Analysis of intracellular feline leukemia virus proteins. I. Identification of a 60,000-dalton precursor of feline leukemia virus p30.

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JouRNALoF VIROLOGY, Oct. 1976,P.96-106

Copyright0 1976 American Society for Microbiology

Vol.20, No.1

Printed in U.S.A.

Analysis

of Intracellular Feline Leukemia Virus Proteins

I. Identification

of a 60,000-Dalton Precursor of Feline

Leukemia

Virus p301

GREGORY F. OKASINSKI AND LELAND F. VELICER*

Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Receivedfor publication 16 March 1976

The synthesis and release of feline leukemia virus p30 was studied using a

permanentlyinfected feline thymus tumor cell line. Disrupted cells were divided into two subcellular fractions, a cytoplasmic extract (CE) representing cellular

materialsoluble in 0.5%NP-40 and a particulate fraction (PF) insoluble in 0.5%

NP-40, but soluble in 0.2% deoxycholate and 0.5% NP-40. Intracellular feline

leukemia virus p30 was isolated from infected cells by immune precipitation with antiserum to p30 and subsequent sodium dodecyl sulfate-polyacrylamide

gel electrophoresis of the precipitated proteins. Cells labeled for 3 h with

[15S]methionine containedequal amounts ofp30 in both the CEandthe PF.p30 synthesis was estimated to be 0.8% of the total host cell protein synthesis. Immune precipitates from cells pulselabeled for 2.5 min contained a labeled

60,000-dalton polypeptide (Pp6O) in the PF and a polypeptide in the CE that

comigratedwithfeline leukemia virusp30insodiumdodecyl sulfate-polyacryl-amidegel electrophoresis.Whencells werechasedafter a pulselabel,there was arapidloss of Pp60 inthe PFandanaccumulation ofp30inthe CE within 30 minfollowedbydistributionofp30 inboth the PF and the CE. Estimation of intracellular and extracellular p30 levels during a 0.5- to 24-h chase period suggestedthat most of the newly synthesizedp30wasincorporatedinto

extracel-lularvirus. Tryptic peptideanalysis oflabeled Pp6O and p30demonstrated the

presenceof 13 of 15 p30peptides within thePp6Omolecule. The trypticpeptide

analysisin concertwiththepulse-chase labeling dataprovidesstrong evidence thatPp6Ois aprecursor ofp30.

The polypeptide composition of both the avian and mammalian oncornaviruses has been thoroughly studied in the past several years (13, 14, 17, 18, 25). The oncornaviruses containfive to seven major structural proteins withmolecularweightsrangingfrom 10,000 to 85,000 (5). Although the polypeptide

composi-tionofthe oncornaviruses has been rigorously studied, information concerning the synthesis

and processing of these polypeptides has ap-peared only recently (1, 10, 23, 24, 31, 33, 34).

Evidence obtained from picornavirus-, par-amxovirus-, and reovirus-infected cells

indi-cates that nononcogenic RNA virus mRNA is

translated from asingle initiation site (4).The mRNA of theseviruses iswellcharacterized (3, 6, 36). All three types of nononcogenic RNA

virus mRNA are translated into polypeptides

thatcorrespondin size withtheviral mRNA (6, 21, 22). In poliovirus-infected cells, the initial

translation product is a large precursor

poly-'Articleno.7557from theMichiganAgricultural

Exper-imentStation.

peptide, which is subsequentlycleaved toyield mature virionpolypeptides(21).

Oncornaviruses contain a

high-molecular-weight genome composed of 28-358 subunits with a molecular weightofapproximately3 x 10 (2, 7, 9). These RNA subunits contain 3'

poly(A) sequences (7, 20, 27). The apparent

abilityofthesesubunitstoserveasmRNA in in vitroprotein-synthesizing systems(24, 30, 35), combined withthe presence on polyribosomes

ofviral-specific RNAwith amolecularweight

similartothat ofgenomicsubunits(12, 16, 32), suggests thatoncornavirus mRNA is very simi-lartogenomic subunits.Ifoncornavirus mRNA istranslated in a manner similarto nononco-genic RNAvirusmRNA,thenonewould expect aninitial translationproductof about 300,000

daltons.

Attempts to isolate the initial translation product ofoncornavirusproteinsynthesishave been directed to in vitro protein-synthesizing

systemsandimmunoprecipitationofviral

poly-peptides from infected cells. Various in vitro protein-synthesizing systems have been used

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FeLV p30 PRECURSOR POLYPEPTIDE 97

withlimited success (8, 30, 35). Recently, how-ever, polypeptides (molecular weights of 140,000 to 185,000 and 50,000 to 75,000) have been synthesized using Rauscher leukemia vi-rus (RLV) genomic RNA in a cell-free protein-synthesizing system (24), and 75,000-to 80,000-dalton polypeptides have been synthesized in

response toadded30-40S RNA of Roussarcoma

virus (35). Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) of im-mune precipitates from avian myeloblastosis virus (AMV) (10, 33, 34)- and RLV (1, 31)-in-fected cells provides evidence fora 76,000-dal-ton precursor in the former and 200,000-, 80,000-, and 65,000-dalton precursor polypep-tides in thelatter. Although precursor

polypep-tideshavebeenisolated, evidencefor a 300,000-dalton precursor polypeptide is lacking.

