Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses.

JOURNALOFVIROLOGY,Nov. 1983,p.352-360

0022-538X/83/110352-09$02.00/0

Copyright©1983,American SocietyforMicrobiology

Vol.48,No. 2

Isolation of Monoclonal Antibodies That Recognize the

Transforming Proteins of Avian Sarcoma Viruses

LEAH A. LIPSICH, ANITA J. LEWIS, ANDJOAN S. BRUGGE*

Department of Microbiology, StateUniversity of New YorkatStonyBrook, Stony Brook, New York 11794

Received9May 1983/Accepted 1 August 1983

Thirteen clones of hybrid cells which synthesizeantibodies directed against the Rous sarcomavirus (RSV) transforming protein, pp605'', were isolated. Mouse

myeloma cells werefused with spleen cells from mice that had been immunized

with purified pp6Osrc from bacterial recombinants which direct the synthesis of the RSV src gene. The hybridomas which survived the selection medium were

screened by immunoprecipitation of pp605'' from 32P-labeled lysates of RSV-transformed cells. Monoclonal antibodies produced by subclones derived from 13 hybridomas recognized pp6O r' encoded by the Schmidt-Ruppin and Prague strains of RSV and the cellularhomolog of pp6O5''. Antibody from clone 261 hada

high affinity for the viralyesgeneproduct, and antibodies from clones 443 and 463

recognized the transforming proteins encoded by viruses containing the related transforming genesfps andros. Several otherclones hadalowaffinity for the viral yes,fps, and rosgeneproducts which could be detected by in vitro

phosphoryla-tion of the transforming proteins after immunoprecipitaphosphoryla-tion with the monoclonal antibody. All of the monoclonal antibodies allowed phosphorylation of pp60S'C and casein inan immune complex-bound reaction.

Rous sarcoma virus (RSV) has proven to be anidealsystem toinvestigate themechanism of oncogenic transformation by retroviruses.

Ge-netic studies have revealed that a single viral gene product is responsible for the events in-volved inoncogenic transformation (reviewedin

reference 26). The protein product of thisgene,

designated

pp6Osrc,

was first identified by

im-munoprecipitation of radiolabeled lysates of RSV-transformed cells,

using

antiserum from rabbits bearingtumors induced by RSV (desig-natedTBR serum) (5).

Thepp6OsrC proteinhas been shown to possess a

tyrosine-specific

kinase activity which has been postulated to be essential for the events

involved in oncogenic transformation by RSV (8, 9, 20, 24, 34). Several substrates of

pp60sr(-mediated phosphotransferase have been

identi-fied; however, the functional significance of phosphorylation of these substrates remains to

be elucidated (4, 6, 10-12, 18, 31, 33).

Uninfected cells containageneproductwhich isstructurally andfunctionally analogoustothe viral transforming protein, designated

pp6Oc-src

(7, 29, 32). It is believed that the transforming

gene of RSVwasderived from the cellular gene

encoding

pp6Oc-rc

(36). The cellular src gene

productalsodisplays tyrosine-specific

phospho-transferaseactivity (29, 32).

Thetransforming proteinsencodedbyseveral other oncogenic avian and mammalian

retrovi-ruseshavealsobeen shownto possess

tyrosine-specific protein kinase activity (1, 2, 13, 14, 21, 39). Recently, it has been shown that the RSV

transforming protein shares considerableamino

acidhomology with these other tyrosine-kinase transformingproteins (19, 23, 35, 37).

The antiserum from tumor-bearing animals

which has beenused for the analysis ofpp6Osrcis apolyclonal antiserum. Although this antiserum hasproven tobeinvaluable fortheidentification of this protein and preliminary characterization of itsphosphotransferase activity, thereare sev-eral drawbacks to its use. First, TBR serum

contains antibodiestoviral structuralproteinsas well as topp6Osrc. Second, most of the antisera

raised in rabbits infected with the

Schmidt-Ruppin (SR) strain of RSV do not recognize

pp6Osrcfrom other strains of RSVorthenormal cellular homolog of

pp6Osrc,

and none of the TBR serarecognizenon-gag-encoded regionsof thetransforming proteins fromviruses carrying

the related transforminggenes,fps,yes, orros.

Third,theantigenicdeterminantsrecognized by

TBR are extremely sensitive to denaturation.

Finally, most TBR-derived antibody molecules inactivate the phosphotransferase activity of

pp6Osrc onexogenous substrates.

To circumvent many of theseproblems asso-ciated with TBR serum, we have prepared monoclonal antibodies to the

pp6Osrc

protein. In this study, we report the isolation of13 hybrid

352

on November 10, 2019 by guest

http://jvi.asm.org/

353

myeloma cell lines producing monoclonal

anti-bodies against pp60src. We have tested these antibodies for precipitation of pp60src derived from other strains of RSV, the transforming

proteins encoded by thefps, yes, androsgenes,

and thecellularhomolog ofpp60srcinavianand mammalian cells. We have also examined the ability of these antibodies to allow

pp605sr,_medi-atedphosphorylation ofcasein.

MATERIALS AND METHODS

Cellsand viruses. Chickenembryo fibroblasts were prepared from 11-day-old gs-minus embryos (SPAFAS, Inc., Norwich, Conn.). The Prague (PR) (subgroup A) strain of RSV was obtained from T. Parsons; the SR (subgroup A) strain of RSV and Yamaguchi 73 (Y73) sarcoma virus were obtained from H. Hanafusa; PRCIIsarcomaviruswasobtained from K. Beemon; and UR-2 sarcoma virus was ob-tained fromP. Balduzzi.SRD-3T3 cells were obtained by infection of BALB-3T3 cells withSRD-RSV,using polyethyleneglycol.

