Excess wild-type p53 blocks initiation and maintenance of simian virus 40 transformation.

Wild-type (wt) murine p53 has been tested for its ability to block and reverse the transforming effects of simian virus 40 (SV40) large T antigen. Established and precrisis mouse cells overexpressing exogenously introduced wt p53 became resistant to SV40 transformation. The introduction of excess wt p53 into SV40-transformed precrisis cells reverted their transformed phenotype. However, the phenotype of SV40-transformed established cells was not reverted by excess wt p53. We conclude that an antioncogenic action of wt p53 is exerted during SV40 transformation and that in precrisis cells, the antitransforming action of wt p53 can be exerted both at initiation and during the maintenance of transformation.     

Cooperation of simian virus 40 large and small T antigens in metabolic stabilization of tumor suppressor p53 during cellular transformation.

Metabolic stabilization of the tumor suppressor p53 is a key event in cellular transformation by simian virus 40 (SV40). Expression of the SV40 large tumor antigen (large T) is necessary but not sufficient for this process, as metabolic stabilization of p53 complexed to large T in abortively SV40-infected cells strictly depends on the cellular systems analyzed (F. Tiemann and W. Deppert, J. Virol. 68:2869-2878, 1994). Comparative analyses of various cells differing in metabolic stabilization of p53 upon abortive infection with SV40 revealed that metabolic stabilization of p53 closely correlated with expression of the SV40 small t antigen (small t) in these cells: 3T3 cells do not express small t and do not stabilize p53 upon infection with wild-type SV40. However, ectopic expression of small t in 3T3 cells provided these cells with the capacity to stabilize p53 upon SV40 infection. Conversely, precrisis mouse embryo cells express small t and mediate metabolic stabilization of p53 upon infection with wild-type SV40. Infection of these cells with an SV40 small-t deletion mutant did not lead to metabolic stabilization of p53. Small-t expression and metabolic stabilization of p53 correlated with an enhanced transformation efficiency by SV40, supporting the conclusion that at least part of the documented helper effect of small t in SV40 transformation is its ability to promote metabolic stabilization of p53 complexed to large T.     

Stabilization of the tumor suppressor p53 during cellular transformation by simian virus 40: influence of viral and cellular factors and biological consequences.

To understand the process and biological significance of metabolic stabilization of p53 during simian virus 40 (SV40)-induced cellular transformation, we analyzed cellular and viral parameters involved in this process. We demonstrate that neither large T expression as such nor the cellular phenotype (normal versus transformed) markedly influence the stability of p53 complexed to large T in SV40 abortively infected BALB/c mouse fibroblasts. In contrast, metabolic stabilization of p53 is an active cellular event, specifically induced by SV40. The ability of SV40 to induce a cellular response leading to stabilization of p53 complexed to large T is independent from the cellular phenotype and greatly varies between different cells. However, metabolic stability was conferred only to p53 in complex with large T, whereas the free p53 in these cells remained metabolically unstable. Comparative analyses of cellular transformation in various cells differing in stability of p53 complexed to large T upon abortive infection with SV40 revealed a strong correlation between the ability of SV40 to induce metabolic stabilization and its transformation efficiency. Our data suggest that metabolic stabilization and the ensuing enhanced levels of p53 are important for initiation and/or maintenance of SV40 transformation.     

Identification of the p53 protein domain involved in formation of the simian virus 40 large T-antigen-p53 protein complex.

An expression vector utilizing the enhancer and promoter region of the simian virus 40 (SV40) DNA regulating a murine p53 cDNA clone was constructed. The vector produced murine p53 protein in monkey cells identified by five different monoclonal antibodies, three of which were specific for the murine form of p53. The murine p53 produced in monkey cells formed an oligomeric protein complex with the SV40 large tumor antigen. A large number of deletion mutations, in-frame linker insertion mutations, and linker insertion mutations resulting in a frameshift mutation were constructed in the cDNA coding portion of the p53 protein expression vector. The wild-type and mutant p53 cDNA vectors were expressed in monkey cells producing the SV40 large T antigen. The conformation and levels of p53 protein and its ability to form protein complexes with the SV40 T antigen were determined by using five different monoclonal antibodies with quite distinct epitope recognition sites. Insertion mutations between amino acid residues 123 and 215 (of a total of 390 amino acids) eliminated the ability of murine p53 to bind to the SV40 large T antigen. Deletion (at amino acids 11 through 33) and insertion mutations (amino acids 222 through 344) located on either side of this T-antigen-binding protein domain produced a murine p53 protein that bound to the SV40 large T antigen. The same five insertion mutations that failed to bind with the SV40 large T antigen also failed to react with a specific monoclonal antibody, PAb246. In contrast, six additional deletion and insertion mutations that produced p53 protein that did bind with T antigen were each recognized by PAb246. The proposed epitope for PAb246 has been mapped adjacent (amino acids 88 through 109) to the T-antigen-binding domain (amino acids 123 through 215) localized by the mutations mapped in this study. Finally, some insertion mutations that produced a protein that failed to bind to the SV40 T antigen appeared to have an enhanced ability to complex with a 68-kilodalton cellular protein in monkey cells.     

