JOURNAL OF VIROLOGY, Sept. 1984, p. 670-6810022-538X/84/090670-12$02.00/0Copyright ©D 1984, American Society for Microbiology

Immunoprecipitation of Some Forms of Simian Virus 40 Large-TAntigen by Antibodies to Synthetic Peptides

EVA PAUCHA, ROBERT HARVEY, AND ALAN E. SMITH*

Biochemistry Division, National Institiute f6r Medical Research, Loncdon NW7 /AA, United Kingdom

Received 20 December 1983/Accepted 1 June 1984

Antibodies were raised against six synthetic peptides corresponding to overlapping amino acid sequences

(106 through 145) from a putative DNA binding domain in simian virus 40 (SV40) large-T antigens. All sixantipeptide sera immunoprecipitated large-T from crude extracts of SV40-transformed cells, but the efficiencyvaried widely; in general, antibodies to the longer peptides produced the strongest anti-large-T activity.Antisera were purified by immunoaffinity chromatography on immobilized peptide. The purified antiserarecognized only some forms of large-T; full-sized large-T from transformed cells, super-T from SV3T3 C120cells, and 70,000-dalton T-antigen from Taq-BamHI cells were immunoprecipitated, whereas large-T fromproductively infected cells reacted irreproducibly, and the full-sized protein, synthesized in vitro or eluted fromsodium dodecyl sulfate-containing gels, and the 33,000- and 22,000-dalton truncated large-Ts from SwissSV3T3 and MES2006 cells, respectively, were not immunoprecipitated. This pattern of reactivity was explainedwhen extracts were fractionated by sucrose density centrifugation, and it was found that only rapidlysedimenting forms of large-T were immunoprecipitated by (he antipeptide sera; that is, large-T complexed withnonviral T antigen was detected, whereas lighter forms were not detected. Cascade immunoprecipitations did

not support the view that this result was caused by the low affinity of the peptide antisera for large-T, andWestern blotting experiments confirmed that the peptide antisera react directly with immobilized, monomericlarge-T but not with nonviral T antigen. Immunoprecipitation assays to detect large-T:nonviral T antigencomplexes bound specifically to fragments of SV40 DNA showed that under conditions of apparent antibodyexcess, DNA still bound to the complex.

Simian virus 40 (SV40) large-T antigen has been shown tobe both necessary and sufficient for the initiation of transfor-mation by SV40 virus and perhaps for the maintenance ofcells in the transformed state. Large-T is found predominant-ly in the nucleus of transformed cells. Several biochemicalactivities have been attributed to large-T, including an

ATPase activity and the ability to bind to DNA and to a hostcell protein called nonviral T antigen (NVT) or p53 (3, 6, 19,21, 25, 48, 49). It is not yet known which, if any, of theseactivities is responsible for transformation.The best-characterized property of SV40 large-T is its

DNA binding activity. The protein binds to well-character-ized sequences around the origin of DNA replication on

SV40 DNA (48, 49). This binding plays a role in controllingviral DNA replication and transcription in productivelyinfected cells. Large-T also binds to cellular double-strandedDNA in vitro (3), but whether precise cellular sequences are

involved in this interaction is not known. It is also not yetestablished whether the binding of large-T to cellular DNA,mediated by its ability to bind either specifically or non-

specifically, plays any role in the transformation process. Theobservation that transformation by SV40 results in theactivation of transcription of specific cellular genes does notresolve the issue, since such activation could be direct or

indirect (39, 41).Attempts to map on SV40 large-T amino acid sequences

that make up a region with the ability to bind to DNA havelocalized at least one putative binding domain. Most of thesestudies have measured the ability of various large-T-relatedmolecules to bind to immobilized calf thymus double-strand-ed DNA (3) or to origin-containing fragments of SV40 DNA

* Corresponding author.

in solution (26, 48). The large-T-related molecules reportedinclude: (i) the D2 protein coded for by an adeno SV40hybrid virus (48); (ii) various forms of SV40 large-T synthe-sized in vitro with unspliced SV40 cRNA as template (33);(iii) various truncated forms of large-T isolated from mutant-infected cells and from different transformed cell lines (4, 7,35); (iv) large-T from pseudorevertants of SV40 origin-defective mutants (43); (v) large-T coded for by altered SV40sequences recovered from various SV40-transformed celllines (11, 37, 46); (vi) various shortened forms of large-Tcoded for by the ND series of adeno SV40 hybrid viruses(28, 32); (vii) fragments of large-T produced by partialproteolysis (29). Although the amino acid sequences presentin all the forms of large-T are not known precisely and not allof the data obtained are consistent, most evidence suggeststhat the binding domain is within the proximal portion of thesecond exon in the large-T coding region; that is, almost alldata are consistent with a domain mapping between aminoacids 83 and about 250.We have attempted to raise antibodies that recognize

determinants in this region of SV40 large-T in the hope thatthey would prove useful in further studies on DNA binding.Although fine mapping of the available monoclonal antibod-ies (14, 15) has not yet been reported, evidence to datesuggests that few, if any, have specificity for this region.Rather than isolate and map further monoclonal antibodies.we have used the alternative procedure of raising antibodiesto synthetic peptides from this region. This method hasalready been used successfully to raise antisera againstamino- and carboxy-terminal amino acid sequences fromSV40 large-T (51) and against internal sequences from a widerange of other proteins (47). Here we report the properties ofantibodies raised against six synthetic peptides covering theregion between amino acids 106 and 145.

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MATERIALS AND METHODSPeptide antibodies. Peptides were synthesized by the solid-

phase Merrifield method (27) as described previously (16,

17). After synthesis, the peptides were eluted from the resin

with hydrogen bromide in trifluoroacetic acid, desalted on

G15 Sephadex in 50% acetic acid, and freeze dried. PeptidesA. B, E, and F were not purified further. Peptides C and D

purified on SPC25 columns with a 0 to 2 M NaCI

gradient. All peptides were coupled to bovine serum albumin

(BSA) carrier protein by benzidine 6. 51). In addition.peptides A and D were coupled to ova.lumin, and peptides

B and F were coupled to BSA by glutaldehyde (16, 17, 51).

The degree of coupling was estimated with '25I-labeledpeptide as tracer followed by chromatography on Sephadex

G50. In all cases, the molar ratio of peptide to carrier was

greater than 10.Antibodies were raised in rabbits. In each case, two

animals were injected successively on days 0, 7, and 35 with

1 to 2 mg of conjugate. Serum (25 to 50 ml) was collectedweekly from weeks 6 to 20 after the first injection.

Purification. Peptides B and F were coupled to CH-

Sepharose (Pharmacia Fine Chemicals, Piscataway, N.J.)

under the conditions recommended by the manufacturer(17). This results in peptide coupled to the resin via the N-

terminal a.-amino group. Yields were 88 and 75% peptide

bound, respectively. Peptides B, C. and F were coupled to

AH-Sepharose (Pharmacia) under the conditions recom-

mended by the manufacturer. This resulted in peptide cou-

pled via its carboxy terminal end. Yields were 29, 19, and

75C'( peptide bound, respectively.A crude immunoglobulin G fraction was prepared by

precipitation of 10 ml of crude serum with 18% Na2SO4.

