Subclasses of simian-virus-40 large tumor antigen. Partial purification and DNA-binding properties of two subclasses of tumor antigen from productively infected cells

Two major subclasses of simian virus 40 tumor antigen were prepared from productively infected monkey cells. These subclasses can be distinguished by their sedimentation properties: one tumor antigen form sediments at 5-6S and the other at 14-16S. The DNA-binding properties of these subclasses were investigated by two different experimental procedures. In the first procedure, the DNA binding of subclasses of crude tumor antigen, separated by zone velocity sedimentation, were assayed by immunoprecipitation of the DNA-protein complexes. In the second procedure, the two tumor antigen forms were partially purified by column chromatography and DNA binding was tested in a filter binding assay. Both procedures gave comparable results. (a) The 5-6-S and the 14-16-S tumor antigen bound specifically to a DNA restriction fragment containing the viral genome control regions. (b) At low salt concentrations, both subclasses bound to specific and to nonspecific DNA sequences; competition experiments in the presence of nonspecific DNA showed, however, that the affinity of both tumor antigen forms for the viral genome control region was at least 10-fold higher than their affinity for nonspecific DNA sequences. (c) The binding of the 5-6-S subclass to viral control region DNA was optimal at 60-80 mM NaCl while specific DNA binding of the 14-16-S form was optimal at 150-200 mM NaCl; however, binding of the 14-16-S form to nonspecific DNA sequences was also more resistant to high salt concentrations than that of the 5-6S form. (d) Both tumor antigen forms bound well to specific and to nonspecific DNA at pH 6-6.5; with increasing pH values, binding to nonspecific DNA decreased while binding to specific DNA reached an optimum at pH 7-7.5. Binding of the 14-16-S form to viral origin DNA was more resistant to pH values above 7.5 than binding of the 5-6-S form.     

Quantitative analysis of the binding of simian virus 40 large T antigen to DNA

SV40 large T antigen (T-ag) is a multifunctional protein that successively binds to 5'-GAGGC-3' sequences in the viral origin of replication, melts the origin, unwinds DNA ahead of the replication fork, and interacts with host DNA replication factors to promote replication of the simian virus 40 genome. The transition of T-ag from a sequence-specific binding protein to a nonspecific helicase involves its assembly into a double hexamer whose formation is likely dictated by the propensity of T-ag to oligomerize and its relative affinities for the origin as well as for nonspecific double- and single-stranded DNA. In this study, we used a sensitive assay based on fluorescence anisotropy to measure the affinities of wild-type and mutant forms of the T-ag origin-binding domain (OBD), and of a larger fragment containing the N-terminal domain (N260), for different DNA substrates. We report that the N-terminal domain does not contribute to binding affinity but reduces the propensity of the OBD to self-associate. We found that the OBD binds with different affinities to its four sites in the origin and determined a consensus binding site by systematic mutagenesis of the 5'-GAGGC-3' sequence and of the residue downstream of it, which also contributes to affinity. Interestingly, the OBD also binds to single-stranded DNA with an approximately 10-fold higher affinity than to nonspecific duplex DNA and in a mutually exclusive manner. Finally, we provide evidence that the sequence specificity of full-length T-ag is lower than that of the OBD. These results provide a quantitative basis onto which to anchor our understanding of the interaction of T-ag with the origin and its assembly into a double hexamer.     

Mutational analysis of simian virus 40 large T antigen DNA binding sites.

We have tested the effects of various mutations within SV40 T antigen DNA recognition sites I and II on specific T antigen binding using the DNase footprint technique. In addition, the replication of plasmid DNA templates carrying these T antigen binding site mutations was monitored by Southern analysis of transfected DNA in COS cells. Deletion mapping of site I sequences defined a central core of approximately 18 bp that is both necessary and sufficient for T antigen recognition; this region contains the site I contact nucleotides that were previously mapped using methylation-interference and methylation-protection experiments. A similar deletion analysis delineated sequences that impart specificity of binding to site II. We find that T antigen is capable of specific recognition of site II in the absence of site I sequences, indicating that binding to site II in vitro is not dependent on binding of T antigen at site I. Site II binding was not diminished by small deletion or substitution mutations that perturb the 27-bp palindrome central to binding site II, whereas extensive substitution of site II sequences completely eliminated specific site II binding. Analysis of the replication in COS7 cells of plasmids that contain these mutant origins revealed that sequences both at the late side of binding site I and within the site II palindrome are crucial for viral DNA replication, but are not involved in binding T antigen.     