Complex formation between the lymphotropic papovavirus large tumor antigen and the tumor suppressor protein p53

The simian B-lymphotropic papovavirus (LPV) encodes a large tumor antigen (T antigen) which is 45% identical to both the simian virus 40 (SV40) and the polyomavirus (PyV) large T antigens. In transgenic mice, the transforming properties of the LPV T antigen are similar to those of the SV40 T antigen. However, little is known about its biochemical activities. Since SV40 T antigen forms a complex with and stabilizes the host cell tumor suppressor protein p53 while the PyV large T antigen does not, we characterized the LPV T antigen for its ability to complex p53. We demonstrate an association between LPV T antigen and p53 in both a tumor-derived cell line and BALB/c 3T3 cells transformed in culture. A third protein of approximately 68 kDa which was found associated with the LPV T antigen-p53 complex in tumor-derived cells appears to be heat shock protein 70 (hsp70). The half-life of p53 in all LPV T-antigen-transformed cells was extended significantly; i.e., it was 3 to 7 h compared with 19 minutes in BALB/c 3T3 cells. The half-life of the LPV T antigen itself was 5 to 9 h depending on the cell line origin. That p53 was stabilized because of association with LPV T antigen and not because of mutation was demonstrated with the p53 conformation-dependent monoclonal antibody PAb246. This antibody distinguishes between wild-type p53 (PAb246+) and mutant, oncogenic p53 (PAb246-). Sequential immunoprecipitation showed all detectable p53 to be of the PAb246+ class in each LPV-transformed cell line, suggesting that the stable p53 was indeed wild type.     

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.     

Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

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 immortalization-competent 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.     

Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

To analyze the proposed growth-inhibitory function of wild-type p53, we compared simian virus 40 (SV40) DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidney cell line LLC-MK2, which expresses a mutated p53 that does not complex with large T antigen. SV40 DNA replication proceeded identically in both cell types during the course of infection. Endogenously expressed wild-type p53 thus does not negatively modulate SV40 DNA replication in vivo. We suggest that inhibition of SV40 DNA replication by wild-type p53 in in vitro replication assays is due to grossly elevated ratios of p53 to large T antigen, thus depleting the replication-competent free large T antigen in the assay mixtures by complex formation. In contrast, the ratio of p53 to large T antigen in in vivo replication is low, leaving the majority of large T antigen in a free, replication-competent state.     

The ability of simian virus 40 large T antigen to immortalize primary mouse embryo fibroblasts cosegregates with its ability to bind to p53.

The large T antigen encoded by simian virus 40 (SV40) plays essential roles in the infection of permissive cells, leading to production of progeny virions, and in the infection of nonpermissive cells, leading to malignant transformation. Primary mouse embryo fibroblasts (MEFs) are nonpermissive for SV40, and infection by wild-type SV40 leads to immortalization and transformation of a small percentage of infected cells. We examined the ability of an extensive set of mutants whose lesions affect SV40 large T antigen to immortalize MEFs. We found that immortalization activity was retained by all mutants whose lesions are located upstream of codon 346. This includes a mutant lacking amino acids 168 to 346. We previously showed (M. J. Tevethia, J. M. Pipas, T. Kierstead, and C. Cole, Virology 162:76-89, 1988) that sequences downstream of amino acid 626 are not required for immortalization of primary MEFs. Studies by Thompson et al. (D. L. Thompson, D. Kalderon, A. Smith, and M. Tevethia, Virology 178:15-34, 1990) indicate that all sequences upstream of residue 250, including the domain for binding of tumor suppressor protein Rb, are not required for transformation of MEFs. Together, these studies demonstrate that the immortalization activity of large T antigen for MEFs maps to sequences between 347 and 626. Several mutants with lesions between 347 and 626 retained the ability to immortalize at nearly the wild-type frequency, while others, with small insertions at amino acid 409 or 424 or a deletion of residues 587 to 589, failed to immortalize. The abilities of mutant T antigens to form a complex with tumor suppressor protein p53 were examined. We found that all mutants able to immortalize retained the ability to complex with p53, while all mutants which lost the ability to immortalize were no longer able to bind p53. This suggests that inactivation of the growth-suppressive properties of p53 is essential for immortalization of MEFs.     

Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53.

We have analyzed the biochemical properties of a nonviable simian virus 40 (SV40) mutant encoding a large T antigen (T) bearing an amino acid substitution (Pro-584-Leu) in its hydrophobic region. Mutant 5080 has an altered cell type specificity for transformation (transforming mouse C3H10T1/2 but not rat REF52 cells), is defective for viral DNA replication, and encodes a T that is unable to form a complex with the cellular p53 protein (K. Peden, A. Srinivasan, J. Farber, and J. Pipas, Virology 168:13-21, 1989). In this article, we show that 5080-transformed C3H10T1/2 cell lines express an altered T that is synthesized at a significantly higher rate but with a shorter half-life than normal T from wild-type SV40-transformed cells. 5080 T did not oligomerize beyond 5 to 10S in size compared with normal T, which oligomerized predominantly to 14 to 20S species. In addition, the 5080 T complex had significantly decreased ATPase activity and had a 10-fold-lower level of in vivo phosphorylation compared with that of normal T. Two-dimensional phosphopeptide analysis indicated several changes in the specific 32P labeling pattern, with altered phosphorylation occurring at both termini of the mutant protein compared with the wild-type T. Loss of p53 binding is therefore concomitant with changes in ATPase activity, oligomerization, stability, and in vivo phosphorylation of T and can be correlated with defective replication and restricted transformation functions. That so many biochemical changes are associated with a single substitution in the hydrophobic region of T is consistent with its importance in regulating higher-order structural and functional relationships in SV40 T.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

Monoclonal antibodies displaying a novel species specificity for the primate transformation-related protein, p53

SV40 large T antigen associates with a cellular phosphoprotein, p53, in virus-transformed cells. We have raised three new monoclonal antibodies, PAb1101, PAb1102 and PAb1103, to this cellular protein, derived from SV40-transformed human fibroblasts. These define at least two non-overlapping determinants on human p53 that are in different areas of the molecule from those recognised by previously available antibodies. Unlike those antibodies, PAb1102 and PAb1103 do not react with rodent p53. PAb1101 reacts far more weakly with rodent p53 than with primate p53. All three antibodies show a preference for binding to the large T-associated form of p53, an effect that is particularly marked with PAb1102. The novel specificity of these antibodies allows further probing of the nature and function of the large T/p53 complex in human cells.     

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen.

To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1-521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyT1-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens.     

Primate's p53 inhibits SV40 DNA replication in vitro

Previous reports indicated that rodent p53 inhibits simian virus 40 (SV40) DNA replication in vitro as well as in vivo while that from primate cells does not (1-4). Here we report the evidence that p53 of primate origin also inhibits SV40 DNA replication in vitro. p53-SV40 large tumor antigen (T antigen) complex purified from SV40 infected COS-1 cells had little replication activity and inhibited SV40 DNA replication in vitro. These results suggest that inhibition of SV40 DNA replication by p53 should be regarded as general property of the protein and does not determine the mode of species specific replication of SV40 DNA.     

Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53

The primary transforming functions of simian virus 40 large T antigen (SV40 LT) are conferred primarily through the binding and inactivation of p53 and the retinoblastoma family members. Normal p53 function requires an association with the CREB binding protein (CBP)/p300 coactivators, and a ternary complex containing SV40 LT, p53, and CBP/p300 has been identified previously. In this report, we have evaluated a secondary function of p53 bound to the SV40 LT complex in mediating the binding of human CBP/p300. We demonstrate that p53 associated with SV40 LT was posttranslationally modified in a manner consistent with the binding of CBP/p300. Furthermore, expression of SV40 LT induced the proportion of p53 phosphorylated on S15. An essential function for p53 in bridging the interaction between SV40 LT and CBP/p300 was identified through the reconstitution of the SV40 LT-CBP/p300 complex upon p53 reexpression in p53-null cells. In addition, the SV40 LT-CBP/p300 complex was disrupted through RNA interference-mediated depletion of endogenous p53. We also demonstrate that SV40 LT was acetylated in a p300- and p53-dependent manner, at least in part through the CH3 domain of p300. Therefore, the binding of p53 serves to modify SV40 LT by targeting CBP and p300 binding to direct the acetylation of SV40 LT.     

