Synthesis and processing of precursor polypeptides to murine mammary tumor virus structural proteins.

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Vol. 25, No. 1 Printed in U.S.A.

Synthesis

and

Processing

of

Precursor

Polypeptides

to

Murine

Mammary Tumor

Virus Structural

Proteins

JANIS RACEVSKIS* andNURUL H. SARKAR

Memorial Sloan-Kettering Cancer Center, New York, New York 10021

Received for publication24June1977

Biosynthesisofmurinemammary tumorvirus (MuMTV) proteinswasstudied

inthechronicallyMuMTV-infectedepithelialcellline MuMT-73by using

mon-ospecificantiseratothemajorMuMTVcoreproteinp27 and themajorenvelope

glycoproteingp47. Inpulse-labeling experiments using[35S]methionine,

monospe-cificantiseratop27precipitateda75,000-molecular-weightproteinasthemajor

intracellularcomponent.Analysis of thesamecellularextractswithmonospecific

antiserato gp47 revealed that the gp47 precursor was a 70,000-dalton protein. After chaseperiods,therewas aloss of label from theprecursorsanda

concom-itant increase oflabeled extracellular mature viral proteins. The glycoprotein

precursorincorporated labeled glucosamine and seemed to be processed more

rapidlythan the p27precursor. Considerableamounts ofapparently

nonvirion-associated gp47 andglycoproteinprecursorcould be detected in the extracellular culture fluid.

Themurinemammary tumorvirus(MuMTV) isatype BRNAtumorviruscomposed offive major structural polypeptides (6, 11, 18, 25,30)

andsomeadditionalminorpolypeptides,

includ-ing the reverse transcriptase (14). Two of the five majorpolypeptidesareglycoproteins (gp47 andgp34), which are associated with the viral membrane (20),whereasthe other three major

polypeptides (p27, p16, and p12) are core

con-stituents (20). (Inour gelsystem, which shows

alinear rangefor calibration proteins between molecular weights 25,000 and 200,000 on a log

scale,wefind that themajorMuMTV

glycopro-teinmigratesas awide bandjustbehind cellular

actin [molecular weight 42,000] in a molecular weight range of between 45,000 and 50,000.) Mostof theinvestigationsinto thestructureand

composition of MuMTV have been done by

usingvirus isolated from the milk of mice from

strains withahighmammary tumorincidence.

However, thesuccessfulcultivation of

MuMTV-producing cell lines (13, 15, 19, 25) has made possible the study of intracellular viral protein

synthesis, processing,andassembly.

A number of laboratories have established

that the structural proteins of type C RNA tumorvirusesare firstsynthesizedas high-mo-lecular-weightprecursors that aresubsequently cleavedandprocessedtogiverisetothemature viralproteins(9, 24, 27).Thesestudies havealso indicatedthat themajorviralglycoprotein and

majorinternalcoreproteinsof thetypeC viruses

are initially synthesizedas part oftwo distinct

separate precursors,referredto astheenvand

gaggeneproducts (1), respectively.Tworecent reports provide evidence suggesting that the

synthesisoftype B RNAtumorvirus proteins

mightoccurinananalogous fashiontothose of typeC viruses.By usingananti-MuMTVserum,

Dickson and co-workers (5) reported a

73,000-dalton precursor to MuMTV glycoproteins in

extracts of

primary

cell cultures derived from murine mammary gland tumors. The protein

synthesis inhibition studies carried out by

Schochetman and Schlom (22), usinga MuMTV-producing cellline, haveprovided evidence for

thenoncoordinate synthesis of the glycoproteins and thecoreproteinof MuMTV.

In thispaper wepresent acharacterizationof

theintracellular synthesis of MuMTVproteins

and the identification of theprecursor polypep-tides of the MuMTV structural proteins gp47

and p27byusing MuMTV-producing epithelial celllineMuMT-73. These studieswere carried

outby the application ofimmunoprecipitation

techniques by usingmonospecific antiseratothe major MuMTVglycoproteingp47 andthe major

coreproteinp27.

MATERIALS AND METHODS

Cell cultures. Thesourceand characterization of theepithelialcelllineMuMT-73, which continuously produces MuMTV, have been described previously (19). The cellswerepropagated inasolution of Eagle minimumessential medium supplemented with 10% (vol/vol) fetal calf serum, 100 U of penicillin per ml and 100 ug ofstreptomycin per ml. The Rauscher virus-infected JLSV-9 cells were a gift from P. J. Gomatos of thisInstitute.

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Labeling of cells and extraction ofproteins. Cellswere labeled a day afterchanging the growth medium in cultures that had grown toconfluence in 75-cm2T flasks. The celllayerwaswashedoncewith

5mlofwarmmethionine-deficient minimum essential medium and then incubated withanadditional7ml of thesamemediumfor1h. Afterthispreincubation period, the mediumwaspouredoff andreplacedwith 3 ml of methionine-free medium containing 200yCi of[3S]methionine (NewEnglandNuclear).After the pulseperiod, eitherthecellswereimmediately lysed orthelabelwaschasedbythe addition of 0.5 ml of 1 mMmethionine ingrowthmediumplus0.4ml of fetal calfserum.

