Plasma membrane orientation of simian virus 40 T antigen in three transformed cell lines mapped with monoclonal antibodies

Abstract

Simian virus 40 large T antigen transforms cells from several species. Recent studies show that it is present on the cell surface. As in other tumor virus systems, this may be important for transformation. We have used a radioimmunoassay to map antigenic determinants on living and formaldehyde-fixed transformed cells with six different monoclonal antibodies to T antigen. Nonrelevant monoclonal antibodies of the same subclasses served as controls. With the transformed mouse line SVT2, antibody PAb 101, which reacts with the C-terminal region of T antigen, and PAb 1700, which is directed against an internal region of T, reacted with both formaldehyde-fixed and living cells. Antibodies PAb 402 (C terminus) and 419 (N terminus) reacted only with living cells, their determinants being destroyed upon formaldehyde fixation. Antibodies PAb 405 (C terminus) and 100 (internal) fail to react on either fixed or living cells. Similar results were obtained on the simian virus 40-transformed human line SV80 and the fixed hamster line CHLwt23, although all antibodies failed to react with living CHLwt23 cells. The data suggest that T antigen is inserted into the plasma membrane of transformed cells in a specific, nonrandom manner, with the C and N termini exposed on the cell surface and the midportion either buried in the lipid bilayer, hidden by the tertiary structure of T antigen, or masked by a post-translational modification such as fatty acid acylation.