Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus

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.