Subunit structure of the glycoprotein complex of avian tumor virus.

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Copyright © 1976 AmericanSocietyforMicrobiology Printed inU.SA.

Subunit Structure of the

Glycoprotein Complex

of

Avian

Tumor

Virus

ROBERT N. LEAMNSON AND MICHAEL S. HALPERN*

TheWistarInstituteofAnatomyandBiology,Philadelphia, Pennsylvania 19104 Received for publication 9 February 1976

Envelopeglycoproteinof aviantumorvirus islinked bydisulfide bonds ina

structure that we havedesignated VGP to stand for viral glycoprotein. VGP

appears to contain one molecule ofgp85 and one ofgp37. Undernonreducing conditions, VGP is the only glycoprotein component that is stable in the

pres-ence ofionicdetergent, although in the presence ofnonionic detergenttwoor

more VGPs are associated in discrete complexes. The disulfide bonds linking

viral glycoprotein areformed before release of virus from infectedcells.

The surfaceprojectionsof avian tumorvirus

are assemblages of two constituent

glycopro-teins (3, 6). Theseglycoproteins,which are

des-ignated gp85 andgp37, have beenisolated

pre-viously by treatment ofpurified virions with reducinganddissociating agents. Forthis

rea-son, detailed information pertaining either to

thesubunit structure of theprojectionsortothe

nature of the bonds stabilizing the subunit interactions is lacking. The experiments

de-scribed in this report were therefore

under-taken to more fully characterizethe structure

oftheglycoprotein complex.

MATERIALS AND METHODS

Viruses and cells. Primary cultures of chicken embryo cells were preparedby the method of Vogt

(19) from 10- or 11-day old C/E chicken embryos

(obtained from H & N Farms, Redmond, Wash.)

that were either positiveornegative for chick helper factor [chf(+) or chf(-)]. Clone-purified stocks of avian sarcoma virus B77, subgroup C, and the Prague strain of Rous sarcoma virus, subgroup B (PR-RSV-B), were used for infection of secondary cultures of the chf(-) cells. Maintenance of unin-fected and inunin-fectedsecondary cultures were as pre-viouslydescribed (9).

Purification of isotopically labeled virus. To la-belvirus with amino acid, confluent secondary cul-tures ofRSV-transformed chicken embryo cells in 100-mmpetridishes wereincubated at 37 C for 18 h

in 5 ml ofmedium 199 that was 97% deficient in amino acid but supplemented with 5% calf serum

andeither 50 ,uCi of a[3H]aminoacidmixture or 20 ,uCi of a ['4C]amino acid mixture per ml (both isotopes purchased from New England Nuclear [NEN], Boston, Mass.). To prepare carbohydrate-labeled virus, RSV-transformed chicken embryo cells were incubated for 18 h at 37 C in 5 ml of medium 199 that was supplemented with 5% calf

serum and one ofthe following: 50 ,uCiof [3H]glu-cosamineperml, 50 ,uCiof[3H]fucose per ml, or20

,uCiof[f4C]glucosamine perml (allpurchasedfrom

NEN). All labeling media contained 1% (vol/vol)

dimethylsulfoxide.

To purify labeled virus, tissue culture

superna-tantswerefirstclarifiedby centrifugationfor 30min

at6,000rpm inaSorval RC-5centrifuge.Viruswas

thensedimented in aSpinco SW41 rotorfor 2 h at

25,000 rpm through a 5-ml layer of 20% (wt/vol)

sucrose and onto a 1-ml layer of 65% sucrose (all sucrose solutions were prepared with standard buffer: 0.1 M NaCl, 0.01 MTris-hydrochloride, pH 7.4, and 0.001 M EDTA). The band of virus at the interface of thetwo sucrose solutionswas then di-lutedtolessthan 20%sucrosewithstandardbuffer, layeredon alinear,preformed gradientof24to48%

sucrose, andsedimented toequilibrium for 16 hat 37,000rpm intheSW41rotor.Virus in the

appropri-ate gradient fraction was diluted with standard

buffer,made0.05 M iniodoacetamide, and recovered

by pelleting for 2 h at 40,000 rpm in the Spinco

SW50.1rotor.

Electrophoretic analysis of the structural poly-peptidesofpurifiedvirions.Electrophoresisof viral

polypeptideswascarriedoutontwopolyacrylamide

gel systems, thesodium dodecylsulfate (SDS)

sys-tem described by Laemmli (13) and the alkaline

urea system described by Reisfeld and Small (18).

ForanalysisonSDS-gelofnonreduced viralprotein,

purified virus was disrupted in 0.1 ml of 1%(wt/vol) SDS, 0.05 M iodoacetamide, 10% glycerol, and

enough phenol red to serve as tracking dye; for

analysis of reduced viral protein, purified virus was

disruptedwith0.1mloftheSDS solution, identical

totheaboveexcept for the omission of the iodoaceta-mide and theaddition of1%(vol/vol) 2-mercaptoeth-anol. Priorto application to SDS-gels, reduced and nonreduced samples were incubated in a boiling

waterbath for3min.

Forcertain SDS-gelelectrophoresis experiments, viral protein was reduced by disruption ofpurified virus in 1.0 ml of 1.0 MTris-hydrochloride, pH 8.1, 0.1 Mdithiothreitol, and 1% SDS. After incubation ofthe reduced sample for 3 min in a boiling water bath, alkylation was effected by the addition of iodo-acetamide to 0.30 M. After a furtherincubation for

15 min at20C andthe addition of 100

jig

ofbovine )56

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serum albumin (BSA) to serve as carrier, the pro-tein was recovered by precipitation with 5 volumes

of ethanol and resolubilized with SDS in the

pres-ence ofiodoacetamide, asdescribed above.