Cellular proteins were extracted according tothe method ofShapiro andAugust (23).Inthismethod, the cellsarewashed three times with ice-cold Earle balanced salt solution and thenscrapedinto2ml of

anextractionbuffercontaining5mM Tris-hydrochlo-ride (pH9.2),1mMEDTA,400mMKCI, 1% Triton X-100, 1 mM TPCK

(L-1-tosylamido-2-phenylethyl-chloromethyl ketone),and1mMPMSF (phenylmeth-ylsulfonyl fluoride). The protease inhibitors TPCK andPMSFwereobtained fromSigmaChemical Co. and added to the extraction bufferjust before use

from a 100mMstocksolution made up indimethyl sulfoxide. Theextracts werecentrifugedat25,000xg for 10min, and the pellets werereextracted with 1

ml ofKCl-freeextractionbuffer.Theywerethen

cen-trifugedat 25,000xgfor 10min.Thesupernatants

were pooled and centrifuged at high speed (1 h at

100,000 xg)beforeimmunoprecipitation.

Forlabelingof cells with[3H]glucosamine, growth medium fromaconfluent culture ina75-cm2Tflask

wasreplacedwith 10 ml of minimumessential medium containing25% of the normal concentration ofglucose and supplemented with 50

ACi

of [3H]glucosamine

(NewEnglandNuclear)perml. The[3H]glucosamine

solution was concentrated by evaporation under a stream ofnitrogen toreduce the amountof ethanol (fromtheglucosaminesolution) addedtotheculture medium. Cellswereincubated for 24 h and then

ex-tracted asdescribed above.

Inexperimentswhere the extracellularculture fluid

wasanalyzedfor the presence of virion- and nonvirion-associatedproteins,thegrowthmediumwasclarified bylow-speed centrifugation (10minat 500xg) and then layered onto a discontinuous sucrose gradient made up of1 ml of 60% and 3ml of 20% sucrosein TEN buffer (20 mM Tris-hydrochloride [pH 7.4]-1

mMEDTA-100 mM NaCl) and spun for90min at

38,000 rpm inanSW41rotor.Thesupernatantculture fluid was removed, and Triton X-100 and sodium deoxycholatewereaddedtoafinal concentration of1

and0.5%, respectively. Virusatthe interface of the discontinuous gradient was removed, diluted with

TENbuffer,anddisruptedbytheadditionof Triton X-100sodiumdeoxycholatetoconcentrations of1and 0.5%,respectively.

Immunoprecipitation. The antisera used were

monospecific goat anti-p27 and anti-gp47 sera-(21) and rabbit anti-wholeMuMTVsera.Thepreparation andadsorption of antiserahave been described(18). Rabbitanti-LETS antiserumwas agiftfrom Lan Bo Chen of ColdSpringHarborLaboratory. Portions of

0.5to1.0 mlofcentrifuged cell extracts were incubated

at 4°C with 3 to 8,ul of antisera, depending on the titer ofthe antiserum. After 20min, 20to40

pld

ofa

10%suspensionofstaphylococcal proteinA-antibody adsorbent(10)wasadded andincubated for an addi-tional 20 min at 4°C. Staphylococcus aureus strain Cowan I waskindly provided by H. Oppermann of theUniversityofCalifornia,SanFrancisco. The prep-aration of thestaphylococcal antibody adsorbent and theisolation of theimmunoglobulins were carriedout

accordingtotheprocedure of Kessler (10). The follow-ing modifications to the techniques of Kesslerwere

introduced toreducenonspecific cellular contamina-tionin theimmunoprecipitates: the washed staphylo-cocciweresuspended toa10%suspension in unlabeled cellextract, and cell extracts were preadsorbed once

withcontrol serum andstaphylococcaladsorbent be-foreimmunoprcipitation. After the 20-min incubation withstaphylococcal adsorbent,thesuspensionswere layeredon 1 mlof1 Msucrose-0.5% sodium deoxy-cholate TEN solution and centrifuged at 3,000 rpm for 10min. Theadsorbent antibody pellets were then washed threetimes with0.5% NP-40 TEN buffer. The antigen-immunoglobulin complexes were dissociated from theadsorbentby heating at 100°C for 3minin 50

pil

of 1.5x concentrated electrophoresis sample buffer(12).

