©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
T Antigens Encoded by Replication-defective Simian Virus 40 Mutants dl1135 and 5080(*)

Brenda S. Collins (§) , James M. Pipas (¶)

From the (1)Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We present a preliminary biochemical characterization of two simian virus 40 mutants that affect different T antigen replication functions. SV40 T antigen mutants dl1135 (17-27 amino acids) and 5080 (P-L) have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice. Both mutants are defective for viral DNA replication in vivo. In order to assess in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography. With each of the purified proteins, we examined some of the biochemical activities of T antigen required for replication, viz. ATPase, binding to the origin of replication (ori) and assembly on ori, DNA helicase and unwinding, and replication in in vitro assays. Consistent with previous studies, we found that the 5080 protein is defective for multiple biochemical activities including ATPase, helicase, ori-specific unwinding, and ATP-induced hexamerization. However, this mutant retains some sequence-specific DNA binding activity. In contrast, the dl1135 protein exhibited significant levels of activity in all assays, including the ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one of several mutants with an altered amino-terminal domain which can replicate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affects a T antigen enzymatic function directly required for viral DNA synthesis, dl1135 may alter an activity required to prepare the cell for viral replication.


INTRODUCTION

Simian virus 40 (SV40) has been a useful model for the study of DNA replication and transcription as well as for better understanding the mechanisms of tumorigenesis. The 708-amino-acid large tumor (T) antigen is the major regulator of productive and transforming infections(1, 2, 3, 4, 5, 6, 7) . Many of the biochemical functions of T antigen have been mapped to specific domains of the polypeptide (Fig. 1)(1, 8) . These include: DNA binding (residues 131-259), ATPase/DNA helicase activity(302-627), and DNA polymerase -primase binding (1-82; 260-517)(3, 9) .


Figure 1: Sites of mutations in SV40 large T antigen, functional domains, and regions recognized by relevant monoclonal antibodies. The primary sequence of T antigen is linearly diagrammed and numbered. The vertical lines with a P at the ends indicate positions of clustered phosphorylated serine and threonine residues. Mapped regions of large T antigen indicated include binding sites for products of the retinoblastoma (Rb) and p53 tumor suppressor genes, DNA binding domain, ATPase/helicase domain, and putative zinc (Zn) finger region. Arrows show the location of mutants dl1135 (17-27) and 5080 (P584>L). The regions recognized by monoclonal antibodies specific for SV40 T antigen, PAb419 and PAb108, detect epitopes at the amino terminus, and PAb101 and KT3, specific to epitopes within the carboxyl terminus. HR, host range domain. cr1 and cr2, conserved regions 1 and 2, respectively.



The first step in viral DNA synthesis is the sequence-specific binding of T antigen within the origin of replication. The region of the origin of replication contains three T antigen binding sites (site I, site II, and site III) in decreasing order of affinity(1, 10) . Site II, the core ori, is part of a 64-base pair sequence that is necessary and sufficient for viral DNA replication; site I and site III can stimulate viral replication(10, 11) . Both site I and site II have pentameric 5`-GAGGC-3` sequences, but in different arrangements. Additionally, site II has two other regions, a 15-base pair inverted repeat on the early side and a 17-base pair A/T-rich region on the late side, which are involved in ori melting and untwisting of the DNA helix, respectively(12, 13, 14) .

A monomer of T antigen can bind to one pentameric sequence in the absence of ATP, but ATP not only increases T antigen binding severalfold but it also induces a conformational change in T antigen such that 12 monomers of T antigen become organized into a two-lobed structure spanning the entire core ori(10, 15, 16, 17, 18) . The T antigen dodecamer complex then melts the origin. The ability of T antigen to bind to and melt site II is regulated by phosphorylation (19-21). An intact zinc finger region, which maps between amino acid residues 302 and 320, is required for hexamer assembly on the origin and for melting of the origin DNA(22) . The presynthesis complex consists of T antigen assembled as a double hexamer in the presence of ATP onto a site II duplex subsequently bound by replication protein A (RP-A), a mammalian single-stranded DNA binding protein(7, 16, 17, 23) . With the association of DNA polymerase -primase, hexameric T antigen in complex with RPA unwinds the DNA bidirectionally in a reaction driven by the hydrolysis of ATP(7, 24, 25, 26, 27) .

