Murine mammary tumor virus structural protein interactions: formation of oligomeric complexes with cleavable cross-linking agents.

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JOURNAL OF VIROLOGY, Sept. 1980,p.937-948

0022-538X/80/09-0937/12$02.00/0 Vol. 35,No. 3

Murine

Mammary Tumor Virus

Structural

Protein

Interactions: Formation

of

Oligomeric

Complexes

with

Cleavable

Cross-Linking

Agents

JANIS RACEVSKIS* ANDNURUL H. SARKAR

MemorialSloan-KetteringCancerCenter,New York, New York10021

Murinemammary tumorvirusproteininteractionsinthe intactvirion structure were studied with the use of the cleavable cross-linking reagents

dithi-obis(succinimidyl propionate) and methyl 4-mercaptobutyrimidate

hydrochlo-ride.Cross-linked oligomericcomplexes of murinemammary tumorvirusproteins were analyzed by two-dimensional gel electrophoresis. Among the complexes most consistentlyformedwere a heterodimer of the two glycoproteins gp36 and gp52, the homodimer ofgp36, and the homotrimer of gp52. Avery prominent

oligomer formed athigher concentrations ofdithiobis(succinimidyl propionate)

was a complex of about230,000 molecular weight, made upofthree molecules eachofgp36andgp52. A numberof lines of evidence, including electron

micro-scopic analysis,suggestthatthe230,000-molecular-weightcomplex actually rep-resents themurine mammary tumorvirus spike structure. Of the murine mam-mary tumor virus core proteins, p14 forms homooligomers most readily. Upon

cross-linkingwithmethyl 4-mercaptobutyrimidate hydrochlorideasmallamount

ofwhatseems to bea heterodimer madeupofthe N-terminalgag protein plO

andthehydrophobic membrane glycoprotein gp36can be observed. The mature murine mammary tumor virus

(MuMTV;type Bparticle) isanenvelopedvirus

whose prominent structural features are an

ec-centrically located electron-dense core

sur-roundedby less dense material andaunit

mem-brane covered with projections arranged in a

regular pattern. The structural proteins of MuMTVcan beclassified into two groups: the

gag gene-derived internal core proteins (plO,

p14, p23, and p28), and the env gene-derived

membrane-associated glycoproteins (gp36 and gp52). What ispresently known about the

struc-turallocationorfunction ofsomeof these

pro-teins within the MuMTVstructure canbe

sum-marizedasfollows:gp52 islocatedontheoutside

of the virion envelope (24, 30), gp36 is mostly embeddedwithinthebilayer membrane(15, 24), p14isthe nucleic acid-binding protein (1), and

plOhas an affinity for the membrane fraction

(5, 16).However, the structural roleofall these

proteins isnot fullyunderstood, and it has not yet been possible to define biochemically the structures that are observed in electron

micro-graphsof MuMTVparticles.

One of the most successfulapproaches to the

study of viral protein interactions, especially

those ofenveloped viruses, has been the use of

cross-linkingreagents (7-9, 18, 20, 22,25, 26, 29). The principal conclusions that have emerged from cross-linking studies ofenveloped viruses are that the spike structures observed on the

membrane of these virusesareoligomeric com-plexes of the viral membrane glycoproteins (8, 9, 20,25, 26,29)and that there is oftena demon-strable interaction betweenamembraneprotein

andaninternal coreprotein (8, 18, 26).

The cross-linking reagents that have proven tobemostvaluableinthesetypesof studiesare the bifunctional molecules, such as

dithio-bis(succinimidyl propionate) (DTBSP) (14)and

methyl 4-mercaptobutyrimidate hydrochloride

(MMB) (11), that contain a cleavable internal disulfide bond. The reactive ends of the mole-culesreadilyformcovalent bondsat room

tem-perature and neutral pH with primary amine

groups such as the E amino group of lysine

residues. Thepresenceof thecleavable bonds in

these reagents permits the regeneration and identification of the monomeric components

from the cross-linkedoligomericcomplexes.

To explore MuMTV protein interactions in

greater detail, we used cleavable bifunctional

cross-linkingagents inconjunctionwith

two-di-mensionalgelelectrophoresis.Themostreadily formed cross-linkedcomplexesthatweobserved

wereoligomersof thetwoglycoproteins,with a

veryprominent oneof about230,000 molecular

weight,made up of three molecules each ofgp36 and gp52. On the basis of a number of lines of evidence presented in this report, we propose that the230,000-molecular-weight complex rep-resentsthe MuMTVspike structure.

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938 RACEVSKIS AND SARKAR

MATERIALS AND METHODS Reagents.The reagents DTBSPand MMBwere

purchasedfrom PierceChemical Co. N-ethylmaleim-ide was obtained from Aldrich Chemical Co., and

dithiothreitolwaspurchasedfromCalbiochem. U-'4C-labeled amino acids and [355]methionine were

ob-tained from New England Nuclear Corp. All ofthe reagentsused for sodium dodecyl sulfate (SDS)-poly-acrylamide gelelectrophoresis, including 2-mercapto-ethanol,werefromBio-RadLaboratories. Agarosewas

obtained from Seakem, and the nonionic detergent Nonidet P-40(NP-40)wasobtained from Shell Chem-icalCo.

