The major goal of this project is to investigate the cellular mechanisms involved in the polarized expression of cell surface proteins. Epithelial cells are organized into sheets in vivo, forming barriers that separate body compartments. A major function of these epithelial sheets is to move specific molecules from one side to the other. To accomplish this, very different populations of proteins must be expressed at the two surfaces. Not surprisingly, viruses have evolved strategies to use this feature, and the route of infection and spread of many animal viruses reflect this asymmetry. Viruses which spread through the respiratory tract mature from the epithelial cell surface exposed in the lungs, whereas viruses that are spread by insect hosts infect and mature through the surfaces exposed to the blood. Epithelial cell lines such as MDCK cells form polarized monolayers in vitro, with separate populations of proteins localized at the apical (top) and basolateral (bottom) cell surfaces. The membrane glycoproteins of most enveloped viruses that infect these cells are specifically targeted to one of the surfaces. Several mutant MDCK cell lines have been isolated which, when infected by vesicular stomatitis virus (VSV), exhibit defects in the proper maturation of the viral envelope glycoprotein (G-protein) to the basolateral surface. In contrast, when these mutants are infected with influenza virus, its major envelope glycoprotein (HA) matures properly to the apical surface. Preliminary characterization of four of these mutants indicate that two (clones 32 & 39) allow G-protein to appear at the apical (incorrect) surface, one (clone 43) accumulates G-protein in the endoplasmic reticulum, and one (clone 77) shows no apparent synthesis of G- protein. The phenotypes of these mutants will be further characterized. Analysis of the oligosaccharide chains of G-protein grown in clone 43 will identify the site of its cellular accumulation.l Infected monolayers of clones 32 and 39 will be tested by 3H-inulin diffusion and electron microscopic studies to determine whether the junctional complexes are intact. Infected monolayers of clone 77 will be immunoprecipitated with polyclonal antisera to determine if antigenic altered G-protein is being made. The generality of the mutant phenotype will be examined by studying the maturation of cellular basolateral proteins and those from unrelated viruses. Ultimately, identification and purification of of the cellular components that are defective in clones 43 and 77 will be accomplished using cell-free reconstitution systems as an assay. Sequentially purified extracts of wild-type cells will be used to supplement mutant cell lysates to identify the factors important for """"""""rescue"""""""" of the defect.