Simultaneous Purification of Murine Mammary Tumor Virus Structural Proteins: Analysis of Antigenic Reactivities of Native gp34 by Radioimmunocompetition Assays

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JOURNAL OF VIROLOGY, Aug. 1979,p.341-349 Vol.31,No. 2 0022-538X/79/08-0341/09$02.00/0

Simultaneous

Purification of Murine

Mammary

Tumor

Virus

Structural Proteins: Analysis of Antigenic Reactivities of

Native gp34

by Radioimmunocompetition Assays

STUART L.MARCUS,* REBECCA KOPELMAN, ANDNURUL H. SARKAR

Laboratoryof Molecular Virology, MemorialSloan-KetteringCancerCenter,NewYork, New York 10021 Received forpublication7March 1979

Allthe structural proteins (gp47, gp34, p27, p23, p16, and p12) of the murine

mammary tumor virus (MuMTV) were simultaneously purified utilizing

alkyl-agarose chromatography as the initial fractionation step. Least-hydrophobic

MuMTVpolypeptides (p23, p16) and theslightly hydrophobicp27wereseparated

from moderately hydrophobic proteinsgp47 and p12 bypassage through

octyl-imino(C8)-agarose; thegp47and p12 could be removed from the matrix by elution

withethylene glycol, whereas themosthydrophobic MuMTV protein,gp34, was

eluted using nonionic detergent together withethyleneglycol. Subsequent

puri-ficationstepsinvolvedion-exchangeorgelfiltrationchromatography. The

result-ing proteinpreparations appeared near-homogeneous on analysis by

polyacryl-amide gelelectrophoresisin the presenceofsodium dodecylsulfate. Recoveries

of MuMTVproteins, basedontheirapproximate individual contributiontototal

virus protein, ranged from about 20% forgp47 togreaterthan100% for the minor

structural component p23, the major phosphoprotein of MuMTV. Antiserum

againstpurifiedC3H MuMTVgp34,together withpurified, radioiodinatedgp34,

wasusedtodevelop aradioimmunoassay which showed thatfrom13 to 14% of

total MuMTV protein by weight is gp34. Using this assay system, the

group-specific antigenic reactivity of gp34 was also demonstrated. When solubilized

preparations of O3H,RIII, andGR MuMTV's wereusedascompetingantigens

ingp34radioimmunoassays withanti-C3H MuMTVserum,bothgroup-and

type-specificdifferences inantigenic reactivitywerefound.

The type B murine mammary tumor virus

(MuMTV)containsapproximatelysix structural

polypeptides,includingtwoglycoproteins(gp34

andgp47) and thenonglycosylatedproteinsp27,

p23, p16, and p12 (5, 13, 21-23, 26, 30). The

glycoproteins gp47and gp34appear to serve as

the extramembrane projections and the

intra-membrane component, respectively, based on

structural dissection in the virions (21, 30) as

well as studies on the individual properties of

theglycoproteins (12,22). Thenonglycosylated proteins have previously been assigned to the

viralcorelocation (12, 22), butrecentevidence

(4) suggests that p12 may functionas a

mem-brane-associatedor matrixprotein. MuMTV is

unique among murine oncornaviruses in

con-tainingtwoglycoproteins andtwo

phosphopro-teins. The major phosphoprotein of MuMTV

was recently shown to bethe minor structural

polypeptide p23,and, in addition, a

subpopula-tionofthemajorcoreprotein (p27) appearedto

bephosphorylated (23).

Purification of MuMTV structural

polypep-tideshas been accomplished usingavariety of

classical biochemicalprocedures(1, 6, 15-17, 21,

25,27,29).However, thetechniqueofgel

filtra-tion in thepresenceofguanidinehydrochloride,

which has proven effective for the purification

oftypeC viralproteins (16),appears to

irrevers-iblydenature and inactivate the antigenicityof

someMuMTVproteins (16, 17).Nosingle

non-denaturing procedurehasyetbeendescribed by

whichall of the structural proteinsof MuMTV

may be purified simultaneously. Additionally,

classicalpurification procedurespose aproblem

for purification of the extremely hydrophobic

membrane-associated peptide, gp34,which has

onlybeen purifiedin thepresence of the

dena-turing detergent, sodiumdodecylsulfate (SDS)

(6).

