Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.     

Simian virus 40 large tumor antigen on replicating viral chromatin: tight binding and localization on the viral genome

Pulse-labeled simian virus 40 (SV40) chromatin as well as uniformly labeled viral chromatin are immunoprecipitable by an SV40-specific tumor antiserum and therefore contain bound tumor antigen (T antigen). Single-stranded calf thymus DNA, immobilized on cellulose, competes effectively for T antigen binding with uniformly labeled nonreplicating, but not with pulse-labeled replicating, chromatin. Furthermore, T antigen dissociates in 0.5 M NaCl from nonreplicating chromatin and from purified SV40 DNA, whereas most T antigen remains associated with replicating chromatin even in the presence of 1.2 to 1.5 M NaCl. We used filtration through DNA-cellulose columns and treatment with high salt to prepare pulse-labeled immunoreactive viral chromatin. The viral DNA was digested before, and in other experiments after, immunoprecipitation with the restriction endonuclease HindIII. We found that SV40 DNA sequences, most probably representing the entire genome, remain in the immunoprecipitate after HindIII digestion, indicating an association of T antigen with origin-distal sections of replicating viral DNA. The results suggest that T antigen in replicating chromatin may be bound to regions close to replicating points. We performed control experiments with in vitro-formed complexes of T antigen and SV40 DNA. When these complexes were immunoprecipitated and HindIII digested we found, in agreement with previous studies, that only the origin containing the HindIII C fragment carried bound T antigen.     

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.     

Immunoprecipitation of SV40 replicating minichromosomes complexed with bacteriophage T4 gene 32 protein.

Simian Virus 40 (SV40) DNA replication is a useful model to study eukaryotic cell DNA replication because it encodes only one replication protein and its genome has a nucleoprotein structure ('minichromosome') indistinguishable from cellular chromatin. Late after infection SV40 replicating DNA molecules represent about 5% of total viral minichromosomes. Since gene 32 protein (P32) from bacteriophage T4 interacts with single-stranded DNA and SV40 replication complexes are expected to contain single-stranded regions at the replication forks, we asked whether P32 might be used to isolate replicating SV40 minichromosomes. When nuclear extracts from SV40 infected cells were treated sequentially with P32 and anti-P32 antibodies, pulse-labeled minichromosomes were selectively immunoprecipitated. Agarose gel electrophoresis analysis confirmed that immunoprecipitated material corresponded to SV40 replicative intermediates. Protein analysis of the pelleted material revealed several proteins of viral and cellular origin. Among them, T antigen and histones were found to be complexed with at least other three proteins from cellular origin, to the replicative complexes. Additionally, anti-P32 antibodies were able to detect three cellular proteins of approximately 70, 32 and 13 kDa in western blots. These proteins could correspond to those found as part of an eukaryotic multisubunit single-stranded DNA binding protein. The use of P32 and anti-P32 antibodies thus allows the separation of replicating from mature SV40 minichromosomes and can constitute a novel method to enrich and to study replicative active chromatin.     

Time-dependent maturation of the simian virus 40 large T antigen-p53 complex studied by using monoclonal antibodies.

Newly synthesized simian virus 40 large tumor antigen (T Ag) slowly forms a stable complex with the host tumor antigen, "p53." By the use of immunological and temporal separations and inhibition of aggregation and processing by A locus mutation, we have distinguished specific steps in the reaction sequence leading to formation of the rapidly sedimenting oligomeric complex. The monoclonal antibody PAb101 bound only a fraction of the total soluble pulse-labeled T Ag bound by antitumor serum. After a chase, all T Ag had matured to the form recognized by PAb101. All p53 in the mouse line SVA31E7 was precipitated by the T Ag-specific monoclonal antibody PAb101, even after a short pulse, and is therefore entirely bound to mature T Ag. The p53-specific monoclonal antibody PAb122 precipitates nearly all of the mature T Ag recognized by PAb101, except A locus mutant T Ag, synthesized at the nonpermissive temperature. A locus mutation inhibited entry of newly synthesized T Ag into the oligomeric greater than 28S complex of T Ag and p53.     

Accessibility to topoisomerases I and II regulates the replication efficiency of simian virus 40 minichromosomes.

We determined the effects of chromatin structure on template accessibility to replication factors and used three different templates as substrates for simian virus 40 (SV40) DNA replication in vitro: native and salt-treated SV40 minichromosomes and protein-free SV40 DNA. Native minichromosomes contain histone H1 and numerous nonhistone proteins in addition to the core histones, whereas salt-treated minichromosomes carry essentially only core histones. We reasoned that the less densely packed salt-treated minichromosomes should be more effective replication templates due to their more extended configuration. However, contrary to this expectation, we found that native minichromosomes replicated with significantly higher efficiency than salt-treated minichromosomes, while protein-free DNA was most active as a replication template. The higher replication efficiency of native minichromosomes was due to two activities bound to the chromatin, which were identified as DNA topoisomerases I and II. By using chromatin substrates of different general configurations, we also showed that the overall chromatin structure determines accessibility to topoisomerases I and II and thereby the efficiency of replicative chain elongation.     

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.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Replication of SV40 minichromosomes in vitro

We operationally define two forms of SV40 minichromosomes, a 75S-form, prepared at low salt concentration, referred to as native minichromosomes, and a 50S-form, obtained after treatment with 0.5M potassium acetate, the salt-treated minichromosomes. Both preparations of minichromosomes serve well as templates for replication in vitro. Their respective replication products are strikingly different: replicated native minichromosomes contain a densely packed array of the maximal number of nucleosomes whereas replicated salt-treated minichromosomes carry, on average, half of the maximal number. We conclude that in both cases parental nucleosomes are transferred to progeny DNA, and, in addition, that an assembly of new nucleosomes occurs during the replication of native minichromosomes. This is apparently due to the presence of a nucleosome assembly factor as a constituent of native minichromosomes that dissociates upon treatment with salt. We further show that preparations of minichromosomes usually contain significant amounts of copurifying hnRNP particles and SV40 virion precursor particles. However, these structures do not detectably affect the replication and the chromatin assembly reactions.     