After dialysis against phosphate-buffered saline (PBS). spe-

cific antibodies were purified by sequential chromatographyon a 5-ml BSA-Sepharose column and a 2-ml peptide-

Sepharose column. The columns were then separated. After

being washed extensively with PBS and with 2 M KCI toremove non-specifically bound material, specific antibodieswere eluted with 1 M propionic acid. Fractions were neutral-

ized immediately with 2 M phosphate buffer. Peak fractions

were collected and dialyzed against Tris-buffered saline.

Routinely, from about 400 mg of total serum protein, ca. 10

to 15 mg of purified fraction was obtained in the eluate from

the peptide-Sepharose.Monoclonal antibodies. The monoclonal antibodies

pAb413 and pAb419 were the gift of E. Harlow (15).

Cells. MES2006 cells were a gift from W. C. Topp. Cold

Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

SVA31E7 and SV3T3 cells have all been described previous-

ly (45). Rat cells (RE52) transformed by a microinjected Taq-BarniHI fragment ofSV40 which express a 70,000 (70k)-

dalton form of T-antigen were a gift from A. Graessmann

(12).Labeling and extraction of cells. Labeling and extraction

procedures were carried out essentially as described previ-

ously (44, 45). Briefly, confluent monolayers of transformed

cells were grown in E4 medium with 5% fetal calf serum on

50- or 90-mm-diameter plastic dishes (Nunc). The cells were

labeled for 2 h by replacing the medium with either E4

medium minus phosphate containing 250 p.Ci of [32P]ortho-phosphate (Amersham International) per ml or E4 medium

minus methionine containing 100 p.Ciof [35S]methionine(specific activity >1,000 Ci/mmol; Amersham International)

per ml. After the cells were labeled, the dishes were rinsed

twice with ice-cold Tris-buffered saline, and the cells were

lysed by the addition of 1 ml/90-mm dish or 0.5 ml/50-mm

dish of lysis buffer containing 50 mM Tris (pH 8.0), 120 mMNaCI, and 0.5% Nonidet P-40 (NP-40). The samples werespun at 10,000 rpm for 10 min at 4°C, and the supernatantwas used directly or frozen and stored at -70°C. Monolayersof CV1 cells were labeled as described above for 2 h at 48 hafter infection with SV40.Immunoprecipitation of cell extracts. Samples of 10 to 20 pu1

of a 32pP-labeled extract or of 100 pl of a [35S]methionine-labeled extract were mixed with 0.5 ml of a buffer containing20 mM Tris (pH 7.0). 100 mM NaCl. 2 mM dithiothreitol(DTT), 1 mM EDTA, and 0.05% NP-40 (buffer B) andsamples of S to 10 p[L of antiserum. The samples wereincubated for 60 min at 18°C. Five serum volumes of a 10%suspension of washed Staplhvlococcus (liirelis bacteria wereadded, and incubation continued for 10 min. The bacterialpellets were washed twice with 0.5 M NaCI in a buffercontaining 20 mM Tris (pH 8.0). 2 mM DTT, 1 mM EDTA,and 0.5% NP-40, and then once more with the same buffercontaining 0.1 M NaCl. Bound proteins were eluted in gel-loading buffer (0.0625 M Tris [pH 6.8], 2% sodium dodecylsulfate (SDS). 10% glycerol. 0.1 M DTT, and 0.01% bromo-phenol blue) and analyzed on SDS-containing 15% poly-acrylamide gels. as described previously (44, 45). Autoradi-ography of dried gels was for 1 to 4 days at -70°C on Fuji NifRx film with an Ilford fast-tungstate intensifying screen.

Gradient analysis. Gradient analyses were performed es-sentially as described by McCormick and Harlow (25).Samples (0.2 ml) of cell extracts were loaded onto 5-ml lineargradients of 5 to 20% sucrose over a 0.2-ml cushion of 60%sucrose in a buffer containing 10 mM Tris (pH 8.0), 10 mMDTT, and 140 mM NaCl. In the case of [35S]methionine-labeled extracts, the sample volume was increased to 0.4 ml,and the sucrose cushion was omitted. Centrifugation was at25,000 rpm for 15 h at 4°C in a Beckman SW55 Ti rotor.Fractions were collected, and samples of each were dilutedwith 0.5 ml of buffer B before being immunoprecipitated asdescribed above.DNA binding. DNA from plasmid pSV328 which contains

the larger BamnHl-EcoRI fragment of SV40 inserted betweenthe Ba,nHI and EcoRI sites of pBR328 (a gift from G. C.Grosveldt) was cut with the restriction enzyme BstNI (NewEngland BioLabs, Inc., Beverly, Mass.), treated with calfintestinal phosphatase (Boehringer Mannheim Biochem-icals, Indianapolis, Ind.), and then end labeled with [y-32P]ATP (specific activity 5.000 to 7,000 Ci/mmol; Amer-sham International) and T4 polynucleotide kinase (NewEngland BioLabs) by the procedure of Maxam and Gilbert(23, 24). The specific activity of DNA obtained was generallybetween 1 x 107 and 3 x 107 cpm/pLg. DNA binding was doneessentially as described by McKay (26). In a typical experi-ment, 0.1 ml of cell extraction buffer containing 10 to 20 p. ofcell extract was mixed with 0.4 ml of binding buffer (20 mMTris [pH 7.0], 1 mM EDTA, 2.5 mM DTT, 125 p.g of BSA perml, 0.05% NP-40), and 10 ng of labeled DNA was added.After incubation at 18°C for 40 to 60 min, 5 to 10 p.l ofappropriate antiserum was added, and incubation continuedfor a further 40 to 60 min. Five serum volumes of S. tlir-elisbacteria were added which had first been washed twice inNET.N buffer (50 mM Tris [pH 7.5]. 150 mM NaCl, 5 mMEDTA, 0.05% NP-40 [44]) containing 100 p.g of BSA per mland 10 p.g of sheared, double-stranded calf thymus DNA perml and was then resuspended in NET.N buffer alone. After afurther5 to 10 min of incubation, the pellets were washed 3times in wash buffer (20 mM Tris [pH 7.0], 1 mM EDTA, 2mM DTT, 0.05% NP-40, and 0.2 M NaCl), and the DNA waseluted in 15 p.1 of gel-loading buffer (10 mM Tris [pH 7.5]. 10

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672 PAUCHA, HARVEY, AND SMITH

mM EDTA, and 2% SDS). The samples were then analyzedby electrophoresis in 2% agarose gels (Sigma Chemical Co.,St. Louis, Mo.) in Tris-acetate buffer (23, 42). The gels werefixed in 2.5 volumes of 10% trichloroacetic acid before beingdried and exposed. Autoradiography was for 1 to 4 days at-70°C on Fuji Nif Rx film with an Ilford fast-tungstateintensifying screen. For those experiments with purifiedlarge-T:NVT complexes, sucrose gradients were run asdescribed above with unlabeled SVA31E7 cell extract. Thefractions containing complex (as judged from parallel gradi-ents such as those shown in Fig. 4) were collected, and 100,ul of the pooled fractions was used for DNA binding asoutlined above. The pH of the pooled fraction was adjustedby the addition of 1 M Tris (pH 8.0) to a concentration of 50mM before use.Western blotting. Samples (50 p.1) of unlabeled extracts of

SVA31E7 or F9 teratocarcinoma cells were electrophoresedon 10% SDS-containing acrylamide gels either with or with-out prior immunoprecipitation with hamster antitumor cellserum. Electrophoretic transfer of the separated proteinsonto nitrocellulose was carried out as described by Burnette(2). Immediately after transfer, the filter was washed for 10min at room temperature in 1 M glycine and then for 30 minat 40°C in 20 mM Tris (pH 7.5)-200 mM NaCl-5% BSA.After being rinsed with the same buffer containing 0.05%NP-40, the sheet was incubated overnight at room tempera-ture in this solution containing the appropriate purifiedantiserum (2 to 3 ,ug/ml). The sheet was then washed in 1 Mglycine for 10 min at room temperature and twice in Tris-saline plus 0.05% NP-40 for 30 min at room temperature. Atotal of 5 x 105 cpm of ['251]protein A per ml was added inTris-saline, BSA, and 0.05% NP-40 and incubated at roomtemperature for 2 h, after which the sheet was washed inglycine and Tris-saline plus NP-40 as described above.Autoradiography was for 16 to 48 h.

