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The cellular chromatin is an important target for SV40 large T antigen in maintaining the transformed phenotype
1Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Federal Republic of Germany.
Abstract:
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.
Insights
Simian virus 40 large T antigen (SV40 large T) interaction with cellular chromatin maintains cell transformation. Temperature-sensitive mutants reveal that stable chromatin association, not just expression, is crucial for SV40 large T
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Simian virus 40 (SV40) large T antigen (LT) is a key oncogenic protein driving cellular transformation.
- Understanding LT's cellular targets is crucial for identifying mechanisms of transformation maintenance.
- Temperature-sensitive (ts) mutants of SV40 LT provide tools to study protein function under varying conditions.
Purpose of the Study:
- To identify cellular targets of SV40 large T antigen (LT) critical for maintaining cellular transformation.
- To compare the biological properties of temperature-sensitive (ts) SV40 LT variants in different cellular contexts.
- To elucidate the role of LT's interaction with cellular chromatin in sustaining the transformed phenotype.
Main Methods:
- Comparison of SV40 tsA58 N-type (temperature-sensitive) and A-type (temperature-insensitive) transformants of rat fibroblasts (F111).
- Analysis of cellular phenotype, subnuclear distribution of LT, and DNA-binding properties in vitro.
- Assessment of LT association with nuclear substructures, including cellular chromatin, at permissive and nonpermissive temperatures.
Main Results:
- N-type transformants reverted to a normal phenotype at nonpermissive temperatures, while A-type transformants maintained transformed properties.
- SV40 LT in N-type cells lost stable association with chromatin at nonpermissive temperatures, unlike in A-type cells.
- In vitro DNA-binding to the SV40 origin of replication (ORI) correlated with chromatin association; N-type LT lost binding, A-type LT retained it.
Conclusions:
- The ability of SV40 LT to stably associate with cellular chromatin is essential for maintaining the transformed phenotype.
- Preservation of a biologically active conformation of LT, enabling chromatin interaction, is key for sustained transformation in A-type cells.
- Specific interactions with cellular chromatin, rather than mere expression levels, dictate the oncogenic function of SV40 LT.