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.     

Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane

We have analyzed the plasma membrane association of the SV40 large tumor antigen (large T) in SV40-transformed BALB/c mouse tumor cells (mKSA). Isolated plasma membranes were subfractionated: treatment with the non-ionic detergent Nonidet P40 (NP40) resulted in a NP40-resistant plasma membrane lamina, which could be further extracted with the zwitterionic detergent Empigen BB. Analysis of the different plasma membrane fractions revealed that only about one third of large T associated with isolated plasma membranes could be solubilized with NP40. The residual plasma membrane-associated large T was tightly bound to the NP40-resistant lamina of the plasma membrane from which it was released by treatment with the zwitterionic detergent Empigen BB. Further evidence for a specific interaction of a distinct subclass of large T with the plasma membrane was provided by showing that only T associated with the NP40-resistant lamina of the plasma membrane contained covalently bound fatty acid. Neither nuclear large T nor large T in the NP40-soluble plasma membrane fraction could be labeled with [3H]palmitic acid. Our results indicate that an acylated subclass of large T interacts specifically with a structure of the plasma membrane, suggesting that it might be involved in a membrane-dependent biological function.     

Simian virus 40 T- and U-antigens: immunological characterization and localization in different nuclear subfractions of simian virus 40-transformed cells.

Simian virus 40 (SV40)-transformed cells and cells infected by the nondefective adenovirus 2(Ad2)-SV40 hybrid viruses Ad2+ND1 and Ad2+ND2 were analyzed for SV40 T- and U-antigens, respectively, using individual hamster SV40 tumor sera or serum for which U-antibodies were removd by absorption. These studies showed that (i) T- and U-antigens can be defined by separate classes of antigenic determinants and (ii) the U-antigenic determinants in SV40-transformed cells and in hybrid virus-infected cells are similar. The apparent discrepancy in the subcellular location of U-antigen in SV40-transformed cells (nuclear location) and in hybrid virus-infected cells (perinuclear location) as determined by immunofluorescence staining of methanol/acetone-fixed cells could be resolved by treating hybrid virus-infected cells with a hypotonic KCl solution before fixation. Upon this treatment hybrid virus-infected cells also showed nuclear U-antigen staining. The possibility of an association of T- and U-antigens with different nuclear subfractions in SV40-transformed cells was investigated. Detergent-cleaned nuclei of SV40-transformed cells were fractionated into nuclear matrices and a DNase-treated, high-salt nuclear extract. Analysis of the nuclear matrices by immunofluorescence microscopy with T+U+ and T+U- hamster SV40 tumor serum revealed that U-antigen remained associated with the nuclear matrices, whereas T-antigen could not be detected in this nuclear subfraction. T-antigen, however, could be immunoprecipitated from nuclear extracts of the SV40-transformed cells.     

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Antigenic and Immunogenic Characteristics of Nuclear and Membrane-Associated Simian Virus 40 Tumor Antigen

Antisera were prepared in syngeneic hosts against subcellular fractions of simian virus 40 (SV40)-transformed cells (MoalphaPM, MoalphaNuc), glutaraldehydefixed SV40-transformed cells (HaalphaH-50-G, MoalphaVLM-G), and electrophoretically purified denatured SV40 tumor antigen (T-ag) (RaalphaT). Immune sera were also collected from animals bearing tumors induced by SV40-transformed cells (HaalphaT, MoalphaT, HAF) and from SV40-immunized animals that had rejected a transplant of SV40-transformed cells (HaalphaS, MoalphaS). Immunological reagents prepared against cell surface (MoalphaPM, HaalphaS, MoalphaS, HaalphaH-50-G, MoalphaVLM-G) reacted exclusively with the surface of SV40-transformed cells by indirect immunofluorescence or protein A surface antigen radioimmunoassay. Immunological reagents prepared against the nuclear fraction (MoalphaNuc) or whole-cell determinants (HaalphaT, MoalphaT, HAF, RaalphaT) reacted with both the nuclei and surface of SV40-transformed or -infected cells. All reagents were capable of immunoprecipitating 96,000-molecular weight large T-ag from solubilized whole cell extracts of SV40-transformed cells. The exclusive surface reactivity of HaalphaS exhibited in immunofluorescence tests was abolished by solubilization of subcellular fractions, which then allowed immunoprecipitation of T-ag by HaalphaS from both nuclear and plasma membrane preparations. Specificity was established by the fact that all T-reactive reagents failed to react in serological tests against chemically transformed mouse cells, and sera from mice bearing transplants chemically transformed mouse cells (MoalphaDMBA-2) failed to react with SV40-transformed mouse or hamster cells. Reagents demonstrating positive surface immunofluorescence and protein A radioimmunoassay reactions against SV40-transformed cells were capable of blocking the surface binding of RaalphaT to SV40-transformed cells in a double-antibody surface antigen radioimmunoassay. This blocking ability demonstrated directly that a component specificity of each surface-reactive reagent is directed against SV40 T-ag. A model is presented which postulates that the differential detection of T-ag by the various serological reagents is a reflection of immunogenic and antigenic differences between T-ag polypeptides localized in nuclei and plasma membranes.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Association of viral and plasmid DNA with the nuclear matrix during productive infection