RESULTSProduction of peptide antisera. We examined the amino

acid sequence of the putative DNA binding domain of SV40large-T, seeking a hydrophilic peptide which current knowl-edge of protein structure (38) would predict to have a highprobability of being located on the surface of the moleculeand of being immunogenic. Two computer programs wereused to aid the analysis: one (based on a method of Chou andFasman [5]) predicts secondary structure and hydrophilicityof the protein, and the second (Hopp and Woods [181)predicts immunogenic regions. The profile of hydrophilicityin large-T as predicted from the Hopp and Woods program isshown in Fig. 1. Both analyses indicated the presence of alarge peak of hydrophilicity centered about lysine 128 andincluding a very basic tract of amino acids, plus a smallerpeak centered about aspartic acid 113.

Further evidence suggests that this region may be exposedin the large-T molecule. First, the bond between arginine 130and lysine 131 is known to be cleaved preferentially bylimiting amounts of trypsin (40). Second, this region of theprotein is heavily phosphorylated with sites identified atserine 106, serine 111, serine 112, threonine 123, and serine124 (36) and at least some of the phosphate has a short half-life (50). Presumably, these sites are readily accessible toprotein kinases. We therefore concentrated our initial stud-ies on region 106 through 145 and synthesized the sixpeptides shown in Fig. 1.The peptides were all synthesized by the Merrifield solid-

phase method (27). After hydrolysis from the resin, thepurity of the peptides was examined by amino acid analysis

S E E M P 9 S 0 D E A T ADS 0 H S T P P K K K R K V E D P K D F P S E L LS F106 145

(Y)(Y)

(Y)(Y)

Peptide A

Peptide BPeptide CPeptide D

Peptide EPeptide F

FIG. 1. Profile of hydrophilicity of large-T antigen as predictedby the program of Hopp and Woods (18). The bars on the abcissaindicate amino acid numbers in hundreds; the scale on the ordinateranges from +3 (top) to -3 (bottom) as defined in reference 18. Theamino acid sequence is that predicted between positions 106 and145; its location is indicated by the horizontal bar. A, alanine; D,aspartic acid; E, glutamic acid; F, phenylalanine; H, histidine; K,lysine; L, leucine; M, methionine; P, proline; Q, glutamine; R,arginine; S, serine; T, threonine; Y, tyrosine; and V, valine. Thelower horizontal bars indicate the sequences of the six peptides wehave synthesized. Where a tyrosine residue was added to one end ofthe peptide to facilitate coupling, that residue is shown in brackets.

after amino-peptidase digestion and acid hydrolysis, and byhigh-pressure liquid chromatography.

Initially, the peptides were purified extensively by ion-exchange chromatography before being attached to thecarrier proteins (peptides C and D). However, in studies inwhich a variety of peptides were used (16, 17; unpublisheddata), we and others (47) found antibodies could be raisedsuccessfully without purification of the peptide. Since thepurification step is the most time consuming, subsequentpeptides were cleaved from the resin, desalted, and coupleddirectly to the carrier protein without further purification.Analyses of peptide E revealed that some degradation hadoccurred during preparation. In spite of this, the partiallydegraded mixture of peptides was processed without furtherattempts at purification. The peptides were all coupled toBSA and in the case of peptide D also to ovalbumin. Serumwas collected weekly and tested by immunoprecipitation foranti-BSA and antipeptide activity. Because during thecourse of these and similar studies (16, 17; unpublished data)we found little correlation between the activity of a givenserum against its peptide and the corresponding parentprotein, sera were routinely tested for their ability to immuno-precipitate labeled SV40 large-T from extracts of SV40-transformed cells, irrespective of the antipeptide activitydetected.

Anti-peptide sera recognize SV40 large-T. Mouse cellswhich had been transformed by SV40 (SVA31E7) werelabeled with 32P, and cell extracts were prepared. Theseextracts were immunoprecipitated with the different antipep-tide sera, and the precipitates were analyzed on SDS-containing polyacrylamide gels (Fig. 2A). The positions ofauthentic large-T and the associated host cell phosphopro-tein NVT (p53) as precipitated by hamster antitumor cellserum are shown in Fig. 2A, lanes 2 and 11. All of theantipeptide sera were capable of immunoprecipitating SV40

17

17

il 4 NI VI V'

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PEPTIDE ANTIBODIES THAT RECOGNIZE LARGE-T 673

A. had very weak anti-large-T activity. Some of the antisera1 2 3 4 5 6 7 8 9 10 11 M precipitated additional proteins, for example, the ca. 110k-

and 33k-dalton proteins precipitated by anti-F serum (lane5). We assume these species are host cell proteins that cross-react with the antipeptide sera. They have not been studied

Large-T-~ ~ ~ ~ ~ ~ ~ -further.Partial proteolytic fingerprinting (44) verified that the 94k-

dalton proteins precipitated by anti-B and anti-F sera were- 60 indistinguishable from large-T immunoprecipitated by ham-

NVT- - -54 ster antitumor cell serum (Fig. 2B). Similar studies with theother antipeptide sera gave identical results (data not

-40 shown).We conclude that six independently synthesized peptides

elicited antibodies in rabbits capable of recognizing SV40large-T. However, the anti-large-T activity in the sera variedwidely, and almost all subsequent studies utilized only themost effective sera, that is, those against peptides B, C, andF.

94-*

b32 1 1T

c2 3 4

S.

Antibody purification. The antipeptide sera against B, C,D, and F were purified ca. 40-fold by affinity chromatogra-phy with immobilized peptide coupled to Sepharose. Anti-BSA activity was removed by a BSA-Sepharose precolumn.Anti-large-T activity was detected exclusively in the peptide-bound fraction, providing indirect evidence that the antiserawere indeed recognizing SV40 large-T through the peptidesequence against which they were raised. A more directdemonstration of this was provided by experiments such asthat shown in Fig. 3, in which immunoprecipitation of large-T was inhibited by addition of peptide.

A B C

I1 2 2 ---IPIVw

Large-T- *a

_l~~~~~~1 **

D

4 *G..