Preparation of pp605rc for immunization. The src gene product waspurified from the particulate fraction ofEscherichia coli cells which carry aplasmid con-tainingthe srcgenefusedto aplasmid containing the UV5 lac operator-promoter and24nucleotides of the

P-galactosidase

gene. Bacteriafrom 500 mlof culture

medium werelysed, treatedwith DNase, and solubi-lizedasdescribedpreviously (15-17). Theparticulate cellular material was pelleted by centrifugation at 16,000 x gfor30min. (This materialwasgenerously provided by Raymond Erikson.) The pellet material wassolubilized byboilingfor1min inelectrophoresis sample buffer (16). pp60src was separated from other bacterialproteins by electrophoresison a7.5% poly-acrylamidegel. pp60sr" wasdetectedby

electrophore-sisof32P-labeled markerpp60srcadjacenttothe bacte-rial matebacte-rial. pp60srcwaseluted from thegel in 0.1% sodiumdodecylsulfate(SDS)-50mMammonium car-bonate.

Preparationofmonoclonal antibodies. A mouse my-eloma cell line of BALB/c origin designated X63-Ag8.653(obtained from the SalkInstitute,SanDiego, Calif.) was used for the fusions. This line does not expressimmunoglobinheavyorlightchains and there-forepermits thegeneration ofhybrids secretingpure monoclonal antibodies. The cells are maintained in culture inRPMI 1640medium(GIBCO,GrandIsland,

N.Y.) supplemented with 10% fetal calfserum(Flow Laboratories, McLean, Va.) and 100 U ofpenicillin and 100 ,ug of streptomycin per ml. Frozen stocks were kept inliquid nitrogen in 10%dimethyl sulfox-ide-90% fetal calfserum, andcells were thawed ap-proximately1 weekbeforeusefor fusion.

Mousespleencells. BALB/c mice (3to 5 weeksold) were injected intraperitoneally three times at weekly intervals with ca. 20 to 50 ,ug of the purified viral pp6Osrc protein cloned in E. coli. The final injection was given intravenously and contained ca. 5 ,ug of

pp6W"rc in phosphate-buffered saline. Fourdays after thefinal injection, the mice were bledand sacrificed by cervical dislocation, and their spleens were re-moved.Spleencells were thenpreparedby teasingthe spleen apart with needles into RPMI 1640 medium.

Erythrocytes were lysed by treatment with 0.83% ammoniumchloride, and spleen cells were then count-edonahemacytometer.

Cell fusionwascarriedoutbyamodification of the method ofLevy and Dilley (25). Briefly, spleen and myeloma cellswerepelleted together inaratio offour spleen cellsto onemyeloma cell. The pelletwasgently suspended in 2 ml of 35% polyethylene glycol (molecu-lar weight; 1,000; Koch-Light, Coinbrook,

Bucking-hamshire, England) in RPMI 1640 medium, and the cells were immediately centrifuged at 230 x g for 6 min.Thepolyethylene glycolwasthen aspirated,and thefused cellsweresuspended in RPMI1640medium supplemented with 20% fetal calf serum and 10% NCTC 109 medium(M.A. Bioproducts, Walkersville,

Md.). The cells were placed in a T-150 flask and incubated overnight at 37°C underan atmosphere of 5% C02-95% air. The following day, hypoxanthine, aminopterin, and thymidine were added to the medium togiveafinal concentration of10-4Mhypoxanthine,4 x 107 M aminopterin, and 1.6 x 10-5M thymidine, and the cells were portioned into 96-well microtiter dishes, allowing ca. 105 spleen cells per well. Two weeks after the fusion, medium samples were taken from the wells containing clones and assayed for antibody. Medium from 500 clones was taken and tested forimmunoprecipitation of radiolabeled pp6Osr' asdescribed below. Of the 500 clones tested, 16 were foundtobepositive for precipitation ofpp60Orc. Posi-tiveclones were transferred to 24-wellmicrotiter dish-esandfed with hypoxanthine-thymidine medium (as above, butomitting the aminopterin). When the cells weresufficiently dense, the media were sampled and again assayed. Clones remaining positive were then grown up, and frozenstocks were made.Cells retained in culturewereroutinely grown in RPMI 1640 medium with 10% fetal calf serum andantibiotics. To ensure monoclonality and long-term stability ofantibody pro-duction, strongly positive clones were subcloned by limiting dilution.

Concentration of monoclonal antibodies from medi-um and preparation of purified IgG. Medium was harvested fromhybridoma cells grownatadensity of ca. 106 cells per ml. This medium was stored under sterile conditionsat4°C until furtherprocessing. Con-centration of the monoclonal antibodies was per-formed by precipitation with60%o ultrapure ammoni-um sulfate (Schwarz/Mann, Orangeburg, N.Y.) and dialysis inone-tenth of the starting volume of phos-phate-buffered saline with 0.02% sodium azide. The antibodieswere storedat 4°C. Freezing and thawing results in a considerable reduction in the titer of the antibody. Immunoglobulin G (IgG)waspreparedfrom the ammonium sulfate-concentrated medium by the followingmethod. Concentrated medium(10ml) was incubated for2h at4°C with 200,i.1 ofswollenprotein A-Sepharose beads (Sigma Chemical Co., St. Louis, Mo.). The beadswere then washed three times with 100mMTris-hydrochloride (pH 8). IgGwaselutedby incubationwith 0.5ml of100mMglycine (pH 3). After sedimentation ofthe beads, the supernatant fraction wasneutralized immediately with 1 M Tris-hydrochlo-ride (pH 8.0). IgG was stored at 4°C and was never subjectedto freezingandthawing. Thefinal concen-tration ofIgGwaslessthan 0.1 p.g/p.l.

Sera. Monoclonal antibody to p19 was obtained from David Boettiger. TBR serum was prepared as

on November 10, 2019 by guest

http://jvi.asm.org/

354 LIPSICH, LEWIS, ANDBRUGGE

describedpreviously (5) fromarabbitbearingatumor induced by RSV. This antiserum containsantibodyto pp6Osrc aswell as tothe gag gene products. Rabbit antiserum directed against the E. coli-produced

pp6Osr, proteinwas agenerousgift fromR. L. Erikson (16).

