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.     

Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens.

We used a murine retrovirus shuttle vector system to construct recombinants capable of constitutively expressing the simian virus 40 (SV40) large T antigen and the polyomavirus large and middle T antigens as well as resistance to G418. Subsequently, these recombinants were used to generate cell lines that produced defective helper-free retroviruses carrying each of the viral oncogenes. These recombinant retroviruses were used to analyze the role of the viral genes in transformation of rat F111 cells. Expression of the polyomavirus middle T antigen alone resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were highly tumorigenic, whereas expression of the polyomavirus large T resulted in cell lines that were unaltered by the criteria of morphology, anchorage-independent growth, and tumorigenicity. More surprisingly, SV40 large T-expressing cell lines were not tumorigenic despite the fact that they contained elevated levels of cellular p53 and had a high plating efficiency in soft agar. These results suggest that the SV40 large T antigen is not an acute transforming gene like the polyomavirus middle T antigen but is similar to the establishment genes such as myc and adenovirus EIa.     

Simian virus 40 can overcome the antiproliferative effect of wild-type p53 in the absence of stable large T antigen-p53 binding.

In simian virus 40 (SV40)-transformed cells, a tight complex is formed between the viral large T antigen (large T) and p53. It has been proposed that this complex interferes with the antiproliferative activity of p53. This notion was tested in primary rat fibroblasts by assessing the ability of SV40-mediated transformation to be spared from the inhibitory effect of wild-type (wt) p53. The data indicate that relative to transformation induced by myc plus ras, SV40-plus-ras-mediated focus formation was indeed much less suppressed by p53 plasmids. A majority of the resultant cell lines made a p53 protein with properties characteristic of a wt conformation. Furthermore, cell lines expressing stably both SV40 large T and a temperature-sensitive p53 mutant continued to proliferate at a temperature at which this p53 assumes wt-like properties and normally causes a growth arrest. Surprisingly, at least partial resistance to the growth-inhibitory effect of wt p53 was also evident when transformation was mediated by an SV40 deletion mutant, encoding a large T which does not bind p53 detectably. In addition to supporting the idea that SV40 can overcome the growth-restrictive activity of wt p53, these findings strongly suggest that at least part of this effect does not require a stable association between p53 and large T.     

Large T antigens of simian virus 40 and polyomavirus efficiently establish primary fibroblasts.

Recombinant retroviruses that transduce the simian virus 40 (SV40) large T antigen or the polyomavirus large T antigen as well as encoding resistance to antibiotic G418 were used to investigate whether these genes alone were sufficient for immortalization of primary cells. The results provided definitive evidence that either viral gene can efficiently establish primary fibroblasts. The capability of the SV40 large T antigen to establish primary fibroblasts was undiminished by a mutation that alters its binding to sequences within the origin of replication. Surprisingly, most of the primary cells established by the expression of the SV40 large T antigen did not have a transformed phenotype. This suggests that transformation by SV40 is not simply due to a high level of expression of the SV40 large T antigen and stabilization of cellular p53.     

Failure of simian virus 40 small t antigen to disorganize actin cables in nonpermissive cell lines.

Mouse C3H 10T1/2 cell lines expressing the simian virus 40 (SV40) small t antigen were obtained by cotransfection of pSV2neo and plasmids which encode small t. Cell lines derived from two plasmids which encode small t in the absence of stable deletion fragments of the large T antigen were morphologically normal and grew to slightly higher saturation densities in low serum than control cell lines. Unexpectedly, the clones had highly organized actin cables, as did parental 10T1/2 cells infected with wild-type SV40. These observations and comparisons of rat F111 cells infected with either polyomavirus or SV40 suggest that the SV40 small t antigen does not directly affect cytoskeletal organization.     

Binding of p300/CBP co-activators by polyoma large T antigen.

Small DNA tumor viruses such as simian virus 40 (SV40) and polyomavirus (Py) take advantage of host cell proteins to transcribe and replicate their DNA. Interactions between the viral T antigens and host proteins result in cell transformation and tumor induction. Large T antigen of SV40 interacts with p53, pRb/p107/p130 family members, and the cyclic AMP-responsive element-binding protein (CREB)-binding protein (CBP)/p300. Py large T antigen is known to interact only with pRb and p300 among these proteins. Here we report that Py large T binds to CBP in vivo and in vitro. In co-transfection assays, Py large T inhibits the co-activation functions of CBP/p300 in CREB-mediated transactivation but not in NF-kappa B-mediated transactivation. p53 appears not to be involved in the functions of CREB-mediated transactivation and is not essential for large T:CBP interaction. Mutations introduced into a region of Py large T with homology to adenovirus E1A and SV40 large T prevent binding to the co-activators. These mutant large T antigens fail to inhibit CREB-mediated transactivation. The CBP/p300-binding Py mutants are able to transform established rat embryo fibroblasts but are restricted in their ability to induce tumors in the newborn mouse, indicating that interaction of large T with the co-activators may be essential for virus replication and spread in the intact host.     

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells

As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.     

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.     

A mathematical model of homologous recombination in cultured cells.

This work presents a model describing the rate of recombination between homologous segments of DNA stably integrated into the genome of cultured cells. The model has been applied to rat cell lines carrying the polyomavirus middle T oncogene and a functional origin of viral DNA replication. Introduction of the gene coding for the polyoma large T antigen or the SV40 large T antigen into cells by DNA transfection promotes homologous recombination in the resident viral inserts with rates varying between 0.1 x 10(-3) and 3.7 x 10(-1) per cell generation.     

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.     

Structural prerequisites of simian virus 40 large T antigen for the maintenance of cell transformation.