Immunological characterization of MuMTV

polypeptideshas beenreported forgp47(25, 26),

p27 (27), and p16 (1). The major glycoprotein,

gp47, was the first soluble MuMTV antigen

shown to containgroup specificity (17, 21),and

subsequent studies have shown that antisera

prepared againstgp47, p27,andp14do not

cross-react with any other than their homologous

341

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342 MARCUS, KOPELMAN, AND SARKAR

MuMTV protein and that they all contain

group-specific antigenic determinants (1, 15,24,

25, 27). Biological distinctions in the

tumori-genicity and hormonedependenceofMuMTV's

fromdifferentmousestrains(14) have prompted

asearch for the presence oftype-specific

anti-genic determinantsonMuMTV structural

com-ponents. Several laboratories (1, 10, 15) have

reported that the use ofradioimmunoassay

pro-cedures served to identify only group-specific

reactivities on the variousantigens tested.

Re-cent reports have indicated the presence of both

group- and type-specificantigenicdeterminants

ongp47 (24, 25) andp27(27). Immunochemical

characterization of other MuMTV proteins, in-cluding gp34, has not been reported.

We have recently reported the use of

alkyl-agarose derivatives in determiningthe relative

hydrophobicity(potential forhydrophobic

inter-action) of both type B and type C murine

on-cornavirus structural proteins (12) and reverse transcriptases (11). In this paper, we report the

development of a protocol for thepurificationof

all six MuMTV structuralproteins fromasingle

batchof C3H mouse tissue culture cell-derived

virus usinghydrophobic chromatography as the

initial step in the purification, and without the

use of protein-denaturing reagents. Purified

gp34 was radioiodinated and used in the

devel-opment of a radioimmunocompetition assay.

Competition studies carried out using homolo-gous virus and solubilized preparations of RIII mouse-derived and GR mouse cell-derived

viri-ons revealed the presence of both group- and

type-specificantigenic determinants on the gp34

polypeptide.

(A preliminary reportof this work has been

published [S.L. Marcus and N.H.Sarkar, Abstr.

Annu. Meet. Am.Soc. Microbiol. 1979, S(H)21,

p.

297].)

MATERIALS AND METHODS

Viruses. The MuMTV that was used for all protein purification wasfrom the Mm5mt mousemammary tumorvirus-producingcellline (7), and was obtained as double isopycnically banded, concentrated prepa-rations from the Virus Cancer Program. The GR mouseMuMTV strain was purified fromsupernatant medium of the GR-3A cellline grown as previously described(19). The RIII MuMTV was obtained from RIIImousemilkandpurified aspreviouslydescribed (21).Avianmyeloblastosis virus, Rauscher murine leu-kemiavirus, and Mason-Pfizer monkey virus wereall obtainedasdoubleisopycnically banded and concen-trated preparations through the Virus Cancer Pro-gram.

Reagents.Alkyl-agarosematriceswerepurchased from MilesLaboratories, Inc. Nonionic detergent P-40 (NP-40) was from Particle Data Co. 125I forprotein iodinationwaspurchased fromNewEngland Nuclear

Corp. Sephadex G-75 was obtained from Pharmacia, andphosphocellulose came fromWhatman, Inc.

Antisera. Anti-C3H MuMTV serum prepared against whole, solubilized virions waskindly provided by R. Cardiff (serum no. 1)andJ. Schlom (serum no. 2). Anti-RIII MuMTV serum was prepared against whole, detergent-solubilized virus as previously de-scribed. All of the anti-MuMTV seraused in this study wereshown toprecipitate both of the MuMTV gly-coproteins. Antiserum prepared against C3H-derived MuMTV gp34purified using sodium dodecyl sulfate (SDS) was obtained through thecourtesy of R. Car-diff, andwasalsopreparedin our ownlaboratorywith the participation ofJ. Racevskis.

Radioiodination of protein. MuMTV gp34 or solubilized virus (2to 4jgofprotein),purified using the protocol described herein, was radioiodinated us-ing Iodogen (1,3,4,6-tetrachloro-3a,6a-diphenylgly-couril)obtainedfrom PierceChemicals,aspreviously described (13).

HydrophobicchromatographyofMuMTV. Vi-rusdisruption, in buffer containing 1%(vol/vol) NP-40,0.5%(wt/vol) sodiumdeoxycholate,10mM dithi-othreitol, 0.4 MKCI,10% (vol/vol)glycerol, and 0.05 M Tris-hydrochloride (pH 7.8), was carried out as previouslydescribed (12). Columns of2- to3-ml bed volume containing octylimino (C8)-agarose and de-cylimino(C,o)-agarosewerepreparedandequilibrated with wash buffercontaining 0.05 M Tris-hydrochloride (pH 7.9),1mMdithiothreitol, 10%(vol/vol) glycerol, and1MKCIat4°C.Theprotein concentration of the finaldisrupted virion solution was kept tobelow 10 mg/ml, and to avoidoverloading no more than5mg ofprotein was applied toany one column of alkyl-agarose matrix. After collection of column flow-through fractions, proteins were eluted with wash buffercontaining8.5 Methylene glycol,and themost tightlybound MuMTV structuralproteinswerefinally eluted from the columns with wash buffercontaining both 8.5 Methylene glycol and 1% (vol/vol) NP-40. Detailedhydrophobicchromatographyproceduresfor thefractionation of oncornaviralproteinsaregivenin references12and13.