Viruses. Unlabeled MuMTVproduced bythe

tis-sueculturecelllineMm5mt/c,wasobtained fromJ. Gruber through the Virus Cancer Program of the National Cancer Institute. MuMTV from RIIImouse

milkwaspurifiedaspreviously described(24).Labeled viruswasobtained fromcelllineMm5mt/c, by

label-ingthe cells with "4C-amino acids as previously

de-scribed(24).

Cross-linking of MuMTV proteins. (i) DTBSP. Fresh stocksolutions of DTBSP indimethyl sulfoxide

wereprepared before each experiment,ata

concentra-tionof 40mg/ml. The stock solutionwasthen diluted with phosphate-buffered saline to a concentration

which would yield the final desired concentration whenaddedtothe virussample inaratioof1:5.Viral

samplesweresuspendedinphosphate-buffered saline, andthecross-linkingreactionwascarriedout at23°C for 20min. The reaction was terminated by adding

SDS-polyacrylamide gel electrophoresis loadingbuffer andheatingthesamplesfor 3minat100°C.

(ii)MMB.Cross-linkingwith MMBwascarriedout withamodificationofthemethod of Kennyetal.(11). A 200 mMstocksolutionofMMB in 500 mM trieth-anolamine hydrochloride, pH8.0, wasadded to the viral sample togive afinal concentration of20 mM MMB. After the reactionwas allowedtoproceed for 20 min at 23°C, hydrogen peroxide was added to a concentration of 40 mM topromotecross-linking be-tween adjacent sulfhydryl groups by disulfide bond formation. Theoxidationwasallowed toproceedfor 15 minat 23°C, after which thereactionwas termi-natedasdescribed above.

Gel electrophoresis. SDS-polyacrylamide gel electrophoresisin the first dimensionwascarriedout in0.75-mm-thick, 5 to 20% gradient gels, using the discontinuousbuffersystemofLaemmli(13) as

pre-viously described(21). Foranalysisofsamples

cross-linked by the cleavable reagent DTBSP or MMB,

reducingagents(2-mercaptoethanolordithiothreitol) were omittedfrom the loading buffer. At the end of

the electrophoretic run, lanescontaining samples to

be analyzed in the second dimension were excised, sealed in SaranWrap, and storedat-20°C.For anal-ysisin the seconddimension, thegelstripswere

in-cubated inLaemmlistacking gelbuffercontaining5% 2-mercaptoethanol for 30 min at 37°C or in buffer

containing 100mMdithiothreitol for 10 minat60°C. Both techniques yielded identical results. After the reducing step, thegel stripwaswashed for5 min in

loadingbuffertogetrid ofexcessreducingagentand toabsorbsomebromophenolblue trackingdye. The

gel stripwasthenplacedontopofa 1.5-mm-thick, 5

to 20% gradient gelcontaining a1.5-cm-wide stacking gel. The gel strip was immobilized by pouring on stacking gel buffer containing 1% agarose. Slots for application of marker virus or other samples were formed at either end of the gel strip in the agarose solution. Gels were fixed and treated by the method of Bonnerand Laskey (4) for analysis by fluorography.

'4C-labeledmolecular weight markers (with molec-ular weights in parentheses) used for calibration pur-poses werethefollowing: ovalbumin (43,000), bovine serumalbumin (68,000), phosphorylase B (94,000), 8-galactosidase (116,000), and myosin (200,000). Fibro-nectin, for use as a marker, was obtained by immuno-precipitating tissue culture medium supernatant of

'4C-aminoacid-labeledMm5mt/c, cellsafter virus har-vestwithanti-fibronectin serum (21). Electrophoresis of the immunoprecipitates in the presence of reducing agentyields fibronectin monomer (molecular weight, 230,000), andelectrophoresis in the absence of reduc-ing agent displays the predominant disulfide-bonded dimer form (molecular weight, 460,000) (12).

Electron microscopy. Virus suspension was placed on a microscopic grid covered with carbon-coated Formvarfilm. After20s,theexcessfluidwas withdrawnbytouchingthegridwith apieceof filter paper. The grid was then washed three times by

flotation on 3 drops of double-distilled water and stained for30swith 2% sodiumphosphotungstic acid,

pH 7.0 (23). Grids were examined in a Philips 300 electronmicroscope.

RESULTS

Cross-linking of MuMTV. These studies were initially performed with unlabeled MuMTV isolates derived from tissue culture cells (line Mm5mt/c1) as well as from mouse milk (strain RIII mice), and the results were analyzed on Coomassie brilliant blue-stained

polyacrylamide gels (Fig. 1). Notethatwehave

adoptedthemorewidelyused nomenclature for

MuMTVstructuralproteins (plO, p14, p23, p28,

gp36, and gp52 [2, 27]) in lieu ofour previous

designations (pl2,pl6, p23, p27, gp34,andgp47 [21]). A comparison between the two control viral samples revealed that considerable

differ-encesexisted betweenthe SDS-polyacrylamide

gelelectrophoresisprofilesofthetissue

culture-derived(Fig. lc) and milk-derived (Fig. ld)virus

isolates.Primarily, the two glycoproteins (gp36

and gp52) of the RIII milk virus (Fig. ld)

mi-gratednoticeablyfaster than theircounterparts of the C3H tissue culture virus (Fig. lc). The apparent fastermigrationof themilk virus