Polyacrylamide gel electrophoresis. Gel elec-trophoresis was done in 20-cm-long, 5 to 20% exponen-tialgradient sodium dodecyl sulfate (SDS)-polyacryl-amide slab gels by the discontinuous Tris-glycine buffer system of Laemmli (12). The exponential gra-dientpolyacrylamide slab gels were prepared as de-scribed by Van Blerkom and Manes (26). Protein molecularweights were determined with the Combi-thekmolecular-weightmarker kit(Boehringer Mann-heim), which includes soybean trypsin inhibitor (21,-500), bovine serum albumin (68,000), and Escherichia coli RNApolymerase subunit a (39,000), subunit ,B (155,000), and subunit if (165,000). Myosin heavy chain (molecular weight 200,000) was also used as a marker. Inaddition, a mixture of [3S]methionine-la-beledvesicular stomatitis virus- and reovirus-infected cell extracts was used as amolecular-weight marker on the autoradiograms, as previously described by Racevskis and Koch (16). [3H]amino acid- or ['4C] amino acid-labeled MuMTV marker virus was pre-paredasdescribed by Sarkar et al. (20). After electro-phoresis,gels were treated by the procedure of Bonner and Laskey (2), dried, and then exposed to Kodak RR Royal X-Omat film at -700C.

RESULTS

Immunoprecipitation of intracellular

viralprecursorproteins. The MuMT-73

ep-ithelial cellline, derivedfromspontaneous

mam-marytumorsofBALB/cfC3H mice (19),which continuously produces MuMTV (B particles),

was used throughout these studies. Although

virusproductioncanbeincreasedinthe

MuMT-73 cells by glucocorticoid hormone treatment (19), the studies described in this report were

doneby using untreated culturessoon after they

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376 RACEVSKIS SARKAR

reachedconfluency andadayafter fresh media

had beenadded. Since these epithelial cellsare

fairlyslow-growing cells, and the viral proteins

account for only avery small fraction of total cellular protein synthesis, itwasfoundnecessary

to pulse-label cells for periods of30 to 60 min

with about

200,uCi

of[3S]methionineper T75

flasktoeasily detectviral-specific polypeptides intheimmunoprecipitates.

By using monospecific antisera tothe major glycoprotein gp47and major internalcore

pro-teinp27, wedetectedtwo closely migratingyet

distincthigh-molecular-weightintracellular

pro-teinsbeing specifically immunoprecipitated with the antisera (Fig. lb and c). Figure lb showsa

60-min [3S]methionine-labeled MuMT-73 cell

extractprecipitatedwithanti-gp47serun.This

serumprecipitatedapolypeptideof70,000

mo-lecularweight (pre gp7O). Some gp47 was also precipitated,ascan beseenbycomparing itto

the [3H]amino acid-labeled marker MuMTV pattern in column a.Figure lcshows thesame extract asin lb, but immunoprecipitated with anti-p27serum,which,as canbeseen, detected

a75,000-daltonspecies (pre p75).For

compari-son purposes, Fig. ld and e are 60-min

[35S]_

methionine-labeled cellextractsof Rauscher

vi-rus-producing JLS-V9 cells precipitated with anti-Rauscher p30and gp69/71 serum,

respec-tively. Figure ld shows the 67,000-dalton

pre-cursor to p30, and Fig. le shows the

80,000-dalton glycoprotein precursor, as well as the envelope glycoprotein gp69/71, which

comi-grateswith the MuMTV glycoprotein precursor.

Analysisof the MuMT-73cellextractwith

anti-Rauscher virus antisera showed no detectable viralproteins beingprecipitated (not shown).

Proteins of cellular origin. In both the MuMT-73 and the JLS-V9cellextracts(Fig. 1),

a very high-molecular-weight species (250,000

daltons) wasprecipitatedwithall the different antisera. There was less of thisprotein in the MuMT-73 cellextractsthan in the JLS-V9 ex-tract because the MuMT-73 extracts were

preadsorbedwithcontrolgoat serumand

staph-ylococcal antibody adsorbent before

immuno-precipitation, as described above. We found it necessary to preadsorb the MuMT-73 cell ex-tractssincetheseepithelialcell linessynthesize

great quantities of cytoskeletal-type proteins,

whichinterferewith theresolutionof

viral-spe-cificpolypeptides.Twoadditional high-molecu-lar-weight proteins are seen in the nonpread-sorbed JLS-V9cell extracts.

Anotherprominent proteinband that is pres-ent in all immunoprecipitates in variable

amounts is a species of about 42,000 daltons. Thecellular originof the42,000-dalton protein

M

UMTV

pre

p(75bm

_{-_r~~~~~~0}

pre

gp(C.

gp47

.-

-,1

C

b

c,

d e

R

LV

*

-pre

gp8O

;

-gp69/7l

-Lc,r)e p67

*--

(1ct

ri

FIG. 1. Autoradiograph of SDS-gel electrophore-sisofimmunoprecipitatesfrom MuMT-73cellsand JLS-V9cellspulse-labeledwithf5SJmethionineand

run on a20-cm-long5 to20% exponentialgradient polyacrylamide gel. (a)

