TRANSPORT OF VIRAL PROTEINS IN MAMMALIAN CELLS

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The general aim of our laboratory is to understand the mechanisms which govern the direction and speed with which viral proteins expressed in mammalian cells are transported from their sites of synthesis to their final destinations. We are approaching this problem in two ways (i) by analyzing a series of genetically-engineered mutant and chimeric viral proteins that are defective at different stages of their particular transport pathways (ii) by isolating mutants of mammalian cells that are defective in transporting viral proteins to specific destinations. Recently, two types of amino acid sequences have been shown to be necessary and sufficient to guide proteins to these very different cellular locations. One of these - hydrophobic signal sequences - causes nascent polypeptides to be translocated across the membrane of the rough endoplasmic reticulum, from where they may travel to the Golgi apparatus and then to the cell surface or to cellular vesicular cytoplasmic organelles such as lysosomes. The other, more recently discovered, causes proteins to be transported to and retained in the cell nucleus. These karyophilic signals, like hydrophobic signals, vary greatly in sequence from protein to protein; in their simplest forms they consist of a short tract of largely basic amino acids. The goal of this application is to understand how two viral proteins (SV40 T antigen and influenze virus hemagglutinin) move to their specific destinations: the oncogenic SV40 T antigen is transported both to the nucleus and the plasma membrane of transformed cells. Influenze virus hemagglutinin (HA) is a prototypical integral membrane protein that moves with high efficiency along the conventional exocytotic pathway to the cell surface. In particular we will (i) isolate and characterize cellular mutants that are unable to transport HA to the cell surface along the conventional exocytotic pathway (ii) investigate the properties and intracellular transport of a set of chimeric SV40 T antigens and contain a hydrophobic sequence derived from another protein (influenza virus hemagglutinin) at their N-terminus (iii) test the hypothesis that SV40 T antigen associated with the plasma membrane reaches the cell surface, not by the conventional secretory pathway, but by transport through the cell nucleus (iv) analyze the transport of HA along the exocytotic pathway in lines of mutant CHO cells that display grossly altered patterns of glycosylation.