gly-coproteins mighthave been dueto a decreased polysaccharidecontentof these proteins

result-ing from the action of glycosidases present in milk. Inaddition,theRIII milk virus containsa

number ofproteinsnot seen in the C3Hvirus, with aprominentoneofabout68,000molecular

weight and anothermigratingbetweenplOand

p14.Theseadditionalproteinsareprobablymilk

proteins which eitheradsorb onto the virus or

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CROSS-LINKING OF MuMTV 939

a b c d e f g

_- -- -- ON ". _f

cre c

u

fin CD

CD,

CL

urVz

SOK

80~~~~4Km of

gp52

.9p36 -L ~ .e

p28 _

p23- _ _ ___

p14 _ _-:t

P1O-FIG. 1. Coomassie brilliant blue C3H tissue culture andRIII milk M, tions cross-linked with DTBSP. (a) DTBSP (100,g/ml); (b)C3H virusI

pg/ml); (c) untreated C3Hvirus; (d)

virus; (e) RIII virus plus DTBSP (30 virus plus DTBSP (60,tg/ml); (g) DTBSP (100pLg/ml);(h) RIII virus pi pg/ml); (i) RIII virus plus 0.1% NP-(800 pg/ml); (j) C3H virus plus 0. DTBSP (800pg/ml); (k)C3H virus p

,ug/ml). 80K, 80,000 molecular weight

arepartofmilkvesicles thatcopu (17).We haveobserved that these proteinprofilesarenotduetovirf

ences, but aretypical ofmilk-dei

latesversustissueculture-derivec

regarrdlessof strain.

Treatmentof intact MuMTVv

linking agent DTBSP followed 1

SDS-polyacrylamide gel electroi

nonreducing conditions resulted

anceofnewprotein bands and the

ordecreasedintensityofsomeM bands.Treating the virusprepara

ativelylowconcentrations ofDTE resulted in a marked decrease i intensities of the glycoprotein (g

bands (Fig. lbande),whereas tk bands remained relatively unch

band ofabout80,000apparent mc

appeared in the cross-linked sar

and e) which was not present

samples (Fig. lc and d). This ne

tissue culture-derived virus (Fig

h j k withaslightly higherapparentmolecular

weight

thanthenewbandseenin the milk-derivedvirus

(Fig. le). At higher DTBSP concentrations (100

,ug/ml),

we noticed an increase of Coomassie t W briHiantblue

staining

materialat very

high

ap-parentmolecularweightranges (near thetopof theseparating gel [Fig. laandg]).At aDTBSP concentration of400,ug/ml, all of the

glycopro-teinshad reacted, andacross-linkedcomplex of

greater than 200,000 molecular weight had

be-come veryprominent (Fig. lh andk). Note that in all of the cross-linked samples, and even in the nonreducedcontrols, therewas a consider-ableamountof Coomassie brilliantblue-stained material thatwaspresent atthe bottom of the loading slots that had not entered the stacking gel. Furthermore, in the cross-linked samples,

there was adiffusion ofmost of the minor

dis-cretenonviralprotein bandsseenincontrol

sam-ples, especially in the higher-molecular-weight ranges.

Cross-linking ofNP-40-lysed virus. One

possible explanation for the apparent lack of

reactivity of thecoreproteins in thepresenceof

-stanVed

gel of DTBSP might have been that the MuMTV

C3H

vipruesplruas

envelope was

impermeable

to the cross-linking

i)lus

DTBSP (30

agent.

To test this

possibility,

we

began

studying

Iuntreated RIII NP-40-lysed

viral

preparations for cross-linking

pg/ml);

(f) RIII with DTBSP.

Treating

MuMTV

preparations

RIII virusplus with NP-40causes

disruption

of the viral mem-,lusDTBSP(400 brane,freeingof thecores,andthe formation of

*40 plus DTBSP rosettes which consist chiefly of a viral

mem-*1%

NP-40

plus

branecontaining thetwoglycoproteinsgp36and

,lusDTBSP (400 gp52 and exhibiting the surface spikes (24). We

observed, however, that under theseconditions,

higher concentrations of DTBSPwererequired

frifywith virus toformdetectable oligomeric proteincomplexes.

differences in The most striking cross-linked complex

ob-alstrain differ- served in theNP-40-lysed viralpreparationswas

rived viral iso- thespeciesofveryhighmolecular weight, which iviralisolates, onlymigrated about1to 2 cmintotheseparating

gel(Fig. liandj).

with thecross- Useof

14C-labeled

MuMTV.Afterthe initial

by analysis on studieswithunlabeled MuMTVisolates, all

fur-)horesis under ther cross-linking experiments were conducted in theappear- with

14C-amino

acid-labeled MuMTV

prepara-disappearance tions because autoradiography of isotopically

uMTVprotein labeled viral preparations is a more sensitive

itions with rel- technique ofanalysis than is staining of unla-3SP(30,g/ml) beled viralprotein.Thegreatlyreduced concen-in the relative tration of virus required in reactions with 14C-p36 and gp52) amino acid-labeled MuMTV also reduces the ie coreprotein potential for artifacts that may occur when

anged. A new cross-linking virus at therelatively high

concen-)lecular

weight trations needed for Coomassie brilliant blue

mples (Fig. lb staining.