Ionexchangeandgelfiltration chromatogra-phy. A column ofphosphocellulose (0.5 by8cm)was preparedandequilibratedwithbuffercontaining0.05 M Tris-hydrochloride (pH 7.8), 1 mMdithiothreitol, 10%(vol/vol)glycerol,and0.05MNaCl. The flowrate of thecolumnwasmaintainedat12ml/hwith theuse of a Pharmacia MP-3 peristaltic pump. Molecular sievechromatographywascarriedoutusingSephadex G-75,whichwasswelledtofinal volume inwaterand equilibrated with buffercontaining50mM Tris-hydro-chloride(pH 7.8), 1mMdithiothreitol,0.1%(vol/vol) NP-40, 10% (vol/vol) glycerol, and0.1 MNaCl. De-scendingchromatographywasperformedwitha pres-sure head of25 cm. The dimensions of the column usedwere 1.6by92 cm.

SDS-PAGE. Polyacrylamide gel electrophoresis (PAGE) wascarriedout inthe presence of SDS and the Laemmnli buffer system in 20-cm-long, 5to 17% (wt/vol) polyacrylamideexponential gradientslabsas previouslydescribed(18)orincylindricalgelsas pre-viously described(22).Ifnecessary,proteinswere

con-centratedbefore SDS-PAGEbyeitherlyophilization J. VIROL.

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MuMTV PROTEIN PURIFICATION: gp34 RIA 343

orprecipitation withtrichloroacetic acid,aspreviously described (12).

Radioimmunoassays. Normalrabbit serum and goatanti-rabbit serum werepurchasedfrom Poconos Rabbit Farms. After radioiodination of MuMTV gp34, free iodine was removedbychromatography on Seph-adexG-25,and the materialelutingatthe voidvolume was usedimmediately for immunoassay or stored at 00C.Titration of theimmunoprecipitation of'"I-gp34 was carried out in a volume of 500 yt using serial dilutionsof antiserum in a buffercontaining1%(vol/ vol)normalrabbit serum, phosphate-bufferedsaline, 0.1% (vol/vol) TritonX-100, and a fixed amount of labeledprotein (12,000 to 20,000 cpm).Incubation was carried out for2h at370Candovernightat40C.The next morning, 50

pl

of goat anti-rabbit serum was added to each reaction,mixed,and incubated for 1 h at370Cand then3 h at 40C.Precipitated protein was pelUeted by centrifugation for 5 min in a Beckman microfuge, and theresultingpelietwaswashed once withphosphate-buffered saline solution. "Ilabel in theprecipitatewascounteddirectlyin a gamma spec-trometer. The concentration of the various antisera necessary toprecipitate50%ofmaximallyprecipitable label was determined in this manner and, unless oth-erwisestated,wasroutinely used for radioimmunoas-say. In competition radioimmunoassays, serial

dilu-tions of unlabeled antigens, as detergent-disrupted virus orunlabeledC3HMuMTV gp34, were used as described above, both with a fixed concentration of antiserum.Virionsweredetergent disrupted, in a total volume of 100 to 200,uland at a proteinconcentration of1 to 1.8 mg/ml, by the addition of NP40 and sodium deoxycholate to final concentrations of 0.5% (vol/vol) and 0.1%(wt/vol),and the mixture was incubated for 30min at370C.Proteinconcentration wasdetermined usingthe Bio-Rad protein assay (3).

RESULTS

Purification of MuMTV structural

pro-teins. A flow chart of the protocols used to

obtain the simultaneous purification of all six

MuMTVstructuralproteinsfromC3H MuMTV

is shown in Fig. 1. We have previously used

(C1O)-agarose for the purification of the

least-hydrophobic proteins p23 and p16 from

MuMTV, in aprocedurewhichcanalso be used

toisolateR-MuLVppl2and plO(13).Toextend

the purification procedure using hydrophobic

chromatographytoall of theMuMTV proteins,

(C8)-agarose

wasusedasthe initial column step

because (i) under theconditions used,p27, p23,

MuMTV

FIG. 1. Flow chart showing the protocolsfor the simultaneous purification of MuMTV proteinsfrom a single preparation ofpurified virions. For details ofpurification refer to the text. Column matrices used in purificationareindicated with rectangles. Thebuffer constituents used for column elution or development are showntotheleft side of each appropriate arrow, and the protein(s) which represents the major constituents of each column fraction is showninparentheses. Circled polypeptide designations represent the final product of the purification.

