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.     

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.     

Oligomerization of oncoprotein p53

Cellular phosphoprotein p53, which seems to be a multifunctional protein, may be assigned to different structural subclasses. Recently established immortalized or transformed cell lines that overexpress p53 allowed us to perform a detailed analysis of the quaternary structure of p53. By means of sucrose density gradient centrifugation, we found in simian virus 40-transformed cells that overexpress p53, in addition to high-molecular-weight T-p53 complexes, low-molecular-weight forms. The level of T-p53 complexes within simian virus 40-transformed cells seemed to be determined by the intracellular concentration of p53. However, the presence of uncomplexed T antigen and p53 indicated that an appropriate modification of at least one of the two proteins appears to be necessary for complex formation. Using different monoclonal antibodies that distinguish between (i) p53 associated with T antigen or heat shock proteins and (ii) p53 in apparently free form, we found p53 from transformed cells always in high-molecular-weight forms. p53 from normal and immortalized cells, however, was found mainly in low-molecular-weight forms. Pulse-labeling experiments revealed that oligomerization of p53 is a very rapid process. Monomeric forms of p53 which could be detected only by 2 min of pulse-labeling were rapidly converted to stable, high-molecular-weight oligomers. Furthermore, our data indicate a correlation between the occurrence of p53 in high-molecular-weight forms and the transformation state of the cell.     

Complex formation of simian virus 40 large T antigen with cellular protein p53

We investigated the formation of native complexes between simian virus 40 large T antigen and the cellular protein p53 (T-p53) by using simian virus 40 tsA58-transformed mouse fibroblasts (tsA58 F2b). We observed that newly synthesized p53 bound to all structural subclasses of large T antigen detectable on sucrose density gradients. This led to various intermediates of T-p53 complexes which converted within 2 h into typical mature aggregates. The final levels of stable T-p53 complexes seemed to be determined by p53 rather than by large T antigen.     

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.     

Isolation of a simian virus 40 T-antigen-positive, transformation-resistant cell line by indirect selection.

In an attempt to identify cellular genes that might be involved in simian virus 40 (SV40) transformation, we have set out to isolate cells which express T antigen but are not transformed. SV40 DNA and the herpes simplex virus thymidine kinase gene were cotransfected into tk- 3T3 fibroblasts. Of 72 colonies screened that were resistant to hypoxanthine-aminopterin-thymidine, 57 were T antigen positive as judged by immunofluorescence. One of these lines, A27, had a normal growth phenotype in monolayer overgrowth and soft agar assays. It contained intact SV40 sequences that could be rescued by fusion to permissive cells. This rescued virus was fully capable of transforming nonpermissive cells to the same extent as did wild-type virus. The A27 cells, however, were not transformable by infection with SV40 or by transfection of SV40 DNA. It is likely that these cells were altered in a cellular function required for the establishment of the transformed state.     

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.     

Origin binding by a 100,000-dalton super-T antigen from SVT2 cells.

The SVT2 line of simian virus 40-transformed mouse cells expresses little or no wild-type-size A protein (T antigen). Instead, a variant form is produced in these cells that is larger than normal-size A protein. This variant form has an Mr of 100,000 (100K super-T antigen) and is found primarily in complexes with the host-cell-coded p53 protein. Binding of the 100K super-T antigen to simian virus 40 origin region DNA was assayed by immunoprecipitation of super-T antigen-DNA complexes and then digestion with DNase I. DNA sequences associated with super-T antigen were protected from digestion and retained in the immune complex, while unprotected sequences were digested and released. The 100K super-T antigen efficiently protects DNA sequences in the previously defined regions I and II (P. Tegtmeyer, B. A. Lewton, A. L. DeLucia, V. G. Wilson, and K. Ryder, J. Virol. 46:151-161, 1983). Within region II (the origin of replication), the pattern and size of protected fragments are identical for super-T antigen and purified wild-type A protein. Thus, even though super-T antigen is larger than wild-type A protein, both must bind with the same alignment on origin DNA. Furthermore, complexes between the host-cell-coded p53 protein and the 100K super-T antigen also retain the ability to bind in regions I and II.     

A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells.

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA.     

The amino-terminal functions of the simian virus 40 large T antigen are required to overcome wild-type p53-mediated growth arrest of cells.