Immunoprecipitation. Forthepreparation of animal cell extracts, cultures were labeled for4 h with32Pi

(0.5 mCi/ml, carrier-free; ICN Pharmaceuticals Inc., Irvine, Calif.) in phosphate-free medium. Cellswere lysed and clarified asdescribedpreviously (5). Either ammonium sulfate-concentrated mediaorpurified IgG

from the hybridoma cell lines were utilized for im-munoprecipitation of radiolabeled lysates. Lysates

were incubated 45 min with each monoclonal anti-body, 20 min with goatanti-mouseIgG (Meloy Labo-ratories, Springfield, Va.), and precipitated with the proteinA-containing bacteria,Staphylococcusaureus (22). The bacterial-bound immune complex was washed three times with radioimmunoprecipitation

(RIPA) buffer (5), and the immunoprecipitated pro-teins were eluted and analyzed on 7.5%

SDS-poly-acrylamide gels.

Detection ofphosphotransferaseactivity.i. Phosphor-ylationof casein. Protein kinase activitywas assayed

by phosphorylation of casein. pp6O,r' bound to S. aureus which had been immunoprecipitated with monoclonal antibodies was suspended in 5 mM

MgCl2-20 mM Tris-chloride (pH 7.2) and incubated with 5 pg of casein(Sigma). After theaddition of10 p.Ciof[y-32P]ATP (ICN), thereactionwas incubated at4°C for 20 min. The reactionwasterminatedbythe addition ofSDS-sample buffer and subjectedto elec-trophoresison10%SDS-polyacrylamidegelsfollowed byautoradiography todeterminethephosphorylation

ofcasein.

ii. In vitro phosphorylation of pp6osrc, pp90Yes,

ppllofPs,

and

pp68srS.

In vitro phosphorylation of

pp6O-src wasassayed byutilizingpp6o0-sr, boundtoS. aureus which had been precipitated from a lysate prepared from a12-day embryonic chicken brain as described above. The immunecomplex was suspend-ed in 5 mM

MgCl2-20

mMTris-chloride (pH 7.2)-10

,uCi of[-y-32P]ATPandallowed toincubateat4°Cfor 20min. TheprecipitateswerewashedoncewithRIPA medium and thensuspendedinSDS-samplebuffer and subjected toelectrophoresis on 7.5%

SDS-polyacryl-amidegels followed byautoradiography todetermine

phosphorylation ofpp6Osrc. Asimilarprocedure was utilizedfor detection ofphosphorylation of the trans-forming proteins from Y73-, PRCII-, or UR-2-trans-formed chicken cells, except that 5 mM MnCI2 re-placed MgCl2.

RESULTS

Weisolated13hybridomacelllinesproducing antibodies which recognize

pp6Osrc.

These cell

lines were prepared byfusion ofmyelomacells with spleen cells of BALB/c mice immunized with thesrcproteinextracted fromE. coli cells

carrying

aclonedsrcgene.Five hundred clones

survived the selection procedure, and medium from each clone was screened for antibody directedagainstpp6Osr, byimmunoprecipitation of

lysates

from

32P-labeled

PR-RSV-transformed chicken cells. Sixteen wells contained clones

J. VIROL.

pp9O- ..S...

Pr76-

pp5O-1 2 3 4 5 6 7 8 9 10 11 12 13 14 FIG. 1. Proteins immunoprecipitated from 32P-la-beled Prague RSV-transformed chicken cells. Alysate from a 100-mm culture of 32P-labeled Prague RSV-transformed chicken cells was immunoprecipitated with 5 ,ul of TBR serum (lane 1) or 100,u1 of concen-trated medium from hybridoma cells as described in the text. Lane2,monoclonal antibody 69; lane 3, 78; lane 4,127; lane 5, 191;lane 6, 199; lane 7, 200; lane 8, 261; lane 9, 273; lane 10, 327; lane 11, 443; lane12, 450; lane 13; 463;lane14, 492.

positive for pp6Osr( precipitation. All 16clones

weresubcloned by limiting dilution to assure the

monoclonality of each hybridoma. Of the origi-nal 16clones, only 3 clones have ceased

produc-ingantibodymolecules which recognize

pp60src.

Figure 1 displays an autoradiogram of a gel

containingthe

32P,-labeled

proteins

immunopre-cipitatedfrom Prague RSV-transformed chicken

cells, usingTBR serum(lane 1) or concentrated

medium from the antibody-producing hybri-doma cells (lanes 2 through 14). TBR serum

immunoprecipitated pp60src as well as Pr76, the

initial translation product of the gag gene, and twocellular proteins of Mr 90,000 (90K) and 50K which are associated in a complex with a small percentage of pp6Osr( molecules (3, 28). The

monoclonal antibodies immunoprecipitated

pp60src aswell asthe two cellular proteins pp9O andpp5O. Antibodies443 and 463 (lanes 11 and 13)consistently precipitated lesspp9O andpp5O relative to pp6O

rc,

suggesting that pp9O and

pp5Omayinterferewith recognition of the deter-minants recognized by these antibodies. Anti-bodies 443 and 463 also precipitated another

32p-labeled protein ofMr 68K which was found to contain phosphoserine and phosphotyrosine

(datanot shown). The identity of this protein is underinvestigation. Allof the monoclonal anti-bodies which recognized pp6Osr, from cells in-fected with PR-RSV also recognized SR virus-encoded pp6osrc (including mutant viruses derived from the PR and SR strains of RSV which carry temperature-sensitive defects in thesrc gene; data not shown). The identity of

pp601S1C was confirmed by partial proteolytic

cleavagewith V8 protease (data not shown). Cross-reactionwith cellularpp6OS'. Figure 2A

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.491.255.451.64.201.2]

displays theimmunoprecipitation of32P-labeled proteins fromuninfected chicken cells.IgGfrom all of the hybridomas precipitated a protein which comigrated with the 60K protein precip-itated with serum directed against the pp60src protein expressed in E. coli (16). Theidentity of

these proteins aspp6Osr( wasconfirmed by par-tial proteolytic peptide analysis with V8 prote-ase (data not shown). In addition, monoclonal

antibodies 273 and 327 (lanes 7 and 8) were

found to specifically immunoprecipitate the hu-man and mouse cellularsrc proteins (data not

shown; other antibodies not tested).