We have investigated the functional roles of two structural subsets of simian virus 40 (SV40) large T antigen, namely homo-oligomers and complexes with the host cellular p53 protein, for the transformed phenotype. We examined T antigen produced in cells transformed by temperature-sensitive SV40 large T mutants: heat-sensitive or unrestricted SV40 tsA58-transformed rat cells and unrestricted tsA1499 transformants. In both unrestricted cell lines, T antigen was temperature-sensitive only for the formation of fast sedimenting homo-oligomers. Corresponding to our recent observations obtained with tsA1499-infected monkey cells, in tsA1499 transformants large T was competent to form stable T-p53 complexes independently of the temperature. However, T antigen coded for by tsA58, which is heat-sensitive for binding to p53, occurred in stable complexes with this protein in unrestricted tsA58 transformants under all conditions. Furthermore, in both unrestricted transformants T-p53 complexes arise in the absence of homo-oligomers of T antigen. In conclusion, T antigen homo-oligomers are not involved in cell transformation, whereas T-p53 complexes may be involved in the maintenance of this phenotype.     

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.     

Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.     

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.     

Human papillomavirus E6 proteins bind p53 in vivo and abrogate p53-mediated repression of transcription.

The transforming proteins of DNA tumor viruses SV40, adenovirus and human papillomaviruses (HPV) bind the retinoblastoma and p53 cell cycle regulatory proteins. While the binding of SV40 large T antigen and the adenovirus E1B 55 kDa protein results in the stabilization of the p53 protein, the binding of HPV16 and 18 E6 results in enhanced degradation in vitro. To explore the effect of viral proteins on p53 stability in vivo, we have examined cell lines immortalized in tissue culture by HPV18 E6 and E7 or SV40 large T antigen, as well as cell lines derived from cervical neoplasias. The half-life of the p53 protein in non-transformed human foreskin keratinocytes in culture was found to be approximately 3 h while in cell lines immortalized by E6 and E7, p53 protein half-lives ranged from 2.8 h to less than 1 h. Since equivalent levels of E6 were found in these cells, the range in p53 levels observed was not a result of variability in amounts of E6. In keratinocyte lines immortalized by E7 alone, the p53 half-life was found to be similar to that in non-transformed cells; however, it decreased to approximately 1 h following supertransfection of an E6 gene. These observations are consistent with an interaction of E6 and p53 in vivo resulting in reductions in the stability of p53 ranging between 2- and 4-fold. We also observed that the expression of various TATA containing promoters was repressed in transient assays by co-transfection with plasmids expressing the wild-type p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study of the Functional Activities Concomitantly Retained by the 115,000 Mr Super T Antigen, an Evolutionary Variant of Simian Virus 40 Large T Antigen Expressed in Transformed Rat Cells

Simian virus 40 (SV40) transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) do not synthesize normal-size large T antigen (M(r), 90,000); instead, they produce a 115,000 M(r) super T antigen (115K super T antigen). This super T antigen is SV40 virus coded, and its synthesis results from rearrangement and amplification of integrated viral DNA sequences in subclone 7 (May et al., Nucleic Acids Res. 9:4111-4128, 1981). In this study the functional activities of 115K super T antigen were compared with the functional activities of SV40 large T antigen. Transfection experiments were performed with (i) cosmid SVE 5 Kb and plasmid pSVsT, both containing the super T antigen gene and (ii) plasmids pSV1 and pSV40, both containing the large T antigen gene. Transfection of pSVsT DNA or SVE 5 Kb DNA into secondary cultures of rat kidney cells induced the formation of transformed cell foci with an efficiency that was about 50% of the efficiency of pSV1 DNA or pSV40 DNA. Concomitant with the transforming activity, two other activities were also retained by super T antigen, namely, the ability to enhance the level of host cellular protein p53 and the capacity to bind to p53. In contrast, pSVsT and SVE 5 Kb DNAs were markedly deficient in the capacity to support tsA58 DNA replication in CV1-P cells at a nonpermissive temperature (41 degrees C), as shown by cotransfection experiments. The yield of virus produced in these experiments was 400-fold less than the yield obtained in parallel experiments with pSV40 or pSV1. However, SVE 5 Kb and pSVsT have a functional SV40 replication origin, as shown by their efficient replication in COS 1 cells which provided functional large T antigen. Super T antigen also possesses a specific affinity for sequences of SV40 viral origin. Our results suggest that under certain conditions, evolutionary changes in T antigen take place and that these changes could be restricted to the phenotypic requirement of maintaining a structure that is able to induce cell transformation, to form a complex with p53, and to enhance the cellular level of p53. Therefore, there appears to be a close relationship among the activities of T antigen involved in transforming cells, in binding to p53, and in enhancing the p53 cellular level. Moreover, this set of activities appears to be separable from the replicative ability of T antigen, based on the observation that 115K super T antigen is markedly defective for initiating viral DNA synthesis.     

The influence of SV40 immortalization of human fibroblasts on p53-dependent radiation responses

The simian virus 40 large tumor antigen (SV40 Tag) has been ascribed many functions critical to viral propagation, including binding to the mammalian tumor suppressor p53. Recent studies have demonstrated that SV40-transformed murine cells have functional p53. The status of p53 in SV40-immortalized human cells, however, has not been characterized. We have found that in response to ionizing radiation, p53-dependent p21 transactivation activity is present, albeit reduced, in SV40-immortalized cells and that this activity can be further reduced with either dominant negative p53 expression or higher SV40 Tag expression. Furthermore, overexpression of p53 in SV40-immortalized ataxia-telangiectasia (A-T) cells restores p53-dependent p21 induction to typical A-T levels. All SV40-immortalized cell lines exhibited an absence of G1 arrest. Moreover, all SV40-immortalized cell lines exhibited increased apoptosis relative to primary cells in response to ionizing radiation, suggesting that SV40 immortalization results in a unique phenotype with regard to DNA damage responses.