in the s- ntrol Cross-linking ofa type C virus. In

struc--w band in the tural studies of type C viruses (20, 25), it has

lb) migrated been reported that oligomeric complexes of

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greater than 200,000molecular weightare also formedas aresult of cross-linking. Becausetype

B andtype C viruses have considerable

struc-tural similarity,wethought it would be of

inter-est tocomparethecross-linkedproducts formed

with both types of viruses. To that end, "4C-aminoacid-labeled Rauscher leukemia virus and

"4C-amino acid-labeled C3H MuMTV were

re-acted with DTBSP under identical conditions and analyzed. To get a better estimate ofthe

molecular weights of the cross-linked complexes, the analysis wasperformed on a 30-cm-long, 3

to20%hyperbolic gradient gel formedinsucha way as to expand the high-molecular-weight range. In addition to our standard molecular

weight markers, fibronectin in its reduced mon-omer (molecular weight, 230,000) and

nonre-duced dimer (molecular weight, 460,000) forms (12), obtained as described above, was run on

thegelaswell.Asreported by Pinter and

Fleis-sner(20), oxidation of the type C virus with

N-ethylmaleimide resulted in the formation ofa

90,000-molecular-weightcomplex (Fig. 2i) which

was composed of the two membrane proteins gp7O and pi5E linked by disulfide bonds. Oxi-dation ofMuMTV withN-ethylmaleimide (Fig. 2c) caused the disappearance of p14 and of the

smallbandmigrating just above p28. Formation

ofnew complexes afterN-ethylmaleimide

oxi-dation of MuMTVwasnotobserved, however. Further analysis ofN-ethylmaleimide-oxidized

MuMTV preparations (data not shown)

re-vealed that p14was complexed intoaggregates too large to enterthe gel. Furthermore, itwas

foundthat,even inuntreated virions,a portion

ofthe p14 molecules existed in an aggregated

statethat could be dissociatedbyreduction with

2-mercaptoethanolordithiothreitol. Treatment

ofRauscher leukemia virus witha low

concen-tration (10lg/ml) of DTBSP caused the ap-pearance ofaprominent band of 60,000

molec-ularweight (Fig. 2h) whichwas shown tobea

dimer of p30 (20). A similar cross-linking of MuMTV (Fig. 2d) resulted in the formation of the80,000-molecular-weightcomplex, whichwas

also observed at a higher (150

fig/ml)

DTBSP concentration(Fig. 2e). Ifadimeric form ofp28 wasformed, it might have been masked by

co-migration with gp52. However, as will be

dis-cussed later, two-dimensional gel analysis did not reveal any such complexes (Fig. 3).

Cross-linking ofNP-40-disrupted Rauscher leukemia viruswith DTBSP resulted in the formation of a complex of greater than 200,000 molecular weight (Fig. 2g) which migrated just slightly above the complex of a similarly treated

MuMTVpreparation (Fig.2f).Notethat in the NP-40-lysed MuMTV sample (Fig. 2f)

cross-linkedatahigh DTBSP concentration (400,ug/

k.:i

a

:v -

WI..;.-..-FIG. 2. Cross-linking of "C-amino acid-labeled MuMTV and"C-amino acid-labeled Rauscher leu-kemia virus with DTBSP. (a) Nonreduced immuno-precipitated fibronectin (molecular weight, 460,000);

(b)untreatedMuMTV;(c) MuMTV oxidized with N-ethylmaleimide; (d)MuMTVplusDTBSP(20ug/ml);

(e) MuMTVplus DTBSP (150

pg/ml);

(f) MuMTV plus 0.1% NP-40 plus DTBSP (400 yg/ml); (g)

Rauscher leukemia virus plus 0.1% NP-40 plus

DTBSP (400 pg/ml); (h) Rauscher leukemia virus plus DTBSP(10 tLg/ml); (i)Rauscherleukemia virus oxidized with N-ethylmaleimide; () untreated Rauscher leukemia virus; (k) "C-labeledmolecular weight markers (followed by molecular weights in parentheses): myosin (200,000), /8-galactosidase

(116,000), phosyphorylase B (94,000), bovine serum albumin(68,000), andovalbumin (43,000);(1)reduced, immunoprecipitated fibronectin (molecular weight,

230,000).80K,80,000molecularweight.

ml) thegp52bandwas very broad andextended

toward a lower-molecular-weight range. This

phenomenon was probably caused by internal

cross-links formed within gp52 molecules,

thereby decreasing their relative size and

per-mitting migration with an apparent lower

mo-lecular weight in the SDS-polyacrylamide gel. The coreproteinp28 showed asimilar,although

less pronounced, altered migration aswell (Fig.

2f). Astreakingto a lowerapparent molecular

weight range was also exhibited by both the

80,000-molecular-weight complex (Fig. 2f) and the complex with a molecular weight greater than 200,000 and was again probably due to

internal cross-links in themolecules making up

thecomplexes. Although it is notdiscerniblein

Fig.2, an examination of theoriginal X-ray film

clearly shows that most ofthe material in the

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CROSS-LINKING OF MuMT'V 941

MuMTV complex with a molecular weight

greater than 200,000 (Fig. 2f) is located at the top of the smeared band, in aregion no larger

thanthe analogous complex of Rauscher leuke-mia virus (Fig. 2g).