SV40 mutants dl1135 and 5080 have been studied extensively with regard to their ability to transform cells in culture and induce tumors in transgenic mice(28, 29, 30, 31, 32) . The mutant 5080 carries an amino acid substitution of P584L located within a stretch of hydrophobic residues near the carboxyl-terminal boundary of the ATPase/p53 binding domain. Previous studies indicated that the 5080 T antigen expressed in transformed murine cell lines was defective for ATPase activity and showed an aberrant pattern of oligomerization and phosphorylation(33, 34, 35, 36) .

The dl1135 mutant carries an in-frame deletion that results in the expression of a T antigen missing amino acids 17-27(29) . This mutant was of interest since it had been reported to be defective for both ATPase and sequence-specific DNA binding, activities thought not to require the amino-terminal domain(37) . Additionally, dl1135 is defective for transformation of some cell types although it retains the ability to complex with pRb and p53. The mutation appears to abolish a third transforming activity of T antigen that can functionally replace the adenovirus E1A 300K binding function (38).

To investigate in more detail the molecular basis for the in vivo replication defects of 5080 and dl1135, we expressed both of the mutant proteins using the baculovirus system and purified them by immunoaffinity chromatography(39, 40, 41) . Purified baculovirus T antigen has been shown to be comparable to T antigen purified from infected monkey cells in all assays tested(42) . Using the purified mutant proteins, we examined some of the biochemical activities of T antigen required for replication.


EXPERIMENTAL PROCEDURES

Recombinant Baculovirus Vectors That Express Wild-type and Mutant T Antigens

Recombinant baculovirus 941T which expresses wild-type SV40 large T antigen was kindly provided by Robert Lanford(39) . Both wild-type (wt) and mutant T antigens were expressed in BTI-TN-5B1-4 (``High 5'') insect cells (Invitrogen). The T antigen mutants dl1135, which has a deletion of amino acids 17-27, and 5080 which has an amino acid substitution of P584L have been described(28, 29) . The plasmids pdl1135 and p5080 contain the complete mutant viral genomes inserted at the BamHI site of pBR322 (28) or a derivative(29) . Recombinant baculovirus containing these mutants were constructed by digesting the pdl1135 plasmid or the p5080 plasmid with BamHI and StuI and ligating the purified SV40 fragment (2657 base pairs) into the BamHI and SmaI sites of the baculoviral transfer vector pVL1393. Subsequently, the recombinant transfer vectors containing the cDNAs of the mutants were made in an exchange reaction with 941T following digestion of both the wild-type and mutant constructs with EcoNI. The new recombinant transfer DNA was cotransfected with purified wild-type baculovirus DNA into insect cells followed by identification and purification of the recombinant virus by three rounds of dot-blot hybridization.

T Antigen Purification

High 5 insect cells were infected with the above recombinant baculoviruses, and the infected cells were lysed 36-42 h later using a buffer containing 0.2 M LiCl, 20 mM Tris, pH 8.0, 1 mM EDTA, 0.5% Nonidet P-40, 0.2 mM dithiothreitol (DTT),()and a protease inhibitor mixture. The T antigens were immunoaffinity purified from the lysates using a protein A-Sepharose (Pharmacia) column cross-linked to either PAb416 (43) or PAb101 (44) as described previously(45, 46) . After high pH elution (pH 11.4), the proteins were dialyzed overnight against a buffer containing 10 mM HEPES, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 50% glycerol, 1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride.

Plasmid DNAs

pUC.HSO contains SV40 nucleotides 5171 to 128 (47). The pDV.XH plasmid carries the SV40 minimal origin of replication corresponding to 64 base pairs of sequence between SV40 nucleotides 5211 to 32(48) . pTBS1 contains T antigen binding site I within SV40 nucleotides 5171 to 5228(49) .

In Vitro Replication of SV40 DNA

The reactions were performed according to the methods previously described(47, 50) . HeLa cell extracts, kindly provided by Dr. Thomas J. Kelly (Johns Hopkins University), were prepared as described previously(51) . The DNA replication products were ethanol-precipitated, subjected to agarose gel electrophoresis, and visualized by autoradiography.

