The cellular chromatin is an important target for SV40 large T antigen in maintaining the transformed phenotype.
To identify cellular targets of simian virus 40 large T antigen (SV40 large T) important for the maintenance of cellular transformation, we have compared biological properties of SV40 tsA58 mutant large T antigens expressed in cells of a matched pair of SV40 tsA58 N-type (temperature-sensitive) and A-type (temperature-insensitive) transformants of the normal rat fibroblast line F111 (D. Pintel et al., J. Virol. 38, 518-528, 1981). Characterization of the selected cell lines demonstrated that cells of the N-type transformant [FR(tsA58)A] exhibited properties similar to those of the corresponding SV40 wild-type transformant [FR(wt648)] at the permissive growth temperature (32 degrees ), but reverted to a phenotype indistinguishable from the parental F111 cells at the nonpermissive growth temperature (39 degrees). At both growth temperatures, cells of the A-type transformant [FR(tsA58)57] were very similar to FR(wt648) cells in all properties analyzed. Both mutant-transformed cell lines expressed authentic tsA58 mutant large T antigens at comparable steady-state levels. Analysis of the subnuclear distribution of large T antigens in wild-type and in mutant-transformed cells kept at permissive or at nonpermissive growth temperature, respectively, revealed an important biological difference between the mutant T antigens in N- and A-type transformants: Whereas the subnuclear distribution of wild-type large T in FR(wt648) cells remained unchanged at both growth temperatures, mutant large T in FR(tsA58)A cells (N-type transformant) already 1 day after the shift to the nonpermissive growth temperature no longer stably associated with nuclear substructures, notably the cellular chromatin. In contrast, mutant large T in FR(tsA58)57 cells (A-type transformant) retained this ability. The ability (or inability) of the mutant T antigens to associate with the cellular chromatin in vivo was paralleled by different DNA binding properties of the mutant large T antigens in vitro. Large T in FR(tsA58)A cells no longer bound to the SV40 ORI in vitro after the shift to the nonpermissive growth temperature, whereas large T in FR(tsA58)57 cells at the elevated growth temperature had preserved this activity to a degree similar to its ability to associate with the cellular chromatin. We suggest that in the system of matched pairs of N- and A-type transformants analyzed in this study, expression of the transformed phenotype in FR(tsA58)57 (A-type) cells at the nonpermissive growth temperature is due to the preservation of a biologically active conformation of the mutant large T, allowing it to maintain its interaction with specific targets at the cellular chromatin.
Richter
W
W
Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Federal Republic of Germany.
Deppert
W
W
D000199:Actins / Q000032:analysis; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D002455:Cell Division; D002472:Cell Transformation, Viral; D002843:Chromatin / Q000502:physiology*; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D006358:Hot Temperature; D009154:Mutation; D010641:Phenotype; D013539:Simian virus 40 / Q000235:genetics*
D000199:Actins; D000952:Antigens, Polyomavirus Transforming; D002843:Chromatin; D004279:DNA, Viral