Molecular weight estimation of the large cross-linked complexes. By plotting the ob-served mobilities of the molecular weight markers in Fig. 2onasemilogarithmic plot,we

obtainedalinearrelationship for mobilityversus molecular weight from ovalbumin (molecular

weight, 43,000) through fibronectin monomer

(molecular weight, 230,000) (data not shown). Themigration of thefibronectin dimer

(molec-ular weight, 460,000), however, departed from

linearityrelative tothe other marker proteins. Asameansofobtainingarough estimate of the molecular weights of the large cross-linked MuMTV and Rauscher leukemia virus

com-plexes, weplottedtheirmigrationonaline

join-ing the fibronectin monomer position to the fibronectin dimer position. Such an estimate gave approximate apparent molecular weights of270,000 forthe MuMTV complex and 300,000 fortheRauscher leukemia virus(RLV) complex. It has beenobserved previously (18) that large oligomeric complexes of cross-linked polypep-tides migrate in SDS-Laemmli discontinuous gels withanapparentmolecularweight approx-imately 20% greater than that calculated from theirpolypeptidecomposition. Ifweapply such a correction factor to the large MuMTV and RLV cross-linked complexes, then we obtain

molecularweights ofapproximately 250,000 for theRLVcomplex and 230,000 for the MuMTV complex.

Two-dimensional gel analysis of cross-linked MuMTV polypeptides. Cross-linked MuMTVsampleswereanalyzed by cuttingout

the appropriate strip from the first-dimension gel, incubating it in the presence of reducing

agents toreversecross-linking, and then running theentire strip inasecond dimension. Figure 3

showsatypical two-dimensional gel of MuMTV

proteinscross-linked with DTBSPata

concen-trationof 30,tg/ml.Theorigin ofelectrophoresis

inthe firstdimension is in the upperleft-hand corner, with the direction of migration to the right. Electrophoresis in the second dimension is from top to bottom. The gel strip shownon

topisaduplicate of the cross-linked sample that was reduced and run in thesecond dimension.

The MuMTVpattern seen on the rightside is an MuMTVstandardthatwasrunadjacentto the first-dimension gel strip in the second-di-mensiongel.

Afterreduction andelectrophoresis in the

sec-ond dimension, the polypeptides thatwere not affectedby the cross-linkingreagentarelocated

on a diagonalline extendingfrom upperleftto

lower right. Spots running below this diagonal

identify polypeptides that were components of

higher-molecular-weightcross-linkedcomplexes

in thefirst dimension.Polypeptidespotslocated above thediagonal indicate that these polypep-tidesmigrated withanapparentlowermolecular weight undernonreducing conditions,probably because of internal cross-linking. The most

prominentspotsbelowthediagonalin Fig. 3 are gp52andgp36, whicharevertically aligned be-neath the80,000-molecular-weight bandseenin cross-linked samples. This alignment indicates that the 80,000-molecular-weight cross-linked complex isaheterodimer ofgp52andgp36.Only

agp36 spot canbe observed beneath the

65,000-molecular-weight band, identifying itas a

hom-odimer of gp36. Note that all of the major MuMTV protein bands show streaks beneath the diagonal. These streaksprobably represent

cross-links inthe first dimension with the

con-taminating cellular proteins, which are present

in MuMTV preparations, as in all enveloped viruses.Two-dimensional gels of MuMTV

prep-arationscross-linkedathigher DTBSP

concen-trations (data not shown) showed increased streaking, withmore materialat

higher-molec-ular-weightranges,butstilldisplayed the

prom-inentspotsderived from the homodimer gp36-gp36 and theheterodimer gp36-gp52as seenin Fig. 3. At the higher DTBSP concentrations, oligomeric complexes of about 140,000 molecular weight, made up of gp52, also became more

noticeable.

Two-dimensionalgel analysis ofan MuMTV preparation lysed with NP-40 and cross-linked

at ahigh DTBSP concentration (800,ug/ml) is shown inFig.4. Aswasdiscussedabove, under these conditions a large complex of about 230,000molecularweightwasformed.Asshown

in Fig. 4, this large cross-linked complex was

composedofgp52 and gp36. The

faster-migrat-ing portion of the gp52 spot probably

repre-sented internally cross-linked gp52 molecules thatwere notfullyreduced. A similar phenom-enon canbeobserved in thesquare-shapedgp52

"spot" runningonthediagonal. These

observa-tionssuggestthat theinternalcross-linksarethe

mostresistanttocleavage by reduction.

Whenthespotsderived from the

230,000-mo-lecular-weight complex were cut out of the gel

and theradioactivitywaseluted andcounted,it was found that theratio ofgp52 to gp36 in the

230,000-molecular-weightcomplexwasthesame

asthatfound in the whole virus. Asmallamount oftheheterodimergp36-gp52 can be seen inFig.

4 aswell.

Cross-linkingwith MMB. Thecross-linking

agent MMB was alsousedin thesestudiessince

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942

I

le

*

4_ -gp52

-gp36

_ p28

_ - p23

plo

FIG. 3. Two-dimensional gelofaMuMTVpreparationcross-linkedwithDTBSP(30pg/ml).Theband on topisanexactduplicateofthestripthatwaselectrophoresedin the seconddimension. The lane on theright

side isanMuMTV marker whichwas runsimultaneouslywith thesecond-dimensionelectrophoresis.

w9

o ZP 5 ta P

SI

gp52 _

gp36 _

p28- _

p23- _

p14 _ plO 2

FIG. 4. Two-dimensional gelofanNP-40-lysedMuMTVpreparation,cross-linked with DTBSP(400pg/

ml). Thestripontop isan exactduplicateofthegel lane thatwas runin thesecond dimension. The laneon theleft side isanMuMTV marker.

