Isolation of monoclonal antibodies against avian oncornaviral protein p19.

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JOURNAL OF VIROLOGY, July 1981,p.325-329 Vol. 39,No.1 0022-538X/81/070325-05$02.OO/O

Isolation

of Monoclonal Antibodies Against Avian

Oncornaviral

Protein p19

I.GREISER-WILKE, K. M. OWADA, AND K. MOELLING*

Max-Planck-Institut

ffir

Molekulare Genetik,

D-X1000

Berlin 33, West Germany

Received 5 January 1981/Accepted 6April 1981

For the production of monoclonal antibodies against pp60src and the gag precursor protein Pr769"9, the spleens of mice bearing tumors that had been induced by avian sarcoma virus Schmidt-Ruppin D-transformed cells were used.

Onehybridoma culture produced antibodies that were directed against the p19

portion of thegag precursor. However, no antibodies directed against pp6Osrc

could be detected in any of the hybridomasupernatants. The anti-p19-producing hybridoma culture was cloned twice in soft agar, and a stable clone was used for the production of high-titer ascites fluid in mice. The monoclonal antibodies

belonged to the immunoglobulin G subclass 2b. The antibodies precipitated

Pr76eagand the processed virion-associated p19, aswell as the 75,000-molecular-weight gag fusion protein from avian erythroblastosis virus-transformed bone

marrow cells. Also, viral ribonucleoprotein complexes were specifically

precipi-table, indicatingthat they contain p19 molecules.

Avian sarcoma viruses (ASV) code for genes

involved in replication (gag, pol, and env) and in transformation (src) (1). Aviancells, such as chicken embryo fibroblasts (CEF), permit rep-lication andtransformationand expressallviral

proteins encoded bythe ASV genome (25). In contrast, in mammalian cells transformed by

ASV,thetransforminggeneis expressed, but no virusreplicationtakesplace (6). Two

virus-spe-cificproteins having molecular weightsof 76,000

(76K) and60,000 (60K) arefound in thesecells (3, 6).The76Kprotein, Pr76gag,isthe precursor totheinternalcoreproteins of

ASV, however,

it

is notprocessed tothe structural proteins p19, p12, p27,andp15 (6). The 60Kprotein,

pp60src)

is the product of the src gene and has been

identified as a

phosphoprotein

with

protein

ki-nase activity (5, 13, 22). Parsons et al.

(20)

showedthat,whenASV-transformedmouse

tu-mor cellsare

injected

into

syngeneic mice,

spe-cific antisera to pp60src can be obtained and suggestedtheuseof the

spleen

cellsfrom

tumor-bearing mice for the establishment of

hybrid-omas

producing

monoclonal antibodies

against

this protein bythe method ofKohler and Mil-stein(10).Forthis purpose,we

injected BALB/

c 3T3 (A31) cells transformed with ASV

Schmidt-Ruppin

D

(SR-D)

into

syngeneic

new-born mice

(purchased

from

Bomholtgard, Ry,

Denmark). Fourof five animals

developed

pal-pable tumorswithin 4 weeks. Serum from the

tumor-bearing mice

precipitated

the

sarcoma-specificprotein

pp60Brc

and the

unprocessed

gag precursor Pr769`9 from SR-D-transformed

chickencelllysates labeled with [35S]methionine for3h.Figure1Ashows theimmunoprecipitates

after gel electrophoretic analysis and au-toradiography. A second bleeding (6 weeks post-inoculation) was analyzed to monitor the

in-creaseinantibodytiter.Onemousediedbecause

of its tumor; mice 2 and 3 were sacrificed for

hybridoma production(10, 11). Atotal of1 x 108 spleen cellswerefused with 2 x 107cellsof the

8-azaguanine-resistant plasmacytoma cell line

P3-NS1-1-Ag4-1 (NS-1) by the procedure

de-scribedby Kennett (9), using polyethylene glycol

anddimethyl sulfoxide.