Foranalysis on alkaline urea gel of nonreduced viral protein, purified virus wasdisrupted in 0.1 ml of10 M urea, 0.05 M iodoacetamide, and enough bromophenol blue to serve as dye marker. Before application to the alkaline urea gels, samples were incubated for 1 h at 37 C. For electrophoresis of reduced and alkylated viral protein on alkaline urea

gel,purified viruswas disrupted for 1 h at 37 C in 1.0 ml of 1.0 M Tris, pH 8.1, 0.1 Mdithiothreitol, and 10 M urea. After the addition ofiodoacetamide to 0.30M, the protein (together with the carrier BSA) was recovered by precipitation with 5 volumes of ethanol and resolubilized in 10 M urea in the

pres-enceofiodoacetamide.

Allgelsweresliced into 2-mm sections on stacked razor blades. Direct determination of radioactivity in individual slices was carried out as previously

described (11). In certain experiments, labeled

pro-teinwas recovered by elution from the individual

gel slices. Elution under nonreducing conditions

waseffected by incubation of the slices for 18 h at 37C in 1.5 ml of 0.01 M Tris, pH 7.4, 0.1% SDS, and 0.05 Miodoacetamide; elution under reducing condi-tionswaseffected by incubation of the slices for 18 h at 20C in 1.5 ml of 0.01 MTris, pH 7.4, 0.1%SDS,

and1%2-mercaptoethanol. Toidentifypeaksof

la-bel,the radioactivity in individualeluates was

de-terminedby scintillation counting of small aliquots

inAquasol (NEN). Protein wasrecovered from the eluate by precipitation with 5 volumes of ethanol

(aftertheadditionof carrierBSA) and then

solubi-lizedasdescribed above.

Sedimentation analysis of the structural

poly-peptidesofpurifiedvirions.Sedimentationanalyses

of viralpolypeptideswereperformed using5to25% linear sucrose gradients containing either ionic or nonionicdetergent. For theformer,the sucrose solu-tions were prepared in 0.2 M NaCl, 0.01 M

Tris-hydrochloride, pH7.4, 0.001 MEDTA,and1%SDS.

Viruspelletsweresuspendedin 0.5mlofstandard

buffer containing1%SDS,sonicated, and incubated

for 30 min at 37C. The radioactivity in a small

aliquotwasdeterminedandanappropriatevolume

layered onto the gradient. Centrifugation was at

37,000 rpm at 18 C inthe SW41 rotor for 18 h. The

gradientwasfractionated bybottom punctureand

aliquotswerecounted inAquasol.Tween20 sucrose

gradientswereprepared andanalyzedinan

identi-cal fashion except that1% (vol/vol) Tween 20 was

substituted forSDS, both in the sucrose solutions

and in the buffer used forsuspensionof viruspellets.

[14C]aminoacid-labeledimmunoglobulinG(IgG),

generously provided by Sherrie Morrison,

Depart-ment of Microbiology, Columbia University, was

cosedimentedin certain of thegradients. The IgG

had beensynthesized byP3 murineplasmacytoma

cells (a tissue culture linederived from theMOPC

21 plasmacytoma). Aliquots of thedialyzed tissue culturesupernatant fluidweredirectly layeredon

gradients sincegreater than 90% of the

trichloro-acetic acid-precipitable radioactivity in the super-natantrepresentedlabeledimmunoglobulin.

Immune precipitation. Anti-gp85 immune precip-itateswere prepared from lysates of secondary cul-tures of[3Hlglucosamine-labeled, uninfected chf(+)

or B77-transformed chicken embryo cells. The

method of immuneprecipitation was as described in Halpern et al.(10), except that the lysing buffer was made 0.05 M in iodoacetamide. The anti-gp85 serum waskindly provided by Dani Bolognesi, Duke Uni-versity MedicalCenter, Durham, N.C. Immune pre-cipitates weresolubilized with SDS in the presence of either iodoacetamide or 2-mercaptoethanol and thenelectrophoresed on SDS-gels.

RESULTS

Covalentlylinked subunits of the glycopro-tein complex. Figure la shows the two peaks of label, corresponding to gp85 and gp37, that are resolved when [3Hlglucosamine-labeled B77

vi-rus isdisrupted with SDS in the presence of the reducing agent2-mercaptoethanol and electro-phoresed on an SDS-gel. In contrast, a single major peak of label is resolved on an SDS-gel when virus is disrupted with SDS in the pres-ence of the alkylating agent iodoacetamide (Fig.lb). This latter peak, which has a slightly lower electrophoretic mobility than gp85 (Fig. lc),will bedesignated as VGP to stand for viral

glycoprotein (the peaks of label which comi-grate with thedye marker at 134 to 138 mm in Fig. lc are presumed to representglycolipidor free carbohydrate since these peaks are not

detected when viral glycoprotein is first ex-tracted with phenol). If values of 85,000 and

37,000 areassumedto representthe molecular weights, respectively, of the major (gp85) and minor(gp37)glycoprotein, the apparent molec-ularweightofVGP, asdetermined bythe mi-gration of the viral glycoproteins on the SDS-gel, is 96,000. This value must beregarded as an approximation, however, since in general

the migration of glycoproteins on SDS-gel is not a strict function ofmolecular weight (4),

and in any case themolecular weights of the major and minor glycoprotein are not known with great accuracy (1).

The absence ofgp37 inthepherogramofFig.

lb suggestedthat one or moredisulfidebonds

covalently link gp37 inVGP. This supposition

was confirmed by analysis of VGP that had been eluted (with SDS inthe presenceof iodo-acetamide) from an SDS-gel. The eluate was

dividedinto twoaliquots, and VGPwas recov-ered from each by ethanol

precipitation.

The VGP fromonealiquotwassolubilized with SDS inthepresence ofiodoacetamide and from the other with SDS inthepresence of

2-mercapto-ethanol. Electrophoresisof the former

(Fig.