Figure 2B displays the phosphorylation of pp6O'-src'afterimmunoprecipitationwithIgG

puri-fied from the hybridoma medium. This assay wasperformed withahighlyconcentratedlysate from fresh chicken brain tissue and appears to be a more sensitive assay of pp60'-sr, than immunoprecipitation of32P-labeled chicken cell

lysates.Allofthemonoclonalantibodies

precip-itated 60K protein which was phosphorylated aftertheadditionof[-y-32P]ATP tothe immuno-precipitated proteins (antibody 199 notshown). The identity of this 60K protein which was

phosphorylated in this assay as

pp6O'-sr(

was

confirmedbypartial

proteolytic peptide

analysis

with V8 protease. These results suggest that all

ofthemonoclonal antibodiesrecognize determi-nants which are shared with pp6O(sr. In both

assays shown in Fig. 2, antibodies 273 and 327

precipitatedhigherlevels ofpp6Osr' than didthe

othermonoclonal antibodies.

Cross-reaction with other viral transforming proteins. Recentinvestigations havefound that the

protein

products ofthe

transforming

genesof other avian sarcoma viruses bear considerable

amino acid sequencehomology with

pp6Osr(

(19, 23, 35, 37). To determine whether any of the

monoclonal antibodies to

pp6Osrc recognize

these related

proteins,

we examined

lysates

fromchicken cells infected with avian sarcoma

viruses containing the yes,fps, and ros genes. These viruses included Yamaguchi 73 (Y73)

(21), UR-2 (14), and PRCII (27), which encode

transforming proteins of 90K (pp9Oyes), 68K

(pp68ros)and 110K

(pp110JfPs)

molecular

weight,

respectively. These

transforming

proteins

are

gag gene fusion

products,

and

antibody

which

recognizes thegag portion was used as a

posi-tive control in these experiments.

Figure 3 shows the immunoprecipitation of

32P-labeled

proteins from Y73-transformed chicken cells. Itcan be seen that

antibody

261

(lane 8) precipitated greater levels of

ppgoYes

thandid monoclonal

antibody

to

p19

(lane 1),the

gag-encoded protein. Three other monoclonal

antibodies (443, 450, and492;lanes 11, 12, and 13, respectively) precipitated lesseramounts of

pp9Oyes

MONOCLONAL ANTIBODIES TO pp60r'( 355

A

-pp6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 B

o -ppGO

1 2 3 4 5 6 7 8 9 10 11 12 13

FIG. 2. Proteins immunoprecipitated from

32P1-la-belednormal chicken cells. (A) Autoradiogram of a gel containing the proteins immunoprecipitated from a

lysate prepared from three 100-mm cultures of32p;

labeled normal chicken cells with 20 ,ul of IgG from hybridoma cell line 69 (lane 1), 78 (lane 2), 127 (lane 3), 191(lane 4), 200 (lane 5), 261 (lane 6), 273 (lane 7), 327 (lane 8), 443 (lane 9), 450 (lane 10), 463 (lane 11), 492 (lane 12), anti-bacterial

pp6O"s

(lane 13), orantimouse IgG alone (lane 14). (B) Proteins phosphorylated in vitroafter immunoprecipitation of alysateofchicken brain with purified IgG as described in the text. Lane 1,monoclonal antibody 69; lane 2, 78; lane 3, 127; lane 4, 191;lane 5, 200; lane 6, 261; lane 7, 273; lane 8, 327; lane 9, 443; lane 10, 450; lane 11, 463; lane 12, 492; lane 13,antimouse IgG alone.

To determine whether the monoclonal

anti-bodies which were negative for

pp9O

I'

precipi-tation in the above experiment could indeed

recognize

pp9oYe"

in a more sensitive assay, we

performed an invitrophosphorylation reaction.

Figure 3B demonstrates the

autophosphoryla-tion of

pp90'ez

bound to the monoclonal anti-bodies. Unlabeled lysates of Y73-transformed

chicken cellswere immunoprecipitatedwith the monoclonal antibodies followed by incubation with

[_y-32PIATP

and MnCl,. In this in vitro

reaction,

pp9oYes

wasphosphorylated.

Precipita-tion with monoclonal antibody to p19 and 261 (lanes 1 and 8) allowed the greatest level of

pp90We3

phosphorylation; however, lesser amountsofphosphorylationweredetectedafter precipitation of monoclonal antibodies443,450,

A

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.491.254.441.66.339.2]

356 LIPSICH, LEWIS, AND BRUGGE

A

pp90- so

1 2 3 4 ' 8 9 lO1I 2 13

1 2 3 4 5 67 810 V12 1419M

FIG. 3. Proteins immunoprecipitated from Y73-transformed chicken cells. (A)Autoradiogramofagel

containing the proteins immunoprecipitated from a

100-mmculture of

3'Pi-labeled

Y73-transformed

chick-en cells as described in the text. Lane 1, anti-p19 antibody; lane 2, concentrated medium from

hybri-doma 69; lane 3, 78; lane4, 111; lane 5, 127; lane 6, 191;lane7, 200;lane8,261;lane9, 273;lane10,327;

lane 11, 443;lane12,450;lane13,492.(B) Autoradio-gramofa gel containingthe proteins phosphorylated

afterimmunoprecipitationfrom alysateof Y73-trans-formed chicken cellsasdescribed in thetext. Lane1,

anti-p19 antibody; lane 2, concentrated medium from monoclonalantibody69;lane3, 78;lane4, 127;lane5, 191; lane6, 199;lane 7, 200; lane 8, 261; lane9, 273;

lane 10, 327;lane 11, 443;lane 12, 450; lane 13, 463;

lane14, 492; lane 15, antimouseantibodyalone.