The association of simian virus 40 (SV40) DNA or plasmid DNA in subcellular fractions from either infected or transfected cells was examined. In lytically infected cells, approx. 25% of viral specific DNA during the infection cycle was retained in nuclei after washing with low ionic strength buffer and 1% Triton X-100. Viral replicating DNA found in the nuclear matrix was capable of performing limited DNA synthesis by the endogenous DNA polymerase in vitro. Viral DNA synthesized in vitro hybridized preferentially to SV40 Hind-III B and C fragments which are in proximity to the origin of replication. In plasmid-transfected COS-7 cells (SV40-transformed cells), the amount of plasmid DNA found in the nuclear matrix was related to its replication efficiency in cells. More than 80% of the plasmid DNA was tightly associated with subnuclear structures. Little or no plasmid DNA was found in the cytoplasmic fraction. The results suggest that, in extrachromosomal model systems, the association of DNA with nuclear matrix is important for the regulation of DNA replication.     

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.     

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.     

Only a minor fraction of plasma membrane-associated large T antigen in simian virus 40-transformed mouse tumor cells (mKSA) is exposed on the cell surface.

The bulk of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed cells localizes within the cell nucleus, while a minor fraction specifically associates with the plasma membrane (PM) and is exposed on the cell surface. PM-associated large T seems to span the lipid bilayer but, on the other hand, does not display typical features of a transmembrane protein. To further characterize the postulated transmembrane orientation of large T, we asked whether all large T molecules associated with the plasma membrane indeed are exposed on the cell surface. We compared the amount of cell surface-exposed large T, determined on living cells by a sensitive 3H-protein A-binding assay and by external immunoprecipitation, with that of total PM-associated large T extracted from isolated PM. We demonstrate that in mKSA cells (SV40-transformed BALB/c mouse fibroblasts), total PM-associated large T accounted for a substantial portion (ca. 2%) of total cellular large T. However, only 0.1 to 0.2% of it could be detected on the cell surface. Thus, only a minor fraction of PM-associated large T (less than 10%) is exposed on the surface of these cells. Interior PM-associated large T is stably associated with the plasma membrane, while the small fraction of surface-exposed large T is rapidly released from the cell surface.     

Cell density-dependent increase in chromatin-associated ADP-ribosyltransferase activity in simian virus 40-transformed cells.

ADP-ribosyltransferase activity associated with chromatin is two- to tenfold higher in simian virus 40 (SV40)-transformed cells than in untransformed cells. When confluent transformed cells were subcultured, their specific enzyme activity first decreased two- to fourfold and the rapidly increased during the logarithmic phase of growth. This increase ceased or slowed down when the cells entered the stationary phase. In contrast, the activity in the untransformed cells remained low throughout the growth cycle. In SV40tsA-transformed cells (ts = temperature sensitive), this density-dependent increase in the enzyme activity was observed when the cells were cultivated at the permissive temperature, whereas the activity remained low at the restrictive temperature. The enzyme activity did not increase during induction of cellular DNA synthesis in quiescent cells either by addition of fresh medium or by infection with SV40. The chromatin-associated enzyme activity extracted with 1 m NaCl was eluted together with almost all the DNA-binding proteins from a phosphocellulose column with 0.6 m NaCl. The enzyme activity in this fraction from transformed cells, measured with or without added DNA and histones, was higher than that in a similar fraction from untransformed cells, reflecting the difference in the original activities present in the nuclei of these cells. The chain lengths of poly(ADP-ribose) formed by chromatin from SV40-transformed and untransformed cells were not significantly different. These results suggest that the number of initiation sites for ADP-ribosylation is increased in the chromatin of SV40-transformed cells compared to that of untransformed cells.     

Antigenic structure of simian virus 40 large tumor antigen and association with cellular protein p53 on the surfaces of simian virus 40-infected and -transformed cells.

The antigenic structure of simian virus 40 (SV40) large tumor antigen (T-ag) in the plasma membranes of SV40-transformed mouse cells and SV40-infected monkey cells was characterized as a step toward defining possible biological function(s). Wild-type SV40, as well as a deletion mutant of SV40 (dl1263) which codes for a truncated T-ag with an altered carboxy terminus, was used to infect permissive cells. Members of a series of monoclonal antibodies directed against antigenic determinants on either the amino or the carboxy terminus of the T-ag polypeptide were able to precipitate surface T-ag (as well as nuclear T-ag) from both SV40-transformed and SV40-infected cells. Cellular protein p53 was coprecipitated with T-ag by all T-ag-reactive reagents from the surface and nucleus of SV40-transformed cells. In contrast, T-ag, but not T-ag-p53 complex, was recovered from the surface of SV40-infected cells. These results confirm that nuclear T-ag and surface T-ag are highly related molecules and that a complex of SV40 T-ag and p53 is present at the surface of SV40-transformed cells. Detectable levels of such a complex do not appear to be present on SV40-infected cells. Both the carboxy and amino termini of T-ag are exposed on the surfaces of SV40-transformed and -infected cells. The possible relevance of the presence of a T-ag-p53 complex on the surface of SV40-transformed cells and its absence from SV40-infected cells is considered.     