2a) yielded predominantly VGP

although

rela-tivelyminor amountsofgp85 (40to44mm)and

gp37 (118 to 124 mm) were

detected,

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958 LEAMNSON AND HALPERN

0

in

a-0

0

N

a-cu

0

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 1. Electropherogramson 10%SDS-gelsof (a) reduced[3H]glucosamine-labeledB77virus;(b)

nonre-duced [3H]glucosamine-labeled B77 virus; (c) reduced andalkylated ['4C]glucosamine-labeled B77 virus

coelectrophoresed with nonreduced[3H]glucosamine-labeled B77virus. The reduction and alkylationwere

carriedoutinthepresenceofSDS,asdescribedinMaterials and Methods.

ably in consequence of spontaneous disulfide

reduction that had occurred during elution.

Electrophoresis ofthe reduced VGP (Fig. 2b) yieldedgp85and gp37 in relativeamounts

com-parable to those detected when purified [3H]glucosamine-labeledviruswasreduced and

analyzed directly (Fig. la). Furthertreatment

withreducingagentof gp85 andgp37thathad

been eluted from an SDS-gel ofreduced virus didnotsignificantlyalter themobility of either glycoprotein (datanotshown).

The experiments illustrated in Fig. 1 and 2

were then repeated, using for analysis

[3H]fucose-labeled

B77 virus and

[3H]glucosa-mine-labeled PR-RSV-B (data not shown). In each case, the absence ofreducing conditions

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STRUCTURE 959

0

6

a-0

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 2. Electropherogramson 10%SDS-gels of(a)nonreduced[3H]glucosamine-labeled VGP; (b)reduced

[3H]glucosamine-labeledVGP. The VGP used for theseanalyseswasrecoveredbyethanol precipitationafter

itselutionfromanSDS-gelequivalent to thatrepresentedinFig.lb.

resulted inthe detection of VGP. Subsequent

reduction of the eluted VGP resulted in the

detectionof gp85and gp37.

Sincecarbohydrate-labeledviruswasusedin all the experiments describedabove, the

possi-bility that one or more of themajor nonglyco-sylatedviral structuralpolypeptideswasalsoa

constituent of VGP could not have been

as-sessed. Because the intensityonSDS-gelof the stained bandcorrespondingtothe

nonglycosyl-atedpolypeptide p19hasalreadybeenshownby

Bolognesi andBauer (2)tobeloweredwhen the structuralpolypeptidesof avianmyeloblastosis

virus are analyzed under nonreducing

condi-tions, thepresence inVGP ofp19,inparticular,

wouldnothave beenunexpected. Totest, there-fore, foranonglycosylated polypeptide constit-uentofVGP, structural analyses of amino acid-labeled viruswerecarriedout.

For this purpose, the polypeptides of

[3H]aminoacid-labeledvirusthathad been dis-rupted with SDS in the presence of

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960 LEAMNSON AND HALPERN

tamide wereresolved on anSDS-gel (Fig. 3a).

Incontrast tothe gelsused earlier (Fig. 1 and 2),all ofwhich contained10% (wt/vol) acrylam-ide, the gels used in Fig. 3 were polymerized with 12% acrylamide because the higher

per-centageofacrylamide ismoresuitable for

reso-lution of nonglycosylated virion structural polypeptides(11).The materialinthepeak cor-respondingto VGPwastheneluted, recovered by ethanol precipitation, and solubilized with

SDS inthepresence ofmercaptoethanol.

Coelectrophoresis of this material with re-duced [14C]amino acid-labeled virus indicated that the major peaks of 3H radioactivity cor-responded to gp85 and gp37 (Fig. 3b). Only small ortrace amountsof3H-labeledp19, p15, orp12weredetectableinFig. 3b(because gp37 isnotwell resolved fromp27,theamountof 3H-labeled p27 in Fig. 3b is difficult to estimate; nevertheless, the displacement of the 3Hpeak

at 70 to 84 mm from the peak fraction of

14C-20 a

'5

-I

0-_5- VGP

- S- b

x

C-

4-labeled p27 suggests that the bulk of the 3H

labelrepresentsgp37). This resultimpliesthat

notoneof thesenonglycosylated polypeptidesis

amajorstructuralcomponentofVGP. It should be noted, nevertheless, that, in agreement

withtheearlier results ofBolognesiandBauer

(2),disruptionofvirusin theabsenceof reduc-ing agentresults in a lowering of the amount ofdetectable p19 (Fig. 3a, comparisonwith the pattern of 14C label in Fig. 3b). Neither this phenomenon nor the apparentreduction ofp12 in the absence ofreducing conditions was in-vestigated further in thisstudy, however.

The finding that VGP comprised predomi-nantly, if not exclusively, viral glycoprotein

raised thequestion ofthestoichiometry of gp85

andgp37inthedisulfide-linked VGP complex. Electrophoretic analysesonalkaline urea-poly-acrylamide gel were undertaken to gain

evi-dencebearingon this point.

[14C]glucosamine-labeledvirus, disruptedwith 10 M urea inthe

P15

p27

p27 5

p15 4

p12

l

30

p19 2 a..

DISTANCE OF MIGRATION (mm)

FIG. 3. Electropherograms on 12%SDS-gels of(a) nonreduced [3H]amino acid-labeled B77 virus; (b)

reduced[3H]aminoacid-labeledVGPcoelectrophoresedwith reduced[14C]aminoacid-labeledB77 virus. The

VGP usedfor the analysis in (b) was recovered by ethanol precipitation after its elution from an SDS-gel

similartothat representedin (a).

J. VIROL.

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presenceofiodoacetamide,wasinitially

coelec-trophoresed with [3H]glucosamine-labeled vi-rus that had been disrupted with 10 M urea under conditions of reductive cleavage of disul-fide bonds. As shown in Fig. 4, all the detecta-ble 14C label remained at or near the origin of the gel, whereas the bulk of the 3H label

mi-grated well within the gel (24 to 52 mm) and only asmallamountof3Hlabelremained at the origin. Similar results to those shown in Fig. 4 were obtained for viral glycoprotein that had been recovered by ethanol precipitation from

SDS-disrupted virus and then resolubilized

with 10 M urea beforeanalysis.