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and p16are all obtained in the initial column

wash (flow-through) fractions (12), and (ii)

in-complete recovery of gp34wasobserved in

stud-ies with (Clo)-agarose (12). The peak

flow-throughfractions from the (C8)-agarose column

were immediately pooled and passed over a

(C1o)-agarosecolumn, which retainedonlyp27.

The p23 and p16 in the (Clo)-agarose column

wash fractions were initially separated by gel

filtration, andcomplete purification of p16 was

achieved bychromatography on

phosphocellu-lose carried out inbuffercontaining0.3MKCl.

The basic p16protein bindstophosphocellulose

at 0.3 MKCl and thencanbeeluted in a single

stepwith1MKCI.Themajorinternal MuMTV

protein, p27, is eluted from (C1o)-agarose with

ethylene glycol-containing buffer and purified

byafinal gelfiltration step. It isimportant to

note, forallof the proceduresinvolving

hydro-phobicchromatography,that thistechnique

uti-lizes 1 M salt in all washbufferstominimize the

possibility of ionic interactions. Before storage

orsubsequentchromatographyonion-exchange

resins or Sephadex, all fractions were dialyzed

againstlow-ionic-strengthbuffer andlyophilized

aspreviouslydescribed for murine oncornaviral phosphoproteins (13).

Of the MuMTVproteinsthatremained bound

tothe (C8)-agarose column,those thatare

mod-erately hydrophobic (gp47, p12) were eluted

with ethylene glycol-containing elution buffer

(12). After dialysis to remove ethylene glycol

andsalt (13), theethyleneglycol eluatefraction

wasconcentratedbylyophilization andapplied to a phosphocellulose column equilibrated as

described in Materials and Methods. Themajor

MuMTVglycoprotein,gp47,wasobtained in the

phosphocellulose column wash fractions, and

thep12 fraction was eluted with buffer

contain-ing 0.3 M NaCl. Final purificationofboth

poly-peptides was achieved by chromatography throughSephadexG-75 as described in Materi-als and Methods.

The strongly hydrophobic MuMTV gp34

re-mainedbound to (C8)-agarose during elution of

moderately hydrophobic proteins with 8.5 M

ethylene glycol (12). The gp34 was removed

from the column by application ofbuffer

con-tainingbothethyleneglycol and detergent (see

MaterialsandMethods).Aquantityofgp47was

also removed with the gp34, butcould be

sub-sequently separated (after dialysis and

lyophili-zation) by gel filtration in the presenceof

non-ionic detergent. Under these conditions the

stronglyhydrophobic gp34 wasobtainedinthe

voidvolume of the Sephadex G-75 column (data

notshown). The individual preparations of

pu-rifiedMuMTV structural proteins obtained as

outlinedinFig.1weresubjectedtoSDS-PAGE

J. VIROL.

analysis asdescribed in Materials and

Methods,

and theCoomassie blue-stained gel is sho*n in

Fig.2.From the appearance of thegel the

struc-tural proteinpreparations were nearly

homoge-neous, indicating that hydrophobic

chromatog-raphy is efficacious for the purification of all

MuMTV structural proteins, and that the

ex-tremehydrophobicity of gp34, which creates a

problemwhen other fractionation methods are

attempted, can actually be exploited as an aid to

itspurification.

Recovery of purified MuMTV structural

proteins. To determine theefficiency of

purifi-cation and the recoveries of the purified

poly-peptidesshown inFig. 2, the percent

contribu-tion of each protein to the total amount of

protein present in the virus should be known. The percentage of amino acid label or the

inten-sity of Coomassie blue stainingassociated with

proteinbandsseparatedbySDS-PAGEanalysis

hasbeen previously reported as useful in

esti-matingthe fraction of virion protein represented

by eachpolypeptide for MuMTV (5, 22, 26, 30).

For the major internal core protein, p27,

esti-matesobtained in this manner werefound to be

ingood agreement withvalues obtained by the

more reliable method ofcompetition

radioim-munoassay (13). For the other five MuMTV

structural proteins, however,

radioimmunoas-sayseither

v7,;

havenot

M

beendevelopedorwere not

r 3. >!'