The cell suspension wasdistributed into five

24-well tissue culture plates and cultured in HAT medium for the selection ofhybrid cells

(14). After 14 days, thesurviving cultureswere

tested for antibody production in an indirect

antibody-binding assay (17, 24, 26). A 96-well platewasfirstcoated with variousantigensand then incubated with the supernatant of each

culture. Positive cultures were identified

by

meansofaniodinated rabbit anti-mouse

immu-noglobulin G

(IgG).

The

antigens

used were

disrupted

transforming

virus, SR-D,

which has been showntocontain

pp6o.rc

(18;M. K.

Owada,

P. Donner,A.Scott, and K.Moelling, Virology,

in press), a

nontransforming virus,

avian mye-loblastosisvirus, as anegative

control,

the

SR-D-transformedBALB/c3T3cells that had been used for immunization of the

mice,

andnormal

BALB/c 3T3 cells

(Fig.

1B).

One

hybridoma

supernatant, from culture no. 52, was found to

bind 10- to 20-fold more radioactive

anti-IgG

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326 NOTES

thanall others but was notspecificfor the src-containing antigens. The antibody-producing

culture no. 52 wasanalyzedfurther for its spec-ificity. Variouspurifiedviralproteinswereused asantigensin anindirect

antibody-binding

assay (Fig. 2A). Theresultsshowed that the antibodies were directed against p19,the N-terminal

por-tionof thegag precursor

Pr769a-, only.

Precipi-tation of Pr769a5 from [35S]methionine-labeled

cell lysatesby the culture supernatant (in

par-ticular, after a 100-fold concentration by 50%

ammonium sulfateprecipitation) supportedthis

result (Fig. 2B).

The antibody-producing culture no. 52 was cloned twice insoftagaruntil 100%of the

sub-cloneswereantibody

producers.

One stable

sub-clone,c152:38:38,wasgrown inmassculture and

:E

-2

- s :

1m

E -Zz I- F- I- F- =

J. VIROL.

used for further characterization. To increase theantibody titer,

107

cellsfrom c152:38:38were injected intraperitoneally into (BALB/c x

C57BL)F1 hybrid mice (termed CD6) that had been primed with pristane (0.25 ml) 10 and 3 days before to enhance ascites fluid formation (21). Theantibody titer of the ascites fluid was about 103-fold higher than that of the tissue culture fluid from c152:38:38. A 10- to 100-fold increase in titer could also beachieved by

con-centrationof culture fluid with50% ammonium

sulfate(Fig. 2C).

ImmunoprecipitationofPr76sas from

[tOS]me-thionine-labeled SR-D-transformed CEF with the various culture supernatants reflected the titersoftheantibodies (Fig. 2B).It wasnotable

that the clonal supernatants and ascites fluids

B

15

10

-pr76

-pp60src

T

-Qctin EE CL Ln U`4

4 2

-p27

_~~~~~~

4~~~~~~~~~~~~~~~~~~~~~~~~

SR-D AMV

SR-D-3T3

3T3

2-L

20 40 60

#ofculture

FIG. 1. (A) Characterization of tumor-bearingmouse (TBM) sera. [35S]methionine-labeled

SR-D-trans-formed CEFwerelysedandtreated with5ulofserumfor indirect immunoprecipitationasdescribedelsewhere

(15). TBM,-5, TBMseraof five mice (first bleeding); TBM, 4, secondbleeding (onemousedied); TBM2v,

mice usedfor fusion atthetime of the second bleeding; Mapl9, monospecific anti-p19mouse serum; TBR,

tumor-bearing rabbitseruminduced by SR-D (19); NMS, normalmouseserum;Hap27, sheep anti-p27serum.