Immunoprecipitation DNA Binding

Modified McKay()assays were done with site I and site II DNAs using two different buffers and conditions: McKay buffer and conditions and replication buffer with conditions appropriate for in vitro replication(52) . The McKay reaction buffer contained 10 mM HEPES, pH 7.3, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mg/ml bovine serum albumin, 2 mM DTT, and radiolabeled DNA. T antigen was then added, and the reactions were incubated on ice for 45 min to 1 h. Two hundred microliters of a 1:1 mixture of KT3/PAb101 hybridoma supernatant was added and the mixture was incubated for 30 min on ice, followed by the addition of 50 µl of protein A-Sepharose. The precipitated proteinDNA complex was washed three times with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40. The replication buffer contained 30 mM HEPES, pH 7.5, 7 mM MgCl, 1 mM DTT, 40 mM creatine phosphate, 10 mg/ml bovine serum albumin, plus radiolabeled DNA. These reactions were started by first adding T antigen and then 1 mM AMP-PNP (Sigma). The reactions were incubated at 37 °C for 30 min, followed by the addition of 200 µl of KT3/PAb101 (44, 53) and incubation for 20 min at 37 °C. The protein A-Sepharose was then added and incubated for 15-20 min. The immunocomplex was washed with 1 M Tris-HCl, pH 7.5, 0.5 M EDTA, and 10% Nonidet P-40. In both instances, the final pellet was resuspended in 10 mM Tris-HCl, pH 7.5, 7.5 mM EDTA, 0.5% SDS, 10% glycerol and heated to 65 °C for 2 min. The protein A-Sepharose beads were pelleted for 2 min, and the supernatant was analyzed by electrophoresis on a 1% or 1.4% agarose gel followed by autoradiography.

Nondenaturing Polyacrylamide Gradient Gel Electrophoresis

The procedure has been described by Loeber et al.(22) . T antigen was incubated at 37 °C for 20-30 min in a reaction buffer consisting of 30 mM HEPES, pH 7.5, 7 mM MgCl, 1 mM DTT, and 4 mM ATP. The protein was then cross-linked for 10-15 min at 37 °C with 0.1% glutaraldehyde. Sample buffer containing 15% 0.5 M Tris-HCl, pH 6.8, 50% glycerol, and 0.0025% bromphenol blue at pH 6.8 was added, and the oligomers were analyzed on a 4-22% nondenaturing polyacrylamide gradient gel in Tris glycine buffer for 11 h at 4 °C at 35 mA.

ori-specific Band Shift

The assay was performed as described by Virshup et al.(48) . A 67-base pair fragment containing the ori (site II) was obtained by digesting the pDV.XH plasmid with ApaLI and SnaBI, purified and radiolabeled. T antigen was added to an assay mixture containing HEPES, pH 7.8, 7 mM MgCl, 40 mM creatine phosphate, 1 mM DTT with 4 mM AMP-PNP (Sigma), and 3 fmol (10,000-15,000 cpm/reaction) of P-labeled DNA fragment and the reactions were incubated at 37 °C for 30 min after which the cross-linker glutaraldehyde was added to a final concentration of 0.05% and the reactions were incubated at 37 °C for 5 more min. The DNAprotein complexes were electrophoresed on 0.5% agarose, 2.4% acrylamide gel in 89 mM Tris borate, 2 mM EDTA (TBE) for approximately 1 h at room temperature at 200 V. The gels were dried on DE81 Whatman paper and then exposed to XAR film at -70 °C with an intensifying screen.

ATPase Assays

The assay buffer consisted of 25 mM HEPES, pH 7.0, 5 mM MgCl, 0.1 mM EDTA, 0.05% Nonidet P-40, 1 mM DTT, and 1.3 µM ATP including [-P]ATP. Each reaction mixture contained 10% glycerol. The reaction was started by the addition of T antigen and incubated at 37 °C. At timed intervals, 1-8 µl of reaction mixture was pipetted into tubes containing an equal volume of 0.75 M KHPO, pH 3.5. Samples of 1-4 µl were subsequently spotted onto polyethyleneimine-cellulose plates and developed in 0.75 M KHPO at room temperature for approximately 2 h. The plates were then exposed to XAR film, and the corresponding ATP and ADP spots were cut out and quantitated in a liquid scintillation counter, or the appropriate spots were quantitated by the AMBIS radioanalytic imaging system.