;4jW-El -lp4:'

Elk gpv34-40

p2-r "'

-3p3, -p2'f7

53)I,

FIG. 2. Purified MuMTV structural protein prep-arations.SDS-PAGE in the slab gelwascarriedout asdescribed in thetext. (a) 60pgof C3HMuMTV protein usedasmarkerfor the other lanes; (b)8pgof MuMTVp12; (c) 6ptgof MuMTV p16; (d) 10pgof MuMTVp23; (e) 9pg ofMuMTVp27;

(t)

12pgof MuMTV gp 34; (g) 13pgof MuMTV gp47. Proteins werevisualizedbystainingwithCoomassie blue.

_.

gF

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used to calculate the percentage of individual

protein in isolated virions. We have therefore

used the values obtained from SDS-PAGE,

which admittedly may be subject to variations

due to the methods used, to approximate the

percentrecovery of virionpolypeptides purified

using hydrophobic chromatography (Table 1).

The bestrecoveries were observed for the

low-molecular-weightMuMTVproteins, with

recov-ery of p23equalingorexceeding 100% insome

preparations, as previously reported (13). Due

tothe addition ofagelfiltration steptoremove

acontaminating18,000-dalton proteinof

uncer-tain origin, recovery of p27 wasless with this

procedure than in an earlier report (13),

al-though recoveries in excess of50% were rou-tinely observed. Recovery of the moderately

(p12 and gp47)andstrongly(gp34)hydrophobic

MuMTVproteinsrangedfrom20 to50%(Table

1). This lower recovery relative to the other

MuMTVstructuralproteinsmayreflect the fact

that theseproteins bindtoandmustbe eluted

from alkyl-agarose columns, with subsequent

ion-exchange and/or gelfiltration

chromatogra-phy, and the useof severalsteps decreases

re-coveryof the viralproteins.

Development of radio'mmunoassay for

MuMTVgp34. We havepreviouslyshown (13) thatpurification ofp27using alkyl-agarose

de-TABLE 1. Recovery ofMuMTVproteinspurified using hydrophobic chromatography

Protein in final Individual Approx % Poly- prepn polypeptide/ recovery of peptide p totalprotein individual

Mga centb inv c(%) proteind

gp47 0.7 7 26-31 22-27

gp34 0.6 6 16-21 29-37

p27 0.9 9 16-18 50-56

p23 0.24 2.4 1-3 >100

p16 0.7 7 7-9 80-90

p12 0.3 3 6-8 37-50

a

Purified

MuMTV preparations (10 mgof protein)

wereused inarepresentative purification. The final purified productsobtainedwereanalogoustothe in-dividualpolypeptide preparationsshown inFig.2.

bVariations of10to20%wereobserved indifferent experiments.

cValues for the approximate percentage of total virionproteinrepresented byeachpolypeptide were obtainedfrom the percentage of totalradioactivity in eachprotein peak after SDS-PAGE using virions la-beled in vivo with amino acids (30) and from the relativeintensity of Coomassie blue stain associated witheach protein band insimilarly analyzed virions (22).

dExpressedasthe ratio of percentinputproteinto thepercentcontributionof the individualpolypeptide tototal viralprotein.

rivatives yielded preparations which did not

have alteredantigenic properties, as determined

by their ability tocompete with p27 inwhole,

detergent-disrupted, radioiodinated MuMTV preparations. We therefore attempted to

de-velop a radioimmunocompetition assay that

would allow the precise quantitation of gp34

within MuMTVvirions.

MuMTV gp34 purified asdescribed above was

radioiodinated in anIodogen-mediated reaction

as described in Materials and Methods. After

removal of the majority of free iodine by gel

filtration, the radioiodinated protein was

ana-lyzed by SDS-PAGE in cylindrical gels. The

1"I-labeled gp34 migrated as a single peak, consist-entwiththepurityof the gp34 preparation (Fig.

2),and indicating that little or no breakdown of

the protein had occurred during the iodination

process (data notshown).

Radioimmunoprecip-itation of

'"I-gp34

wascarried out asdescribed

inMaterials and Methods, and all anti-MuMTV

andanti-gp34 seraused in thisstudy produced

a sigmoid radioimmunoprecipitation titration

curve similar or identical to those previously

described (24, 25, 27). From 60 to 90% of the

labeled gp34 could be precipitated with

appro-priate antisera. In the absence ofspecific

anti-serum, however, <2% of inputradioactivity was

found in the pellet afterincubation with goat

anti-rabbit serum (data not shown).