FIG. 2. Immunoprecipitation of large-T antigen with differentantipeptide sera. (A) Equal amounts of a 32P-labeled extract ofSVA31E7 cells were immunoprecipitated with a 10-,ul sample ofeach serum, and the precipitates were analyzed on SDS-containingpolyacrylamide gels. Lanes: 1, Normal hamster serum: 2 and 11,hamster antitumor cell serum: 3, anti-peptide B serum: 4. anti-C: 5.anti-F; 6, normal rabbit serum; 7, anti-ovalbumin-conjugated D; 8.anti-BSA-conjugated D; 9, anti-E; 10, anti-A. M. Marker proteinswith apparent molecular weights in thousands. (B) Partial proteoly-sis mapping of large-T precipitated by antipeptide sera. Labeledprotein was precipitated by hamster antitumor cell serum (a), byanti-B serum (b), or by anti-F serum (c) and excised from a

preparative-scale polyacrylamide gel. Samples (50 ,ul) of each were

then incubated with 0.1, 1.0, or 10 p.g of V8 protease (a. b, and c,lanes 2 through 4, respectively), as described in detail previously(44). Control incubation with no added enzyme is shown in lanes 1.M, Molecular weight markers as above.

large-T. Anti-peptide B, anti-peptide C, and anti-peptide Fsera (Fig. 2A, lanes 3, 4, and 5) had activity against large-Tcomparable to that of hamster antitumor cell serum. Theactivity in serum raised against peptide D, conjugated toBSA (lane 7) or ovalbumin (lane 8) and against peptide E(lane 9) was significantly lower, but specific immunoprecipi-tation was readily detected. Anti-A serum (Fig. 2A, lane 10)

NVT-

FIG. 3. Inhibition of immunoprecipitation of large-T with puri-fied antipeptide sera in the presence of peptide. Anti-B and anti-Fsera were purified by affinity chromatography over Sepharosecolumns to which the appropriate peptides had been coupled byeither end (see text for details and definitions). Samples of 32P-labeled SVA31E7 cells were immunoprecipitated by anti-amino B(A), anti-carboxy B (B), anti-amino F (C). and anti-carboxy F (D) inthe presence of either 20 ,ug of BSA (lanes 2). 132 ,ug of peptide B(lanes A3 and B3), or 123 jig of peptide F (lanes C3 and D3).Immunoprecipitation without any addition is shown in lanes 1.Other experiments have shown that 5 ,ug of appropriate peptide was

sufficient to inhibit precipitation with anti-amino B or anti-amino F.whereas 50 ,ug of peptide B was required with anti-carboxy B.

a2 1

4B. MT

60- *

54-

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674 PAUCHA, HARVEY, AND SMITH

For this, the antisera were further fractionated by usingimmobilized peptide which was coupled to activated Sephar-ose by either its amino or its carboxy terminus. Peptide Bwas originally coupled to BSA in two reactions, one usingbenzidine, which should react preferentially with the amino-terminal tyrosine residue, and a second using glutaralde-hyde, which should react with amino groups including thoseon the carboxy-terminal lysines. Peptide F was also coupledby using both benzidine and glutaraldehyde, and it tooshould have been anchored to BSA by either end of thepeptide. Before injection into rabbits, equal samples of thetwo fractions were mixed. In this way, we hoped to ensure

that the rabbit was exposed to a large number of configura-tions of coupled peptide.The use of peptide coupled to Sepharose through one end

of the molecule was designed to enrich for populations ofantibodies to the opposite, that is the exposed, end of thepeptide. Thus, that fraction of the antiserum raised againstpeptide B retained by Sepharose, with peptide B attachedthrough its amino terminus, is referred to as anti-carboxy B.The four purified fractions obtained against peptides B and

F retained activity against large-T (Fig. 3, lanes 1). Thisactivity was unaffected by addition of BSA to the immuno-precipitation reaction (Fig. 3, lanes 2), whereas addition ofpeptide B inhibited immunoprecipitation of large-T by bothanti-amino B and anti-carboxy B (Fig. 3A and B, lanes 3). Bycontrast, only the immunoprecipitation of large-T by anti-amino F (Fig. 3C, lane 3) was blocked by the addition ofpeptide F, and anti-carboxy F still precipitated large-T in thepresence of peptide (lane D3). This result provides evidencethat the separation of the antisera by using the differentforms of immobilized peptide does achieve fractionation ofthe antibody into different subpopulations. The result thatadded peptides does not inhibit the immunoprecipitation oflarge-T, even though the anti-large-T activity was retainedon immobilized peptide, was also obtained with anti-C andanti-D sera. We interpret this to mean that in the case ofantibodies recognizing the carboxy-terminal end of peptidesF, C, and D, the affinity of the antibody for large-T is higherthan for peptide alone.

Characterization of the antipeptide sera. (i) Specificity ofantisera. To examine the spectrum of activity of the sera andto confirm independently the position of large-T of determi-nants recognized by them, we prepared extracts of a numberof SV40-transformed cell lines, some of which containedabnormal large-T molecules, and immunoprecipitated themwith the different antisera. The data are summarized in Table1. The peptide antibodies were capable of immunoprecipitat-ing full-sized large-T from all the transformed cell extractsexamined. The 145k-dalton super-T from SV3T3 C120 cells(22) could also be immunoprecipitated. However, large-Tfrom cells productively infected with SV40 could not beimmunoprecipitated reproducibly by the peptide antibodies.In addition, the antibodies tested did not immunoprecipitatelarge-T synthesized in a rabbit reticulocyte cell-free systemin response to mRNA from infected or transformed cells(30), nor were they capable of precipitating large-T fromtransformed cells which had been electrophoresed in andeluted from SDS-containing polyacrylamide gels (20).

Swiss SV3T3 and MES2006 cells contain truncated large-T antigens with approximate molecular weights of 33k and22k, respectively (4). Analysis of the structure of the 33k-dalton species has shown that it extends beyond the aminoacid sequences represented in the synthetic peptides used inthis study; similarly, the 22k-dalton protein contains the bulkof the sequences (that is, at least until residue 131). In spite

TABLE 1. Immunoprecipitation of T-antigens with antipeptidesera"

Response with following antipeptide serum":Source of large-T (size)

A B C D E F

SVA31E7 (±) + + + (+) +Gel-eluted (94k) - - -Cell-free (94k)

Infected CV1 (94k)C120 (145k) +SWSV3T3 (94k) + + + +SWSV3T3 (33k) - - - -MES2006 (94k) + +MES2006 (22k) - -Taq-BamHI (70k) + + + +

' Immunoprecipitates of extracts of 32P-labeled infected or transformedcells were analyzed on SDS-containing polyacrylamide gels, as were theimmunoprecipitates of the ['5S]methionine-labeled products of translation ofmRNA in a nucleased rabbit reticulocyte lysate.

h +, Presence of a 94k-molecular-weight band on an autoradiogram of thedried gel: -, no detectable band: (+), weak band; *, irreproducible result, seetext for explanation. Reprecipitation of gel-eluted large-T was monitored byscintillation counting. -, No counts above background. Antitumor cell serumgave 95% reprecipitation. Each symbol represents results of at least threeseparate experiments.

of this, none of the antipeptide sera tested (B, C, D, and F)immunoprecipitated either the 33k- or the 22k-dalton trun-cated large-T species. By contrast, all of the antisera tested(B, C, D, and F) were capable of immunoprecipitating ashortened (70k-dalton) form of large-T synthesized in a lineof rat cells which had been microinjected with the Taq-BamHI fragment of SV40 DNA (12). The size of this proteinis consistent with its being translated from sequences entire-ly within the second exon of the SV40 early region, possiblybeginning at the first ATG corresponding to methionine 109(12). If this is the case, the 70k-dalton protein would containsequences corresponding to all the peptides used in thisstudy, except three amino acids in peptide A.These results suggested that the determinants recognized

by the antipeptide sera were not available or accessible insome forms of large-T known to contain the appropriateamino acid sequence and that the determinants could bedestroyed by denaturation with SDS. We therefore subfrac-tionated normal large-T from transformed cells to determinewhether the antibodies recognized only a certain subpopula-tion of molecules.

