Our long term goal is to understand the molecular mechanisms by which viruses occasionally jump from their normal host species to a new species, a process that may lead to emerging viral diseases in the new host species. We will focus on the interactions of the spike glycoprotein of murine coronavirus MHV with host cell receptors in the CEA family of glycoproteins. Several labs have shown that persistent infection of murine cell lines with MHV leads to markedly reduced expression of the CEACAM1a receptor glycoprotein. In the persistently infected cultures, viruses with mutations in their spike glycoproteins and some other genes rapidly replace the wild type virus. The mutations in the spike genes are associated with acquisition of the ability of the virus to replicate in cell lines from cats, pigs, rats, monkeys and humans. We will identify amino acid residues in both the viral spike glycoprotein and the CEACAM1a receptor that determine the specificity of binding virus. We will study the effects of mutations in S and in the receptor upon the specificity of virus-receptor interactions. We will continue our work on determining the crystal structures of CEACAM1 proteins to learn how the functional CEACAM1a receptor differs from CEACAM1b proteins from MHV-resistant mice and human CEACAM1. We will engineer spike and receptor proteins that will form co-crystals in order to determine the crystal structure of the complex. We will analyze the conformational changes in the viral spike protein induced by soluble receptor or by pH 8 at 37 degrees C that are associated with membrane fusion and virus entry. To develop strains of inbred mice that are resistant to MHV infection, we will manipulate the Ceacam1gene in mice to reduce or eliminate expression of CEACAM1 proteins. We will also substitute chimeric Ceacam1a/b genes for the normal Ceacam1 gene in mice. The animals will be tested for MHV-susceptibility and immune responses. These studies will provide important information about the mechanism of changing receptor specificity in a model virus that causes disease in its natural host.
