Domains of simian virus 40 large T-antigen exposed on the cell surface

Abstract

The orientation of SV40 large T on the surface of SV40-transformed mouse cells and of human cells infected with nondefective adenovirus 2 SV40 hybrid viruses has been studied. Using antibodies against a synthetic peptide corresponding to a region of 11 amino acids at the carboxyterminus of large T, the surface of formaldehyde-fixed SV40-transformed cells could be specifically stained by indirect immune fluorescence. Staining was inhibited by an excess of the peptide. These data suggest that the carboxyterminus of large T is exposed on the surface of formaldehyde-fixed cells. Antibodies against the carboxyterminus of large T also stained the surface of cells infected with the hybrid viruses Ad2 +ND1, Ad2 +ND2, and Ad2 +ND4. Thus, the carboxytermini of the SV40-specific proteins synthesized in hybrid virus-infected cells are also exposed on the cell surface. When analyzed with an antiserum against purified denatured large T, which among many other determinants also recognizes the large T carboxyterminus, surface fluorescence was observed in cells infected by all three hybridviruses. The surface fluorescence of Ad2 +ND1-infected cells, expressing an SV40-specific protein of 28 K, and Ad2 +ND2-infected cells expressing SV40-specific proteins of 42 K and 56 K molecular weight, was completely inhibited by carboxyterminal peptide. However, the surface fluorescence of Ad2 +ND4-infected cells, expressing SV40-specific proteins up to nearly full size large T, was unaffected by carboxyterminal peptide. Our data suggest that a major portion of large T, located between a region near the carboxyterminus and a region corresponding to the aminoterminus of the 56 K protein, is not exposed on the surface of hybridvirus-infected cells. However, some parts of the aminoterminal one-third of large T appear to be exposed again. We conclude that SV40 large T on the surface of SV40-transformed cells is oriented in a specific manner, suggesting that it is specifically associated with the plasma membrane.