I3H]amino

acid-labeled MuMTV marker virus. (b) 60-min f5S]methionine

pulse-labeledMuMT-73 cellextract immunoprecipi-tated withanti-MuMTV-gp47serum.(c) Sameas(b)

but immunoprecipitated with anti-MuMTV-p27

se-rum. (d) 60-min

f6S]methionine

pulse-labeled JLS-V9cellextractimmunoprecipitated with

anti-RLV-p30serum. (e) Same as(d) but immunoprecipitated withanti-RLV-gp69/71serum.

and the nonspecificity of the precipitation is indicated by the observation that the quantity

present in the immunoprecipitates is inversely

proportional tothe extent that the precipitates

are washed. This cellular protein is probably actin, which has been estimated to make up some5to10% of theprotein of nonmuscle cells

(3) and identified as being present in purified preparations ofseveral different enveloped vi-ruses (28). Onthebasis of thereport that

non-muscle actinshave thesamemobility on SDS-acrylamide gels as muscle actin (3), we have

identifiedthe42,000-daltonproteininour

prep-aration as actin bythe observation that it

co-migratedwith mouseskeletal muscle actin(Fig. 2b). MuMTV purified from RIII mouse milk

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a

b

-

myosin

gp47-

*

- -

actin

p27-FIG. 2. Coomassie brilliant blue-stained

SDS-polyacrylamide gel. (a) Purified RIII mouse milk MuMTV. (b)RIIImouseskeletalmuscleextract.

alsocontainedaprotein bandwhichcomigrated with actin extracted from RIII mouse skeletal muscle(Fig. 2a).

Specificityofthe antisera. The

monospec-ificity of the antisera used in this study was demonstrated by Sarkarand Whittington (21) by the application of the double antibody tech-nique against purified ['4C]amino acid-labeled MuMTV. Wehaverepeated the testfor speci-ficity by employingthestaphylococcal antibody adsorbenttechnique against purified [3S]methi-onine-labeled MuMTV(Fig.3c).Goat antiserum against p27 precipitated only p27 (Fig. 3a), and goat antiserum against gp47 precipitated only gp47 (Fig. 3b) when tested against [3S]methio-nine-labeled, purified MuMTV (Fig. 3c). It is

also quite clear from Fig. 3d and e that the

intracellularviralprotein species designatedas

prep75 andpregp7O precipitatedfrom 90-min

pulse-labeled cell extracts by each antiserum

werequite distinct and thatnocross-reactivity

was observed. The anti-p27 serum also seems tohaveprecipitatedaless prominent protein of about 100,000 daltons (Fig. 3d), whichwas not

observed in the sample precipitated with

anti-gp47 serum (Fig. 3e). Analysisof the same la-beled cellextractwith nonimmune goat serum

(Fig. 3f) reveals that only the

high-molecular-weight (250,000 daltons) species was

precipi-tated. No other prominent species were ob-served.

Identification of the 250,000-dalton

cel-lular protein. On the basis of a number of

observations, includingmolecularweight,release

into tissue culture medium, and labeling with glucosamine, wesuspected that the large,

250,-000-molecular-weightprotein observed in all the

immunoprecipitates might be thelargeexternal

transformation-sensitiveglycoproteinknownby

theacronym LETS(7).Immunoprecipitationof the

[3S]methionine-labeled

cell extract with anti-LETS antiserum (Fig. 3g) confirmed our

suspicion. All theimmunoprecipitations shown

in Fig. 3 d (anti-p27), e (anti-gp47), f (control serum), andg(anti-LETS)weredone with iden-tical aliquots of the same [3S]methionine-la-beled cellextractusing identical volumes of the respective antiserum andstaphylococcal

adsorb-ent.Toquantitate theamountof

high-molecu-lar-weight (250,000 dalton) protein in each of

the above immunoprecipitates, densitometer

scans of the top half of these four lanes (d

through g) were done (Fig. 4). The amount of

250,000-dalton protein in the sample

immuno-precipitatedwith anti-LETSserum(Fig.4a)was

abouteightfoldgreater than that inany oneof the other three samples (Fig. 4b through d),

thusestablishingitsidentityasLETS

glycopro-tein. It may also be noted that in additionto

the LETS glycoprotein, anti-LETS serumalso

brought down many other

high-molecular-weight proteins (Fig. 3g). This observation is

not surprising in view ofthe fact that LETS

has been shown to be extensively disulfide bondedtomanyotherproteinsonthe

mamma-lian cell surface (8). Since the cell extraction

andimmunoprecipitation arecarriedoutunder

nonreducingconditions, thedisulfide bonds

be-tweenLETSandothercellularproteinsremain

intact,resultingin thecoprecipitationof various LETSaggregates.