Dose-response of two cellular proliferation phenotypes produced by simian virus 40 large T antigen

Abstract. A set of cell lines was constructed by infection of established murine fibroblasts with recombinant retroviruses encoding the simian virus 40 large T antigen (Tag) gene. By immunofluorescence flow cytometry, it was shown that these cell lines expressed Tag over at least a 20-fold concentration range. Using these cells, the dose-response relationship between Tag concentration and a phenotype detected by flow cytometry that measures the rate at which proliferating cells transit the cell cycle (i.e. cell-cycling phenotype) was determined. This relationship between Tag concentration and phenotype was not linear. Instead, the cell-cycling phenotype became saturated at relatively low Tag concentrations, i.e. a further increase in Tag concentration did not change the phenotype. The dose-response relationship between Tag and a second phenotype, colony formation in soft agar, was also determined. Colony formation in soft agar is a measurement of cell transformation. In contrast to the cell-cycling phenotype, transformation was linearly related to Tag over the entire 20-fold Tag concentration range. This phenotype did not saturate at high Tag concentrations. Therefore, the dose-response relationship between Tag concentration and the cell-cycling phenotype was different from that between Tag concentration and cellular transformation. Since the Tag gene is comprised of multiple genetic domains that independently affect cellular proliferation, one possibility is that the differences in dose-response of the two phenotypes indicate that different genetic domains of the gene are necessary for production of each phenotype.     

Discrete regions of simian virus 40 large T antigen are required for nonspecific and viral origin-specific DNA binding.

The nondefective adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses, Ad2+ND2 and Ad2+ND4, have been used to determine which regions of the SV40 genome coding for the large tumor (T) antigen are involved in specific and nonspecific DNA binding. Ad2+ND2 encodes 45,000 M4 (45K) and 56,000 Mr (56K) T antigen-related polypeptides. The 45K polypeptide did not bind to DNA, but the 56K polypeptide bound nonspecifically to calf thymus DNA, Ad2+ND4 encodes 50,000 Mr (60K), 66,000 Mr (66K), 70,000 Mr (70K), 74,000 Mr (74K), and 90,000 Mr (90K) T antigen-related polypeptides, all of which bound nonspecifically to calf thymus DNA. However, in more stringent assays, where tight binding to viral origin sequences was tested, only the 90K protein specified by Ad2A+ND4 showed specific high affinity for sequences at the viral origin of replication. From these results and previously published experiments describing the SV40 DNA integrated into these hybrid viruses, it was concluded that SV40 early gene sequences located between 0.39 and 0.44 SV40 map units contribute to nonspecific DNA binding, whereas sequences located between 0.50 and 0.63 SV40 map units are necessary for specific binding to the viral origin of replication.     

Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells.

Two cellular proteins are found to be complexed with simian virus 40 small-t antigen in cellular extracts. The complex is a relatively unstable but dynamic one which can dissociate and reform in extracts. In extracts of permissive monkey kidney cells, the small-t antigen appeared to be present in excess, whereas the cellular proteins were nearly entirely committed to the complex in permissive monkey kidney cells.     

New properties of simian virus 40 large T antigen

An 8,000-molecular-weight (8K) T antigen was found in all cells transformed by simian virus 40. The 8K T antigen was weakly labeled in vivo with [35S]methionine or 32Pi. A deletion in the human papovavirus BK genome, in the region coding for the carboxy-terminal end of the large T antigen, reduced the size of the 8K T antigen. The last 80 amino acids of the large T antigen include the sequence Asp-Asp-Asp-Asp unique to the activation peptide of trypsinogen. Large T antigen bound diisopropyl fluorophosphate and was retained by D-phenylalanine coupled to Sepharose beads, an affinity adsorbent that can retain chymotrypsin. The large T antigen and the recA protein of Escherichia coli, a known protease, have several properties in common as well as several similar sequences. Antibodies against large T antigen interacted with native recA protein.     