Radioim-munocompetition assaysfor gp34using

antise-rum to SDS-purified C3H gp34 and purified,

unlabeled C3H MuMTV gp34 or

detergent-dis-rupted virions of C3H MuMTV, RIII, MuMTV,

and GR MuMTV as competing antigens are

shown inFig. 3.Thisstudy indicated that gp34

comprises approximately 13 to 14% of the total

MuMTV protein, a value in good agreement

with the more conservative approximations

reached by the analysis of stained gels after

SDS-PAGE (22,30). The anti-C3H gp34 serum

used in thisstudydidnot reactwithsolubilized

preparationsof avianmyeloblastosis virus,

Ma-son-Pfizer monkey virus, or Rauscher murine

leukemia virus, even athigh virus protein

con-centrations. This result indicates the presence of

stronggroup-specific antigenic determinantson

gp34.

Analysisoftype-specific antigenic

deter-minantson gp34. Previousreports have

dem-onstrated that, when usingantiserum directed

against the purifiedMuMTVgp47 (24, 25) and

p27 (27) in radioimmunocompetition assays,

type-specific aswell asgroup-specific antigenic

determinantsmaybefoundonthesestructural

proteins. The factthat, in radioimmunoassays

utilizing antisera against SDS-purified C3H

MuMTV gp34, all three strains of MuMTV tested showed identical immunocompetition VOL. 31,1979

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346 MARCUS, KOPELMAN, AND SARKAR curves

with C absenc

the po

presen

altered

10( 8(

6(

4( 2(

,aswellasabilitytocompletely compete that,when antiserawasraised againstit, a

pop-'3H gp34 (Fig. 3) suggested a possible ulation ofantibodies against type-specific

deter-eoftype-specific determinants.However, minants may have been absent.

Radioimmu->ssibility existed that purification in the noassays utilizing antisera against whole

IceofthedenaturingagentSDSmayhave MuMTV andpurifiediodinatedgp47have been

I the conformation of the protein such shown effective for the identification of

type-specific reactivity (24, 25). Therefore, several

preparations qf anti-C3H MuMTVserumwere

.___. __._ obtained(see Materials andMethods) and used

:)

C3H

§

- in the

radioimmunoassay

analysis.

Because

pre-GR vious studies indicated that low dilutions of

an-RZ

t\iXntiC3H-9p34

tiserum

provided

increased

sensitivity

to

type-(SDS-purified) specific differences (24), we used two dilutions

D of

antiserum,

one of which was sufficient to

precipitate 50% of the maximally precipitable

quantity of

1251-gp34

whereas the otherwas

10-O _ to 15-fold less diluted. Anti-C3H MuMTV serum

no. 2 used in this study (Fig. 4) showed strong

O _ / \ \<^S _ group

specificity

on

gp34

atlowserumdilution

C3H-gp34 (Fig. 4A). With this specific anti-C3H MuMTV

serum at this concentration, differences were

0oo

lo] 102 103 observed both in

slopes

ofthe

immunoprecipi-10o

102lOl° tation competition curves among MuMTV of

Competing protein (ng) C3H, GR, and RIII mouse origins and in the

3. Competition radioimmunoassay for C3H extentoffinal

competition

withlabeled

antigen

'Vgp 34 using antiserum prepared against at

high concentrations

of competing antigens.

trifiedC3Hgp34 and1251-gp34purified as de- Although the homologousC3Hvirus was ableto in Fig. 1. Antiserum was used at a final completely compete with labeled antigen for

a of1:15,000, which was sufficient to precipi- antibody, detergent-disrupted GR and RIII

%ofmaximally precipitable label. Detergent- MuMTV only competed toamaximumof 60%.

ted, solubilized avian myeloblastosis virus When the same anti-C3H MuMTV antiserum zuscher murine leukemia virus(V), andMa- was used at a final dilution of 1:180,000 (the ,zermonkey virus (O) were used as competing concentration capable of precipitating 50% of Istodetectgroup-specificantigenic reactivity. maximally precipitable labeled antigen), the

ized C3H(0),GR (0), and RIII (A) MuMTV of peting antigen ),

the

Rso used ascompetingantigens in addition to slopes ofcompetingantigens from all threetypes iC3Hgp34

(U)

toquantitate the percentcon- of MuMTVappeared quite similar.However, it )nof thatpolypeptide to totalMuMTVprotein. was evident that only the homologous C3H mmunocompetition assays and virus solubili- MuMTVprovidedsufficient antigenic determi-were carried out as described inthe text. nants to allow complete competition with the

5 Anti C3H-MuMTV

E 100

0~~~~

" 80 \

S 60 -G

X5 40

-1 20 -R

II IC3H-gp34

101 102 103 100 101 102 103

Cornpeting protein (ng)

FIG. 4. Radioimmunocompetition assayfor MuMTVgp34using anti-C3H MuMTVserumno. 2 (see the

text) at (A) 1:10,000and (B) 1:180,000final dilution (that dilution ofserumallowing50%precipitationof labeledantigen). Competing antigenswere asdescribedin thelegendtoFig. 3.

c

C

0

Q_E

-N

C

0

2

c--)

c

a.