Pulse-chase studies-kinetics of

precur-sorprocessing.Theresultsofpulse-chase

ex-periments usingthe MuMT-73 cell lineare

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378

a b c d e f g

200 -100

-

80-70

-60

-50

-40

-30

-a -a m

#

-

LETS

.

r

pre

p75

doft

-Lpre

gp7O

-

gp47

-

gp34

[image:5.505.151.382.68.366.2]

-

p27

FIG. 3. Analysis of antiserum specificity. (a)P5S]methionine-labeledpurifiedMuMTVimmunoprecipitated with monospecific anti-gp27serum. (b) Same as (a) but immunoprecipitated with monospecific anti-gp47 serum. (c) 65S]methionine-labeledpurified MuMTV. (d) 90-minPS]methioninepulse-labeledcell extract immunoprecipitated with anti-p27 serum. (e) Same as (d) but immunoprecipitated with anti-gp47 serum.

(f)

Same as (d)butprecipitatedwith controlnonimmune goat serum. (g) Same as (d) but immunoprecipitated withanti-LETS serum.

picted in Fig. 5 and 6. Figures 5b through d

show immunoprecipitates obtained with

anti-p27 sera incellextractsof60-min

[35S]methio-nine-pulsed and 90- and 180-min-chased

cul-tures, respectively. It canbeseenthat the

pre-sumed75,000-molecular-weight polypeptide pre-cursorofp27isfairly stable and thata consid-erablequantityofprecursoris stillpresentafter

a chaseperiod of3h.Inotherexperiments, we

have found that the precursor protein is still

detectable after a chase period of 6 h. During thechaseperiod,the pre p75appearedtodisplay

aslightlyloweredmobilityintheSDS-gel (Fig.

5candd). Wehave found that thisslowed

mi-gration of pre p75 after the chase periods is caused by a post-translational modification of theprecursorinvolving phosphorylation (N.H.

Sarkar, E. S.Whittington, J.Racevskis, andS. L. Marcus, manuscript in preparation). The

100,000-molecular-weight protein,whichis

con-siderablylessprominentthan prep75and seems to be p27 specific, is chased as well (Fig. 5b

through d).Amoreprominentband in theregion

of 34,000daltons ispresentin allthreesamples shown inFig.5bthrough d andmightrepresent anintermediate precursor of p27. The amount

of intracellularp27 isbarelydetectable.Analysis ofadditionalportions ofthe same cellextracts

withanti-gp47serumrevealed that the glycopro-teinprecursor pregp7Oisprocessedmorerapidly

than prep75,and thatlargequantities ofmature

gp47 are present intracellularly after a60-min

pulse and after ensuing chase periods (Fig. 5e

through g). Some protein speciesthat seem to be gp47 specific are visible in the molecular-weight region of about 60,000 (Fig. 5e through

g). The fact that theseproteins decreasein con-centration after chase periods and display smearing, which is characteristic of

glycopro-teins electrophoresed on SDS-gels, indicates

thattheymay beprocessing intermediatesfrom pregp7O.Thediffuse dark arearunningbehind pre gp7O (Fig. 5e) mightrepresent fuly glyco-sylated speciesoftheprecursor(Fig. 7k).These

species, however, make up only a very small

fraction of total pregp70, asshownbythe

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lected and likewise immunoprecipitated with whole MuMTV antisera (Fig. 6d through f). After a90-minchaseperiod (Fig. 6e),five viral proteins were recovered from the media: the majorglycoprotein gp47, the majorcoreprotein

p27, twosmall viral proteins of less than 20,000

molecularweight, and one diffuseband that

co-migrated with glycoprotein precursor pre gp7O. Inadditiontothese viralproteins, large

quanti-ties of LETS glycoproteinand other

high-molec-ular-weight cellular proteins

(probably

LETS

a

b c d e f

g

h

200-

150-

100-

80-

70-

60-

50-200 100 80 70

FIG. 4. Densitometerscansofthe upper portion of columnsd, e,f,and gofthegelshown inFig.3. (a) Column (g) of Fig. 3. (b) Column (D of Fig. 3. (c) Column(e)ofFig.3.(d)Column(d)ofFig.3. Approx-imatemolecularweightsin kilodaltonsareshownat

thebottomofthefigure.

sitometer scan (Fig. 4c). The glycoprotein

het-erogeneityisespeciallynoticeable on these

20-cm-long high-resolution

gradient

gels,

and it

may be noted that gp34 of the marker virus (Fig. 5a) isactuallyaheterogeneouspopulation ofsomethreeorfour discrete bandsrunningin

amolecular-weightrangeof between32,000and

37,000. Wecanalso seefromFig. 5thatLETS

proteinis decreased in concentration after the

chase periods, indicating that it is

probably

transportedtotheoutersurface of thecell

mem-branefrom where it isshedinto themedia. Extracellulartransportof viralproteins. Analysis of anti-MuMTV immunoprecipitates obtainedfromextractsofcellspulsedfor60min

and eithernotchasedorchasedfor either90 or 180 minis shown in Fig. 6a throughc. Allthe