J. VIROL.

13) and which was identical to

pp110PS

by partial proteolytic peptide analysis with V8 pro-tease (data not shown). The phosphorylated protein of Mr 60K did not show any partial peptides identical to those of pp60(-5r. Longer

exposureof thisgel revealed faint protein bands

in other lanes which comigrated with

ppllOfPS.

Thisexperiment was repeated with more

mono-clonal antibody anda more highly concentrated lysate of PRCII-infected chicken cells to

in-creasethesensitivity of thisassay. Under these conditions, several monoclonal antibodies

pre-cipitated a 110K protein which wasidentical to

ppllOfPs

by peptide analysis. Thelevel of phos-phorylation of

ppllOWPS

wasconsistently at least

A

B

.

4

1 2 3 45

-pp110

6 7 8 910 11 12 13 14 15

9I .e

.S

vo. * pp68

and 492(lanes 11, 12,and 14,respectively). The

identity of these proteins as pp90We' was

con-firmed by peptide analysis with V8 protease (datanotshown). Theprotein migrating slightly faster than pp9OYes in lanes 4, 5, and 7 did not share any peptides with pp9OX'e, whereas pro-teins precipitated by antibodies 261, 443, 450,

and 492 were identicaltopp9OYe.

Since the in vitro reactiondescribed above for

Y73-transformed chicken cells proved to be moresensitive than the direct immunoprecipita-tionof32P-labeledlysates,the

autophosphoryla-tion reacautophosphoryla-tion wasutilized fordetermining

cross-reactivitywith other viraltransforming proteins. Figure4Ashows theautophosphorylationofthe

PRCIItransforming protein, pp11OfPs, boundto the monoclonal antibodies. Antibodies 443 and

463(lanes10and12)precipitateda110Kprotein

whichcomigratedwith the

pp1l0fPS

protein

pre-cipitated by monoclonal antibody to p19 (lane

1 2 3 4 5 6 78 9 10 11 12 1314 15

FIG. 4. Proteins immunoprecipitated from PRCII-or UR-2-transformed chicken cells. (A) Autoradio-gramof a gel containing the proteins phosphorylatedin vitro after immunoprecipitation from a lysate of PRCII-transformed chicken cells as described in the text. Lane 1,purified IgG from hybridoma69; lane 2, 78; lane 3, 127; lane 4, 191; lane 5, 199; lane 6, 200; lane7, 261; lane 8, 273; lane 9, 327; lane 10, 443; lane 11, 450; lane 12, 463; lane 13, 492; lane 14, anti-p19 antisera;lane15, antimouseIgGalone. (B) Autoradio-gram of agel containing theproteinsphosphorylated after immunoprecipitation from a lysate of UR-2-transformed chicken cells as described in the text. Lane 1, purified IgG fromhybridoma 69; lane 2, 78; lane3, 127; lane 4, 191; lane 5, 199; lane 6, 200; lane 7, 261;lane 8, 273; lane 9, 327; lane 10, 443; lane 11, 450; lane 12, 463; lane 13, 492; lane 14, TBR antiserum; lane 15, antimouse IgGalone.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.491.54.242.57.302.2] [image:5.491.260.448.241.511.2]

MONOCLONAL ANTIBODIES TO pp603'' 357

10-fold less than thatfound with antibodies 443 and 463, and the relative levels of phosphoryla-tion varied for each monoclonal antibody in multiple experiments. The 110K protein band detected in the control reaction of Fig. 4A was

notrelated toppllOPs and was not reproducibly detected in other experiments. These results suggest that some of themonoclonal antibodies have a low affinity for

ppllfP

and that these

reactions are carried out at the lower limit of detection. When PRCII cells are labeled with 32p;, ppllOP can only be detected with antibod-ies 443 and 463.

Figure 4B shows asimilar in vitro phosphor-ylation of the UR-2 transforming protein, pp68r"s, bound to the monoclonal antibodies. Antibodies 443, 463, and 492 (lanes 10, 12, and 13) precipitateda68Kprotein which comigrated with pp68r"s precipitated with TBR serum. TBR

serum contains antibodies togag-specific

deter-minants ofpp68r0s and was used to precipitate

pp68r0S because the monoclonal antibodytop19 didnotprecipitate pp68r"S efficiently.The identi-tyofpp68r"swascorroborated by peptide

analy-sis with V8 protease. We alsofound low levels

ofprecipitationofpp68r"swithmonoclonal anti-bodies other than 443 and 463 when this assay was performed under the moresensitive

condi-tions described above for ppllOfPS. pp68ros

could also be detected by immunoprecipitation

of [35S]methionine-labeled lysates of UR-2-in-fectedchickencells(data notshown).

Protein kinaseactivity. Todetermine whether anyof the monoclonal antibodies interfere with the phosphotransferase activity ofpp6Osrc,

pro-tein kinaseactivitywasassayed by phosphoryla-tionof exogenous casein inanimmune

complex-bound reaction (Fig. 5A). An identical plate of 32P-labeled cells was immunoprecipitated with the same amount of IgG to demonstrate the

relativeamountofpp60fsr precipitated with each monoclonalantibody (Fig. 5B). Figure5Ashows phosphorylation of casein after immunoprecipi-tation ofunlabeled lysates of SRD-RSV-trans-formed 3T3 cells. It can be seen that

pp6OsrC

bound to all ofthe monoclonal antibodies was

abletophosphorylatecasein. The relative levels

of casein phosphorylation generally reflect the level of 32P-labeled

pp6O"r

shown in Fig. 5B.

Theprotein of Mr60Kwhichwas phosphorylat-ed in the in vitro reaction (Fig. SA) is

pp6Osrc.

Differences in the level of phosphorylation of

pp60Jrc

also appearto reflect the ability ofthe monoclonal antibody to precipitate

32P-labeled

pp605rc. Although antibodies 273 and 327 (lanes 7and8) allowedphosphorylation of casein to a

lesserextentthanthe other monoclonal

antibod-ies, this was not reproducible in other

experi-ments. Monoclonalantibodies 127, 199, and 200 showedasimilarphosphorylationof

pp60s1'

and

A

*I IgG

4_.__ -VW pp60

46 -I _ -casein

B

1 2 3 4 5 6 7 8 9 1011 12 13

6-.

to.- gm

,

-4

-F

6~~~~~~~~~~~~~~~~~~~~~~.