Toobtainlabeledmaterial with a higher

spe-cific activity, the electrophoresis on alkaline urea-gel with nonreduced viral glycoprotein

was then repeated using

[3H]glucosamine-la-beled virus. The labeled material in the first fractionwasthenelutedwith SDS in the pres-ence of mercaptoethanol. Analysis of this eluate on SDS-gel resolved gp85 and gp37 in relative amountscomparableto thosedetected

afterdirectanalysis of reducedvirus (Fig. 5a, comparison with Fig. la).By contrast, electro-phoresis on SDS-gelofthepeakelutedfrom the first fraction of the alkaline urea-gel of the

reduced and alkylated viral glycoprotein

yielded predominantly gp37 (Fig. 5b); electro-phoresis of the majorpeak elutedfrom the frac-tions between 24and 52 mm yielded

predomi-nantly gp85 (Fig. 5c).

The resultspresentedinFig.4and5indicate that gp85andgp37 canbeseparatedonalkaline urea-gel only under reducing conditions. The implications ofthese results forelucidation of

the subunit structureofVGP will beconsidered

inthe Discussion.

Noncovalentlybonded subunits ofthe

gly-3

-

a-ol-1

coproteincomplex. The experiments described heretofore, which used SDS or urea as a disso-ciating agent, could not serve to establish whether two or more molecules of VGP are linked in whole virus bynoncovalent bonds. To detect putative VGP complexes,

[3H]glucos-amine-labeled B77 virus was disrupted with Tween 20 and then analyzed on a Tween 20 sucrosegradient. As shown in Fig. 6a, the bulk ofthe radioactivity was detectable in two peaks

(designatedAand B), both of which sedimented morerapidlythan IgG. In contrast, as shown in Fig. 6b, only a single peak of label (designated C), which sedimented more slowly than IgG, was detected when SDS-disrupted [3H]gluco-samine-labeled virus was analyzed on an SDS sucrosegradient. (To insure nonreducing condi-tions, all the gradients represented in Fig. 6 wereformed with iodoacetamide and the virus samples analyzed on these gradientswere dis-rupted in the presence ofiodoacetamide.) Cal-culations madeusing the relationship derived by Martin and Ames (16), relating the sedimen-tationcoefficients of proteins to their distances ofmigration on sucrosegradients, served to as-sign approximate S values of 12, 9, and 5 for

peaks A, B, and C, respectively(these calcula-tions weremade assuming that the sedimenta-tioncoefficient of IgG is 6.6).

Experiments werethen undertaken todefine therelationship of thematerialinpeaksA, B,

and C to VGP. Electrophoresis on SDS-gel of the peak C material, after its recovery from

SDSgradient byethanolprecipitationand sub-sequentsolubilizationwithSDSinthepresence

of mercaptoethanol, resolved gp85 and gp37 (Fig. 7b). Electrophoresis of recovered peak C material

after

solubilization with SDS and

io-doacetamide resolvedonlyasinglepeak of label

+ 3

0

o

K

2O

CL

I!

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 4. Electropherogram on a4% alkaline urea-gel ofnonreduced['4C]glucosamine-labeled B77 virus

coelectrophoresedwith reduced andalkylated[3H]glucosamine-labeled B77virus. The reduction and

alkyla-tion werecarriedoutinthe presenceofurea,asdescribedinMaterials and Methods.

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962 LEAMNSON AND HALPERN

0

.

0 I -T

of

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 5. Electropherograms on 10% SDS-gels of

(a)material elutedfromthefirst fractionofan alka-line urea-gel (similar to that shown in Fig. 4) of nonreduced [3H]glucosamine-labeled B77 virus; the eluate wasreducedbeforeelectrophoresis; (b)

mate-rialeluted from thefirst fraction of an alkaline

urea-gel ofreduced and alkylated

[3H]glucosamine-la-beledB77 virus; (c) material eluted from the peak

(correspondingtothe 3H labelat 24to52 mm inFig.

4) ofanalkaline urea-gel ofreduced andalkylated [3H]glucosamine-labeledB77virus.

that migrated with the mobility expected of

VGP (Fig. 7a) and, hence, peak C must be

equivalenttoVGP.

Additionalsedimentationanalysis then

indi-cated thattheglucosamine-labeled material in

peaksA and B (isolated from aTween20

gra-dient) migrated on SDS sucrose gradient with

the mobility ofpeak C (datanot shown),

sug-gesting that both peaks represented discrete

complexes of VGP. Direct evidence for the

pres-ence of VGP in peaks A and B was provided

by SDS-gel electrophoresis. The

electrophero-gramsof the nonreduced and reduced forms of

peaksA (Fig. 7e and f) and B (Fig. 7c and d) were indistinguishable from those ofpeak C (Fig. 7aand b). Inaddition, onlyasmall frac-tionof the total labelin[3H]aminoacid-labeled virussedimentedinpeaksAandB(Fig. 6c).As

analyzed by SDS-gel electrophoresis of the amino acid-labeled material recovered from

pooled gradient fractions (data not shown),

a

6 6

4 4

2 -2

b

IgG

128- { t6

8

4t

a. a.

Io 0

10 20 30 40

FRACTION

FIG. 6. Sedimentation analysis on sucrose

gra-dients of (a) Tween 20-disrupted [3H]glucosamine-labeled B77 virus; (b) SDS-disrupted

[3H]gluco-samine-labeled B77 virus; (c) Tween 20-disrupted

preparations of[3H]glucosamine-labeled and

['4C]-amino labeled B77 virus. [14C]amino acid-labeled IgG was cosedimented with virus on the

gradientsin(a) and (b).The gradients in (a) and (c) contained Tween 20 and the gradient in (b) con-tainedSDS. Tops of gradients are to the right.

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0

-2-10. d

gP85

8-VGP

6-

4-2- gp3?