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CROSS-LINKING OF MuMTV 943 it penetrates the viral membrane morereadily

than does DTBSP(8) and might thereforereact

more extensively with internal core proteins.

The major discrete spots visible in the

two-di-mensional gel of MMB cross-linked MuMTV (Fig. 5) again corresponded to the complexes gp36-gp52and gp36-gp36.Amajorspotofgp52

wasvisibleunderneathamolecularweightrange

of about 140,000, whichmight have represented

a cross-linkedhomotrirner of gp52. As wehave

seen, gp52 tended to form internal cross-links, which might have accounted for the elongated

form of thespotand itsrelativelyfastmigration.

On the left side of Fig. 5 is a lane of marker MuMTV, and on the right side is a lane that

represents material which was cross-linked in such large complexes that it did not enter the firstdimensiongel,as wasshown inFig.1.These highly cross-linkedpolypeptidecomplexeswere

recovered bycuttingoutthetop portion of the stacking gel, cleaving the disulfide bonds with reducingagent,andrunningtheproducts

along-side the two-dimensional gel. Most MuMTV proteinswerepresentin theselargecomplexes,

including most of the p14; however, what was

strikingwasthatnogp52wasdetectable.Similar analysesweredoneaftercross-linkingMuMTV

~4)1

at high DTBSP concentrations (data not

shown), in thepresence andabsence of NP-40, and the same observation was made; namely,

gp52 did not participate in the formation of

cross-linkedcomplexestoolarge to enter the gel

undernonreducing conditions.

Underneathamolecularweight range of about 55,000,therewas anelongatedspotof p28, which

might have been derivedfrom dimeric forms of p28. On the bottom of Fig. 5 we can observe

threeplO spotsunder the diagonal; the firsttwo nearestthediagonal(no. 1and2)probablyarose

from dimerand trimer forms ofplO.The third

rather faintspot ofplO (no. 3) coincided

verti-cally with a distinct gp36 spot, underneath a

molecular weightrangeof about 45,000,raising the possibility of their derivation from a gp36-plOheterodimer. The longer distance between spots 3 and2, relative tothe distance between

spots 1 and2, also suggests that the third plO spot did notarise from a tetramer ofp10, but

probably derived fromaheterodimer ofplOand

gp36.

Morphologyof cross-linkedMuMTV.

Tis-sue culture-derived MuMTV preparations

treated with DTBSP (400

Ag/ml)

under

condi-tions which cross-linkgp36 and gp52 togenerate

ry

~(b

2

gp52-

%k

gp36- p28- p23- p14-

plO--gp52

-gp36 -p28 -p23 -P'4 -plO

FIG. 5. Two-dimensional gelof a MuMTV preparationcross-linkedwith MMB (20mM). Thestrip on top isaduplicate of theoneanalyzed in the second dimension. The lane on the left side is marker virus. MuMTV proteins that were cross-linked into oligomericcomp7exestoo large to enter the separating gel in the

first-dimensiongelwereremoved, reduced, and run in the lane shown onthe right sideofthetwo-dimensional gel. VOL. 35,1980

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the 230,000-molecular-weight component were

examined under the electron microscope. Most of the cross-linked virions appeared spherical

(Fig. 6A andB), and about 40to50% of the viral particles were penetrated by phosphotungstic

acid (Fig. 6A). Whereas the virionspenetrated

by stain usually formed large aggregates

(Fig.

6A), theunpenetrated particleswereuniformly

distributed(Fig. 6B). Unliketheuntreated

par-ticles (Fig.6D),thecross-linkedvirions (Fig. 6A,

B,and C) didnotshow head and tail configura-tions but, instead, were found to exhibit small

blebs. Themorphology of the projectionsof the

cross-linked virions didnotappear to have

un-dergone any drastic alteration, particularly in

thoseparticles thatwere notpenetratedby stain.

* L e--':. .tJ--,,,

FIG. 6. Appearance of tissueculture-grownMuMTV after treatment with thecross-linkingreagent DTBSP (400pg/ml) (AtoC). The membrane (m)of theparticlesin(A) is clearlyvisible.Animmature particle(im)is shown in(B). Most ofthe particles in (B) are notpenetratedbystain; they show a small bleb (b). Pdenotes projection. Theparticle in (C) shows areticular structure (arrow). (D) shows the morphologyofuntreated MuMTV(control).Mostoftheuntreated particlesshow head (h) and tail(t)configurations. (A)X40,000; (B) X100,000;(C)x280,000; (D)x80,000.

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The knob portion of the projections of many

stained particles appeared slightly smaller in size, whereas the stalks that connectthe knobs

to the viralmembranewere muchmorevisible

(Fig.6C) than those inuntreated virions.These

observations suggestthat the knobs and stalks formed stable aggregates after treatment with cross-linking reagents. Furthermore, the previ-ously described (23) surface reticularstructure

of sixfoldsymmetry wasmuch morefrequently exhibited by the cross-linked virions (Fig. 6C) thanby the control viralpreparations.