Monoclonal antibodies as probes for a function of large T antigen during the elongation process of simian virus 40 DNA replication.

Various monoclonal antibodies specific for simian virus 40 large tumor antigen (T antigen) inhibit the elongation process of viral DNA replication in an in vitro system. The results provide strong evidence for a function intrinsic to T antigen during ongoing replicative-chain elongation. The antibody inhibition studies were further used to establish a correlation between the known biochemical activities of T antigen and its function during the elongation phase. The data demonstrate that, in addition to DNA binding and ATPase, a third function of T antigen is required for replicative chain elongation. This function is most probably related to the recently described DNA helicase activity of T antigen. This conclusion is based on the following results: aphidicolin treatment of actively replicating simian virus 40 minichromosomes causes a partial uncoupling of parental DNA strand separation and DNA synthesis; the strand separation reaction is blocked by the same monoclonal antibodies which strongly inhibit the elongation process. DNA helicase activity of isolated T antigen is equally well inhibited by the same set of monoclonal antibodies that affect minichromosome replication in vitro.     

A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.

Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.     

Preferential association of newly synthesized histones with replicating SV40 DNA.

The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA.     

Free and viral chromosome-bound simian virus 40 T antigen: changes in reactivity of specific antigenic determinants during lytic infection.

Simian virus 40 (SV40) large T antigen (TAg), both free and bound to mature 70S and replicating 90S SV40 chromosomes, was prepared from lytically infected cells. The relative reactivity of the different TAg-containing fractions toward 10 monoclonal antibodies directed against three different regions in SV40 TAg and toward an antibody against the p53 protein was measured. The results for free TAg indicated that all of the determinants in both the amino-terminal (0.65 to 0.62 map units) and carboxy-terminal (0.28 to 0.17 map units) regions were highly reactive, whereas all five determinants located between 0.43 and 0.28 map units in the midregion of TAg were poorly reactive. For TAg bound to replicating chromosomes, all but one of the antibodies specific for TAg were highly reactive. Thus, antigenic sites in the middle of TAg, the region important for nucleotide binding and ATP hydrolysis (an activity required for viral DNA replication), were more accessible in TAg-replicating DNA complexes. As replicating molecules matured into 70S chromosomes, three or more determinants at different locations in TAg bound to chromatin became two- to fivefold less reactive, indicating other changes in TAg structure. Overall, at least nine different antigenic determinants in the TAg molecule were identified. Anti-p53 was reactive with about 10% of the free TAg and the same amount of SV40 chromosomes of all ages, suggesting that p53-TAg complexes are not preferentially associated with either replicating or mature viral chromosomes. When the reactivity of both mature and replicating labeled SV40 chromosomes with polyclonal tumor anti-T was measured as a function of time after purification, TAg bound to mature chromosomes appeared to dissociate about fourfold faster than that bound to replicating chromosomes. The relative amount of TAg in various subcellular fractions was measured by an enzyme-linked immunosorbent assay. Approximately 1.3% of the total TAg was estimated to be associated with SV40 chromosomes in infected cells. Based on the relative amounts of TAg and viral DNA in the 70S and 90S fractions, replicating chromosome-TAg complexes were estimated to bind 4.8 times more TAg per DNA molecule, on the average, than mature chromosome-TAg complexes. Together, these results are consistent with major differences in TAg structure when free and associated with replicating and nonreplicating SV40 chromosomes.     

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.     

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.     

Minichromosome of simian virus 40: presence of histone HI.

In contrast to conclusions of previous studies /I-3/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. In addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.     

Re-oxygenation of hypoxic simian virus 40 (SV40)-infected CV1 cells causes distinct changes of SV40 minichromosome-associated replication proteins.

Hypoxia interrupts the initiation of simian virus 40 (SV40) replication in vivo at a stage situated before unwinding of the origin region. After re-oxygenation, unwinding followed by a synchronous round of viral replication takes place. To further characterize the hypoxia-induced inhibition of unwinding, we analysed the binding of several replication proteins to the viral minichromosome before and after re-oxygenation. T antigen, the 34-kDa subunit of replication protein A (RPA), topoisomerase I, the 48-kDa subunit of primase, the 125-kDa subunit of polymerase delta, and the 37-kDa subunit of replication factor C (RFC) were present at the viral chromatin already under hypoxia. The 70-kDa subunit of RPA, the 180-kDa subunit of polymerase alpha, and proliferating cell nuclear antigen (PCNA) were barely detectable at the SV40 chromatin under hypoxia and significantly increased after re-oxygenation. Immunoprecipitation of minichromosomes with T antigen-specific antibody and subsequent digestion with micrococcus nuclease revealed that most of the minichromosome-bound T antigen was associated with the viral origin in hypoxic and in re-oxygenated cells. T antigen-catalysed unwinding of the SV40 origin occurred, however, only after re-oxygenation as indicated by (a) increased sensitivity of re-oxygenated minichromosomes against digestion with single-stranded DNA-specific nuclease P1; (b) stabilization of RPA-34 binding at the SV40 minichromosome; and (c) additional phosphorylations of RPA-34 after re-oxygenation, probably catalysed by DNA-dependent protein kinase. The results presented suggest that the subunits of the proteins necessary for unwinding, primer synthesis and primer elongation first assemble at the SV40 origin in form of stable, active complexes directly before they start to work.     

DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.