High levels of the p53 tumor suppressor protein can block progression through the cell cycle. A model system for the study of the mechanism of action of wild-type p53 is a cell line (T64-7B) derived from rat embryo fibroblasts transformed by activated ras and a temperature-sensitive murine p53 gene. At 37 to 39 degrees C, the murine p53 protein is in a mutant conformation and the cells actively divide, whereas at 32 degrees C, the protein has a wild-type conformation and the cells arrest in the G1 phase of the cell cycle. Wild-type simian virus 40 large T antigen and a variety of T-antigen mutants were assayed for the ability to bypass the cell cycle block effected by the wild-type p53 protein to induce colony formation at 32 degrees C. The results indicate that two functions within the amino terminus of T antigen are essential to induce cell growth: (i) the ability to bind to the retinoblastoma protein, Rb, and (ii) the presence of a domain in the first exon that appears to interact with the cellular protein, p300. Thus, the cell cycle arrest triggered by wild-type p53 may be overcome by formation of a T-antigen complex with Rb, p300, or both that could then function to either remove p53-mediated negative growth regulatory signals or promote a positive cell growth signal. Surprisingly, T antigen-p53 complexes are not required to overcome the temperature-sensitive p53 block to the cell cycle in these cells. These data suggest that simian virus 40 T antigen associated with Rb, p300, or both proteins can communicate in a cell with the functions of the wild-type p53 protein.     

Intracellular location and kinetics of complex formation between simian virus 40 T antigen and cellular protein p53

The intracellular location and kinetics at which the simian virus 40 T antigen and the cellular protein p53 associate with one another were determined for simian virus 40-transformed mouse (215) and rat (14B) cells. Cells were labeled under pulse-chase conditions and fractionated into nuclear and cytoplasmic components, and the proteins were immunoprecipitated with monoclonal antibodies (pAb 416, 101, and 122). We found that newly made T antigen and p53 migrated to the nucleus of these cells independently; that is, in uncomplexed form. Newly made p53 was transported to the nucleus more rapidly than T antigen in both cell lines and formed a complex with a mature form of T antigen recognizable by pAb 101. This association was very rapid in both cell lines (t 1/2, 5 to 15 min). In contrast, the time course of complex formation between newly made T antigen and the p53 in the nucleus varied with the ratio of T antigen to p53 of the cell line studied. In 215 cells, where the ratio was 3.6, the kinetics were quite slow (t 1/2, 30 min), whereas in 14B cells, where the ratio was 1.7, they were quite rapid (t 1/2, 5 min). We suggest that a competition between newly made and uncomplexed T antigen for the p53 in the nucleus is the major determinant of the rate of complex formation for newly made T antigen. Our studies indicate that this macromolecular interaction is extremely dynamic.     

Virus-derived platforms for visualizing protein associations inside cells

Protein-protein associations are vital to cellular functions. Here we describe a helpful new method to demonstrate protein-protein associations inside cells based on the capacity of orthoreovirus protein muNS to form large cytoplasmic inclusions, easily visualized by light microscopy, and to recruit other proteins to these structures in a specific manner. We introduce this technology by the identification of a sixth orthoreovirus protein, RNA-dependent RNA polymerase lambda3, that was recruited to the structures through an association with muNS. We then established the broader utility of this technology by using a truncated, fluorescently tagged form of muNS as a fusion platform to present the mammalian tumor suppressor p53, which strongly recruited its known interactor simian virus 40 large T antigen to the muNS-derived structures. In both examples, we further localized a region of the recruited protein that is key to its recruitment. Using either endogenous p53 or a second fluorescently tagged fusion of p53 with the rotavirus NSP5 protein, we demonstrated p53 oligomerization as well as p53 association with another of its cellular interaction partners, the CREB-binding proteins, within the inclusions. Furthermore using the p53-fused fluorescent muNS platform in conjunction with three-color microscopy, we identified a ternary complex comprising p53, simian virus 40 large T antigen, and retinoblastoma protein. The new method is technically simple, uses commonly available resources, and is adaptable to high throughput formats.     

Dynamic nature of the association of large tumor antigen and p53 cellular protein with the surfaces of simian virus 40-transformed cells

A molecular complex of simian virus 40 large tumor antigen (T-Ag) and p53 cellular protein is present on the surface of simian virus 40-transformed mouse cells. The stability of the association of the two proteins with the cell surface was characterized. Cells were either surface iodinated by the lactoperoxidase technique or metabolically labeled with [35S]methionine, and surface antigens were detected by differential immunoprecipitation with specific antibodies immediately after labeling or after incubation at 37 degrees C. A rapid, concomitant disappearance of T-Ag and p53 from the cell surface was observed. The half-life of iodinated surface T-Ag was less than 30 min, whereas that of [35S]methionine-labeled surface T-Ag was 1 to 2 h. Although T-Ag and p53 were rapidly lost, both were also rapidly replaced on the cell surface, since newly exposed molecules could be detected when cells were reiodinated after a 2-h chase period. Control experiments established that the loss of the surface molecules was not induced by the iodination reaction. The appearance of surface T-Ag was prevented when cellular protein synthesis was inhibited with cycloheximide. The disappearance and replacement of T-Ag and p53 appeared to be energy-independent processes, as neither was inhibited by sodium azide or 2,4-dinitrophenol. Incubation of iodinated cells at 4 degrees C did block the loss of T-Ag and p53. These observations suggest that T-Ag and p53 are coordinately turned over in the plasma membrane. The nature of the association of the T-Ag-p53 complex with the cell surface can best be described as highly dynamic.     

