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,

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...

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalizationcompetent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.

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We have investigated the mechanism by which the simian virus 40 large T antigen (SVLT) interferes with the differentiation of C2 myoblasts. SVLT mutants, defective either in the Rb binding site, near the N-terminal end, in a region that affects binding to p53, or in the nuclear transport signal, were also employed to determine whether the interference was especially dependent on these functional domains. It was found that wild-type (wt) SVLT strongly inhibited the terminal differentiation of mouse C2 myoblasts, but this arrest occurred only after the synthesis of myogenin, an initial step in biochemical differentiation. Neither the synthesis nor some basic activities of MyoD appeared to be affected by wt SVLT. In these transformants, mitogen depletion elicited an increase in the Rb level comparable to that in normal C2 cells; wt SVLT, however, promoted the phosphorylation of a large part of the induced Rb. Mutations affecting nuclear transport were far more critical for the ability to interfere with myogenic differentiation than were those affecting the transforming potential; cytoplasmic SVLT expression was fully compatible with the terminal differentiation of C2 cells, despite enabling them to grow in semisolid medium, thus showing that the myogenesis-inhibiting property can be dissociated from transforming competence. The remaining SVLT mutants presented different degrees of ability to inhibit differentiation (as shown by the expression of tissue-specific markers in transformants). The inhibiting mutants, including the Rb binding site mutant, were able to promote a higher state of Rb phosphorylation than that observed in either normal cells or cytoplasmic-SVLT transformants.