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.