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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 ceUls, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 ceUls) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 ceUs 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 ceUs, 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 ceUls of the same population, as if the nuclear uptake of some molecules depended on individual cel 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.The nuclear localization of a number of eucaryotic proteins is known to depend on the presence in their polypeptide chains of determinants that vary in sequence and number; these are generally referred to as nuclear signals (3,

Two main modifications of the simian virus 40 chromatin were found to occur during the lytic cycle. One was the progressive increase in the acetylation level in the four non-H1 histones as the 75S deoxynucleoprotein complexes (minichromosomes) became assembled into heavier structures. The other was the final elimination from viral chromatin of histone H1. An important stage in the course of these changes was represented by an intracellular simian virus 40 particle, in which the virus-coded proteins were already assembled, but properties distinct from those of mature virions were still present. This particle resembled the mature virions in morphology, sedimentation rate, and buoyant density. It was distinguished by the instability, the presence of histone H1, the uptake of radioactive acetate, and the lower infectivity. Its significance appears to be that of an immature virion on the basis of these characters and of the consistent kinetic behavior during the lytic cycle.

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.The large tumor antigen (large T) of simian virus 40 (SV40) is a multifunctional protein of 88 kDa encoded by the viral early gene A. This protein regulates the transcription of viral RNA and the replication of viral DNA during productive infections and the initiation and maintenance of the transformed state in transformed cells (1, 2). In both types of interaction, the large T shows a predominantly nuclear ("karyophilic") localization.A large body of evidence indicates that several functions of the SV40 large T are critically dependent on particular regions of the molecule, rather than on the integrity of the whole protein. A very precisely localized activity of the large T is the adenovirus helper function (3, 4), which also appears to play some role in productive SV40 infections (5). The lytic and transforming competence of the large T are genetically separable (6,7), and single nucleotide changes may result in the loss of the former but not of the latter (8, 9). The COOHterminal half of the protein was shown to be dispensable for the stimulation of cellular DNA synthesis (10, 11) and, more recently, for the transformation of some cultured cells (12)(13)(14)(15)(16). Further progress in the dissection of the SV40 large T activities has been recently achieved by constructing in vitro a series of deletion mutants altogether covering almost the entire A gene (17).In polyoma virus, the essential functions carried in SV40 by the large T are naturally separated in two proteins: the large T, residing in the nucleus, and the middle-sized T, residing in the cytoplasm. An important distinction concerning the transforming function of this virus has been added recently, when it was discovered that the middle-sized T is both n...