T antigen origin-binding domain of simian virus 40: determinants of specific DNA binding

To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator protein of the SV40 virus, and cognate and noncognate DNA oligomers. The results reveal two structural effects associated with "origin-specific" binding that are absent in nonspecific DNA binding. The first is the formation of a hydrogen bond (H-bond) involving His 203, a residue that genetic studies have previously identified as crucial to both specific and nonspecific DNA binding in full-length T antigen. In free T-ag-obd, the side chain of His 203 has a pK(a) value of approximately 5, titrating to the N(epsilon)(1)H tautomer at neutral pH (Sudmeier, J. L., et al. (1996) J. Magn. Reson., Ser. B 113, 236-247). In complexes with origin DNA, His 203 N(delta)(1) becomes protonated and remains nontitrating as the imidazolium cation at all pH values from 4 to 8. The H-bonded N(delta1)H resonates at 15.9 ppm, an unusually large N-H proton chemical shift, of a magnitude previously observed only in the catalytic triad of serine proteases at low pH. The formation of this H-bond requires the middle G/C base pair of the recognition pentanucleotide, GAGGC. The second structural effect is a selective distortion of the A/T base pair characterized by a large (0.6 ppm) upfield chemical-shift change of its Watson-Crick proton, while nearby H-bonded protons remain relatively unaffected. The results indicate that T antigen, like many other DNA-binding proteins, may employ "catalytic" or "transition-state-like" interactions in binding its cognate DNA (Jen-Jacobson, L. (1997) Biopolymers 44, 153-180), which may be the solution to the well-known paradox between the relatively modest DNA-binding specificity exhibited by initiator proteins and the high specificity of initiation.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

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.     

Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA

The large T antigen (T-ag) protein binds to and activates DNA replication from the origin of DNA replication (ori) in simian virus 40 (SV40). Here, we determined the crystal structures of the T-ag origin-binding domain (OBD) in apo form, and bound to either a 17 bp palindrome (sites 1 and 3) or a 23 bp ori DNA palindrome comprising all four GAGGC binding sites for OBD. The T-ag OBDs were shown to interact with the DNA through a loop comprising Ser147-Thr155 (A1 loop), a combination of a DNA-binding helix and loop (His203-Asn210), and Asn227. The A1 loop traveled back-and-forth along the major groove and accounted for most of the sequence-determining contacts with the DNA. Unexpectedly, in both T-ag-DNA structures, the T-ag OBDs bound DNA independently and did not make direct protein-protein contacts. The T-ag OBD was also captured bound to a non-consensus site ATGGC even in the presence of its canonical site GAGGC. Our observations taken together with the known biochemical and structural features of the T-ag-origin interaction suggest a model for origin unwinding.     

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.     

The binding of simian virus 40 large T antigen to the polyphosphate backbone of nucleic acids

Simian virus 40 (SV40) large tumor antigen (T antigen), a phosphoprotein found in nuclei of SV40-infected and -transformed cells, binds nonspecifically to DNA. To study this mechanism the binding properties of T antigen to double-stranded (ds) and single-stranded (ss) DNA-cellulose as well as to phosphocellulose were compared. After incubation of [35S] methionine or [3H] leucine/[32 P] phosphate radioactively-labeled cell extracts at different pH values (6.0, 7.3, 9.0) with DNA- or phosphocellulose, bound and unbound species of T antigen were purified and analyzed by SDS-polyacrylamide gel electrophoresis for both the yield and the possible correlation with protein phosphorylation. T antigens bound with comparable affinities to ds- and ss-DNA-cellulose and phosphocellulose. These results suggest the binding of T antigen to the polyphosphate backbone of DNA as a molecular mechanism for its nonspecific binding. The evidence for this observation was supported by blocking the binding of T antigen to DNA-cellulose by divalent cations (Ca2+, Mg2+). 3H/32P ratios of T antigen obtained by double-labeling cells for various times imply that higher phosphorylated forms of T antigen bound more strongly to ds- and ss-DNA as well as to phosphocellulose. Thus, in the presence of cellular proteins and other components the binding activity of T antigen to the polyphosphate backbone of DNA seems to be positively correlated with its phosphorylation. These observations are consistent with the hypothesis that the binding affinities of SV40 T antigen to host cell DNA may be regulated by its phosphorylation.     

Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity.

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)] appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is involved in nonspecific DNA binding, we found that up to 96% of this latter activity was specifically inhibited in wild-type T antigen by several monoclonal antibodies which collectively bind to the region between residues 269 and 522. In order to investigate the relationship between the origin-binding domain and the second region, we performed origin-specific DNA binding assays with increasing amounts of calf thymus DNA as competitor. The results suggest that this second region is not an independent nonspecific DNA binding domain. Rather, it most likely cooperates with the origin-binding domain to give rise to wild-type levels of nonspecific DNA binding. Our results further suggest that most of the nonspecific binding to double-stranded DNA is involved in a function other than direct recognition and binding to the pentanucleotides at the replication origin on simian virus 40 DNA.     

Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication

We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication.     

ATP enhances the binding of simian virus 40 large T antigen to the origin of replication.

Simian virus 40 large T antigen initiates DNA replication by binding to the origin of replication. We examined the binding of T antigen to origin regions I, II, and III under conditions designed for efficient in vitro replication functions. We found that 4 mM ATP enhanced the binding of T antigen to regions I and II of the origin DNA by 4- to 20-fold. DNase-footprinting and fragment assays showed that ATP extended the DNase protection domain of T antigen bound to region II by 5 to 10 base pairs at both ends of the core origin of replication. This alteration suggests a change in the conformation of T antigen, bound DNA, or both.     

Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen.

We isolated 16 new monoclonal antibodies that recognize large T antigen of simian virus 40 and mapped the epitopes to three distinct regions of the large T antigen. Also, 3 of the 16 recognized the large T antigen of the human papovavirus BKV.     

The zinc finger region of simian virus 40 large T antigen

Simian virus 40 large T antigen contains a single sequence element with an arrangement of cysteines and histidines that is characteristic of a zinc finger motif. The finger region maps from amino acids 302 through 320 and has the sequence Cys-302LeuLysCys-305IleLysLysGluGlnProSerHisTyrLysTyrHis- 317GluLysHis-320. In a conventional representation, the binding of zinc to the cysteines and histidines at positions 302, 305, 317, and 320 would form two minor loops and one major loop from the intervening amino acids. We made single amino acid substitutions at every position in the finger to identify possible functional elements within the putative metal-binding domain. Amino acids in the zinc finger could be divided into three classes characterized by distinct roles in DNA replication and transformation. Class 1 consisted of amino acids in the two minor loops of the finger and in the amino-terminal part of the major loop. Mutations here did not affect either replication or transformation. Class 2 consisted of the SerHisTyrLysTyr amino acids located in the carboxy terminus of the major loop of the finger. Mutations in this contiguous region reduced replication of the mutant viruses to different degrees. This clustering suggested that the region is an active site important for a specific function in DNA replication. With the exception of a mutation in the histidine at position 313, these mutations had no effect on transformation. Class 3 consisted of the proposed zinc-binding amino acids at positions 302, 305, 317, and 320 and the histidine at position 313 in the major loop of the finger. Mutations in these amino acids abolished the viability of the virus completely and had a distinctive effect on the transforming functions of the protein. Thus, the five cysteines and histidines of class 3 may play an important role in determining the overall structure of the protein. The histidine at position 313 may function both in the active site where it is located and in cooperation with the proposed zinc-binding ligands.