My part of this grant application concerns structural studies of small, single-stranded, RNA viruses and their structural components using cryo=electron microscopy (cryoEM) and X-ray crystallography. The basic concept, which we and others have been exploring in the last decade, is that, whereas larger biological units are generally unlikely to crystallize, their three-dimensional structures can frequently be determined to 9 A resolution or better by cryoEM. At the same time, the atomic structures of some of the component proteins can be determined by X-ray crystallography to near atomic resolution (probably to better than 3 A resolution). These two types of results can be combined to give """"""""quasi-atomic"""""""" structures of whole viruses or virus complexes. Although more than half of our effort will be directed to a continuation of our previous investigation of alphaviruses, in particular of Sindbis virus, I plan to extend our efforts to the study of larger and probably less symmetric, enveloped, RNA viruses, including rubella virus (another genus of alphavirus for which it is not clear whether the virions have reproducible icosahedral symmetry), coronaviruses, and possibly also measles virus (an orthomyxovirus) and Hantaan virus (a bunyavirus). The work on alphaviruses will include the determination of the still structurally unknown E2 glycoprotein; extension of the cryoEM map of Sindbis virus to possibly 6 A resolution; study of the immature, pre-maturation, E2-E3 uncleaved virus; analysis of the RNA structure in the core; examinations of the virus bound with cellular receptor molecules and neutralizing antibodies; and further attempts to crystallize Sindbis virus. Work on other ssRNA viruses will need to go hand-in-hand with the development of methods to determine whole virus structure when the virions are asymmetric and pleomorphic, albeit similar in localized surface features. At the same time it will be necessary to start exploring the crystal structures of the component structural proteins. The major emphasis and the major challenge will be the study of the pleomorphic, non-symmetrical enveloped viruses. Tentatively, we plan to use cryoEM tomography to identify regions of local symmetry in the viral membrane and then to average (if appropriate) these regions between different particles. Alternatively, it might be possible to search cryoEM images of a pleomorphic virus for common patterns. These might represent different orientations of a locally recurring structure.
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