(B) Indirectantibody-binding assayfor screening ofculturefluids. Virus (SR-D andavian myeloblastosis virus[AMV]) and cells(SR-D-transformed 3T3cells and 3T3 cells) weredisrupted in lysis buffer (50 mM

Tris-hydrochloride [pH 9.2], 400 mMKCl,ImMEDTA,1% Triton X-100,2mMphenylmethylsulfonylfluoride) anddialyzed againstphosphate-bufferedsaline.A50-/lamountcontaining2.5,ug ofprotein was bakedas

antigens in Limbro plates with 96 wells (14 h at 37°C). The wells were then treated with wash buffer

(phosphate-buffered saline containing 5% fetalcalfserum, 0.1I%Triton X-100,0.02%7osodium azide) for 90min at37°C.A50-,ulamountofclarified hybridomasupernatantwasthen added and allowedtobindfor 60min at37°C. The wellswerethenwashed three times with200,ulof wash buffer. Subsequently,50,ulof iodinated rabbitanti-mouseIgG (0.2 ug/ml), correspondingto3x105cpm,prepared accordingtoHunter(8)wasadded

to each wellfor 60 min at37°C. After washing as above, the radioactivity of the individual wells was

solubilizedin100/l of 2 MNaOH (30min,37°C) andcountedinagammacounter.

80

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NOTES 327

A

T

x

ao

B 0w ii o

in

anaN CL ^

X * w 4 xZ m x >

-p180

-pr76 °

-a

E CL

-actin

-P32

-p27

c

-1 -2 -3 -4 logdil.

FIG. 2. (A)Indirectantibody-binding assay with purified antigens. A0.25-,ugamount ofpurifiedp27,p19,

pl5, p12, andgp85/37(16)was usedas antigens (Ag)according to the procedure described in the legend to

Fig.lB. The supernatant of the hybridoma culture (no.52) wascomparedwith amonospecific mouse anti-p19

serum(Mapl9)andnormal mouse serum (NMS). (B) Immunoprecipitation of viral polyproteins from

trans-formed CEF as in Fig.IA, using 50

j.l

ofculture fluid from culture no. 52, from a clone,c152:38, derived from

it, andfromasubclone,c152:38:38. The culture fluid of no. 52 was also tested after 100-foldconcentration by

ammoniumsulfate(AS). Asc,, Ascites fluid (5td);NS-1,culture fluid (5,uI) serving as a negative control for

the hybridoma supernatants;Rapl9,monospecific rabbitanti-p19 antibodies; NRS,normalrabbit serum; V,

[3S]methionine-labeledSR-D as a marker; NMS, normal mouse serum; TBM, tumor-bearing mouse serum. (C) Comparison of antibody titers in an indirect antibody-binding assay. Tenfold serial dilutions of the

hybridoma supernatantno. 52 (-4*) and the subclones c152:38 (A A) and c152:38:38 (A A) and

serumfrom tumor-bearing mice (M,EI-F) wereprepared. Theirantibody-binding ability was compared

with thatof supernatantsafter10-fold(A---A, ----A) and 100-fold(-- 4)concentrations by ammonium

sulfate precipitation (50%, vol/vol).Ascites fluid(O-O) was obtained from F, hybrid mice afterpristane

treatmentandinjectionofclone52:28:38cells.

less efficiently precipitated p19-related inter-mediategagprecursors, three of whichranged

between 50 and 70K. These

polyproteins

were

readily detected with the monospecific rabbit anti-p19serumusedasa control. Since the

pu-tativegag-pol fusionprotein

Prl80

was precipi-tated by the ascites

fluid,

it appears that the recognition of fusion

proteins

may

depend

on

the

accessibility

oftheir

p19 portion

tothe anti-bodiesthatare directed

against

one

specific

an-tigenic determinant.InSR-D-transformed CEF labeled for3h, theamountoflabeledprocessed viral structural protein (in

particular,

of

p19)

was

relatively

small.

To analyze whether the monoclonal anti-bodies

recognized

the

processed

virion-associ-ated

p19

molecule, [35S]methionine-labeled

virus waslysedand usedfor

immunoprecipitation.

