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Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences
D Tedesco1, L Fischer-Fantuzzi, C Vesco
1Instituto di Biologia Cellulare del CNR, Rome, Italy.
Abstract:
Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.
Insights
Altering the SV40 large T protein
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- The SV40 large T antigen is crucial for viral replication and cellular transformation.
- Key functional domains include the retinoblastoma protein (Rb) binding site and nuclear transport signal.
- Understanding these domains' roles is vital for dissecting viral oncogenesis.
Purpose of the Study:
- To investigate the functional significance of the SV40 large T antigen's Rb binding site and nuclear transport signal.
- To determine how specific amino acid substitutions in these regions affect cellular transformation and immortalization.
- To elucidate the interplay between different functional domains of the large T antigen.
Main Methods:
- Site-directed mutagenesis was used to introduce amino acid substitutions into the SV40 large T antigen.
- Mutant activities were assessed using transformation assays in rat embryo fibroblasts (REFs) and other rodent cell lines.
- Phenotypic changes, including focus formation and anchorage-independent growth (agar colony formation), were evaluated.
- Immortalization potential of mutants in pre-crisis REFs was also examined.
Main Results:
- Mutations in the Rb binding site reduced transforming ability, preventing agar colony formation in REFs but allowing focus formation.
- Some Rb binding site mutants retained the ability to induce agar colonies in other cell lines (NIH3T3, F2408).
- All tested mutants efficiently immortalized pre-crisis REFs.
- Cytoplasmic mutants' transforming activity depended on Rb-binding integrity, similar to wild-type, but in vivo complexes were not detected.
- The presence of the small t antigen was critical for mutants with limited transforming abilities.
Conclusions:
- The Rb binding site and nuclear transport signal are critical for distinct aspects of SV40 large T antigen-mediated transformation.
- Rb binding site integrity is essential for anchorage-independent growth but not necessarily for immortalization or focus formation.
- The small t antigen plays a significant role in modulating the transforming potential of large T antigen mutants.
- These findings provide insights into the structure-function relationships of SV40 large T antigen and its oncogenic mechanisms.