species precipitated with the individual

mono-specificantisera inFig.5 arealsorecognized by

the antisera to disruptedwhole MuMTV. The

culturemedia from the three cultureswere

col-40-

I

was fI

3-,-ma

m-30-9

20-FIG. 5. Autoradiograph of SDS-gel

electrophore-sis of immunoprecipitates frompulse-chase studies

in MuMT-73 cells. (a) '4C]amino acid-labeled

MuMTVmarker virus. (b) 60-min f5SJmethionine

pulse-labeled cell extract immunoprecipitated with

anti-p27serum.(c)Sameas(b)butchasedfor90min

with excess cold methionine. (d) Same as (b) but chased 180min. (e)Sameas(b) but

immunoprecipi-tated withanti-gp47serum.(/)Sameas(e) but chased

for90min. (g) Sameas (e) but chasedfor 180min.

(h)Mixtureofvesicularstomatitis virus-and

reovi-rus-infected cell extracts labeled with f5SJmethio-nine(molecular-weight markers).

a LETS

b

C pregp7O

d prep75

A

pre

p75

pre

gp70

-g

p

47 -gp34

S

-

p27

-p16

-pl2

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a

b

c

d e f

200

-100 - etoe

80-70--

*

60

-

^2

50-40-

*4

30

-pre

p75

1-pre

gp7O

gp47

"N i"4U

p27

FIG. 6. Polyacrylamide gelelectrophoresis analy-sis of the immunoprecipitates oftheMuMT-73 cell extractsand extracellular media reacted with

anti-whole MuMTVantisera. (a) 60-minI5SJmethionine

pulse-labeledcellextractsimmunoprecipitated with

anti-MuMTVserum. (b)Sameas(a), but cellswere

chasedfor90 minwith excess coldmethionine. (c)

Same as (a), butcellswerechasedfor 180min. (d) Cell-free culture media from same cells as in (a) immunoprecipitated with anti-MuMTV serum. (e)

Culturemediafrom 90-min-chasedcells immunopre-cipitated withanti-MuMTVserum.(f) Culturemedia

from 180-min-chased cellsimmunoprecipitated with anti-MuMTVserum.

aggregates) were found adhered to the immu-noprecipitates (Fig. 6d through f). Two factors that probably contributed to the high level of these cellular contaminantswere(i)the cell-free

mediainthe experimentwerenotpreadsorbed before immunoprecipitation, and (ii) relatively large volumes ofanti-MuMTV sera as well as staphylococcal adsorbentwere used to precipi-tateviralproteinsfromthe media.

Virion- and nonvirion-associated extra-cellular viral proteins. Inview ofthe

unex-J. VIROL.

pected detection, with anti-MuMTV sera, of what seems to be pre gp7O in cell-free media (Fig. 6e and f), we attempted to identify the

origin of the pre gp70 and determine whether

the other extracellular viral proteins were, or were not, virus associated. Cells were labeled

with [3S]methionine in media containing

one-twentieth thenormal concentration of methio-nine for12h and thenchased foranadditional

12 hby the addition ofexcess,cold methionine.

The media from these cultures was collected

andlayeredontopofa20to60%discontinuous

sucrosegradient andcentrifugedat100,000 x g for 2 h. The remaining virus-free media were

saved, and the viral fraction at the 20 to 60% interface was removed and treated with lysing buffer. Immunoprecipitation of thecell extract

with anti-p27 and anti-gp47 sera is shown in Fig. 7b and c,respectively. Itcanbe seenthat thepregp7O andprep75precursors werebarely detectable after the long chase period. There

was a verysmallamountof p27 inthe cell (Fig. 7b);however, largeamounts ofgp47 were pres-ent(Fig.7c). It should be noted thatnolabeled cell-associated LETSproteinwasobserved after the long chase period. Immunoprecipitation of the virus-free media withanti-p27sera (Fig. 7d) revealed thatsomenonvirion-associatedp27 was presentinthe mediatogether withaprotein of less than 20,000 molecular weight. Analysis of thesame mediawithanti-gp47 serum (Fig. 7e) revealedthat, in additionto alarge amountof nonvirion-associated gp47, precursor gp7O was

also present in the media. The

low-molecular-weight proteindetected withanti-p27serum was

also observed. This protein does notcomigrate withanyof thesmallviral proteins in the marker MuMTV (Fig. 7n) and probably represents a

cellularspecies whichmaybeabreakdown

prod-uct or anassociated protein ofthe

high-molec-ular-weight cellular contaminants. Ananalysis

of the viral fraction withmonospecific antisera revealed the presenceof p27 and gp47 (Fig. 7f

andg,respectively).In contrast tothe virus-free

media, nopre gp7Owasfound in the viral

frac-tion. Some p27 was unexpectedly observed in

the viral fraction precipitated with anti-gp47 serum (Fig. 7g). We have observed that when

