV). Results of the arenavirus cryo-EM studies are being prepared for publication. Several protein specimens have been prepared as vitreous ice layers suspended over holey carbon substrates. Datasets have been acquired from several varieties of corona virus using Leginon. The data is being analyzed by the investigator. This work should be completed within the next month and will then be written up for publication.