No abstract

The possible interaction between simian virus 40 (SV40) large tumor antigen (T-ag) and cellular proteins in the plasma membrane of SV40-transformed mouse cells was investigated. The presence of SV40 T-ag, 53,000 (53K) cellular protein, and histocompatibility (H-2) antigens on the surface of SV40-transformed cells was demonstrated by immunofluorescence. The use of lactoperoxidase-catalyzed cell surface iodination and a differential immunoprecipitation technique established that large T-ag is associated with the 53K host-coded protein on the surface of the transformed cells. In contrast, no detergent-stable complex between large t-ag and H-2 antigens was detected. Both labeled T-ag and 53K protein were coprecipitated from surface-iodinated SV40-transformed cells by monoclonal antibodies directed against either the viral or the cellular protein. Based on the unique antigenic sites recognized by the anti-T monoclonal antibodies, it appears that both the carboxy and amino termini of the T-ag polypeptide are exposed on the surface of SV40-transformed mouse cells. The nature of the association between surface T-ag and 53K protein, as well as that between the molecular complex and the plasma membrane, remains to be determined. The possible effect of the surface-associated T-ag/53K complex on cellular proliferation is considered.

The antigenic structure of simian virus 40 (SV40) large tumor antigen (T-ag) in the plasma membranes of SV40-transformed mouse cells and SV40-infected monkey cells was characterized as a step toward defining possible biological function(s). Wild-type SV40, as well as a deletion mutant of SV40 (d11263) which codes for a truncated Tag with an altered carboxy terminus, was used to infect permissive cells. Members of a series of monoclonal antibodies directed against antigenic determinants on either the amino or the carboxy terminus of the Tag polypeptide were able to precipitate surface Tag (as well as nuclear Tag) from both SV40transformed and SV40-infected cells. Cellular protein p53 was coprecipitated with Tag by all Tag -reactive reagents from the surface and nucleus of SV40-transformed cells. In contrast, Tag , but not Tag -p53 complex, was recovered from the surface of SV40-infected cells. These results confirm that nuclear Tag and surface Tag are highly related molecules and that a complex of SV40 Tag and p53 is present at the surface of SV40transformed cells. Detectable levels of such a complex do not appear to be present on SV40-infected cells. Both the carboxy and amino termini of Tag are exposed on the surfaces of SV40-transformed and-infected cells. The possible relevance of the presence of a Tag -p53 complex on the surface of SV40-transformed cells and its absence from SV40-infected cells is considered. Many biochemical, physiological, and morphological changes take place after cell transformation induced by a variety of agents, including viruses (for a review, see reference 38). Viruses are ideal models with which to dissect the molecular mechanisms involved in cell transformation since their genetic content is small, well defined, and amenable to manipulation. Nonstructural viral proteins, termed tumor antigens (T-ag), are synthesized by simian virus 40 (SV40)infected and-transformed cells (for reviews, see references 35, 63, and 65). Large Tag , a phosphoprotein with an apparent molecular weight of 94,000 (94K), appears to be responsible for initiation and maintenance of the transformed phenotype (2, 4, 29, 37, 41, 59). Large Tag may be considered a multifunctional molecule because of the many functions that it mediates in both SV40-transformed and-infected cells. In productively infected cells, large Tag is required for initiation of viral DNA replication (7, 58) and is involved in autoregulation of transcription of the early region of the viral genome (1, 28, 43, 45). Induction of cellular DNA and rRNA synthesis in SV40-transformed cells also appears to be dependent on the presence of a functional large Tag (3, 8, 19-21, 26, 50-53, 62, 66). Several of the recognized biological activities of Tag have been assigned to relatively small, specific coding sequences in the A gene of SV40 and, by extrapolation, localized to defined regions of the Tag molecule (44). Therefore, the Tag molecule can be visualized as being composed of different functional domains which can be related to the structure of the po...