DNA Helicase Assay

This assay is based upon the method of Stahl et al.(54) . The annealed substrate consisting of M13mp18 (Life Technologies, Inc.) with a 17-nucleotide forward-sequencing primer (U. S. Biochemical Corp.) was extended from the primer using Klenow DNA polymerase in the presence of [-P]dATP, dGTP, and dTTP as described by Stahl et al.(54) . The assay mixture consisted of 0.3 fmol of annealed primer, 25 mM Tris, pH 7.5, 5 mM MgCl, 2 mM ATP, 1 mM DTT, 40 mM phosphocreatine, 0.5 µg of creatine phosphokinase, 0.5 µg of poly(dI-dC) (Boehringer Mannheim). The reaction was started with the addition of 100 to 500 ng of T antigen followed by incubation at 37 °C for 1 h. The reaction was stopped by the addition of an equal volume of 1.5% SDS, 0.2 M EDTA. Subsequently, 5 µl of sample buffer was added to each reaction, and the reactions were electrophoresed on a 12% acrylamide gel in TBE buffer for 2 h at 200 V.

ori-dependent Unwinding Assays

The unwinding assays were performed as described by Virshup et al.(55) . The plasmid pUC.HSO, containing the minimal origin of replication, was linearized with HindIII, labeled using the Klenow fragment and [-P]dCTP, and digested with BamHI and HinfI. This results in two labeled fragments, an ori and an ori fragment. The ori fragment served as an internal control for the unwinding reactions. T antigen was incubated with 0.5 fmol of labeled substrate DNA in a buffer containing 30 mM HEPES, pH 7.8, 7 mM MgCl, 4 mM ATP, 40 mM creatine phosphate, and single-stranded DNA binding protein (United States Biochemical Corp.). The reactions were incubated for 1 h at 37 °C and then stopped with 0.5% SDS, 10 mM Tris, pH 7.5, 5 mM EDTA, and proteinase K (10 mg/ml). The incubation was continued for 1 h at 37 °C. DNA sample buffer was then added, and the reactions were heated at 65 °C for 2 min. The reactions were then analyzed on an 8% acrylamide gel, dried on DE81 Whatman paper, and autoradiographed with XAR film.


RESULTS

Purification of T Antigens

Recombinant baculoviruses expressing 5080 and dl1135 were constructed as described under ``Experimental Procedures,'' and the T antigens were subsequently immunoaffinity-purified from infected insect cells. Samples of each protein were assessed for purity and integrity by silver staining and Western blot analysis (Fig. 2). The silver stain shows a predominant band of approximately 97 kDa for both mutant and wild-type T antigens. Based on a visual assessment of this gel, we estimate that 5080 and dl1135 T antigens are greater than 90% pure. Fig. 2B shows a Western blot of purified dl1135 protein, and wild-type T antigen demonstrates that the monoclonal antibodies PAb101 and KT3, which recognize epitopes within the carboxyl-terminal region of T antigen, detect both mutant and wild-type proteins. However, the dl1135 deletion results in a loss of reactivity for PAb419 and PAb108. Since both PAb419 and PAb108 recognize denaturation-resistant epitopes, it seems likely that the deletion of residues 17-27 encompasses the actual epitopes for these monoclonal antibodies. The mutant protein 5080, like wild-type T antigen, is detected by antibodies PAb101 and KT3 as well as PAb419 and PAb108 (not shown).


Figure 2: Immunoaffinity-purified baculovirus-expressed wild-type and mutant T antigens. A, silver-stained SDS-PAGE with electrophoresed proteins (1 µg). The molecular masses of marker proteins (Bio-Rad) are shown at the left in kilodaltons. B, Western blot of denaturing polyacrylamide gel with electrophoresed wild-type and mutant baculovirus T antigens. Antibodies used for detection are indicated below the immunoblot. In the lanes labeled KT3/PAb101, a mixture of these two antibodies was used. Immunoblot detection employed enhanced chemiluminescence (ECL; see ``Experimental Procedures'').