FIG. MuMT SDS-pu scribed dilutioA tate50f: disrupto (x), Ra son-Pfi antigen Solubil wereal

purifiec

tributio Radioir zationi

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MuMTV PROTEIN PURIFICATION: gp34 RIA 347 labeledgp34.The GR and RIII MuMTV's

com-petedto85and90%,respectively(Fig. 4B),even

at 10-fold higher protein concentrations than

those showninthefigure. Therefore,

type-spe-cific antigenic determinants onthe gp34

mole-cule were observed using anti-C3H MuMTV

serum no. 2,and the presence ofsuch

determi-nantscould be amplified through theuseof low

dilutions of antiserum.

Using rabbit serum no. 1 to C3H MuMTV

(see Materials and Methods) at low (1:10,000)

and high (1:100,000) dilution, the slopesofthe

immunocompetition curves for C3H, RIII, and

GRvirusesappearedidentical(Fig. 5).The

fail-ureofGRMuMTVtocompetecompletelywith

labeled antigen was the only apparent signof

type-specific antigenic differences, although

group-specificreactivitywasstill apparent(Fig.

5A). Two other anti-C3H MuMTV sera

pre-pared and used in this study at high and low

dilution revealed notype-specific

immunoreac-tivity ongp34for the viruses examined.

There-fore, our studies using various rabbit antisera

prepared against C3H MuMTV show that the

abilitytoidentify type-specific antigenic

deter-minantsonMuMTVgp34 is dependent on the

specific antiserum used.

DISCUSSION

Hydrophobic chromatography has only

re-cently been appliedtothe study ofoncornavirus

proteins (11-13). We have shown that these

mat-ricescanbe usedtodetermine thepotential for

hydrophobic interaction (relative hydrophobic-ity) of structural proteins, and have arbitrarily

defined fourcategories ofincreasingrelative

hy-drophobicity based on these procedures (12).

The fractionation of viral structural proteins

observed onthese matricesnaturallysuggested

X Arin-C3H-MuMTV E

80

- 60

GM

0

20-theiruse asreagentsinprotein purification. The

lack of binding of both murinetype C andtype

B phosphoproteins to (Clo)-agarose has been

reported as a rapid and effective method for

theirpurification (13).Modificationof this

pro-tocol, as described above (Fig. 1), has allowed

the simultaneous purificationofallthe MuMTV

proteins, whichappearnear-homogeneousupon

SDS-PAGEanalysis,asdetermined by

Coomas-sieblue staining (Fig. 2).Traces of

contaminat-inglow-molecular-weight proteins ofuncertain

originare,however, sometimesseenin p23

prep-arations (13). The fact thatgp34 aggregates in

aqueous solution prompted other investigators

to resort totheuseof SDS in its purification(6).

Methods suchaslectin-agarosechromatography

(20, 29)aswe7lasion-exchange chromatography

(6),which haveprovensuccessfulin the

purifi-cation ofgp47, are notuseful forgp34isolation.

Indeed, gp47andgp34 copurify througha

vari-etyoffractionationsteps(29) and doso even to

a degree during hydrophobic chromatography

(12). Therefore, in addition toprovidinga

pro-cedurefor the simultaneous isolation of all six

MuMTV structural proteins in good yields

(Ta-ble 1), the protocol shown in Fig. 1 represents

the first method for purifyinggp34 without the

useofdenaturingreagents, through the

exploi-tation of its extremely hydrophobic nature. It

should be mentioned, however, that the

MuMTV DNApolymerase is not a likely

can-didate for future purification on alkyl-agarose

matrices, since,incontrast tostructuralproteins,

it bindstoboth(C8)-and

(Clo)-agarose

with such

avidity that neither ethylene glycol-containing

buffernorthesamebuffer alsocontaining

deter-gentsisabletoelute active enzyme(11).

By preparing aradioimmunocompetition as-saywithpurifiedgp34,wewere ableto

quanti-o0 o10' 102 103 lo-, 100 lol 102

Competing protein (ng)

FIG. 5. Radwimmunocompetition assayforC3HMuMTVgp34usinganti-C3H MuMTVserum no.1 (see

thetext)atdilutionsof (A) 1:10,000and(B) 1:100,000 (thatdilutionofantiserumallowing50%precipitation

oflabeledantigen).Symbols for competing antigensareidenticaltothosedefinedin thelegendtoFig.3. VOL. 31,1979

http://jvi.asm.org/

[image:7.504.112.405.486.633.2]

(8)

tatethe amount ofgp34present inmaturevirus.