Overproduction of protein p53 contributes to simian virus 40-mediated transformation.

The possible involvement of p53 overproduction in simian virus 40 (SV40)mediated transformation was studied by using the rat embryo fibroblast focus formation assay. Transformation by wild-type SV40 was enhanced two- to threefold by cotransfection of a plasmid overexpressing mouse p53. More significantly, such a plasmid could partially complement a transformation-defective deletion mutant of SV40. Hence, the ability of SV40 T antigen to induce high p53 levels may indeed be directly relevant to the viral transforming potential.     

Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53.

The cellular phosphoprotein p53 is presumably involved in simian virus 40 (SV40)-induced transformation. We have monitored changes in the state of phosphorylation of p53 from normal versus SV40-infected or -transformed cells. In normal cells, p 53 was hardly phosphorylated. Upon infection or transformation, a quantitative and qualitative increase in p53 phosphorylation was observed as revealed by two-dimensional phosphopeptide analysis. This increase was dependent on a functional large T antigen. In rat cells, enhanced phosphorylation of p53 resulted in conversion to a second, electrophoretically distinct form. In cells transformed with transformation-defective mutants, phosphorylation of p53 was reduced and conversion to form 2 was inefficient. These data suggest (i) that SV40 large T antigen induces or activates a protein kinase, one substrate of which is p53, (ii) that transformation-defective mutants are impaired in kinase induction, and (iii) that either a certain phosphorylation state of p53 or the SV40-induced kinase is critical for efficient transformation.     

Transformation of a continuous rat embryo fibroblast cell line requires three separate domains of simian virus 40 large T antigen.

Mouse C3H 10T1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 10T1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 10T1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.     

Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells.

To determine functional subcellular loci of p53, a cellular protein associated with cellular transformation, we analyzed the nucleoplasmic, chromatin, and nuclear matrix fractions from normal mouse 3T3 cells, from methylcholanthren-transformed mouse (MethA) cells, and from various simian virus 40 (SV40)-transformed cells for the presence of p53. In 3T3 and MethA cells, p53 was present in all nuclear subfractions, suggesting an association of p53 with different structural components of the nucleus. In 3T3 cells, p53 was rapidly turned over, whereas in MethA cells, p53 was metabolically stable. In SV40-transformed cells, p53 complexed to large tumor antigen (large T) was found in the nucleoplasmic and nuclear matrix fractions, as described previously (M. Staufenbiel and W. Deppert, Cell 33:173-181, 1983). In addition, however, metabolically stable p53 not complexed to large T (free p53) was also found in the chromatin and nuclear matrix fractions of these cells. This free p53 did not arise by dissociation of large T-p53 complexes, suggesting that stabilization of p53 in SV40-transformed cells can also occur by means other than formation of a complex with large T.     

The phosphorylation at Thr 124 of simian virus 40 large T antigen is crucial for its oligomerization

SV40 large T antigen is phosphorylated at up to ten different amino acids clustered in an N-terminal and a C-terminal part of the polypeptide chain. The N-terminal phosphorylated residues include Ser 123 and Thr 124. We have analyzed the oligomerization, the complex formation with the cellular oncoprotein p53 and the DNA-binding properties of T antigen from two different SV40 transformed cell lines which have either an amino acid exchange at Ser 123 to Phe (W7) or Thr 124 to Ile (D29). In comparison to wild-type T antigen both mutant T antigens have a slightly reduced binding affinity for both binding sites, I and II, of SV40 DNA. Phosphorylation at both residues of T antigen is not essential for formation of the complex with p53. Only the phosphorylation at Thr 124 seems to be critical for the formation of high molecular mass oligomers. Our data support the hypothesis that the oligomerization of T antigen seems to be implicated in viral DNA replication.