-pp6O

1 2 3 4 5 6 7 8 9 10 11 12 13

FIG. 5. Phosphorylation of casein by pp6Osrc

bound to monoclonal antibodies. (A) Autoradiogram

of a gel representing the phosphorylation of casein afterimmunoprecipitationofpp6o0r5 from SR-D-trans-formed 3T3 cells as described in the text. Lane 1, antimouse IgGalone;lane2,purifiedIgGfrom

hybri-doma69; lane 3, 78; lane 4, 191;lane 5,200; lane 6, 261;lane 7,273; lane 8, 327; lane9, 443; lane 10,450; lane11, 463; lane12, 492; lane 13,TBR-Bantiserum. (B) Autoradiogram of a gel containing the proteins

immunoprecipitated from an equivalent amountofa

32Pi-labeled

SRD-3T3 cell lysate as that used in (A) Lane1,antimouseIgGalone;lane2,purified IgGfrom hybridoma69; lane 3, 78; lane 4,191; lane5, 200;lane 6, 261; lane7, 273; lane 8, 327; lane 9, 443; lane 10, 450; lane 11, 463; lane 12, 492; lane 13, TBR-B antiserum.

casein(datanotshown). The TBR serum used in this assay to precipitate pp6Osr, also allowed caseinphosphorylation. IgGwasnot phosphory-lated after immunoprecipitation of pp6Osrc by any of the monoclonal antibodies. In a similar experiment with lysates from Y73-transformed chicken cells, it was found that

pp90Ye`

which was immunoprecipitated with antibody 261 also phosphorylated casein.

VOL.48, 1983

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.491.250.445.77.420.2]

[image:7.491.50.244.72.175.2]

358 LIPSICH, LEWIS, ANDBRUGGE

TABLE 1. Recognition of proteins by monoclonal antibodies

Protein Recognized by

monoclonal antibodies'

pp60.src. All

pp60 ....273, 327> 69, 78, 443,

463 >other

pp90yes 261 >443, 450 >492

ppllOfvs

... 443,463

pp68r0s...43,463

U> Indicatesmoreefficientimmunoprecipitation.

DISCUSSION

In this study, we report the isolation and preliminary characterization of monoclonal anti-bodies to pp605rc from 13 hybridoma cell lines. To determine the extent of cross-reactivity of thesemonoclonal antibodies andtoestimate the number ofdifferent antigenic determinants that

are recognized by these reagents, we screened

normal cells and cells infected with viruses carrying transforming genes other than the src gene(Table 1). All of the monoclonal antibodies

recognized pp6f051 from Prague and SR RSV-infected chicken cells and the cellular homolog

ofpp601rt. Antibodies 327 and 273 appeared to precipitate pp6O'-1'' more efficiently than the

other antibodies. One monoclonal antibody, 443, recognized all of the viral transforming proteins: pp60src,

pp110fPS,

pp68'05, and, to a lesserextent, pp90)'e. Antibody 463 hada simi-lar pattern of cross-reactivity to 443 in that it precipitated

pp110fPS,

pp68r0S, and pp6051s, but

we have not yet detected pp90OeS precipitation

by 463. This suggests that these antibody

mole-cules do not recognize the same epitope on pp6O5"". It is noteworthy that both antibodies precipitated the same 32P-labeled 68K protein

fromRSV-transformed cell lysates (Fig. 1,lanes

11 and 13).

Several of the monoclonal antibodies

ap-peared to have weak cross-reactivity with pp9OYes,

pp110fP',

and pp68r,, that was

detect-able inaninvitrophosphorylationassaybutwas

not detectable by immunoprecipitation of

pro-teinsradiolabeledinvivo. It isnotclearwhether

this weak recognition is due to amino acid

sequence differences or to different

conforma-tional arrangements of each of the epitopes in

thevarious transforming proteins. We are pres-ently attempting to vary the conditions of

im-munoprecipitationtooptimize forrecognition of

these proteinsby the different monoclonal

anti-bodies. Preliminary experiments comparingthe

efficiency ofprecipitation of the pp60" protein

expressed in E. coli with that of pp60"'S from

RSV-transformed chicken cells indicates that several of the monoclonal antibodies recognize

the E. coli-produced protein much more

effi-ciently than the protein from transformed chick-en cells. That is not an unexpected result since the antigen used for immunization was a dena-tured form of the bacterialprotein. This protein is distinct from the RSV src protein in that it containseight amino acids from ,-galactosidase at its amino end. Since all of the monoclonal antibodies recognizeda52Kcleavage product of pp60src which has lost the amino terminal se-quences ofpp60*"' (datanot shown), the mono-clonal antibodies wouldnotappeartorecognize the 3-galactosidase-derived sequences. There-fore, differences in the precipitation of the two

forms ofpp605'' could reflect differences in the conformation ofpp605'' molecules derived from either the bacterial or the animal cell environ-ment.

This battery of monoclonal antibodies should be valuable for investigations of pp6O`r", its normal cellular homolog, and the related

trans-forming proteins from other avian sarcoma vi-ruses. Monoclonal antibodies provide the tech-nology to perform immunodetection assays without contaminating reactivities which are

present in polyclonal serum. Parsons and

co-workers have previously reported the isolation of a monoclonal antibody which recognizes

pp60"-"'' (30).Monoclonal antibodies 273 and 327

willconsiderably improvethemeansfor analysis

of the normal cell src protein since most sera

from animals bearing RSV-induced tumors ei-ther do not recognize pp6O('-5' or have a low titer of antibodies to this protein. None of the

RSV-specific TBR sera which have been

de-scribed previously recognize

transformation-specific regions oftheviral

fps,

ros,oryes gene

products. With the exception of

pp1301Ws

(13),

theseproteins havebeen characterizedby using antibodies directed against the gag-specific

re-gion of these proteins.

