MOLECULAR DISSECTION OF THE CORONAVIRUS SPIKE

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DESCRIPTION (Adapted from investigator's abstract): Neurotropic murine coronaviruses cause a spectrum of central nervous system diseases ranging from acute encephalitis to chronic demyelinating disorders. The latter, persistent viral infection serves as a model for such human diseases as multiple sclerosis and subacute sclerosing panencephalitis. Many of the clinical manifestations of the murine infection are explained by changes in the virion spike glycoprotein, a complex multifunctional molecule which mediates virus binding to cell receptors as well as fusion of viral and cellular membranes. In addition to its importance in understanding the pathogenesis of the infections, the spike has unique properties which set it apart from other viral fusion proteins. The central objective of this proposal will be to study the binding and fusion properties of this important protein in assays in which each function is examined separately. To this end, spike genes from viruses known to differ in their receptor- binding properties will be compared by sequence analysis. Virus:receptor binding assays will be developed, and the relative affinity of these viruses for receptor(s) will be measured. A soluble and secreted form of the receptor will be expressed, purified and its affinity for the different spikes will be determined. Conformational changes in the spikes that accompany receptor binding will be analyzed. An assay capable of measuring membrane fusion capacity independent of receptor binding function will be developed and used to rank the fusogenicity of spikes with predicted alterations in secondary and tertiary structure. Ph-induced structural changes that are associated with activation of fusion function will be monitored using proteases and antipeptide antibodies as probes. The investigator expects that the results will provide evidence for a unique fusion-active spike conformation. The investigator states that the results of this project will lead to a greater understanding of the essential initial steps in coronavirus infection, and will advance our knowledge of relationships between viral glycoprotein structure and function.