immunoprecipitation is done in concentrated

viral extracts, some p27 will be present in the precipitates unless theyarewashedextensively

andcentrifugedthroughasucrosedeoxycholate

solution asdescribedabove. Thesampleshown inFig.7g was notcentrifugedthroughasucrose

deoxycholate solution. The p27 probably

ad-herestothestaphylococcusadsorbentsincep27 wasalso detected insamples precipitated with nonimmune sera, which were not washed

thor-oughly. We have observed that Rauscher

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200

-a b c d e f g h j k I m n

*4.0

150

-80-

- 70-60- w

50-

40-* -p27

20

-% -p16

- - p12

FIG. 7. Long-term labeling of MuMT-73 cells with f'S]methionine andI3H]glucosamine. (a) Mixtureof vesicular stomatitis virus- and reovirus-infected cell extracts, P3S]methionine labeled (molecular-weight markers). (b) MuMT-73 cell extract labeledfor 24 h with f35S]methionine andimmunoprecipitated with anti-p27serum. (cJ Sameas (b) butimmunoprecipitated withanti-gp47serum. (d)Mediafromcells in (b) after spinningoutvirus andimmunoprecipitatedwithanti-p27serum.(e)Sameas(d),butimmunoprecipitated

withanti-gp47serum. (f) Spun down, solubilized virus isolated from media of 24-h[fS]methionine-labeled

cellsimmunoprecipitated with anti-p27serum.(g) Sameas(f)butimmunoprecipitated with anti-gp47serum.

(h) MuMT-73 cell extract labeledfor 24 h with P3Higlucosamine and immunoprecipitated with anti-p27

serum.(i) Sameas(h)butimmunoprecipitatedwithanti-gp47serum.

OF)

Virus-freemediaofsamecellsasin

(h), immunoprecipitatedwithanti-p27serum. (k)Sameas(j) butimmunoprecipitated withanti-gp47serum.

(1)pH]glucosamine-labeledsolubilized virusisolatedfrommediaofsamecellsasin(h),immunoprecipitated withanti-p27serum.(m)Sameas(1) butimmunoprecipitatedwithanti-gp47serum.(n) "4C-labeledMuMTV

marker virus.

kemia virus p30 also hasan affinity to staphy-lococcus adsorbent (Racevskis and Sarkar, un-published data). LETS glycoproteinis present in the media as well as in the virus samples. Additional, higher-molecular-weight bands are seeninthe viralsamplesas well (Fig. 7f and g).

Glucosamine labeling of viral proteins. Tofurthercharacterize and identify the glyco-sylated viralproteins, [3H]glucosamine labeling of the MuMT-73 cells was done. Cells were labeled for 24 h with[3H]glucosamine in media containing 25% of the normalamountofglucose. Cellswereextracted,mediawerecollected, and viruswasspunoutasdescribedabove.Analysis of the three fractions-cellular extracted (Fig.

7i), virus-freemedia (Fig.7k),and virus fraction (Fig. 7m)-with anti-gp47 serum shows that boththe70,000-molecular-weightprecursorand gp47 were heavily glycosylated and that the glycoproteinsinthe various fractionswere iden-ticaltothoseseenpreviouslywith the [3S]me-thionine-labeled cells. After 24 h oflabeling in low-glucose media,large quantitiesofgp47were present, but not much intracellularpregp70was observed (Fig. 7i).The virus-free medium (Fig. 7k) contains large quantities ofprecursor and matureglycoprotein,whereas the virus fraction (Fig. 7m) contains only mature glycoprotein gp47 associated with it. As seen in Fig. 7h, j, and 1, anti-p27 serum did not precipitate any

30- l _

-pre gp70

-

gp47

-

gp34

I

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[3H]glucosamine-labeled proteins from any of

the three fractions, except for LETS protein,

which isglycosylated.

DISCUSSION

By the application of immunoprecipitation techniques using monospecific antisera and high-resolution polyacrylamide gels, we have found that the MuMTVmajorglycoproteingp47

andmajorcoreprotein p27areinitially synthe-sized as part oftwo separate high-molecular-weightprecursorpolypeptides (pre gp7O andpre p75,respectively).

Dickson et al. (5) described the detection of

a 73,000-dalton precursor to the two MuMTV glycoproteins (gp47 and gp34) by the use of antiserumfor whole MuMTV inpulsedcell ex-tracts. However, these authors failed todetect the major core protein precursor even though their anti-MuMTVserumhadastronganti-p27 activity when tested against purified virus (4). When used in labeled cell extracts, therefore, this same antiserum should have precipitated the p27 precursor,butbecause of therelatively small difference in molecularweights, the p27

precursor was probably not resolved from the glycoproteinprecursorin theirgelsystem.

