Cell surface binding simian virus 40 large T antigen becomes anchored and stably linked to lipid of the target cells.
Previously a new small subclass of SV40 large T antigen with a high-binding affinity to living target cells was characterized (J. Lange-Mutschler and R. Henning, 1983, Virology 127, 333-344.) In the present study the external binding process, particularly the tight linkage of T antigen to lipid of the target cells, was analyzed. Extraction of SV40-transformed target cells (SV80) first by sonification yielded approx 80% of [35S]methionine-labeled T antigen (mechanical extract). A further 20% was obtained by treatment of cellular debris with hydroxylamine (hydroxylamine extract). As shown by an 125I-protein A radioimmunoassay, hydroxylamine extracts contained significantly higher amounts of cell surface binding T antigen. Correspondingly, after incubating [3H]palmitic acid-prelabeled target cells (HeLa) with unlabeled extracts, predominantly T antigen from hydroxylamine extracts became 3H labeled by the target cells, dependent on metabolic or enzymatic conditions. 3H-labeled T antigen became unlabeled after treatment with hydroxylamine indicating a covalent ester linkage between cell surface-bound T antigen and lipid of the target cells. The cell surface localization of in vitro acylated T antigen was demonstrated by mild trypsin digestion of living target cells. These results strongly support the idea about a novel mechanism by which a minor subclass of T antigen after being bound to the cell surface becomes covalently linked to lipid of the living cell.
D000952:Antigens, Polyomavirus Transforming; D002462:Cell Membrane / Q000378:metabolism; D002472:Cell Transformation, Viral; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D013539:Simian virus 40 / Q000378:metabolism*; D014316:Tritium; D014764:Viral Proteins / Q000378:metabolism*
D000952:Antigens, Polyomavirus Transforming; D008563:Membrane Lipids; D010169:Palmitic Acids; D011991:Receptors, Virus; D014764:Viral Proteins; D014316:Tritium; D019308:Palmitic Acid; D011494:Protein Kinases