(ii) Antisera recognize large-T complexed with NVT. A 32p-labeled extract of SVA31E7 cells was analyzed by sucrosedensity gradient centrifugation (25). One-third of each frac-tion from the gradient was precipitated by hamster antitumorcell serum, and the remainder was precipitated by antipep-tide sera (Fig. 4). Figure 4A shows the three peaks of32P-labeled large-T immunoprecipitated by hamster antitu-mor cell serum. The 5 to 6S peak probably contains dimersof large-T, the 11 to 13S peak contains tetramers with somecomigrating NVT, and the 23 to 25S fraction contains thebulk of the labeled NVT in complex with large-T (19, 21). Asimilar distribution has been found in several other studies(1, 8, 10, 13, 25). Figure 4B shows immunoprecipitation ofthe same gradient fractions with anti-B serum, and Fig. 4Cshows it with anti-F serum. Only the rapidly sedimentingfraction of large-T containing the complex with NVT wasprecipitated by the antipeptide sera; fractions sedimenting at5 to 6S and at 11 to 13S were not immunoprecipitated.Identical results were obtained with anti-C and anti-D sera.To establish that this pattern of immunoprecipitation was

not a result of using 32P-labeled extracts, we repeated the

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experiment shown in Fig. 4 with an extract of cells whichhad been labeled with [35S]methionine for 2 h (Fig. 5).Antitumor cell serum (Fig. 5A) immunoprecipitated variousforms of large-T, as with the 32P-labeled extract, but the

relative amounts were different; in particular, the rapidlysedimenting forms of large-T were relatively poorly labeled.A similar result has been reported previously and interpretedto mean that large-T in complex with NVT is heavily

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fractionated on a 5 to 20% linear sucrose gradient as detailed in the text. One-third of each fraction was immunoprecipitated with hamster

antitumor cell serum (A), one-third with anti-B serum (B), and one-third with anti-F serum (C), and the immunoprecipitates were analyzed on

SDS-containing polyacrylamide gels. The direction of sedimentation is from right to left. Lanes C show large-T and NVT immunoprecipitatedfrom a sample of SVA31E7 extract with hamster antitumor cell serum. M, Markers of 94,000, 60,000, 54,000, and 40,000 apparent molecularweight.

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676 PAUCHA, HARVEY, AND SMITH

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FIG. 5. Immunoprecipitation of gradient-fractionated [35S]methionine-labeled large-T antigen with anti-B serum. A [35S]methionine-labeled extract was fractionated on two 5 to 20% linear sucrose gradients as described in the text. Both gradients were collected into one set oftubes, and then half of each of the resulting fractions was immunoprecipitated with hamster antitumor cell serum (A) and half wasimmunoprecipitated with anti-B serum (B). The direction of sedimentation is from right to left. Lanes C show large-T and NVTimmunoprecipitated from a sample of the starting material with hamster antitumor cell serum. M, Molecular weight markers.

phosphorylated and not newly synthesized (13, 25). FigureSB shows the proteins immunoprecipitated with anti-B se-rum. Again, large-T:NVT complexes were the predominantspecies detected. Small amounts of some forms with anintermediate sedimentation rate, but possibly not associatedwith NVT, were present, but light forms of [35S]methionine-labeled large-T were not present. It is known that several ofthe serine residues in the region recognized by the antipep-tide sera are phosphorylated in large-T; however, the experi-ments shown in Fig. 4 and 5 indicated that phosphorylationdid not substantially alter the patterns of immunoprecipita-tion observed. The antipeptide sera recognized rapidly sedi-menting forms of both 35S- and 32P-labeled large-T, and theydid not immunoprecipitate lighter forms.

Similar gradient analyses (data not shown) of the differentextracts listed in Table 1 showed that only that fraction (i) oflarge-T from infected cells, (ii) of the 145k-dalton super-Tfrom SV3T3 cells (22), and (iii) of the 70k-dalton large-T

from Taq-BamHI transformed rat cells (12) which was foundsedimenting rapidly in complex with NVT was immunopre-cipitated by the antipeptide sera. Lighter forms of large-Twere never immunoprecipitated. Earlier analysis had shownthat the 33k- and 22k-dalton truncated large-T molecules donot complex with NVT (4). Thus, all data were consistentwith the conclusion that the antipeptide sera only immuno-precipitate heavy forms of large-T, predominantly large-T:NVT complexes, not other forms of the protein.The unexpected specificity of the antipeptide sera prompt-

ed a number of control experiments to confirm that thedeterminant(s) recognized by the antibodies was indeedlocated on the large-T component of the complex.

(i) Western blotting (2) in which proteins present inextracts of SV40-transformed cells were separated by elec-trophoresis, transferred to cellulose nitrate, and reacted withpurified anti-B and anti-F demonstrated that the antiserareact directly with immobilized, 94k-dalton large-T (Fig. 6,

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PEPTIDE ANTIBODIES THAT RECOGNIZE LARGE-T 677

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FIG. 6. Western blots with antipeptide sera. Samples (50 ,ul) ofSVA31E7 cells (lanes 1, 2, and 4) or F9 teratocarcinoma cells (lane3) were either immunoprecipitated with 5 p.l of hamster antitumorcell serum (lanes 1 and 2) or not (lanes 3 and 4), electrophoresed,and transferred to nitrocellulose. The nitrocellulose strips wereincubated with anti-F (lane 1) or anti-B sera (lanes 2, 3, and 4) andthen with 125I-protein as described in the text. Autoradiographywas for 16 h. The faint band on lane 1 and on longer exposures oflane 2 in the apparent molecular weight range of 50k is probably dueto residual protein A binding to immunoglobulin heavy chains, sinceit is not present in the non-immunoprecipitated samples (lanes 3 and4). The ca. 33k-dalton protein in lanes 3 and 4 corresponds to thehost cell protein detected in the immunoprecipitates shown in Fig.2A (lanes 3, 4, and 5).

high-affinity antibody. The assumption made is that a low-affinity antibody will immunoprecipitate a fraction of theantigen in the first round, a further fraction in the secondround, and so on. Any unprecipitated antigen is recovered inthe final step by the high-affinity antibody.

In the experiment shown in Fig. 7, samples of a 32p_labeled extract of SVA31E7 cells were immunoprecipitatedwith hamster antitumor cell serum (Fig. 7A, lane 1), amonoclonal antibody which recognizes the amino-terminalend of the T-antigens, pAb4l9 (lane Bi), anti-carboxy F(lane Cl), anti-carboxy B (lane Dl), and a monoclonalantibody which recognizes a determinant located in thecarboxy-terminal half of large-T, pAb4l3 (lane El). Immu-noprecipitates were collected on S. aureus bacteria, andfurther samples of serum were added to the supernatants.This process was repeated twice (Fig. 7, lanes 2 and 3), andthen hamster antitumor cell serum was added. Both of theantipeptide sera tested were capable of removing the bulk ofthe large-T:NVT complex in a single step, and none wasrecovered by the antitumor cell serum (Fig. 7). The mono-clonal antibody pAb4l9 removed most of the complex in twosteps, whereas pAb4l3 immunoprecipitated only a smallfraction of the complex, and even after three rounds ofprecipitation, the bulk of the labeled complex was recoveredin the supernatant with the antitumor cell serum.Although experiments of this kind give only a rough guide

of relative affinity, the results obtained do not support theview that the antipeptide sera react specifically with large-T:NVT complexes simply because of low affinity.

lanes 1, 2, and 4). NVT present in the same extract or inextracts of normal cells or of F9 teratocarcinoma (21) did notreact with the purified antisera (lanes 1 through 4).