As shown in Fig. 3A

(left),

p19

was

precipitated

fromvirusparticlesandwas

efficiently

competed

forby theaddition of

purified p19.

In

addition,

the a and ,B subunits of reverse

transcriptase

werealso

precipitated

with the monoclonal anti-bodies. We

thought

that this

phenomenon

might

be due tothe

coprecipitation

with the

ribonu-cleoproteins which are in a

complex

with the

polymerase,

some

p27,

p12,

andasmallamount of

p19

(4,23).Toprove

this,

viruswas

disrupted,

and the soluble

(supernatant)

and insoluble

(pel-let) materials were separated by ultracentrifu-gation,applyingthe methoddescribed by Chen

etal. (4). The monoclonal anti-p19serumcould

efficiently

coprecipitate reverse transcriptase from the pellet, butnot from the supernatant.

Also,

the specific

precipitation

ofp19 with the monoclonal antibodies could not be competed forbypurified p27 or

polymerase

(Fig. 3A).In

addition,

themonoclonal

anti-p19

serumdidnot reactwithpurified polymerase in the

antibody-binding

assay

(data

not

shown).

These

findings

strongly support the data of Sen and Todaro (23), who

reported

that very few molecules of p19are associated with RNA.

The monoclonal

anti-p19

antibodies were

tested further for their

ability

to

precipitate

the

putative

transformation-specific

fusionproteins of acute leukemia

viruses,

the N-terminus of which is p19. In avian

erythroblastosis

virus-transformed bonemarrow

cells,

the virus codes

fora

protein

of

75K,

which is

probably

transfor-mation

specific

and fused to

p19

(2,

7).

This

fusion

protein

is detected in

[35S]methionine-la-beled transformed bone marrow cells by the

monoclonal antibodies. Since these cells also

produced the helper virus protein Pr769"w, this protein wasremoved by preprecipitation. Asit isvery similar insize,it would otherwiseprevent thedetection of the 75Kprotein (Fig. 3B).

VOL. 39,1981

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328 NOTES

S p

z

ap27 apol IP z<I Asc.1 f

I:

w.-CD

c), CL t-r- z z

_==R-pr1pr80

7SK-

-pr76

t_u3 _ _ nrac*in X 6: 00 _

_p

26

'1S~~~~~~1

abbcdbcdabcd a bb'

FIG. 3. (A)Immunoprecipitation ofviralantigens from[35S]methionine-labeledSR-D. The viruswaslysed

with RIPA buffer (1%TritonX-100, 1%deoxycholate, 0.1%sodiumdodecyl sulfate, 0.1 MNaCl,0.05 M

Tris-hydrochloride [pH7.2]) containing 0.8 M NaCl and applied directlyto thegel (V)orincubated with the

indicatedantibodysamples (abbreviationsasinFig. 2). Forthecompetition assay,purified p19 (5,Ig)was

addedtothe ascitesfluid(Asc.,+p19). Alternatively,thelabeled viruswasdisrupted bythe methodofChen

etal.(4)andcentrifugedinanSW50.1 rotor at40,000rpmfor60minat4°Ctoseparate the solublematerial

(S) fromthepellet (P) (4). Samples ofbothwereapplied directlytothegel (SandP)orusedfor

immunopre-cipitation. Absorption ofthesera(5,l each)wasperformedwithnoantigen (a), with1jugof p19 (b),with 10

pg of p19 (b'),p27 (c), andpurified reverse transcriptase (d) asantigens (5 pug each). Mouse ascites fluid

producedwith NS-1cells[Asc. (NS- 1)]servedasthecontrol;aand indicate thetwosubunitsofthereverse

transcriptase. (B) Immunoprecipitation from [35S]methionine-labeled avian erythroblastosis

virus-trans-formedbonemarrowcells(3 h).The lysateswerefirstpreprecipitatedwithnormal rabbitserum(NRS)and

ap27 serum (pre) and subsequently treated with hybridoma supernatants. (C) Agar gel diffusion testfor

identification ofthe immunoglobulin class. The central well contained: (a) 15,ul of 100-fold-concentrated

supernatantofcultureno.52; (b)20,ulof10-fold-concentrated c152:38:38;and(c)20,ul of10-fold-concentrated

c152:38 supernatants.The other wellscontained(starting fromthetop,counterclockwise)10

