Related Experiment Videos
Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the
1Istituto di Biologia Cellulare del Consiglio Nazionale delle Ricerche, Roma, Italy.
Abstract:
We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.
Insights
Researchers studied simian virus 40 large T antigen nuclear import. They found that multiple nuclear localization signals and cell type influence protein localization and function, impacting cell growth.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Simian virus 40 large T antigen (SV40 T-ag) is a potent transforming protein.
- Nuclear localization is crucial for SV40 T-ag's oncogenic functions.
- A naturally occurring mutant lacks the nuclear transport signal.
Purpose of the Study:
- To investigate the requirements for nuclear import of SV40 T-ag.
- To determine the functional consequences of altered nuclear localization.
- To identify the optimal conditions for nuclear accumulation of SV40 T-ag variants.
Main Methods:
- Constructing SV40 T-ag gene variants with inserted nuclear localization signals using synthetic oligonucleotides.
- Analyzing stable transfectant cell populations (mouse NIH 3T3, rat embryo fibroblasts, simian CS cells) for protein localization.
- Assessing the transforming activity of nuclear-localized versus cytoplasmic SV40 T-ag variants.
Main Results:
- Cell type significantly impacts SV40 T-ag nuclear localization efficiency, with CS cells being most efficient and NIH 3T3 cells least efficient.
- Reiterating the nuclear localization signal or modifying its amino acid context enhanced nuclear import.
- Three copies of the signal were sufficient for wild-type-like nuclear accumulation in NIH 3T3 cells.
- Nuclear-localized SV40 T-ag variants showed slightly increased transforming activity in rat and NIH 3T3 cells but decreased activity in CS cells.
Conclusions:
- Nuclear import of SV40 T-ag is influenced by signal number, context, and cell-specific factors.
- Altering SV40 T-ag's subcellular localization affects its transforming competence in a cell-dependent manner.
- Understanding SV40 T-ag nuclear transport provides insights into viral oncogenesis and protein localization mechanisms.