(ii) Uncomplexed NVT from F9 teratocarcinoma cells (21)could not be immunoprecipitated with the purified antiseraunder conditions identical to those described for the experi-ment shown in Fig. 2 (data not shown).

(iii) Experiments with mutants of SV40 with deletions inthe region spanned by peptides B and F (D. Kalderon andA. E. Smith, Virology, in press) showed that onlysmall amounts of large-T could be immunoprecipitated byanti-B or anti-F sera (or both) from extracts of cells trans-formed by appropriately deleted mutants (data not shown).When considered together with the observation that there isno homology between the predicted amino acid sequencesfor SV40 large-T (49) and mouse NVT (52), these datastrongly suggest that, as expected, the peptide antibodiesrecognize large-T directly and that their immunoprecipita-tion of large-T:NVT complex is not due to cross-reactivitywith NVT itself.Cascade immunoprecipitation. One explanation of the re-

activity of the antipeptide sera is that they are all of lowaffinity for large-T, and consequently, although they recog-nize all forms of large-T, they only immunoprecipitatecomplexed forms because the latter have multiple bindingsites which generate sufficient avidity to allow detection. Weattempted to measure the affinity of the antibodies for large-T by the same method that has been used with monoclonalantibodies to SV40 large-T, with which similar questionshave arisen (14, 15). In the so-called cascade assay, a givenextract is subjected to multiple rounds of immunoprecipita-tion with the same antibody followed by a final round with a

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FIG. 7. Cascade immunoprecipitation with antipeptide sera andmonoclonal antibodies. Samples of a 32P-labeled extract ofSVA31E7 cells were incubated with different sera, and the immuno-precipitates were collected on S. aureus bacteria. A further sampleof serum was added to each supernatant, and the process wasrepeated twice; finally, hamster antitumor cell serum was added toeach reaction. The bacterial pellets were washed and eluted, and theimmunoprecipitates were analyzed on SDS-containing polyacryl-amide gels. A, Hamster antitumor cell serum; B, pAb419; C, anti-carboxy F; D, anti-carboxy B; and E, pAb413. Lanes 1 through 3 ofA through E, Three successive rounds of immunoprecipitation witheach serum named; lane 4 of B through E, hamster antitumor cellserum. A preincubation step with either normal hamster serum (A)or normal rabbit serum (B through E) was included. Gradientanalyses of the extracts used in this experiment showed that the bulk(over 80%) of the labeled large-T present was in complex with NVT.This observation, together with losses incurred during the first threerounds of immunoprecipitation, accounts for the absence of freelarge-T in lanes 4 of B through D.

1 M

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678 PAUCHA, HARVEY, AND SMITH

DNA binding. The peptide antibodies recognize sequencesselected because they lie within the putative DNA bindingdomain on SV40 large-T. The finding that theyimmunoprecipitate only heavy forms of large-T made uppredominantly of large-T:NVT complexes was an unexpect-ed complication. Nevertheless, the complex is known tohave the ability to bind to DNA (34, 37), and we used theantisera to study DNA binding in vitro.

All the experiments used an assay in which extracts ofcells containing large-T were reacted with a mixture oflabeled DNA fragments. The large-T and any bound DNAwere subsequently immunoprecipitated, and the DNA frag-ment(s) was identified by gel electrophoresis (26).

Figure 8 shows the result of an experiment in which anEcoRII restriction enzyme digest of a plasmid containing thelarger EcoRI-BamHI fragment of SV40 DNA was end la-beled with [y-32P]ATP, mixed with an extract of SVA31E7cells, and subsequently immunoprecipitated with anti-car-boxy B and anti-amino B sera. Both antipeptide sera werecapable of immunoprecipitating specifically the 311-base-pair fragment which contains the SV40 origin of DNAreplication (Fig. 8, lanes 1 and 3). Immunoprecipitation wasblocked by added peptide B (Fig. 8, lanes 2 and 4). Additionof peptide B to control immunoprecipitation by using amixture of two monoclonal antibodies to SV40 large-T (withbinding sites at the amino and carboxy termini) had no effect(Fig. 8, lanes 5 and 6). Similar results were obtained withanti-F sera. These results indicated that the antipeptide sera

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not inhibit subsequent binding of DNA. Large-T:NVT complex waspurified from SVA31E7 cells by sucrose gradient centrifugation.Samples of purified complex were incubated with antibody before(lanes b) or after (lanes a) the addition of 10 ng of 32P-labeledEcoRII-digested SV40-containing plasmid DNA. Immune complex-es were collected and analyzed as described in the text. 1, Monoclo-nal antibodies pAb419 and pAb413; 2 through 4, 3, 6, and 12 ,ug ofanti-carboxy B, respectively; 5, 12 ,ug of anti-carboxy B withmonoclonal antibodies pAb419 and pAb413 added before DNA.Marker tracks (M) contain 0.5 ng of the DNA digest used in theexperiment with the length of each fragment in base pairs.

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FIG. 8. Immunoprecipitation by anti-B sera of large-T bound toSV40 origin-containing DNA. Samples of extracts of SVA31E7 cellswere incubated with 10 ng of 32P-end-labeled EcoRII digest of a

plasmid containing the larger EcoRI-BarnHI fragment of SV40 DNA(pSV328). Antibody was then added to each sample, and incubationwas continued. The immunoprecipitates formed were collected on

S. aureus bacteria and washed before being analyzed on 2% agarosegels. Lane 1, Anti-amino B serum; 2. anti-amino B plus 132 ,ug ofpeptide B; 3, anti-carboxy B serum: 4, anti-carboxy B plus 132 ,ug ofpeptide B; 5, a mixture of two monoclonal antibodies, pAb419 andpAb413; 6, pAb419 and pAb413 with 132 ,ug of peptide B. Markertracks (M) contain 0.5 ng of the DNA digest used in the experiment.Numbers refer to the length of the fragment in base pairs. The 311-base-pair fragment contains the origin of SV40 DNA replication.

could recognize complexes of large-T:NVT which had DNAbound to them.