20 40 6f0101010 20 4 0 8 0 2 4

VGP

6-

4-2- gp37

20 40 60 100 120 140 20 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 7. Electropherogramson10%SDS-gels of radioactively labeled material recovered from gradientsas inFig. 6. (a) Nonreduced peak C material; (b) reduced peak C material; (c) nonreduced peak B material; (d) reduced peak B material; (e) nonreduced peak A material;

(t)

reduced peak A material. [3HJglucosamine-labeled B77 viruswasused bothfor the isolationon aTween 20sucrosegradient (similartothat showninFig. 6a) ofpeaksAandBandfor the isolationon anSDSsucrosegradient (similartothat shown inFig. 6b) of peak C. The samples ofreduced and nonreduced material for peak C (7a and b), peak B (7c and d) and peakA

(7e andt) wereanalyzedonparallel gels for direct comparison oftherelative mobilities.

VGPrepresentedthepredominant protein

spe-cies detectable inpeaksAandB,whereasonly

nonglycosylated viral structural protein was

detectable in the slowly sedimenting peak at

fractions 28 to 36 ofFig. 6c. This latter result

eliminates the possibility that the greater S

values ofpeaksAandB, relativetoC,weredue to the association ofsingle molecules of VGP withlargeamountsofnonglycosylated protein and implies instead that several molecules of

VGPareassociatedinpeaksAand B.

Synthesisof viralglycoprotein.To ascertain

whetherthe disulfide bondslinkingviral glyco-proteinareformedintracellularly (eitherin the

cytoplasm or on the plasma membrane), im-mune precipitation with a monospecific

anti-gp85serum wasusedas aprobefor thepresence

of VGP inlysatesofglucosamine-labeled

trans-formed cells. ElectrophoresisonSDS-gel ofan

anti-gp85 immuneprecipitate, preparedunder nonreducing conditions, resolvedasingle major

peak of label (Fig. 8a). Thispeak was not

de-tected when nonnal rabbit sera were

substi-tuted for theanti-gp85serum(datanotshown),

implying that the peak material mustpossess

antigenicdeterminants in commonwithgp85.

The lowermobilityof thepeakdetected inFig. 8a, relativetothe mobilityof the cell-associated gp85(resolved, asshown inFig. 8b,on a

paral-lelgel ofareducedanti-gp85immune

precipi-tate), suggested that the peak represented VGP.

The equivalence of VGP and the peak

de-tected inFig. 8awasdirectlyconfirnedby

elec-trophoretic analysisof the elutedpeak. An

ali-quotof theeluate,resolubilizedin thepresence

of iodoacetamide, comigrated with

[14C]glu-cosamine-labeled viral VGP (Fig. 9a),whereas

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964 LEAMNSON AND HALPERN

25

20

15

0 a-0

x

2

*6)

10.

5-1

5-I0

5 gp37

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 8. Electropherograms on 10% SDSgels of anti-gp85 immune precipitates, eachprepared with 106

trichloroacetic acid-precipitable countsper minute (cpm) ofa lysateof [3H]glucosamine-labeled B7rtrans-formed chicken embryocells:analysis under (a) nonreducing conditions; (b) reducing conditions.

an aliquot ofthe eluate, resolubilized in the

presenceofmercaptoethanol, yieldedtwopeaks

comigrating with viral gp85 and gp37, respec-tively(Fig. 9b). These results establish that the

peak inFig. 8a represents VGP, and we

there-fore conclude that covalent linkage of viral

gly-coprotein in VGP occurs intracellularly, prior

tothe releaseof virus into the culture medium.

Similar experiments were then undertaken

to ascertain ifVGP wasdetectableinlysates of

uninfected chf(+) chicken embryo cells.

Pre-viouswork (9), based on analysis under

reduc-ing conditions of anti-gp85 immune

precipi-tates, hadindicated that these cells synthesize

gp85 that possesses antigenic determinants of

subgroupEviral gp85. These experiments were repeated in this study and the results are

shown in Fig. lOa. A peak corresponding to

gp85 isdetected,as well as a minorpeak that,

on thebasis of its coelectrophoresis with viral

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SUBUNIT

16-12

0

0

a-CL)

0

on

0

0

CY

I

0

x

C<)

C-20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 9. Electropherograms on 10% SDS-gels of the major peak eluted from the SDS-gel (similar to that shown in Fig. 8a) of an anti-gp85 immune precipitate that had been prepared with a lysate of

[3H]glucosamine-labeledB7rtransformedchickenembryocells and analyzed under nonreducing conditions:

(a) eluateanalyzed undernonreducing conditions; (b) eluateanalyzedunderreducing conditions. Nonre-duced ['4C]glucosamine-labeled B77 virus was coelectrophoresed in (a) and reduced

['4C]glucosamine-labeledB77viruswascoelectrophoresedin(b);thepositionsofthe viralVGP,gp85,andgp37areindicated.

gp37, is presumed to represent cell-associated

gp37.

Inthisstudy, anti-gp85 immuneprecipitates were also analyzed under nonreducing condi-tions, and, asshownin Fig. 10b, onlyasingle

major peak of label was detected. Because of

the relativelylow level ofradioactivity associ-ated with this peak, analysis of eluted peak

materialwas notundertaken. Nevertheless, as

evidenced by the coelectrophoresis with viral VGP and the absence from the pherogram of

theputative gp37moietythat isdetectedwhen

reducing conditions areused, thispeakalmost certainlyrepresents VGP.

DISCUSSION

Twomodelsforthesubunit structure of VGP canbereconciledwiththe results of the

analy-ses onSDS-gels. Inone,VGP wouldrepresent

individual molecules ofgp85andgp37 linked by

one or moredisulfide bonds;intheother, VGP

wouldcorrespondto adisulfide-linkedcomplex of

gp37

molecules (most reasonably a trimer, butconceivably adimer) thatis notcovalently bondedtogp85. Any modelinwhich VGP

rep-resents a higher-molecular-weight aggregate

(such as a dimer of gp85) is ruled outby the observation that the mobility on SDS-gel of VGP is only slightly lower than that of gp85.

