Severe acute respiratory syndrome (SARS) is a new disease in humans. SARS is associated with severe atypical pneumonia, which causes diffuse alveolar damage in infected patients and results in a mortality rate between 3 to 10%. A novel coronavirus (SARS-CoV) has been identified as the causative agent associated with SARS. Sequencing of the SARS-CoV has revealed similarities but, more importantly, significant differences between SARS-CoV and other coronaviruses. The spike(s) glycoprotein of coronavirus family members (which now includes the SARS virus) plays a pivotal role in viral infectivity by mediating the specific high affinity attachment of virions to cell surface receptors and the subsequent fusion of viral and cellular membranes. Similar in nature to many other viral fusion proteins, the spike protein is known to undergo a large conformational changeto become active in mediating membrane fusion. Three-dimensional structures of viral fusion proteins, other than coronaviruses (whose structures are still unknown) reveal that the conformational change involves three-stranded coiled-coils collapsing to a 6-helix bundle structure in the fusion-active state. Evidence now suggests that peptides, which bind to these coiled-coil domains or helices, can prevent the formation of the fusion-active 6-helix bundle structure required for membrane fusion. The goals of this project are many. First, to predict, synthesize and characterize the c_-helices that form the coiled-coil structural domains of the SARS-CoV spike glycoprotein S and determine the conformational changes that occur during viral/cell membrane fusion. Second, to prepare site-directed o_-helix-specific antibodies using newly developed technology in our laboratory to map the conformational changes needed for membrane fusion and virus entry. Third, to design and synthesize stable peptides and peptidomimetics that will inhibit SARS virus infection of human cells. Since protein stability is so critical to understanding conformational change, we have developed a novel program called STABLECOIL to predict a stability profile of coiled-coil regions in proteins. This program will be used to assist us in the design of stable peptide inhibitors. We will characterize the coiled-coil domains using a series of biophysical techniques including circular dichroism spectroscopy, analytical ultracentrifugation, polyacrylamide gel electrophoresis and size- exclusion chromatography. The affinity and specificity of our site-directed antibodies to (_-helices will be characterized using Biacore interaction analysis. Conformational constraints and amino acid substitutions will be designed into our peptide inhibitors to enhance affinity and specificity.
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