Subunit structure of the knobs of MuMTV projections. In an earlier study on

the surface structure ofmilk-derived MuMTV (23), itwasobserved that theviral surface

pro-jection knobs seemedtobecomposed ofatleast three subunits. In this study, we examined in detail the knobstructureof tissue culture parti-cles and established that the knobs of MuMTV

are indeed made of three subunits. Figure 7A

shows the morphology ofan MuMTVparticle, thesurface of which is covered withprojections.

In some areasofthe viral surface (squaredarea

in Fig. 7A), the projection knobs are seen to

have a sixfold symmetry; i.e., one projection knob is surroundedby siximmediate neighbors (Fig. 7B). The knobs have the appearance of triangles, andtwo orthree subunitsarereadily

seen insome ofthese knobs; the presence ofa

hole (arrow,Fig. 7B)isalso evident.Toestablish the number ofsubunitsmakinguptheprojection knobs, we employed the technique of image rotationasdescribed earlier (23). Figures 7C, D, Eand Frepresenttheappearanceof the projec-tion knob shown by the arrowin Fig. 7B after

three-, four-, five- and sixfoldrotations,

respec-tively. It appears from these rotation images

that this particular knob is composed of three

units, as only threefold rotation resolved the

subunitsclearly. It should be stressed that the

structures that are analyzed here are close to

the resolution (practical) of the microscope, which contributes to some distortions and

im-perfections of the images. That the knobs are

arranged in a sixfold symmetry on the viral surface is also evident from the sixfold rotation image (Fig. 7F); note the resolution of the six knobs inFig. 7F. The resolutionof the subunits of the knobs is also dependent on how tightly

they are grouped together. When the subunits areverytightly packed, they appear as a

trian-gularstructureand are notresolvedby rotation

(Fig. 7G). In knobs in which the subunits are

spacedapartdue tomechanicalstress, the sub-units could beresolved,and their number could beestimatedfrom threefold rotation pictures as shown in Fig 7H through K. Rotations other

than threefold did not resolve the subunits of the knobs analyzed in these figures. These ob-servations strongly suggest that the knobs of MuMTVprojectionsarecomposed of three

sub-units and support the biochemical results

re-ported above.

DISCUSSION

Cross-linking reagents have become major tools forexploring subunitstructuresand molec-ular associations in various biological systems.

The underlying assumption of this technique, that thecross-linked complexes provide chemi-cal evidence of in situ interactions, has been generally accepted and borne out. The

signifi-cant cross-linked products (those that reflect noncovalent interactions in the unperturbed state) are the ones that are rate favored, i.e., those thatareformedatthelowest cross-linker concentrations andover a wide range of

condi-tions. Among the cross-linked complexes of MuMTV proteins that we have observed that satisfy these criteriaare the heterodimer gp36-gp52 and the homodimer gp36-gp36. The for-mationof these complexes indicates that in the nativestate, inthe MuMTV envelope, thetwo

glycoproteinsgp36and gp52 are inintimate con-tact and thatgp36 moleculesarein close

asso-ciation with each other. This closeassociation of thetwoglycoproteinswassuggested by the

ear-lier observation(28)thatgp36andgp52copurify together through lectin-agarose and molecular sievechromatography. Anotherconsistently ob-served cross-linked complex is whatappears to

be a homotrimer of gp52. This trimer of gp52

exhibitsarather fastmigrationinthe

SDS-poly-acrylamide gels, which is probably caused by

extensive internal cross-links within the gp52

molecule. The most interesting cross-linked

complexthatwehaveobserved, however,isthe

230,000-molecular-weight complex composedof gp36 and gp52. Although the large complex is only detectable at higher DTBSP

concentra-tions, it is formed over a wide range of

cross-linker concentrations and most notably is also formed in thepresenceof thedetergent NP-40. The detergenttreatmentofMuMTVresultsin

the disruption of the virus and formation of rosettes,whichareviral membranefragmentsin the form of vesicles carrying the typical

MuMTV spike structures ontheirsurface (24). On the basis of its size, composition, and the ratio of the twoglycoproteins,weconcludethat the large cross-linked complex is made up of three molecules each of gp52 and gp36. Since the rate-favoredcross-linkingreaction is the for-mation ofgp36-gp52 heterodimers, it might be more accurate to describe the 230,000-molecu-VOL. 35,1980

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I

FIG. 7. Surface morphology of tissue culture-grown MuMTV.The central partof the enclosedareain (A) shows projections in asixfoldsymmetry. Ahighlymagnified viewoftheprojections isshown in(B). Three subunits (C) oftheprojectionknob marked by an arrow in (B) are resolved bya threefold(n= 3) rotation about thecentralpartofthe structure; n=4in(D);n=5in(E); n=6in(F). (G)to(K)showthreefold rotation images of five selectedknobs in which the subunits wereeitherheld together very tightly or loosely. (A) x350,000;(B)x1,800,000;(C to K)x2,900,000.