Figure 9 shows the results of an experiment in whichantibody was bound to complex before DNA to test whetherthe presence of antibody could block subsequent binding ofDNA. The large-T:NVT complex was first purified bysucrose gradient centrifugation, and antibodies were addedeither before (Fig. 9, lanes b) or after (lanes a) incubationwith DNA. Control incubation with monoclonal antibodiesdirected against both ends of large-T showed that less DNAwas precipitated when antibodies were added before theDNA (Fig. 9 lane lb) than after (lane la). This suggests thatantibody binding induced a conformational change whichpartially inhibited subsequent DNA binding. The resultswith antipeptide serum are shown in Fig. 9, lanes 2 through4. Lanes 2a, 3a, and 4a show that the smallest amount ofserum used in this experiment, 3 p.g of purified anti-B (lane2a), was sufficient for maximal precipitation of DNA whenantibody was added after the DNA had been bound. Thisresult implied that each molecule of complex with boundDNA must have had at least one antibody molecule alsobound to it under these conditions. When antibody wasbound before DNA (Fig. 9, lanes 2b, 3b, and 4b), increasingthe amount of antibody added from 3 p.g (lane 2b) to 12 ,ug(lane 4b) did not significantly decrease the amount of DNAsubsequently bound to the antibody:large-T:NVT complex.Other experiments in which the amount of serum added wasincreased 10-fold gave the same result. We have not yetmade a more detailed kinetic analysis to ascertain whethermore subtle effects of the peptide antibodies on DNAbinding can be measured. However, we conclude that underthe conditions used here, which include apparent antibodyexcess, the presence of peptide antibody attached to the

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PEPTIDE ANTIBODIES THAT RECOGNIZE LARGE-T 679

large-T:NVT complex does not prevent it from bindingspecifically to the origin of replication on SV40 DNA.

DISCUSSION

Peptide antibodies. We have raised antibodies against sixpeptides of overlapping sequence between amino acids 106and 145 of the predicted amino acid sequence of SV40 large-T within a putative DNA binding domain. All the antibodiesimmunoprecipitated large-T, but the amount of protein im-munoprecipitated varied widely. In general, the longer pep-tides elicited a stronger anti-large-T activity than the shorterones. The major exception to this, peptide E, was partiallyfragmented during preparation. Since the peptide was notpurified before coupling and injection, the amount of intactpeptide actually injected was low, and this probably ex-plains, at least partially, the poor response obtained. In otherstudies, we found that fragmentation of peptides at prolinelinkages occurs commonly during the sodium-in-liquid am-monia treatment required to remove the tosyl groups used toblock arginine residues (27). If combinations of proline andarginine can be avoided or an alternative chemistry isemployed, we find that purification of peptides before injec-tion is not essential, as already concluded by Sutcliffe et al.(47). However, possible complications that may arise be-cause of this, such as those encountered with peptide E,must always be borne in mind.The spectrum of activity of the peptide antibodies studied

here was puzzling until it became clear that they onlyimmunoprecipitate heavy forms of large-T, composed pre-dominantly of large-T in complex with NVT. Lighter formsof large-T were not detected. This property can account forthe failure to immunoprecipitate truncated T-antigens, be-cause Chaudry et al. (4) showed that neither the 33k- nor the22k-dalton forms of large-T associate with NVT. On theother hand, the 70k-dalton T-antigen found in Taq-BamHIcells does associate with NVT (12) and can beimmunoprecipitated by the peptide antibodies. The failure toimmunoprecipitate noncomplexed large-T may also explainthe variability found when precipitating large-T from ex-tracts of SV40-infected monkey cells. The large-T:NVTlcomplex is known to be unstable in these extracts (15. 25).Control Western blot experiments (2) confirmed that thespecificity for large-T:NVT complexes did not arise becauseof some unexpected cross-reactivity between the peptideantisera and NVT.

It could be argued that the peptide antibodies studied hererecognize the large-T:NVT complex preferentially because itcontains multiple recognition sites and fail to immunoprecip-itate smaller forms because of low affinity. Such a modelwould predict that at any given time during the immunopre-cipitation procedure the probability of at least one antibodybeing attached to and immobilizing a large-T moleculepresent in a large-T:NVT complex is greater than for anisolated large-T molecule. The only experimental data thataddress this question are derived from cascade immunopre-cipitation and do not support the view that the antibodies areof low affinity. If low affinity is not the explanation of theresults obtained, we are forced to conclude that the peptideantisera recognize a feature on large-T that is present inheavy forms but absent or hidden in other forms.DNA binding domain. The peptides used in this study were

selected because they come from a region thought to be partof the DNA binding domain. Indeed, some evidence sug-gests that the limits of the domain can be refined muchfurther. Prives and colleagues (33) showed that an 82k-dalton

protein synthesized in vitro in response to SV40 cRNA bindsto SV40 multiple-origin variant DNA immobilized on cellu-lose. The 82k-dalton protein is believed to initiate at the firstATG distal to the splice in the SV40 large-T mRNA,corresponding to amino acid 109 (31). Morrison and col-leagues (29) reported that an N-terminal fragment of large-Tproduced by limited proteolysis and comprising residues 1 to130 retains the ability to bind to the origin-containing frag-ment of SV40 DNA. If both these molecules contain allsequences necessary and sufficient for binding to SV40origin DNA, one domain may comprise only residues 109through 130. The peptide B used in these studies corre-sponds to amino acids 111 through 129. With this in mind, wehoped that the antibodies that recognize these sequenceswould be of value in future studies of DNA binding.The results obtained show that the presence of antibody

bound to large-T did not prevent DNA from binding. Theinterpretation of these results is complicated by the findingthat the antibodies only recognize heavy forms of large-T,predominantly large-T:NVT complexes. Estimates of thenumber of large-T molecules per unit complex have varied,although four has recently been reported as the number percomplex isolated from rat cells (9). Under the conditionsused to obtain Fig. 9, therefore, the concentration of anti-body should have been sufficient to saturate the total numberof binding sites in the complex. Nevertheless, DNA bindingwas not significantly reduced. There are many possibleexplanations of this result. It could be argued that the affinityof binding of DNA to complex is higher than the affinity ofbinding of the peptide antibody, so that DNA could displaceany antibody bound. It is also possible that a conformationalchange induced by the binding of antibody to one moleculeof large-T excludes subsequent binding at the equivalent sitein at least one of the other large-T molecules present in thecomplex, leaving that site free to bind to DNA. A furtherexplanation of our results is that the DNA binding domain onlarge-T, at least as present in the large-T:NVT complex. isnot within the site defined by the studies outlined above.Further work is required to distinguish among these alterna-tives.

Antibody specificity. We do not know the explanation forthe specificity of the peptide antibodies. The secondary-structure predictions, the accessibility of arginine 130 totrypsin digestion, and the multiple phosphorylation sites inthis area all argue that at least a portion of this region is at thesurface of large-T. Although these properties were deter-mined for unfractionated populations of large-T, it is difficultto understand why the peptide antibody recognition sitesshould be inaccessible or masked in all but the heavyspecies. The finding that several nonoverlapping peptidesyield antibodies with the same properties tends to argueagainst a trivial explanation such as steric hindrance from aside-chain modification at some point in the recognitionsequence.

It is possible that association with NVT stabilizes aparticular conformation of large-T which is required forimmunoprecipitation by peptide antibodies. A similar argu-ment is put forward by Leppard and Crawford (20) to explainthe observation that monoclonal antibodies raised againstgel-purified, denatured NVT (p53) efficiently immunoprecip-itated NVT in complex with large-T but not non-associatedor gel-eluted NVT. In the cases reported here, however, theresults are even more surprising because the antisera usedare polyclonal. If this explanation is correct, it also mustmean that the conformation induced by complexing withNVT is present in immobilized large-T as recognized in the

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680 PAUCHA, HARVEY, AND SMITH

Western blot experiments but not present in, or at least notsufficiently stable to allow immunoprecipitation of, SDS gel-eluted large-T or large-T synthesized in vitro. The difficultyin understanding results of this kind highlights the limitedvalue of computer-assisted secondary-structure and immu-nogenicity predictions based on the primary amino acidsequence of proteins. Even if the predictions were absolute-ly correct, it appears to be impossible at present to predictthe pattern of reactivity of the antibodies likely to beobtained.The peptide antibodies produced in this study may prove

less useful than anticipated for studies on DNA binding.However, they do not have the property of recognizing thelarge-T:NVT complex. We are currently exploiting this topurify the complex by using immunoaffinity chromatographyon immobilized antibody.