The major intracellular protein that is precip-itated withmonospecific anti-p27serumaftera

shortpulseisaspeciesof 75,000daltons,which

has arelatively longhalf-life of about 3to4 h

inthe MuMT-73 cells.Duringthe chaseperiods,

the 75,000-dalton protein displayed a slightly

altered mobility in the SDS-gels. We have de-termined that the alteredmigration results from

post-translational phosphorylation of the

pre-cursor (unpublished data). Two additional

pro-teinsareprecipitatedwithanti-p27 serumfrom the MuMT-73 cells. One isaminor protein of about100,000 molecularweight,which isnearly undetectable aftera 180-minchaseperiod.The otherproteinismoreprominentandhas a

mo-lecular weight of 34,000 daltons. This 34,000-dalton species is observed after the 60-min

la-beling periodaswellasafter the 180-min chase

period,andmightrepresent aprocessing inter-mediate precursor of p27. This polypeptide is not to be confused with the viral glycoprotein

gp34, which showsabsolutelynocross-reactivity

withouranti-p27serum.The34,000-dalton

pro-tein, which isprecipitatedwithanti-p27serum, hasalso beenfoundtobe phosphorylated (un-published data). Very little p27 can be found

intracellularly, even after the chase periods

whenlabeledvirion-associatedp27canbe

read-ily detected in the culture medium, suggesting that the final cleavage step to mature p27 isa very late event in virus maturation.

The major glycoproteingp47 isinitially

syn-thesized as a polypeptide of 70,000 daltons, which is processed about twice as fast as the corresponding p27 precursor pre p75. Large quantities of gp47 are found to be associated with thecells and in the extracellular medium,

both in the viral fraction and in the virus-free media. In addition, largeamountsof the glyco-proteinprecursor pre gp70 are found extracel-lularly in the virus-free media, butno glycopro-teinprecursoris found associated with the iso-lated virus fraction. This observation suggests

that thepre gp7O found in the media doesnot

result from the breakdown of virus, but is prob-ably shed from the cell membrane in a free form. Thematuregp47might also be shed from cell membranes, although it is possible that it derives from breakdown of virus. Our observa-tion of the in vitrosheddingof viral glycoprotein by tissue culture cells might explain the obser-vation of elevated levels of MuMTV glycopro-teinantigensintheplasma of mammary

tumor-bearing mice as reported by Ritzi et al. (17).

Both gp47 and pre gp70 readily incorporate [3H]glucosamine, indicating that both species areheavily glycosylated.

Inspection of the gel profile of the

immuno-precipitated cell extracts reveals that they all containaprominent high-molecular-weight

pro-tein of about 250,000. This protein, therefore,

seems to bea "sticky" cellular contaminant of

the immunoprecipitates since it isalso

precipi-tatedby controlserumandcanbe quantitatively reduced in the cellextracts by a preadsorption

procedure with nonimmune serumand

staphy-lococcal adsorbent. The fact that thislarge cel-lular protein incorporates [3H]glucosamine, is released from cells into the media, and has a

molecular weight of about 250,000 led us to

consider that it is perhaps the large external transformation-sensitive glycoprotein referred

toby theacronymLETS(7).Analysis ofcellular

extracts with anti-LETS serum confirmed its

identityastheLETSglycoprotein.The

MuMT-73epithelialcellssynthesizeconsiderable

quan-tities of LETS and continuously shed it, along

with otheraggregated species, into the culture

medium.

Another protein ofcellular origin, actin, has been identified both in theimmunoprecipitates

ofcellextractsand in purifiedMuMTV

prepa-rations,onthe basis ofcomigrationinSDS-gels

with actin extracted from RIII mouse skeletal

muscle.

Ourfindings oftwo distinct precursor forms

for the two main MuMTV structural proteins,

one for the major envelope constituent and a secondfor themajorinternalcorepolypeptide,

isconsistent with thestudyofSchochetmanand

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Schlom

(22), which indicated noncoordinate synthesisof these two MuMTV proteins. A

sim-ilar mechanism of noncoordinate synthesis of viral structural protein has been well established by a number of laboratories for the better-char-acterized typeC RNA tumor viruses (9, 24, 27). The findings presented in this report, together with the previous findings with MuMTV and type C RNA tumor viruses, suggest that non-coordinate synthesis of viral structural proteins, by way of independently synthesized high-mo-lecular-weight precursors, might be a general characteristic of

all

RNA tumorviruses.

Further studies using antisera to the other MuMTV proteins as well as tryptic peptide anal-yses

will

be required to firmly establish the precursor-product relationship between the var-ious MuMTV proteins.

ACKNOWLEDGMENTS

We are indebted to H. Oppermann of the University of

Californiaat San Francisco for providing us with the Staphy-lococcus aureus Cowan I strain. We thank Lan Bo Chen of the Cold Spring Harbor Laboratory for his gift of anti-LETS serum. We also thank E. Whittington forhis assistance in preparing the labeled, purified MuMTV marker virus.

This work was supported byPublicHealth Service grants CA-08748 and CA-17129 from the National Cancer Institute.

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