Oligomerization and DNA Binding

When electrophoresed through a nondenaturing gradient gel, T antigen resolves as multiple oligomeric forms that appear as a ladder extending from the monomer to dodecamer and higher. At 37 °C in the presence of ATP and magnesium, T antigen undergoes a conformational change such that the predominant species is the hexamer(15, 56) . This reaction can occur in the presence or absence of DNA; however, DNA cooperatively enhances protein to protein association(18, 56) . T antigen assembles on the origin of replication (ori) as a double hexamer in the presence of ATP and participates in localized melting of the origin(7, 12, 13) . Therefore, we wished to see if the mutant proteins formed the normal pattern of oligomeric forms and if they formed hexamers in response to ATP. The dl1135 protein resolved into an oligomeric ladder in the absence of ATP at 4 °C without cross-linker, but unlike wild-type extended only from monomer to apparently hexamer with clear resolution. Some higher oligomeric forms were present, but these appeared fainter than with the wild-type T antigen (Fig. 3). There also seems to be a non-integer band with 1135 which is not present with wild-type, which we have seen in multiple experiments and for which we cannot account. When cross-linker was added at 4 °C, the oligomeric forms appeared to be indistinguishable from wild-type. With the addition of 4 mM ATP at 37 °C, some of the dl1135 protein shifted to the hexameric form, although most of it appeared to be in the monomeric form, suggesting that the response was less efficient than wild-type.


Figure 3: Nondenaturing gradient gel for resolution of oligomeric forms of T antigen. Immunoaffinity-purified wild-type and mutant T antigens were electrophoresed through a 4-22% acrylamide gradient gel following incubation for 20-30 min at 37 °C in a HEPES/Mg buffer with or without 4 mM ATP. The cross-linker glutaraldehyde was added to a final concentration of 0.1% except for a control with T antigens at 4 °C in the same buffer (see ``Experimental Procedures''). The final glycerol concentration in all reactions was 15%. The gel was fixed and silver-stained.



Most of 5080 protein seems to be a smear in the gel with a only few lower molecular weight bands migrating near the level of dimer in the wild-type lane at 4 °C without cross-linker (Fig. 3). With cross-linker, the few lower molecular weight bands are no longer visible, and there appears to be more smearing toward the top of the gel, suggesting more higher oligomeric or aggregated forms. The 5080 mutant also did not show any monomer as did both the wild-type and 1135 proteins. At 37 °C with cross-linker in the presence and absence of ATP, the 5080 protein did not appear to enter the gel but remained at the top of the gel, suggesting that perhaps the protein forms a greater aggregate at the higher temperature or that the smaller oligomers are unstable at the higher temperature. This property of the 5080 T antigen has been reported previously by Lin et al.(36) .

We then examined the ability of the purified mutant T antigens expressed in the baculovirus system for sequence-specific DNA binding activity using an immunoprecipitation assay. A modified McKay assay was done using plasmids containing sites I or II in either a HEPES/EDTA buffer (McKay buffer) on ice or a HEPES/MgCl buffer with ATP and without EDTA (replication buffer) at 37 °C. The wild-type T antigen appeared to bind to site I and site II slightly better in McKay buffer (on ice) than in replication buffer at 37 °C (Fig. 4). The 5080 T antigen bound to site I nearly as well as wild-type in replication buffer. In contrast, 5080 bound to site I very inefficiently in McKay buffer, although it retained some specific DNA binding activity (Fig. 4) The 1135 protein also could specifically bind to site I, but the efficiency of the binding was much less than with wild-type.


Figure 4: Immunoprecipitation DNA binding assay. Affinity-purified T antigen was added to 0.3 ml of reaction buffer in the concentrations as indicated. A, the reaction buffer contained labeled restriction enzyme-digested (HincII) plasmid harboring site I. B, the reaction buffer contained labeled plasmid harboring the minimal origin (site II) digested with restriction enzymes ApaLI and NaeI. HEPES/EDTA (on the right) or HEPES/Mg with AMP-PNP incubated with the T antigen on ice or at 37 °C, respectively, for 30-45 min. The DNA-protein complex was precipitated with a mixture of monoclonal antibodies KT3/PAb101 followed by the addition of protein A-Sepharose. The pellets were washed and resuspended in sample buffer (see ``Experimental Procedures''), then heated for 2 min. The supernatants were electrophoresed through a 1.4% agarose gel, and the gel was subsequently prepared for autoradiography. Arrows indicate the specific band.



