The objective of the proposed research is to define the cellular processes by which membrane glycoproteins are targeted to specific locations as well as the structural features of viral glycoproteins which are recognized by these processes. Particular emphasis will be placed on the involvement of sorting mechanisms in the directional transport and assembly of influenza virus polypeptides. For these studies, we will use vaccinia virus vectors to express glycoproteins in polarized epithelial cells, in order to investigate the effects of specific modifications of viral glycoprotein sequences upon the transport pathway and site of cell surface expression. The intracellular vesicles involved in transport of viral glycoproteins from the Golgi complex to the plasma membrane will be identified by immunoisolation in conjunction with temperature shift experiments, and will be characterized as to their composition and morphology. Mutants of MDCK cells will also be characterized to identify defects in cellular processes involved in glycoprotein transport. We will further characterize nonstructural proteins of influenza virus which has recently been identified as cell surface antigens. Their site of expression in polarized epithelial cells, and their association with sites of virus assembly, will be investigated by immunofluorescence and immunoelectron microscopy. The mobility of viral antigens on surfaces of cells was previously found to differ depending on the host cell and virus type. We will investigate whether these differences are intrinsic properties of the respective protein molecules, or whether they depend on other viral or cellular components. We will also investigate the intracellular localization of components of Punta Toro virus, a virus which assembles by budding at intracellular membranes and attempt to identify specific amino acid sequences which determines the location of the viral proteins.
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