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lar-weight complex as made up of three

gp36-gp52heterodimers. Thepresenceof cross-linked

gp52 homotrimersindicates that the gp52 mol-eculesareallin contactwithoneanother inthe

largercomplex. The absenceofgp52molecules from very large cross-linked complexes (those

too largeto enter thegel) suggests that, in the

viral or rosette structures, gp52 molecules exist in isolated groupings oflimited size, probably clusters of three. All ofthese observations lead

to the conclusion that the

230,000-molecular-weight complex is actually the MuMTV spike that one observes in the electron microscope. Similarly, the 250,000-molecular-weight

com-plex formeduponcross-linking Rauscher leuke-mia virus preparationsmustrepresent atrimer of gp70-p15E heterodimers and isprobably the spike structure. Formation of homotrimers of

gp85-(gp7O-pl5E complexes) after cross-linking of type C virus has been previously reported (25).Furthermore,Takemotoetal.(25)reported

that no complexes larger than the

240,000-mo-lecular-weight homotrimer ofgp85could be de-tected after reaction with various concentrations ofcross-linking agents. A major difference

be-tweentypeC andtype Bvirusenvelope protein

interactionsemergesfrom thesestudies; namely,

in typeC viruses, the envelope proteinsareheld together by disulfide bonds (20), whereas in MuMTV, thetwoglycoproteinsseem tointeract by hydrophobic forces (15, 28).

The above conclusions about thenatureof the 230,000-molecular-weight MuMTVcomplex, ob-tained by biochemical means, complement the electronmicroscopic observation thatMuMTV surfaceprojectionsseem tobecomposed of three subunits. This observationwas originally made

in astudyonthe surfacestructure of RIIImilk virus (23). The present studies on tissue

culture-derived MuMTV show even more clearly the

subunitstructureof the MuMTVsurface projec-tions. Thethree-part substructure oftheknobs

is clearly evident upon analysis by the image

rotation technique. Electron micrographs of

DTBSP cross-linked MuMTV preparations show that the membrane ispermeabletostain and that themorphologyof the viriongenerally

resemblesthat ofglutaraldehyde-fixed virions.

Of the MuMTV core proteins, p14 will most readily form homooligomers; it doesso evenin

the absence of cross-linking agents. In nonre-duced virionpreparations,alargefraction ofp14

molecules iscomplexed in disulfide-bonded oli-gomers too largetopenetrate into the stacking

gel. Upon exposingthe virus preparationto

ox-idizingagents, all of thep14moleculescomplex into these large oligomers. Oxidation of virus also causes the disappearance of the protein

band of 30,000 molecular weight, just above p28, thatis presentin all MuMTV isolates. Tryptic peptidemapstudies of MuMTV proteins (6, 10)

have demonstrated that all of the p14 peptide

spots are contained in the p30 tryptic peptide

map. Although the role of p30 in MuMTV

as-sembly isnotknown, its behavioruponoxidation probably reflects the fact that thep14 sequence

is contained within it. The major core protein

p28 shows a very slight tendency to form ho-mooligomers in the presence of cross-linkers,

much less so than the major core proteins of

typeC viruses.

While this work was in progress, a report

appeared describing DTBSPcross-linking stud-ies withRIIImilk MuMTV (7).Our resultsagree

with and confirmanumberof theirobservations, including that p14 forms large homooligomers

and thatDTBSPcross-linkinggenerates homo-dimers of gp36 and heterohomo-dimers ofgp36-gp52.

However, Dion et al. (7) report that

heterodi-mers of the major core protein p28and

mem-braneglycoproteingp36 are presentbothin

con-trol nonreduced viral preparations and in

DTBSP cross-linked virions. We havenever ob-served suchap28-gp36complex underany

con-ditions and have also not observed the gp52

homodimers which Dion et al. (7) also report.

Furthermore, these workers did not report the formation ofthe230,000-molecular-weight com-plex, even though they studied isolatedrosette

preparations athigh

(400,ug/ml)

DTBSP

con-centrations. Thereason for thesediscrepancies

isnotknown,although it ispossible that theuse

ofhigh concentrationsofvirusneeded for stain-ing andalower-resolution gelsystem (7) might

accountforthediffering interpretations.

As for the interaction of any core proteins with any of the envelope proteins of MuMTV, the only evidence that we have is a possible

formation ofaplO-gp36heterodimer in the pres-ence of the cross-linker MMB. Although the amountof this complex thatweobserveis quite

small,itisthereaction thatone would expect in

view of thestudies (5, 15, 16) which have shown

thatplOhas anaffinity for the MuMTV

enve-lope. Recently, it has been shown that the N-terminal gag region protein of avian and murine typeC virusescan belinkedto viral membrane

lipids with dimethyl suberimidate (19). Since plO seems to be the equivalent protein in the MuMTV system (6, 16), by analogy one would expect that it too is membrane associated and hence possibly in contact with gp36. Failure to detect moreplO-gp36heterodimers is not

unex-pected, however,since a number of studies with type C viruses (3) and a recent report on MuMTV (16) suggest that the recognition

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tween the N-terminalgagprotein and the hy-drophobic membrane protein mightoccuratthe

gagpolyproteinprecursorstage,before

process-ing.

ACKNOWLEDGMENTS

Weacknowledge the contribution ofA.Pinter ofthis

insti-tuteforproviding technical advice, and for sharing withus

theresults ofsomecross-linking experiments donewith

sur-face-labeledMuMTV.

This workwassupported by Public Health Servicegrants

CA-16599, CA-17129,andCA-08748 from theNationalCancer Institute.

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