[lI

of:rabbit anti-totalmouseIgGserum,rabbit anti-mouseIgG2b,rabbitanti-mouseIgG2a,andrabbitanti-mouseIgGl (the

last wellwasempty).Thephotographwastakenafter4hat37'C.

Inan agargel diffusionanalysis with

immu-noglobulin class-specific reagents purchased

from Miles Laboratories, Inc., Elkhart, Ind., it

wasshown thatthe antibodiesproduced bythe

originalculture belongedto twoIgG classes, to

IgG2b and,toaless extent,toIgG1. The

super-natants of thecloneswerespecificand reacted

positivelywithanti-IgG2b only (Fig.3C).

The isolation of monoclonalantibodies against

p19 will prove to be useful for analyzing the

location and function ofp19 in virus particles

and ofp19-containing fusion proteinsin

virus-infected cells. This may be helpful in

under-standing the role of fusion proteins of acute

leukemia viruses in transformation and

differ-entiation.Also, since only monoclonalantibodies

have high concentrations of pure and specific

antibodies,theymaybe used formicroinjection

into cells and allow an analysis ofthe

mecha-nisms of transformation anddifferentiation.

Fur-thermore,monoclonal antibodiesmaybeuseful

forstudying properties of thereverse

transcrip-tase in ribonucleoprotein particles (4) and the interaction of p19 with RNA which has been suggested as being relevant in the splicing of viral mRNA's (12).

Althoughthe first intentionwastomake

mon-oclonal antibodies against the

transformation-specific protein pp6osr, the indirect antibody-bindingassayshowedahigher sensitivityforthe

viralstructural proteins than forpp6Osrc.

There-fore,theprobabilityoffinding hybridomas

pro-ducingmonoclonalantibodiesagainst the

trans-formation-specific pp6Osrc proteinwas verylow.

The use ofpartially purified pp6osrc protein as

anantigen would improve the sensitivity of the assay.

Wethank Mette Strandand J. T.August for introducing

tousthemethod ofpreparing monoclonal antibodies and for theircontinuous advice. Thehelp of T. Bunte in the

prepa-ration of iodinated antibodies and theproduction of ascites by P. Donneraregratefully acknowledged.

A

_,

z

SZ<I Asc.1

a b

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VOL. 39, 1981

I.G.-W. issupportedby theDeutsche Forschungsgemein-schaft.

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Figure

FIG.#offormedsolubilizedmiceandatrabbitantigensattumor-bearingto(15).Tris-hydrochloride(phosphate-buffered(B)virus each 37°C
FIG.#offormedsolubilizedmiceandatrabbitantigensattumor-bearingto(15).Tris-hydrochloride(phosphate-buffered(B)virus each 37°C p.2
FIG. 2.pl5,formed[3S]methionine-labeledFig.theserumsulfateserumhybridomatreatmentammoniumit,with(C) and (A) Indirect antibody-binding assay with purified antigens
FIG. 2.pl5,formed[3S]methionine-labeledFig.theserumsulfateserumhybridomatreatmentammoniumit,with(C) and (A) Indirect antibody-binding assay with purified antigens p.3
FIG. 3.aproducedhydrochloridewithformedindicatedetaddedcipitation.ap27(S)supernatanttranscriptase.c152:38identificationlasttotalpg al
FIG. 3.aproducedhydrochloridewithformedindicatedetaddedcipitation.ap27(S)supernatanttranscriptase.c152:38identificationlasttotalpg al p.4

References

Related subjects : Ascites fluid normal rabbit serum