We estimated that gp34 accounts for

approxi-mately 13to14% of the totalproteininpurified

virions. The absence of deoxycholate, or an

in-crease in virusproteinconcentration in the

sol-ubilization reaction mixture above 1.5 mg/ml,

resulted inunderestimationof the percentage of

gp34 in thevirion (datanotshown). This result

may bedue to the tenacious association of gp34

with the virusmembrane, toitslipophilic

prop-erties, to its tendency to aggregate in aqueous

solution (whichperhapsresults in the masking

of certainantigenicsites),or toall of these. The

factthatlipophilic proteins bindlargequantities

of detergents (9) suggests that theweightratio

ofprotein to detergent may be acriticalfactor

inthe solubilization of certain virus proteins.

Although several reports using differing

im-munological techniques have shown the pres-ence of group-specific antigenic determinants

associated with MuMTV (1, 15, 16, 21),

identi-fication oftype-specificdeterminantsonpurified

MuMTV proteins have onlybeen reported for

gp47(24, 25) and p27 (27).Blair (2) was the first

toreport thepossible presence oftype-specific

differences amongMuMTV's,usingwhole virus

preparations in immunodiffusion analysis. We

have been able to demonstrate the apparent

existence of both group- andtype-specific

anti-genicreactivitiesonthe gp34polypeptide (Fig.

3 to5) usingdetergent-solubilized preparations

of thehomologous (C3H)MuMTVand RIII and

GRmousecell-derivedMuMTV'sascompeting antigens. Ofthree different anti-C3H MuMTV

seratested having similaror identical

radioim-munoprecipitationtiters forgp34,onlyone

(se-rum no. 2, Fig. 4) showeddistinct type-specific

differences in gp34antigenicreactivity;theRIII

and GR viruseswereunabletocompletely

com-pete withC3H gp34 for the available antibody

population. Teramoto and Schlom (28) have

obtained similar results with antiserum no. 2.

Type-specific antigenic determinants on

MuMTV gp34 also appear to bemost

demon-strable at low antiserum dilution (Fig.4A).

Al-though only one of theseratestedshowed clear

type-specific differences,all anti-C3HMuMTV

seraused in this study demonstrated

group-spe-cificreactivities on MuMTV gp34. The finding

oftype-specific antigenic reactivities on a

puri-fied viralproteinwith oneantiserummay

there-forenotnecessarily mean that identical results

willbeachievedwith other antiviralsera, even

ifthey show comparable titers in

radioimmu-noprecipitationassays.

Trypticpeptidefingerprinting analysisof

ra-dioiodinated viral proteins has recently been

used to analyze possible strain-specific

differ-ences in the gp47, gp34, and p27components of

RIII,C3H,andGRMuMTV (8). Differences in

the peptide maps of gp47 and p27 were found

consistent with previous descriptions of type

specificity (24, 25, 27). However, significant

dif-ferences in the gp34 peptide maps from all three

virus strains were notdetected (8). These results

are in contrast to the demonstration of

type-specific determinants by at least one

anti-MuMTV serum in our studies (Fig. 4). It is

possible that this antiserum contains a unique population of antibodies to specific and perhaps

minorchanges inglycosylationofthe C3H gp34

as compared with other gp34molecules of the

RIII and GR MuMTV strains. These changes

would not be detectable in peptide mapping. Alternatively, type-specific differences may

re-side inpeptideslacking tyrosine residues, which

would be undetected by the procedure used for

the analysis of the peptide maps of MuMTV

proteins (8). We are currently preparing antisera

against gp34 purified by the above procedure

(Fig. 1 and 2) to determine whether antisera

prepared against the nativeformof gp34 can be

used to detecttype-specific reactivities on this

protein.

ACKNOWLEDGMENTS

We are grateful to R. Cardiffand J. Schlom for their generousgiftsof MuMTVantiseraandtoC. Sherr forhelpful discussionsregarding radioinununoassay protocol.Wethank J.GruberforprovidingtheC3H MuMTV and nonmammary oncornavirusesused in thisstudy.We also thank J. Racevskis and E. Whittington for anti-C3H gp34 serum and for SDS-PAGEanalysis.

This workwassupported,inpart,byPublic HealthService grants CA-17129 and CA-08748 from the National Institutes of Health.

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