The 5080 T antigen bound very inefficiently to site II under both reaction conditions (Fig. 4). Site II binding by the 1135 protein was barely detectable when assayed in McKay buffer (Fig. 4). Clark et al.(37) also reported detecting no ori binding for the 1135 protein purified from infected CV1 monkey cells in a similar immunoprecipitation DNA binding assay. However, 1135 did bind to site II in replication buffer, but, in addition, it bound two other fragments. This binding is not entirely nonspecific since the fastest migrating band of 458 base pairs was not bound by 1135. This pattern of binding was seen in several independent experiments and with different protein preparations.

We next wished to determine whether dl1135 and 5080 were capable of ATP-dependent assembly on the minimal origin (site II) of SV40 DNA. The ATP-dependent assembly of T antigen on the origin as a double hexamer is required for the presynthesis complex (for a review, see Ref. 7). Virshup et al.(55) showed that in using a plasmid containing the core ori in a DNA mobility shift assay two DNAprotein complexes are retarded, which correspond to a faster-migrating T antigen hexamer bound to the origin and a slower-migrating T antigen dodecamer bound to the origin. In the gel-shift assay, the mutant 5080 gave no distinct band shifts, only a diffuse smear not seen in the control lane (data not shown). The smear suggests some ability of 5080 to bind the core ori; however, it fails to assemble hexamers in the presence of ATP. When the mutant T antigen dl1135 was examined in a gel-shift assay, it bound to site II both as a dodecamer and as a hexamer similar to wild-type (Fig. 5). This is consistent with the results of the nondenaturing gradient gel experiment which showed that dl1135 is able to hexamerize in response to ATP. Thus, despite the different McKay site II result, this assay indicates that 1135 binding to ori is normal although this assay would not detect additional ``nonspecific binding.''


Figure 5: DNA mobility shift assay. The probe was a minimal origin (site II)-containing fragment. T antigen was added to an assay mixture containing the replication buffer with 1.5 mM AMP-PNP and 3 fmol (10,000-15,000 cpm/reaction) of P-labeled DNA fragment. The reactions were incubated at 37 °C for 30 min followed by the addition of the cross-linker glutaraldehyde to a final concentration of 0.05%. The DNA-protein complexes were electrophoresed on an agarose-acrylamide gel and prepared for autoradiography.



In the course of our studies, we noted a disconcerting variability in the biochemical activity profile of different 1135 T antigen preparations. A gel-shift assay using the ori fragment with a preparation different from the one used in Fig. 5showed more hexamer than dodecamer, suggesting a loss of cooperativity between hexamers. This preparation was defective for replication and ori-specific unwinding, but retained ATPase and helicase activity and the same immunological reactivity as the replication-positive preparations of dl1135. Apparently, it is possible to disrupt specific T antigen activities by purification. This emphasizes the need to assay multiple preparations of each mutant protein for each activity.

ATPase Activity of Mutant T Antigens

Wild-type T antigen carries an ATPase activity which along with the helicase activity is required for unwinding DNA strands for replication(54, 58) . The mutant 5080 was very defective for ATPase activity as reported by Tack et al.(33) , retaining only about one-sixth of the ATPase activity of wild-type T antigen (Fig. 6). Increasing the amount of T antigen used in the assay also increases the amount of ATP hydrolysis for both dl1135 and wild-type, although less so for 5080 (Fig. 6). As expected, the dl1137 protein showed no measurable ATPase activity (Fig. 6). The 1135 T antigen exhibited a significant level of ATPase activity ranging from 50-70% of wild-type with different preparations. This is in contrast to the report of Clark et al.(37) , which reported that 1135 was defective of ATPase activity. Possible explanations of the discrepancy are provided under ``Discussion.''


