The enveloped, RNA-containing murine coronaviruses include a large collection of strains. Each strain infects different tissues and thus causes a distinct disease such as hepatitis, gastroenteritis, or chronic encephalomyelitis; the latter serves as a model for human neurodegenerative diseases. This proposal will elucidate coronavirus entry mechanisms, and in doing so will explain how very similar strains cause such dramatically different diseases. A key determinant of coronavirus tropism is the spike (S), a protein that carries out essential virus entry functions. S proteins bind to carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, which cause conformational changes culminating in virus-cell membrane fusion. ? The first aim will characterize the structural changes following interactions between S and CEACAM. We will specifically focus on novel biochemical features of the S proteins, such as their thiol-disulfide reactivities, as they relate to structural changes required for virus entry. We expect to provide new insights into protein-mediated membrane fusion reactions. ? The second aim will determine how cholesterol operates as a cofactor to support coronavirus entry. We will develop in vitro assays for virus fusion and use them to identify cholesterol-dependent stages of the S-mediated fusion process. ? The third aim will connect our biochemical studies of coronavirus entry with in vivo investigations of disease. This will be accomplished by comparing viruses of variable neurovirulence in controlled in vitro measurements of S-CEACAM binding and S-induced membrane fusion. Our results will relate the biochemical properties of the coronaviruses with their pathogenesis. Our collective findings will expand current knowledge of virus entry, and help set the stage for future antiviral drug developments. ? ? ?
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