Monospecific

antibody

directed against the

transformation-specific

re-gions of these proteins willmake it possible to

focus on the transforming protein without the

additionalrecognition ofthe gag geneproducts. Large-scalepurification of theseantigensis pos-sible by using the monoclonal antibodies for

immunoaffinity chromatography.

All of the monoclonalantibodies described in this report allowed

phosphorylation

of casein in

animmunecomplex-boundassay.This

provides

arapidand simplemeansofassaying phosphor-ylation of exogenous substrates. This also pro-videsanassay which canbe usedtoinvestigate

the normal cellular src protein from unlabeled material. We have not yet determined whether

the src protein from lower eucaryotes can be

recognizedbyanyof the monoclonal antibodies.

From the preliminary analysis of the cross-reactivities ofthese monoclonal

antibodies,

we canpredictthatthisbatteryof antibodies recog-J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

nizes at least six different antigenic determi-nants. None of thesedeterminantsappearstobe

within the amino-terminal 8,000 daltons of

pp60"',

sinceall oftheantibodies recognized the 52K cleavage product ofpp6O0"' generated dur-ing lysis of RSV-transformed cells (data not shown). Since all of the monoclonal antibodies recognized thecellular homolog of pp60"'(, they apparently do notrecognize the

carboxyl-termi-nal 12amino acidswhich are unique to pp6o'-"( (38). Finer-detailed mapping and competition assays are required todistinguish other

specific-ities.

ACKNOWLEDGMENTS

We thankJoe Cirrone and Diane Darrow forassistance in

thescreeningandcharacterizationof the monoclonal antibod-ies.WeareindebtedtoTonaGilmerand Raymond Eriksonfor providing pp60"' expressed in E. coli. We thank Piero Bal-duzziandKaren Beemon and Hidesaburo Hanafusafor

sup-plyingus with UR-2, PRCII. and Y73 sarcoma viruses. We

alsothankSandraBurns fortypingthis manuscript.

This work was supported by grant CA 27951 from the NationalCancer Institute. L.L. is supported byafellowship

fromtheMuscularDystrophy Association. LITERATURECITED

1. Barbacid, M., A. V. Lauver, and S. G. Devare. 1980. Biochemicaland immunologicalcharacterization of poly-proteinscodedforbytheMcDonough. Gardner-Arnstein. and Snyder-Theilen strains of feline sarcoma virus. J. Virol. 33:196-207.

2. Blomberg,J.,W.J. M.VandeVen,F. H.Reynolds, Jr., R. P. Nailwaik, and J. R. Stephenson. 1981. Synder-Theilenfeline sarcomavirusP85 contains asingle phos-photyrosine acceptor site recognized by its aissociated proteinkinase.J.Virol. 38:886-894.

3. Brugge, J., E. Erikson, and R. L. Erikson. 1981. The specificinteractionofthe Roussarcomavirus transform-ing protein. pp6tprc. with twocellular proteins. Cell 25: 363-372.

4. Brugge,J. S.,and D.Darrow.1982.Roussarcoma virus-induced phosphorylation ofa 50.000 molecular weight cellularprotein.Nature(London)295:250-253. 5. Brugge, J. S.,and R.L.Erikson.1977.Identificationofa

transformation-specific antigen induced byanavian

sar-coma virus.Nature(London)269:346-348.

6. Burr, J.G.,G.Dreyfuss,S.Penman, and J. M. Buchanan. 1980.Associationof thesrc geneproduct of Rous sarco-ma viruswithcytoskeletal structuresofchicken embryo fibroblasts. Proc.NatI. Acad. Sci. U.S.A.77:3484-3488.

7. Collett,M.S.,E.Erikson,A. F.Purchio, J.S.Brugge, and R. L. Erikson. 1979. A normal cell protein similar in

structureandfunction tothe avian sarcomavirus trans-forming gene product. Proc. Natl. Acad. Sci. U.S.A. 76:3159-3163.

8. Collett, M. S.,and R. L. Erikson. 1978. Protein kinase activityassociatedwiththeaviansarcoma virussrcgene

product. Proc.Natl.Acad. Sci. U.S.A. 75:2021-2024.

9. Collett, M. S.,A. F. Purchio,and R. L. Erikson. 1980. Aviansarcomavirus-transformingprotein.pp6O0. shows proteinkinaseactivity specific fortyrosine.Nature (Lon-don)285:167-169.

10. Cooper, J. A., and T. Hunter. 1981. Changes in

pro-teinphosphorylationin Roussarcomavirus-transformed chickenembryocells. Mol.Cell. Biol. 1:165-178.. 11. Cooper, J. A., and T. Hunter. 1983. Identification and

characterization of cellular targets for tyrosine protein kinases. J. Biol. Chem.258:1108-1115.

12. Erikson, E., and R. L. Erikson. 1980. Identification ofa

cellular protein substrate phosphorylated by the avian

sarcoma virus-transforming gene product. Cell 21:829-836.

13. Feldman,R., and H. Hanafusa. 1980.Characterizationof

protein kinase activity associated with the transforming

gene product ofFujinami sarcomavirus.Cell 22:757-765.

14. Feldman, R. A., L.-H. Wang, H. Hanafusa, and P. C. Balduzzi. 1982. Avian sarcoma virus UR2 encodes a

transforming protein which is associated with a uinique

proteinkinase activity. J. Virol. 42:228-236.

15. Gilmer, T.,and R.L. Erikson. 1981. Rous sarcomia viruIs

transforming protein, pp6O0''. expressed in E. co/i.

func-tionsasaprotein kinase. Nature(London)294:771-773.

16. Gilmer, T. M., and R. L. Erikson.1983. Development of

anti-pp6OG' serum with antigen produced in Esc/erichia

(0/i.J. Virol. 45:462-465.