ACKNOWLEDGMENTS

We thank Ralph Faulkes for synthesis of the peptides and PeterGillett for preparing and purifying the antisera. We also thank DanKalderon for the pSV328 DNA used in these experiments. We aregrateful to him and to Graham Belsham for critical reading of themanuscript, to our colleagues for helpful discussion, and to LydiaPearson for typing the manuscript.

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1. Bradley, M. K., J. D. Griffin, and D. M. Livingston. 1982.Relationship of oligomerization to enzymatic and DNA bindingproperties of the SV40 large-T antigen. Cell 28:125-134.

2. Burnette, W. N. 1981. "Western blotting:" electrophoretictransfer of proteins from sodium dodecyl sulfate-polyacryl-amide gels to unmodified nitrocellulose and radiographic detec-tion with antibody and radioiodinated protein A. Anal. Bio-chem. 112:195-203.

3. Carroll, R. B., L. Hager, and R. Dulbecco. 1974. Simian virus 40T-antigen binds to DNA. Proc. Natl. Acad. Sci. U.S.A.71:3754-3757.

4. Chaudry, F., R. Harvey, and A. E. Smith. 1982. Structure andbiochemical functions of four simian virus 40 truncated large-Tantigens. J. Virol. 44:54-66.

5. Chou, P. Y., and G. D. Fasman. 1978. Prediction of thesecondary structure of proteins. Adv. Enzymol. 47:45-148.

6. Clark, R., D. P. Lane, and R. Tjian. 1981. The use of monoclo-nal antibodies as probes of simian virus 40 T-antigen ATPaseactivity. J. Biol. Chem. 256:11854-11858.

7. Clark, R., K. Peden, J. M. Pipas, D. Nathans, and R. Tjian.1983. Biochemical activities of T-antigen proteins encoded bysimian virus 40 A gene deletion mutants. Mol. Cell. Biol. 3:220-228.

8. Fanning, G., K.-H. Westphal, D. Brauer, and D. Cortin. 1982.Subclasses of simian virus 40 large-T antigen: differential bind-ing of two subclasses of T-antigen from productively infectedcells to viral and cellular DNA. EMBO J. 1:1023-1028.

9. Freed, M. I., I. Lubin, and D. T. Simmons. 1983. Stoichiometryof large-T antigen and pp53 in complexes isolated from simianvirus 40-transformed rat cells. J. Virol. 46:1061-1065.

10. Gidoni, D., A. Scheller, B. Barnet, P. Hantzopoulos, M. Oren,and C. Prives. 1982. Different forms of simian virus 40 largetumor antigen varying in their affinities for DNA. J. Virol.42:456-466.

11. Gluzman, Y., and B. Ahrens. 1982. SV40 early mutants that aredefective for viral DNA synthesis but competent for transforma-tion of cultured rat and simian cells. Virology 123:78-92.

12. Graessman, M., and A. Graessmann. 1982. Simian virus 40cRNA is processed into functional mRNA in microinjectedmonkey cells. EMBO J. 1:1081-1088.

13. Greenspan, D. S., and R. B. Carroll. 1981. Complex of simianvirus 40 large T antigen and 48,000 dalton host tumor antigen.Proc. Natl. Acad. Sci. U.S.A. 78:105-109.

14. Gurney, E. G., R. 0. Harrison, and J. Fenno. 1980. Monoclonalantibodies against simian virus 40 T antigens: evidence fordistinct subclasses of large T antigen and for similarities amongnonviral T antigens. J. Virol. 34:752-763.

15. Harlow, E., L. V. Crawford, D. C. Pim, and N. M. Williamson.1981. Monoclonal antibodies specific for simian virus 40 tumorantigens. J. Virol. 39:861-869.

16. Harvey, R., R. Faulkes, P. Gillett, N. Lindsay, E. Paucha, A.Bradbury, and A. E. Smith. 1982. An antibody to a syntheticpeptide that recognises SV40 small-t antigen. EMBO J. 1:473-477.

17. Harvey, R., B. A. Oostra, G. J. Belsham, P. Gillett, and A. E.Smith. 1984. An antibody to a synthetic peptide recognizespolyomavirus middle-T antigen and reveals multiple in vitrotyrosine phosphorylation sites. Mol. Cell. Biol. 4:1334-1342.

18. Hopp, T. P., and K. R. Woods. 1981. Prediction of proteinantigenic determinants from amino acid sequences. Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828.

19. Lane, D. P., and L. V. Crawford. 1979. T antigen is bound to ahost protein in SV40-transformed cells. Nature (London)278:261-263.

20. Leppard, K., and L. V. Crawford. 1983. Monoclonal antibodiesdisplaying a novel species specificity for the primate transfor-mation-related protein, p53. EMBO J. 2:1457-1464.

21. Linzer, D. I. H., and A. J. Levine. 1979. Characterization of a54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.Cell 17:43-52.

22. Lovett, M., C. E. Clayton, D. Murphy, P. W. J. Rigby, A. E.Smith, and F. Chaudry. 1982. Structure and synthesis of asimian virus 40 super T-antigen. J. Virol. 44:963-973.

23. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning. A laboratory manual, p. 122-123. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

24. Maxam, A. M., and W. Gilbert. 1980. Sequencing end-labelledDNA with base-specific chemical cleavages. Methods Enzymol.65:499-560.

25. McCormick, F., and E. Harlow. 1980. Association of a murine53,000-dalton phosphoprotein with simian virus 40 large-T anti-gen in transformed cells. J. Virol. 34:213-224.

26. McKay, R. D. G. 1981. Binding of SV40 T-antigen relatedprotein to DNA. J. Mol. Biol. 145:471-488.

27. Merrifield, R. G. 1963. Peptide synthesis on solid supports. J.Am. Chem. Soc. 85:2149-2158.

28. Montenarh, M., W. Deppert, and R. Henning. 1982. Mapping ofa DNA-binding domain of simian virus 40 T-antigen using non-defective adenovirus-2-simian virus 40 hybrid viruses. FEBSLett. 142:129-132.

29. Morrison, B., M. Kress, G. Khoury, and G. Jay. 1983. Simianvirus 40 tumor antigen: isolation of the origin-specific DNA-binding domain. J. Virol. 47:106-114.

30. Paucha, E., R. Harvey, and A. E. Smith. 1978. Cell-free synthe-sis of simian virus 40 T-antigens. J. Virol. 28:154-170.

31. Paucha, E., and A. E. Smith. 1978. The sequences between 0.59and 0.54 map units on simian virus 40 DNA code for the uniqueregion of small-t antigen. Cell 15:1011-1020.

32. Prives, C., B. Barnet, A. Scheller, G. Khoury, and G. Jay. 1982.Discrete regions of simian virus 40 large T antigen are requiredfor nonspecific and viral origin-specific DNA binding. J. Virol.43:73-82.

33. Prives, C., Y. Beck, and H. Shure. 1980. DNA binding proper-ties of simian virus 40 T-antigens synthesized in vivo and invitro. J. Virol. 33:689-696.

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