Mutation of the serine 312 phosphorylation site does not alter the ability of mouse p53 to inhibit simian virus 40 DNA replication in vivo.

Two mutations were introduced into the wild-type mouse p53 gene by oligonucleotide-directed mutagenesis. These mutations substituted alanine or aspartic acid for serine at position 312, which is constitutively phosphorylated. Phosphopeptide mapping of the mutant proteins, expressed in COS cells, confirmed the loss of phosphorylation at position 312. There were no changes in the ability of the mutant p53s to express the conformation-dependent epitope for monoclonal antibody PAb246 or to participate in complexes with the simian virus 40 (SV40) large T antigen. Replication of a plasmid containing the SV40 origin of replication was inhibited in COS cells by wild-type p53 and both of the phosphorylation site mutants with equal efficiency. A transforming mutant of p53, encoding valine at position 135, did not inhibit SV40 DNA replication in COS cells.     

Interactions between SV40 T antigen and DNA polymerase alpha

Simian virus 40 large T antigen is the only viral protein required for SV40 DNA synthesis in vivo and in vitro. This complex protein recruits the cellular DNA replication apparatus to the SV40 origin and provides a good model for the initiation of cellular DNA replication. The interaction between SV40 large T antigen (TAg) and DNA polymerase alpha has been shown previously to be inhibited by murine p53, the nuclear protein product of a cellular anti-oncogene. The murine p53 protein will inhibit SV40 replication both in vivo and in vitro. Using monoclonal antibodies to TAg, p53, and polymerase alpha, we developed immunoassays to measure the complexes formed between TAg and polymerase alpha and between TAg and p53. The assays allowed us to detect the TAg-polymerase alpha and TAg-p53 complexes in lytically infected and transformed cells. The amount of TAg complexed to p53 was far lower in infected cells than in transformed cells. We used a large range of monoclonal antibodies to different sites on T antigen and found that antibodies that inhibited the formation of the TAg-polymerase alpha complex also inhibited the formation of the TAg-p53 complex. Finally, we found that the tsA58 and 5080 point mutations in TAg, previously shown to inhibit the binding of TAg to p53, also inhibit its binding to polymerase alpha. Together these results emphasize the specificity and functional importance of the TAg-polymerase alpha complex. The disruption of this interaction by the cellular anti-oncogene p53 provides an interesting model for the normal action of p53 and the effects of its removal on the regulation of cellular DNA synthesis.     

Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells.

Recombinant baculoviruses were constructed which express simian virus 40 large T antigen (SVT-Ag) or murine p53 to high levels in infected insect cells. Characterization of the expressed proteins revealed that they display many properties of the corresponding mammalian-derived proteins. Both proteins are of wild-type size, localize to the nucleus, are recognized by several SVT-Ag- or p53-specific monoclonal antibodies, and are phosphorylated in this system. Complexes are formed between baculovirus-derived SVT-Ag and p53 after coinfection of insect cells with both recombinant viruses. After infection of insect cells with either virus individually, each protein can self-associate to form a variety of oligomeric species. Pulse-chase experiments indicated that both SVT-Ag and p53 are highly stable in insect cells, even in the absence of complex formation.     

Stable T-p53 complexes are not required for replication of simian virus 40 in culture or for enhanced phosphorylation of T antigen and p53.

We generated a number of simian virus 40 (SV40) mutants with single amino acid substitutions in T antigen between residues 388 and 411. All but one mutant (398LV) replicated like wild-type SV40 and gave rise to normal-size plaques. Three different mutations at residue 402 (Asp to Glu, Asn, or His) totally prevented the formation of stable complexes with the cellular protein p53 in monkey cells but had no effect on virus replication. Only one other mutation in this region, involving residue 401 (Met to Thr), slightly inhibited the formation of T-monkey p53 complexes. The three mutant T antigens with substitutions at residue 402 also formed no stable complexes with human p53 but generated low levels of complexes with mouse p53. These results indicate that residue 402 is critical for binding to monkey and human p53 proteins and is important for binding to mouse p53. We suggest that it is one of several points of contact. In cells infected with any one of the three residue 402 mutant viruses. T antigen and p53 became increasingly phosphorylated, as they were in cells infected with wild-type virus. Our data therefore show that stable T-p53 complexes are not required for replication of SV40 in culture or for enhanced phosphorylation of either protein.     

The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen.

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.     

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.