In at least one respect, the results obtained

with electrophoresis on SDS-gel are more

con-sistent with the one gp85-one gp37 model for

VGP structure. If the gp37 trimer model is

correct, then reduction of the interchain disul-fidebondsinthe VGPcomplexshould not have influenced the mobility of gp85. The greater mobility ofgp85 that is infact observed under reducing conditionscould thenonly have been

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966 LEAMNSON AND HALPERN

- b ~~~~-VGP

.-

4

3:

20 40 60 80 100 120 140

DISTANCE OF MIGRATION (mm)

FIG. 10. Electropherograms on10%SDS-gelsof anti-gp85immuneprecipitates,eachpreparedwith4 x

105 trichloroacetic acid-precipitable counts/min (cpm) ofalysate of[3H]glucosamine-labeled, uninfected

chf(+)chickenembryocells; analysisunder(a)reducingconditionsand(b)nonreducingconditions.Reduced

['4C]glucosamine-labeledB77 viruswascoelectrophoresedin(a)and nonreduced['4C]glucosamine-labeledB77

viruswascoelectrophoresedin(b);thepositionsofviralVGP,gp85,andgp37areasindicatedby thearrows.

due to reduction of putative intrachain

disul-fidebondsingp85. Ingeneral,

however,

reduc-tion ofintrachain disulfide bondsinpolypeptide

chains should generatelesscompactmolecules that would beexpectedto migrate on SDS-gel

with a lower mobility than their nonreduced

counterparts; for the caseofatleast one

poly-peptide chain thatis known tocontain

intra-chaindisulfide bonds, the light chain of

immu-noglobulin, a lowered mobility of the com-pletely reduced and alkylatedform isobserved (unpublished observations). These

considera-tionswouldsuggest thatthegreatermobilityof

gp85underreducingconditionsis more

reason-ablyascribed to reductive cleavage ofa disul-fide-linked complex of gp85 and gp37 than to reduction of intrachaindisulfidebondsingp85.

Anargumentbasedonthe relative mobilities

on SDS-gel ofreduced and nonreduced

glyco-proteincanonlybeconsideredsuggestive, how-ever, and so experiments were carried out to obtain additional evidence to distinguish the twomodels for VGPstructure.The assumption wasmadethat,iftheinterchain disulfide bonds

in the VGP complex serve to link only gp37

molecules, then the gp37 aggregate should be separable fromgp85 undernonreducing

condi-tions so long as separation iseffected on the

basis of some property other than molecular

size. Since the alkaline urea-gel system

re-solves proteins on the basis of theircharge, as

well as size, electrophoretic analyses of the

viralglycoproteins werecarried outusingthis

system.

After treatmentof whole virus withreducing

agentandsubsequent alkylationofhalf-cystine

VIROL.

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residues, gp85 was observed to migrate well. into an alkaline urea-gel, whereas gp37 re-mained at the origin of the gel. Similar results were obtained using the acid urea-polyacryl-amide gel systemdescribed by Duesberg et al. (7). The gp85 andgp37 in nonreduced prepara-tions of virus remained at the origin of an acid urea-gel and only the gp85 in reduced prepara-tions migrated well into the gel (unpublished

observations). The behavior of gp37, as ana-lyzed underreducing conditions, is presumably areflection either of the native charge of gp37 or ofthe tendency of gp37 to aggregate even under dissociating conditions. Fleissner (8) had earlier noted that gp37 self-aggregates in the presence of 6 M guanidium-chloride so that ag-gregation in the presence of 8 M urea (the concentration of ureainthe gels)would not be unexpected.

In any case, since gp85 was resolved from gp37underreducing conditions, asimilar reso-lution should have been achieved under

nonre-ducing conditions, if in fact the assumption werecorrect thatgp85 and gp37 are notlinked

by disulfide bonds. The observation that nei-ther gp85 nor gp37 entered the gels under non-reducingconditions would then tend to exclude amodelinwhich gp85 is not covalently bonded to gp37. The formal possibility remains that nonreduction of putative intrachain disulfide bondsingp85 hadprevented(nonreduced)gp85 from entering the gels. Thispossibility seems

unlikely, however. It is known, for example,

that the mobilities onalkaline urea-gel of im-munoglobulin light and heavy chains are not

significantly affected bythe stateof reduction of their intrachain disulfide bonds

(unpub-lishedobservations).

Since the data obtained with alkaline and acidurea-gelelectrophoresisrenderamodelin which VGP represents adisulfide-linked aggre-gate of gp37 unlikely, the onlymodel remain-ing that is consistent with all the data pre-sented here is one in which gp85 is linked by disulfidebondstogp37.Implicitinthis model is theassumption ofanequalmolarratioofgp85

and gp37 in intact virions. Fleissner (8) had earlierestimatedan excessofgp37overgp85in intact virions, but his estimate did notcorrect for carbohydrate content in the two

glycopro-teins and so does not constitute arigorous

argu-mentagainst the model.Forthepurposes ofthe remainder of thisdiscussion, we willtherefore

assume that the one gp85-one gp37 model for

VGP structure is correct. The proviso must,

nevertheless, be added thatproofof thismodel willrequiredirect chemical analysis in which the individual polypeptide chains containing

the half-cystine residues involved in the inter-chain disulfide linkage are identified.

Inaddition, no rigorous evidence yet exists to eliminate the possibility that gp85 and gp37, which have only been detected under reducing conditions (3, 6, 8), are artefactual breakdown products of a protease-nicked VGP that con-tains intrachain disulfide bonds. If this possi-bility is correct, it would follow that VGP must exist as a single polypeptide chain in intact virions. Until evidence bearing directly on this point is obtained, however, we will assume, againfor purposes of discussion, that the detec-tion of gp85 and gp37 does not constitute an artefact.

It should be noted that, in an earlierstudy, Duesberg et al. (6) found that disruption of virionswith Tween 20 resulted in the quantita-tive liberation of gp85 and gp37, which cosedi-mented in an 8S structure. The analyses of Duesberg et al. (6) were carried out under re-ducing conditions. Since their 8S structure is probably analogous to the peak B material

de-tected under nonreducingconditions, it is likely that gp85 and gp37 are bonded by noncovalent forces as wellasbydisulfide bonds.