Figure 6: ATPase assays. The reactions were started by the addition of T antigen, 941T (wild-type), 1135, 5080, 1137, and were incubated at 37 °C. The assay buffer contained both cold and [-P]ATP (see ``Experimental Procedures''). A, samples of each reaction mixture were taken at different time points, and a portion was spotted onto polyethyleneimine plates for thin layer chromatography. B, ATP hydrolysis determined for different concentrations of the mutant and wild-type T antigens; the time interval used was 15 min. The spots corresponding to ATP and ADP were cut out of the polyethyleneimine plates and quantitated in a liquid scintillation counter or the ATP and ADP spots were quantitated by AMBIS radioanalytic imaging. , 941T; , dl1135; &cjs2110;, 5080; dl1137 values were not above background and were not shown.



DNA Strand Displacement Assay

The DNA helicase activity of T antigen was assessed using M13mp18 annealed to a complementary synthetic oligonucleotide (17-mer) as a substrate. Different amounts of T antigen were incubated with labeled annealed substrate in replication buffer at 37 °C for 1 h. The products were then electrophoresed through a 12% polyacrylamide gel in TBE buffer and then prepared for autoradiography. The results are shown in Fig. 7. The dl1135 mutant was active for helicase activity, exhibiting about 40% the level of wild-type. As expected, no helicase activity was detected for the ATPase-defective 5080 protein.


Figure 7: DNA helicase assay. Varying amounts of mutant and wild-type T antigens were incubated under replication conditions with end-labeled oligonucleotide annealed to single-stranded M13 DNA. The products were electrophoresed through a 12% native polyacrylamide gel and prepared for autoradiography.



Origin-specific Unwinding

In order to assess the ability of the dl1135 protein to unwind the origin, a plasmid containing minimal ori was linearized and radiolabeled. The labeled fragment was then digested with two restriction enzymes to generate ori and ori fragments, thus allowing an internal negative control. T antigen was then incubated with the labeled substrate for 1 h, and the products were electrophoresed through an 8% TBE gel. Fig. 8shows that dl1135 unwinds the origin, although less efficiently than wild-type T antigen.


Figure 8: Origin (ori)-specific unwinding assay. T antigen at the amounts indicated was incubated with origin-specific and non-origin DNA in replication buffer containing E. coli SSB at 37 °C for 1 h. The reaction products were electrophoresed in an 8% polyacrylamide gel which was subsequently dried and exposed to film.



In Vitro DNA Replication of Wild-type and Mutant T Antigens

Both dl1135 and 5080 are defective for replication of viral DNA in monkey cells(28, 29) . We next evaluated the ability of purified 5080 and dl1135 to activate DNA replication in an in vitro system. The dl1135 and 5080 proteins were incubated for 2 h in replication buffer with HeLa cell extract at 37 °C. The products were resolved by electrophoresis through a 1% agarose gel and visualized by autoradiography. Incorporation of radiolabel into DNA was measured by the acid-precipitable counts to quantify the amount of replication. The results are shown in Fig. 9. As expected, the 5080 T antigen was completely defective in this in vitro assay. Surprisingly, the dl1135 protein showed significant activity, approximately 40-60% of wild-type.


Figure 9: In vitro SV40 DNA replication by wild-type and mutant T antigens, dl1135 and 5080 using human cell extracts. The reaction mixture included the template, pUC.HSO and the concentrations of T antigen indicated in the conditions described under ``Experimental Procedures.'' The reactions were incubated at 37 °C for 2 h. The replication products were electrophoresed through a 1% agarose gel and then visualized by autoradiography. The control was the reaction mixture without T antigen. A, replication activity of wild-type and 5080; B, replication activity of wild-type and dl1135.




DISCUSSION

The SV40 mutants dl1135 and 5080 are both replication-defective in vivo. Previous studies have suggested that, in both cases, the mutations result in effects on activities thought to reside in different domains. Thus, these mutants might be useful for understanding interdomain communication. The mutant 5080, for instance, which carries a substitution of residue P584L located within the ATPase/p53 domain of T antigen, does not bind to pRb even though the mutation lies outside of the Rb binding region (34). The single amino acid change also renders the protein defective for ATPase and p53 binding(33) . Similarly, dl1135, which carries an in-frame deletion that results in the expression of a T antigen missing amino acids 17-27, appeared to be defective for DNA binding and ATPase activity(37) . Both of these biochemical activities map to a different region of the molecule than the dl1135 mutation. In this paper, we report the characterization of the purified baculovirus T antigen encoded by those mutants.

