Role of simian virus 40 gene A function in maintenance of transformation

Abstract

Mouse, hamster, and human cells were transformed at the permissive temperature by mutants from simian virus 40 (SV40) complementation group A in order to ascertain the role of the gene A function in transformation. The following parameters of transformation were monitored with the transformed cells under permissive and nonpermissive conditions: morphology; saturation density; colony formation on plastic, on cell monolayers, and in soft agar; uptake of hexose; and the expression of SV40 tumor (T) and surface (S) antigens. Cells transformed by the temperature-sensitive (ts) mutants exhibited the phenotype of transformed cells at the nonrestrictive temperature for all of the parameters studied. However, when grown at the restrictive temperature, they were phenotypically similar to normal, untransformed cells. Growth curves showed that the ts A mutant-transformed cells exhibited the growth characteristics of wild-type virus-transformed cells at the permissive temperature and resembled normal cells when placed under restrictive conditions. There were 3to 51-fold reductions in the levels of saturation density, colony formation, and uptake of hexose when the mutant-transformed cells were grown at the elevated temperature as compared to when they were grown at the permissive temperature. Mutant-transformed cells from the nonpermissive temperature were able to produce transformed foci when shifted down to permissive conditions, indicating that the phenotypically reverted cells were still viable and that the reversion was a reversible event. SV40 T antigen was present in the cells at both temperatures, but S antigen was not detected in cells maintained at the nonpermissive temperature. All of the wild-type virus-transformed cells exhibited a transformed phenotype when grown under either restrictive or nonrestrictive conditions. These results indicate that the SV40 group A mutant-transformed cells are temperature sensitive for the maintenance of growth properties characteristic of transformation. Virus rescued from the mutant-transformed cells by the transfection method was ts, suggesting that the SV40 gene A function, rather than a cellular one, is responsible for the ts behavior of the cells. 619 on August 1, 2020 by guest http://jvi.asm.org/ Downloaded from 620

Ras-mediated cell cycle arrest is altered by nuclear oncogenes to induce Schwann cell transformation.

“…Most cells remained viable at 39.50C for 9 days, and 50-80% of cells underwent mitosis within 4 days of shifting the cultures back to 33°C. As observed for other cell types expressing SV40-tsA58 large T (Brugge and Butel, 1975;Brockman, 1978), changes in growth behaviour on altering the temperature occurred slowly, over a period of days. Similar results were obtained with 33-1l ras2 cells (not shown).…”

Detection of simian virus 40 surface‐associated large tumor antigen by enzyme‐catalyzed radioiodination

“…Cells either infected or transformed by tsA mutants of SV40, with few exceptions, express immunologically reactive nuclear T-ag at the nonpermissive temperature (Brugge and Butel, 1975;Osborn and Weber, 1975;Tegtmeyer, 1975;Noonan and Butel, 1978;Alwine and Khoury, 1980), indicating that the tsA lesion does not preclude transport of mutant T-ag to the nucleus. Since tsA T-ag was observed on the surface of tsA-infected cells at the nonpermissive temperature by surface iodination, it appears that transport of the molecule to the cell surface is likewise not abolished by the tsA lesion.…”

“…Members of one class of SV40 temperature-sensitive mutants map in the A cistron (tsA mutants) and exhibit altered thermal stability of immunological and biological properties of T-ag (Carroll et al, 1974;Alwine et al, 1975;Jessel et al, 1975;Tenen et al, 1975). The tsA mutants transform cells at a reduced efficiency at the nonpermissive temperature (Kimura and Dulbecco, 1972;Tegtmeyer, 1972) and most cells transformed at the permissive temperature by the tsA viruses are temperature-sensitive for many growth-related properties associated with the transformed phenotype (Brugge and Butel, 1975;Kimura and Itagaki, 1975;Martin and Chou, 1975;Osborn and Weber, 1975;Tegtmeyer, 1975). The role of small t-ag in cellular transformation is less clear.…”

In vivo and in vitro TSTA‐inducing activity of temperature‐sensitive (TS) mutants of SV40

“…With the use of SV40 ts A mutants, data demonstrating the necessity of A gene function for the maintenance of the transformed state have been presented (Tegtmeyer, 1975 ;Osborn and Weber, 1975;Martin and Chou, 1975;Brugge and Butel, 1975). Butel et al (1975) discovered that the expression of the S antigen in cells transformed with ts A mutants is temperature-dependent and that this antigen is not expressed at the non-permissive temperature.…”