Aquestion that remains to be answered con-cerns the number (if more than one) of VGP subunits linked together to form the spikelike surface projections that are observed upon elec-tron microscope examination of nondefective aviantumor virus (17).Sedimentation analysis onsucrosegradients ofTween20-disrupted vi-rus resolved two discrete complexes of VGP molecules. The sedimentation coefficients of 12S and 9S calculated for the structures in

peaksA and B, respectively, suggest that the former comprises three molecules of VGP, whereas the latter comprisestwo molecules of VGP. It is notknown at present whether these structuresarerandom aggregates producedby

disruptionwith nonionicdetergentor,

alterna-tively, whetherone orthe other of these struc-tures is, in fact, the intact surface projection.

Electronmicroscopevisualization of each struc-turemayclarifythispoint.

The finding that covalent linkage of viral glycoprotein occurs intracellularly raises the

additional question of the mechanism of VGP

biosynthesis [we assume that an equivalent

mechanism to that operative in infected

chickenembryocells isoperativeinuninfected

chf(+) chicken embryocells]. Several possible

mechanismscan be envisioned. In onemodel,

VGP would be formed by disulfide linkage of

gp85 andgp37 moieties (either prioror

subse-quentto fullglycosylation)that wereoriginally

present in separatecytoplasmic pools. The

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968 LEAMNSON AND HALPERN

istenceof separate pools of gp85 andgp37could

eitherreflect the translation of the polypeptide

moieties of each glycoprotein on individual

classes ofmessenger ribonucleic acid or, con-ceivably, the specific cleavage of a fraction of

the nascent gp85 chains to yield the pool of

functional gp37.

Analternative model for VGP biosynthesis is

one inwhichVGP is aprimaryviralgene

prod-uct, the polypeptide moiety of which is

trans-lated on a singleclassofmessenger. Some time

aftertranslation, an intrachain disulfide bond

would be formed in the polypeptide chain

fol-lowedby aproteolytic cleavagestep thatsplits the chaininto (disulfide-linked) smallerchains corresponding to gp85 and gp37. This

mecha-nism for VGP synthesis is analogous to the

pathway of synthesis ofthesubunit of the he-magglutinin spike of influenza virus (12, 14, 15). Itmayalso be analogoustothepathwayof

syn-thesis of the glycoprotein of mouse mammary

tumor virus since anuncleavedprecursor to the

two virionglycoproteinshasrecently been

iden-tified (5). Thepossibilityexists, therefore, that

several different enveloped ribonucleic acid

viruses mayhave evolved a common pathway

for thesynthesis of theglycoprotein subunitsof their surface projections.

ACKNOWLEDGMENTS

We thankBarbaraSeebergerfor excellent technical as-sistanceand WilliamMasonforprovisionof cells and virus stocksaswellasforcriticalreadingof themanuscript.

Thisinvestigationwassupported byPublic Health Ser-vicegrantsCA-16047 and CA-10815 from the National Can-cerInstitute.

LITERATURE CITED

1. August, J. T., D. P. Bolognesi, E. Fleissner, R. V. Gilden, and R. C. Nowinski.1974. Aproposed nomen-clature for the virion proteins of oncogenic RNA vi-ruses. Virology60:595-601.

2. Bolognesi, D. P., and H. Bauer. 1970. Polypeptides of avian RNA tumorviruses. I. Isolationandphysical and chemicalanalysis.Virology 42:1097-1112. 3. Bolognesi, D. P., H. Bauer, H. Gelderbloom, and G.

Htiper. 1972. Polypeptides of avian RNA tumor vi-ruses. IV.Components of the viral envelope. Virology 47:551-566.

4. Bretscher, M. S. 1971. Major humanerythrocyte glyco-protein spans the cell membrane. Nature (London) New Biol.231:229-232.

5. Dickson,C., J. P. Puma, and S. Nandi.1976. Identifica-tion of aprecursor protein to the major glycoproteins ofmousemammary tumor virus. J. Virol. 17:275-282. 6. Duesberg, P. H., G. S. Martin, and P. K. Vogt. 1970. Glycoprotein components of avian andmurineRNA tumorviruses. Virology 41:631-646.

7. Duesberg, P. H., H. L. Robinson, W. S. Robinson, R. J. Huebner, and H. C. Turner. 1968. Proteins of Rous sarcomavirus. Virology 36:73-86.

8. Fleissner, E. 1971. Chromatographic separation and antigenicanalysis of proteins of the oncornaviruses. I. Avian leukemia-sarcoma viruses. J. Virol. 8:778-785.

9. Halpern,M.S., D. P. Bolognesi, R. R. Friis, and W. S. Mason. 1975. Expression of the major viral glycopro-teinof avian tumor virus in cells of chf(+) chicken embryos. J. Virol. 15:1131-1140.

10. Halpern, M. S., D. P. Bolognesi, and L. J. Lewan-dowski. 1974. Isolation of the major viral glycoprotein and a putative precursor from cells trasformed by avian sarcomaviruses. Proc. Natl. Acad. Sci. U.S.A. 71:2342-2346.

11. Halpern, M. S., E. Wade, E. Rucker, K. L. Baxter-Gabbard, A. S. Levine, and R. R. Friis. 1973.Astudy of the relationship of reticuloendotheliosis virus to the avianleukosis-sarcoma complex of viruses. Virol-ogy 53:287-299.

12. Kienk,H. D.,and R. Rott. 1973. Formation of influenza virusproteins. J. Virol. 11:823-831.

13. Laemmli, U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227:680-685.

14. Laver, W. G.1971. Separation of two polypeptide chains fromthehemagglutinin subunit of influenza virus. Virology 45:275-288.

15. Lazarowitz, S. G., R. W. Compans, and P. W. Choppin. 1971. Influenza virus structural and nonstructural proteins in infected cells and their plasma mem-branes. Virology 46:830-843.