17. Gilmer, T. NI., J. T. Parsons, and R. L. Erikson. 1982.

Constructionofplasmidsforexpressionof Roussarcoma

virus transforming protein, p6O'r, in Escherichia (oli.

Proc. NatI. Acad. Sci. U.S.A. 79:2152-2156.

18. Gilmore, T. D., K. Radke, and G. S. Martin. 1982.

Tyrosine phosphorylation of a 50K cellular polypeptide

associated with the Rous sarcoma virus transforming

protein, pp6O Mol. Cell. Biol. 2:199-206.

19. Hampe,A.,I. Laprevotte, F. Galibert, L. A. Fedele, and

C.J.Scherr. 1982. Nucleotide sequencesof feline retro-viral oncogenes (v-fes) provide evidence fora family of

tyrosine-specific protein kinasegenes. Cell 30:775-785.

20. Hunter, T., and B. Sefton. 1980. Transforminggene

prod-uctof Rous sarcoma virusphosphorylatestyrosine. Proc. NatI. Acad. Sci. U.S.A. 77:1311-1315.

21. Kawai, S., M. Yoshida, K. Segawa, R. Sugiyama, R. Ishizaki, and K. Toyoshima. 1980. Characterization of Y73,an avian sarcomavirus: a uniquetransforminggene

and its product, a phosphoprotein kinase activity. Proc. Nat]. Acad. Sci. U.S.A. 77:6199-6203.

22. Kessler, S. W.1975. Rapidisolation ofantigensfromcells

witha Staphylococcalprotein A-antibody absorbent:

pa-rametersofthe interactionof antibody-antigen complexes withproteinA. J. Immunol. 115:1617-1624.

23. Kitamura, N.,A.Kitamura,K.Tovoshima,Y.Hirayama, andM. V'oshida. 1982. AviansarcomavirusY73genome sequenceand structural similarity ofitstransforminggene

producttothat of Roussarcomavirus.Nature (London) 297:205-208.

24. Levinson, A. D., H. Oppermann, L. Levintow, H. E.

Varmus, and J. B. Bishop. 1978. Evidence that the trans-forming geneofavian siarcomia virusencodes a protein

kinaseassociated withaphosphoprotein. Cell15:561-572.

25. Levy, R.,andJ. Dilley. 1978. Rescueofimmunoglobulin secretion from humanneoplasticlymphoid cells by

somat-iccell hybridization. Proc. Natl. Acad. Sci. U.S.A. 75: 2411-2415.

26. Linial, M., and D. Blair.1982. Geneticsof retroviruses.p.

649-784. In J. Weiss (ed.). RNA tumor viruses. Cold SpringHarborPress,Cold Spring Harbor. New York. 27. Neil, J.,NI. L.Breitman, and P. K.Vogt.1981.

Chairacter-ization ofa 105.000 molecular weight gag-related phos-phoprotein from cells transformedby thedefectiveavian sarcomavirus PRCII. Virology 108:98-110.

28. Oppermann, H., A. D. Levinson, L. Levintow, H. E. Varmus, J. M.Bishop, and S. Kawai. 1981.TwocellIlar

proteins that immunoprecipitate with the trzansforming protein of Roussarcomavirus. Virology 113:736-751. 29. Oppermann, H.,A.D. Levinson, H. E. V'armus, I,.

Levin-tow,andJ.Ml. Bishop. 1979. Uninfectedvertebratecells contain itproteinthatis closely relatedtothe productof the avian sarcoma virus transforminggene (src). Proc.

NatI. Acad. Sci. U.S.A.76:1804-1808.

30. Parsons,S. J., D. J. McCarley, C. M. Ely,D. C.Benjamin, and J. T. Parsons.1983. Isolation and partial

characteriza-tionofamonoclonalantibodytothe Rous sarcoma virus

transformingproteinpp6O"'(. J.Virol. 45:1190-1194. 31. Radke,K., T. Gilmore,andG. S.Martin. 1980.

Transftr-mation by Rous sarcoma virus: a cellular substrate for

on November 10, 2019 by guest

http://jvi.asm.org/

360 LIPSICH,LEWIS, AND BRUGGE transformation-specific protein phosphorylation contains phosphotyrosine.Cell 21:821-828.

32. Rohrschneider, L. R., R. N. Eisenman, and C. R. Leitch. 1979.Identification ofaRoussarcomavirus transforma-tion-related protein in normal avian andmammaliancells.

Proc.NatI. Acad. Sci.U.S.A. 76:4479-4483.

33. Sefton, B. M., T.Hunter, E. H. Ball, and S. J. Singer.

1981.Vinculin: acytoskeletal targetof the transforming protein ofRoussarcomavirus.Cell24:165-174.

34. Sefton, B.M.,T. Hunter, K. Beemon,and W. Eckhart.

1980. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma

virus. Cell 20:807-816.

35. Shibuya, M., and H. Hanafusa. 1982.Nucleotidesequence

ofFujinamisarcomavirus:evolutionaryrelationship ofits

transforming genewith thetransforming genes of other sarcomaviruses.Cell30:787-795.

36. Stehelin, D., H. E. Varmus, J. M. Bishop, and P. K. Vogt.

1976. DNArelatedtothetransforminggene(s)ofavian

sarcoma virusis present innormalavian DNA. Nature (London)260:170-173.

37. Takeya, T., R. A. Feldman, and H. Hanafusa.1982.DNA

sequenceof theviraland cellularsrcgeneofchickens.I.

Complete nucleotide sequenceofan EcoRI fragment of recovered aviansarcomavirus which codes for gp37and

pp605".J.Virol.44:1-11.

38. Takeya, T., and H. Hanafusa. 1983. Structure and se-quence of the cellulargenehomologoustothe RSVsrc

geneand the mechanism forgenerating the transforming

virus.Cell 32:881-890.

39. Witte, 0. N., A. Dasgupta, and D. Baltimore. 1980.

Abelsonmurineleukemia virus proteinisphosphorylated

in vitrotoform phosphotyrosine. Nature(London) 283:

826-831.

J.VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/