Tack et al.(33) studied the 5080 T antigen expressed in transformed C3H10T1/2 cells and found that it was defective for ATPase. As expected, we also found that the purified baculovirus-expressed 5080 was defective for ATPase, although it showed some activity, retaining approximately one-sixth of the wild-type activity. Since T antigen ATPase activity is required for its helicase and unwinding functions, the greatly reduced ATPase activity is sufficient to explain the replication defect of 5080. Tack et al.(33) also examined the oligomeric state of 5080 T antigen in C3H10T1/2 cells by glycerol gradient sedimentation. Also Ludlow et al.(34) studied oligomerization using extracts of CV-1P cells transfected with 5080. In both instances, 5080 was found to exist in 5-10 S forms only compared with wild-type 20 S and 14 S forms. Thus, 5080 appeared to be defective for forming higher oligomers.

By electrophoresis in nondenaturing gels, we found that the purified 5080 T antigen was in a high molecular weight form that barely penetrated the gel. Lin et al.(36) reported a similar result with a T antigen harboring the same mutation. The large oligomers which barely enter the gel are reminiscent of zinc finger mutants described by Loeber et al.(22) . Two possible explanations for the discrepancy in the oligomerization results from the different techniques are the following: 1) the higher molecular weight forms represent aggregated T antigen resulting from the purification process and are not reflective of the in vivo situation or a real property of 5080; 2) the lower molecular weight forms reported by Tack et al.(33) are an artifact and a consequence of the aggregated T antigen being discarded in the clarification step of the immunoprecipitation.

We found that the dl1135 T antigen purified from baculovirus-infected insect cells was able to stimulate SV40 ori-dependent DNA replication in vitro. Although less active than wild-type, 1135 exhibited significant levels of ATPase, helicase, and ori unwinding activity. Gel-shift experiments indicated that 1135 could efficiently bind to and assemble double hexamers on the ori. On the other hand, 1135 showed very reduced binding to site I and site II in immunoprecipitation DNA binding assays. One explanation for the difference in the binding of site II by 1135 in the modified McKay assay versus the gel-shift assay may lie in a decreased affinity or a quick on/off rate by the mutant compared with wild-type. The cross-linking agent used in the gel-shift assay may compensate for a quick release of the core ori by ``freezing'' the complex so that it is detectable, whereas without a cross-linking agent, the immunoprecipitation assay does not allow the complex to be detected. This may also account for the results of Clark et al.(37) , who reported little or no origin binding activity by dl1135 as measured by a method similar to the McKay assay.

The dl1135 mutant is one of several mutants in the amino-terminal domain which can replicate DNA in vitro but not in vivo. For example, mutants 3213 (E107K), 5110 (D44N), and GMSV40 (67-82) are all defective for in vivo replication but support replication in vitro(59, 60) . The amino terminus of T antigen plays a role in several functions including transformation and transactivation and interacts with numerous cellular proteins, i.e. pRb, p107, etc.(61, 62) . Although the amino terminus may not be directly involved with the enzymatic/origin binding activities of T antigen in vivo, it may activate a cellular target or affect some preliminary step to the initiation of replication.


FOOTNOTES

*
This study was supported by Grant VM-29 from the American Cancer Society (to J. M. P.) and National Institutes of Health National Research Service Award Grant CA 09077 (to B. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD 20892.

To whom correspondence and reprint requests should be addressed. Tel.: 412-624-4691; Fax: 412-624-4759.

The abbreviations used are: DTT, dithiothreitol; AMP-PNP, adenyl-5`-yl imidodiphosphate.

R. A. F. Dixon, personal communication.


ACKNOWLEDGEMENTS

We thank Robert Lanford for the recombinant baculovirus 941T. We are especially grateful to Tom Kelly and his laboratory for sharing their expertise, protocols, and HeLa cell extract. We thank all members of the Pipas laboratory, especially Tim Kierstead, Ashok Srinivasan, and Alex Castellino, for their advice and comments. Critical reading of the manuscript by Roger Hendrix and Keith Peden is greatly appreciated.


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