Cell growth control appears to be drastically altered as a consequence of transformation. Because the cell surface appears to have a role in modulating cell growth and simian virus 40 (SV40)-transformed cells express large T antigen (T-Ag) in the plasma membrane, we investigated whether surface T-Ag expression varies according to cell growth rate. Different growth states were obtained by various combinations of seeding density, serum concentration, and temperature, and cell cycle distributions were determined by flow microcytofluorometry. Actively dividing SV40-transformed mouse cell cultures were consistently found to express higher levels of surface T-Ag and T-Ag/p53 complex than cultures in which cells were mostly resting. In addition, the T-Ag/p53 complex disappeared from the surface of tsA7-transformed cells cultured under restrictive conditions known to induce complete growth arrest (39.50C), although the surface complex did not disappear from other tsA transformants able to keep cycling at 39.5°C. These results suggest that surface SV40 T-Ag or surface T-Aglp53 complex, or both, are involved in determining the growth characteristics of SV40-transformed cells.Simian virus 40 large tumor antigen (SV40 T-Ag) is a remarkably multifunctional polypeptide (for a review, see reference 43) that has among its functions the ability to induce cellular DNA synthesis (8,20,40,48,55). This ability has been mapped between 0.42 and 0.49 map units on the early region of SV40 DNA (48).T-Ag is found in association with a host-coded cellular protein of about 53,000 molecular weight (p53), both in the nucleus (for a review, see reference 27) and on the surface (4446) of SV40-transformed cells. The specific role of either nuclear-or surface-associated T-Ag/p53 complex in maintenance of transformation is unknown. Increased levels of p53 have been detected in proliferating cells (15,38,39), and serum stimulation of DNA synthesis in Swiss 3T3 cells is inhibited by microinjection of anti-p53 monoclonal antibodies (36, 37). Therefore, a role for p53 in control of normal cell proliferation is implicated. It is possible that T-Ag alters the normal mechanism(s) which control(s) cell division either directly or indirectly through an interaction with p53.A first approach toward determining whether surface T-Ag, either by itself or when complexed with p53, plays a role in regulating proliferation of SV40-transformed cells is described in this study. A correlation was established between cell growth rate and the expression of T-Ag and T-Ag/p53 complex on the cell surface. The expression of T-Ag and T-Ag/p53 complex on the surface of actively dividing SV40-transformed mouse cells was elevated when compared with the levels found on resting cells. In addition, complete growth arrest of tsA7-transformed cells cultured under nonpermissive conditions was preceded by the disappearance of the T-Ag/p53 complex from the surface of those cells. These observations suggest that surface-associated T-Ag or T-Ag/p53 complex is involved in the alteration o...

A molecular complex of simian virus 40 large tumor antigen (T-Ag) and p53 cellular protein is present on the surface of simian virus 40-transformed mouse cells. The stability of the association of the two proteins with the cell surface was characterized. Cells were either surface iodinated by the lactoperoxidase technique or metabolically labeled with [35S]methionine, and surface antigens were detected by differential immunoprecipitation with specific antibodies immediately after labeling or after incubation at 37°C. A rapid, concomitant disappearance of TAg and p53 from the cell surface was observed. The half-life of iodinated surface TAg was less than 30 min, whereas that of [35S]methionine-labeled surface TAg was 1 to 2 h. Although TAg and p53 were rapidly lost, both were also rapidly replaced on the cell surface, since newly exposed molecules could be detected when cells were reiodinated after a 2-h chase period. Control experiments established that the loss of the surface molecules was not induced by the iodination reaction. The appearance of surface TAg was prevented when cellular protein synthesis was inhibited with cycloheximide. The disappearance and replacement of TAg and p53 appeared to be energy-independent processes, as neither was inhibited by sodium azide or 2,4-dinitrophenol. Incubation of iodinated cells at 4°C did block the loss of TAg and p53. These observations suggest that TAg and p53 are coordinately turned over in the plasma membrane. The nature of the association of the TAg -p53 complex with the cell surface can best be described as highly dynamic.