Virus assembly is the final step in the life cycle of a virus prior to its release from the infected cell. Detailed information concerning this process is fundamental for the development of more effective antiviral strategies and will be useful in developing viral vectors that can be used in genetic engineering and medicine. The proposed project will investigate the assembly of alphaviruses. The alphaviruses are a group of arthropod-borne, plus-strand RNA viruses, many of which cause encephalitis, arthritis, myositis and fever in humans. They represent one of the simplest types of enveloped animal viruses and are model systems for assembly, in part, due to the well-defined structural organization of the virion. Dr. Kuhn proposes to investigate the process by which these viruses assemble their inner nucleocapsid core, the association of the core with transmembrane glycoproteins, and the final process of virus maturation that occurs as the virus buds from the infected cell membrane. A multi-disciplinary approach will be employed to investigate this assembly process. This approach will entail the use of molecular genetics, biochemistry and structural techniques to probe the mechanism of virus assembly and to ultimately describe the process in atomic detail. Using cryo-electron microscopy image reconstructions of the whole virus, and the atomic structure of the nucleocapsid protein as a guide, site-directed mutagenesis will be carried out on the nucleocapsid protein and the E2 glycoprotein to probe structure-function relationships. The resulting mutants will be studied by a variety of in vivo and in vitro biochemical assays that will examine proteolytic activity protein-protein and protein-nucleic acid interactions involved in nucleocapsid core formation and the interaction of the nucleocapsid core with the cytoplasmic domain of glycoproteins E2. Biophysical techniques such as x-ray crystallography, nuclear magnetic resonance and cryo-electron microscopy together with image reconstruction will examine wild type and mutant proteins involved in the assembly process.
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