The proposed project will identify and characterize cellular receptors for alphaviruses. The structural basis of the interaction of the alphavirus envelope glycoproteins with their cellular receptors will also be investigated. The alphaviruses are a group of enveloped, arthropod-borne, plus-strand RNA viruses, many of which cause encephalitis, arthritis, myositis, and fever in humans. Genetic and biochemical techniques will be used to identify the cellular receptor(s) used by Ross River virus to enter permissive cells. The receptor cDNA will be used to transform receptor-negative cells and the virus-receptor interaction will be analyzed by in vivo and in vitro binding assays. Immunological and nucleic acid reagents will be developed to study the virus-receptor interactions. Expression of receptor protein in a mouse model system will be correlated with the replication levels of virus in various target tissues. Site-directed and random mutagenesis will be carried out on the receptor cDNA and on the Ross River envelope glycoproteins to probe structure-function relationships. Studies will also be carried out on Sindbis glycoproteins and on the previously identified Sindbis cellular receptor, the high-affinity laminin receptor. In collaboration with Thomas Smith and Timothy Baker, we will examine the structural features of the envelope glycoproteins, their interactions with cellular receptors, and the binding of monoclonal antibodies to both of these using X-ray diffraction and cryo-electron microscopy. Protein for these studies will be produced using a variety of expression systems: bacterial expression, baculovirus expression, and mammalian expression using a Sindbis-based expression system. The proposed studies will provide a detailed description of the initial events in the entry of alphaviruses into target cells and further our general knowledge into virus entry and pathogenesis.
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