In vitro initiation of DNA replication in simian virus 40 chromosomes

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.     

Monoclonal antibodies against simian virus 40 nuclear large T tumour antigen: epitope mapping, papova virus cross-reaction and cell surface staining.

Thirty six cloned hybridomas have been isolated which produce monoclonal antibodies directed against simian virus 40 (SV40) large T tumour antigen. They have been shown to recognize at least six different epitopes along the T antigen polypeptide according to their reaction with the various truncated forms of T antigen expressed by adenovirus-SV 40 hybrid viruses. Sixteen antibodies cross-react with cells infected by the closely related human BK virus. Only two antibodies, PAb1604 and PAb1614, directed against different epitopes of the SV40 T antigen, cross-react with polyoma large T tumour antigen which has a more limited amino acid sequence homology. This cross-reaction is rarely seen with polyclonal antibodies. Monoclonal antibody PAb1620 gave nuclear immunofluorescence only with murine cells transformed by SV40 and was found to react with a complex of T-antigen and 53 000-dalton host-coded protein. All the monoclonal antibodies react with nuclear T antigen and all but four antibodies stained the surface of SV40-transformed cells. These were four of the five antibodies directed against the central third of the T antigen. Thus the monoclonal antibodies show that cell surface T antigen differs from nuclear T antigen, either in accessibility or structure.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Effects of the cellular p53 protein on Simian-virus-40-T-antigen-catalyzed DNA unwinding in vitro

It is known that large T antigen, the regulatory protein encoded by Simian virus 40 (SV40), forms tight complexes with the cellular p53 protein in SV40-transformed rodent cells. Using immunoaffinity procedures we have purified large T antigen and, in separate experiments, the cellular p53 protein. The two proteins formed complexes in vitro which bound well to double-stranded DNA fragments although in a sequence-unspecific manner. Free, uncomplexed T antigen readily converted double-stranded DNA into a single-stranded form whereas in-vitro-formed p53-T-antigen complexes were inactive in this reaction. We conclude that one function of p53 in SV40-transformed mouse cells could be the inhibition of the replication initiating activity of T antigen.     

Transformation of precrisis human cells by the simian virus 40 cytoplasmic-localization mutant pSVCT3 is accompanied by nuclear T antigen.

pSVCT3 is a cytoplasmic-localization mutant of simian virus 40 (SV40) isolated from the SV40 adenovirus 7 hybrid virus (PARA) and cloned into plasmid PBR. The large T antigen of pSVCT3 accumulates in the cytoplasm of infected monkey cells instead of being transported to the nucleus. The sole change in CT3 large T antigen is amino acid residue 128 (Lys----Asn). Transformation of precrisis rodent cells by pSVCT3 is negligible, whereas the frequency of transformation of established rodent cell lines by pSVCT3 is comparable to that of wild-type SV40. According to the model, in which transformation of precrisis cells involves the combined oncogenic action of both nuclear and cytoplasmic gene products, we predicted that pSVCT3 would localize in the cytoplasm of human cells and would therefore at most only partially and rarely transform precrisis human cells. We have found that pSVCT3 is able to transform precrisis human cells at high frequency. Furthermore, pSVCT3-transformed human precrisis cells relocalized T antigen to their nuclei. The relocalization of large T antigen was not dependent on cell growth. Wild-type and pSVCT3-transformed human cell lines both have about five copies of integrated SV40 DNA. SV40 virus-specific proteins, including the 100,000-molecular-weight super large T antigen, were expressed in pSVCT3-transformed human cells. Our results suggest that molecules in precrisis human cells, but not cells of other species, are able to complement the cytoplasmic-localization defect of the CT3 mutant large T antigen.     

Heterogeneity in distribution and content of p53 in SV40-transformed mouse fibroblasts

The high level and intranuclear location of the cellular phosphoprotein p53 are usually regarded as invariant features of SV40-transformed fibroblast lines. During the development of improved methods for immunocytochemical detection of p53 using SVA31 E7 mouse fibroblasts, we have observed unexpected and marked variations in its distribution. Cells grown on plastic coverslips were fixed in acetone and the content and distribution of p53 and SV40 large T-antigen analysed by an indirect immunoperoxidase procedure using monoclonal antibodies PAb122 and PAb 248 for p53, and PAb416 for large T. First, we observed in all cultures an apparent reversal of intracellular compartmentalization, with strong cytoplasmic and absent nuclear/chromatin positivity in mitotic cells and young daughter cells. More importantly, for a short period, between 18-24 h after trypsinization and cell passage, we observed a marked overall reduction in detectable nuclear p53 content in all cells with both PAb122 and PAb248 antibodies. The first observation also held for SV40 large T-antigen, the second only for p53. These variations have important practical implications for the immunocytochemical analysis of cellular content and intracellular compartmentalization of p53. The biological implications of our findings are also discussed.