16. Martin, R. G., and B. N. Ames. 1961. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. 236:1372-1379.

17. Ogura, H., and R. Friis. 1975.Further evidence for the existence ofa viral envelope protein defect in the Bryan high-titer strain of Rous sarcoma virus. J. Virol. 16:443-446.

18. Reisfeld, R. A., and P. A. Small, Jr. 1966. Electropho-reticheterogeneity of polypeptide chains of specific antibodies. Science152:1253-1254.

19. Vogt, P. K.1970. "Envelope classification of avian RNA tumor viruses," Comparative Leukemia Research. Bibl. Haematol. (Basel) 36:153-167.

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Figure

FIG.1.carriedducedcoelectrophoresed Electropherograms on 10% SDS-gels of (a) reduced [3H]glucosamine-labeled B77 virus; (b) nonre- [3H]glucosamine-labeled B77 virus; (c) reduced and alkylated ['4C]glucosamine-labeled B77 virus with nonreduced [3H]glucosami
FIG.1.carriedducedcoelectrophoresed Electropherograms on 10% SDS-gels of (a) reduced [3H]glucosamine-labeled B77 virus; (b) nonre- [3H]glucosamine-labeled B77 virus; (c) reduced and alkylated ['4C]glucosamine-labeled B77 virus with nonreduced [3H]glucosami p.3
FIG. 2.[3H]glucosamine-labeledits elution Electropherograms on 10% SDS-gels of(a) nonreduced [3H]glucosamine-labeled VGP; (b) reduced VGP
FIG. 2.[3H]glucosamine-labeledits elution Electropherograms on 10% SDS-gels of(a) nonreduced [3H]glucosamine-labeled VGP; (b) reduced VGP p.4
FIG. 3.reducedVGP Electropherograms on 12% SDS-gels of (a) nonreduced [3H]amino acid-labeled B77 virus; (b) [3H]amino acid-labeled VGP coelectrophoresed with reduced [14C]amino acid-labeled B77 virus
FIG. 3.reducedVGP Electropherograms on 12% SDS-gels of (a) nonreduced [3H]amino acid-labeled B77 virus; (b) [3H]amino acid-labeled VGP coelectrophoresed with reduced [14C]amino acid-labeled B77 virus p.5
FIG. 4.coelectrophoresedtion Electropherogram on a 4% alkaline urea-gel of nonreduced ['4C]glucosamine-labeled B77 virus with reduced and alkylated [3H]glucosamine-labeled B77 virus
FIG. 4.coelectrophoresedtion Electropherogram on a 4% alkaline urea-gel of nonreduced ['4C]glucosamine-labeled B77 virus with reduced and alkylated [3H]glucosamine-labeled B77 virus p.6
FIG. 5.gel[3H]glucosamine-labeledeluate4)line(correspondingrial(a)belednonreduced of Electropherograms on 10% SDS-gels of material eluted from the first fraction ofan alka- urea-gel (similar to that shown in Fig
FIG. 5.gel[3H]glucosamine-labeledeluate4)line(correspondingrial(a)belednonreduced of Electropherograms on 10% SDS-gels of material eluted from the first fraction ofan alka- urea-gel (similar to that shown in Fig p.7
FIG. 6.preparationsgradientsaminocontainedsamine-labeledlabeledlabeledtaineddients Sedimentation analysis on sucrose gra- of (a) Tween 20-disrupted [3H]glucosamine- B77virus;(b)SDS-disrupted [3H]gluco- B77 virus; (c) Tween 20-disrupted of [3H]glucosamine-l
FIG. 6.preparationsgradientsaminocontainedsamine-labeledlabeledlabeledtaineddients Sedimentation analysis on sucrose gra- of (a) Tween 20-disrupted [3H]glucosamine- B77virus;(b)SDS-disrupted [3H]gluco- B77 virus; (c) Tween 20-disrupted of [3H]glucosamine-l p.7
FIG. 7.peak6a)inreducedlabeled(7e Fig. Electropherograms on 10% SDS-gels ofradioactively labeled material recovered from gradients as 6
FIG. 7.peak6a)inreducedlabeled(7e Fig. Electropherograms on 10% SDS-gels ofradioactively labeled material recovered from gradients as 6 p.8
FIG. 8.formedtrichloroacetic Electropherograms on 10% SDS gels of anti-gp85 immune precipitates, each prepared with 106 acid-precipitable counts per minute (cpm) of a lysate of [3H]glucosamine-labeled B7rtrans- chicken embryo cells: analysis under (a) nonreducing conditions; (b) reducing conditions.
FIG. 8.formedtrichloroacetic Electropherograms on 10% SDS gels of anti-gp85 immune precipitates, each prepared with 106 acid-precipitable counts per minute (cpm) of a lysate of [3H]glucosamine-labeled B7rtrans- chicken embryo cells: analysis under (a) nonreducing conditions; (b) reducing conditions. p.9
FIG. 9.duced[3H]glucosamine-labeledshownlabeled(a) Electropherograms on 10% SDS-gels of the major peak eluted from the SDS-gel (similar to thatin Fig
FIG. 9.duced[3H]glucosamine-labeledshownlabeled(a) Electropherograms on 10% SDS-gels of the major peak eluted from the SDS-gel (similar to thatin Fig p.10
FIG. 10.105['4C]glucosamine-labeledchf(+)virus Electropherograms on 10% SDS-gels ofanti-gp85 immune precipitates, each prepared with 4 x trichloroacetic acid-precipitable counts/min (cpm) of a lysate of [3H]glucosamine-labeled, uninfected chicken embryo ce
FIG. 10.105['4C]glucosamine-labeledchf(+)virus Electropherograms on 10% SDS-gels ofanti-gp85 immune precipitates, each prepared with 4 x trichloroacetic acid-precipitable counts/min (cpm) of a lysate of [3H]glucosamine-labeled, uninfected chicken embryo ce p.11

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