The workreported herewasundertaken(i)to

determine whether a precursor polypeptide of feline leukemia virus (FeLV) p30 existed and (ii) tomonitortheincorporationof intracellular p30 intoextracellular virus. Data are presented thatdemonstratea60,000-daltonprecursor pol-ypeptide (Pp6O) of FeLV p30 and suggest that mostof thenewly synthesized intracellularp30 isincorporatedintoextracellularFeLV.

(Most ofthis work was submitted by G. F. Okasinski inpartialfulfillment of the require-ments for the Ph.D. degree, Michigan State Univ., EastLansing, 1976. Thiswaspresented in part at the 75th Annual Meeting of the American SocietyforMicrobiology, 27April-2 May 1975, New York, N.Y., and at the Cold

Spring Harbor meeting on RNA Tumor Vi-ruses, 28 May-1 June 1975, Cold Spring

Har-bor, N.Y.)

MATERIALS AND METHODS

Source of cells and virus. The permanently

in-fectedfeline thymustumorcellsuspension (F-422)

was used throughout these experiments. Thiscell

lineproducesthe Rickard strain of FeLV and was

propagatedaspreviously described(7).

Radioactive labelingofcellsand FeLV.Labeled

intracellular proteinand extracellular FeLV were

obtained from cells incubated for3 or 20h,

respec-tively, with either [35S]methionine, 3H-amino acid

mixture,or 14C-amino acidmixture (NewEngland

NuclearCorp.). All labelingwasdoneat astarting

celldensityof 2 x 106 cell/mlwith 1,uCiofisotope

per 106 cells. Labeling with 3H- or 14C-amino acid mixtures wasdone in mediumcontaining10% of the normalsupplementof aminoacids. [35S]methionine

labelingwasdone in growth mediumcontaining5%

of the normalsupplementof methionine.

Pulse-chase labeling was done with cells

previ-ously incubated in methionine-deficient or amino

acid-deficientgrowth media for 45 min todepletethe

aminoacidpools.The cells were then labeled for 2.5 min with[35S]methionineor

"4C-amino

acidmixture

(1,Ci/100 cells)at acelldensity of50x 106cells/ml.

The pulse was terminated by placing the labeled

cellsonfrozen mediacontaining 10 times thenormal

concentrationofmethionineor aminoacids (chase medium), adding 20 volumes of cold chase medium,

andcollectingthecellsbycentrifugation.

Cells to bechasedwerethen incubated in warm

chasemedium forvarioustimes at acelldensityof

106cells/ml.

Purification ofvirus. Cells were removed from

the growth mediumby centrifugation at 1,000 rpm

for5min inan International PR-6 centrifuge. The

growth mediumwasfurther clarifiedby

centrifuga-tionat10,000rpmfor 10 min in a SorvallGSArotor.

Clarified mediumwasthenoverlaid onto a

discon-tinuousgradient consisting of 5 ml of 40%sucrose (wt/wt) inTNEbuffer(0.01 MTris-0.1 MNaCl-0.001

MEDTA,pH 7.5)and 5 ml of 20% sucrose (wt/wt) in

TNE. The virus was banded on the 40% sucrose

layerbycentrifugation at 25,000 rpm for 1.5 h in an

SW27 rotor (Beckman). The bandedvirus was

col-lected, dilutedwith anequal volume of TNEbuffer,

andpelleted by centrifugationat25,000 rpm for 1.5h

inanSW27rotor.The viral pellet wasresuspended insamplebufferorlysisbuffer(see below and Fig. 1)

for SDS-PAGE and detergent disruption,

respec-tively.

Preparationof subcellular fractions. Cells were

collected by centrifugation, washed inHanks

bal-anced salt solution, resuspended in lysis buffer (0.5%NP-40-0.15MNaCI-0.01MTris,pH7.4), vor-texed for 20 s, andthenincubatedfor5min at4°C.

Thedisrupted cells were thencentrifuged at 2,400

rpmfor 5 min in anInternationalPR-6centrifuge.

The supernatant was removed and centrifuged at

100,000 xgfor 1 h in anSW50.1 rotor(Beckman).

The 100,000 x gsupernatant (cytoplasmicextract)

was carefully removed and the pellet was

resus-pendedinlysis buffer containing0.2%deoxycholate.

The cytoplasmic extract(CE) was also made 0.2%

deoxycholate in oneexperiment (seeFig. 6). Both

theresuspended pellet andCEwere rapidly

freeze-thawed eight times. The solubilizedpellet andthe

CE werethencentrifugedat 100,000 x gfor1h in an SW50.1 rotor. The supernatantfrom the solubilized

pellet wastermedthe particulate fraction (PF) or

NP-40-insoluble fraction, whereasthe CE was also

termed theNP-40-soluble fraction.

Preparation ofantisera. Antiserum to p30 was

prepared as previouslydescribed (17). Antisera to

bovineserum albumin(BSA)wasobtainedfromE.

Sanders(Michigan State Univ., East Lansing). Immunodiffusion analysis. Double diffusion was

performed, using 2%Noble agar (Difco), as

previ-ouslydescribed (17).

Immune precipitation. Antiserum used for

im-mune precipitation was clarified by centrifugation

at100,000 x g for 0.5 h in an SW50.1 rotor.Clarified antiserumwas added tosubcellular fractionsor

dis-rupted virusandincubatedfor 30minat37°C and

then overnight at4°C. Immune precipitates were

collectedbylayeringtheincubation mixtureover 1 ml of 5%sucrose (wt/wt)in lysisbuffer, followed by

centrifugationat2,000rpm for 20min.The immune

precipitates were resuspended in 0.5 ml of lysis

buffer, layeredover 5% sucrose, and centrifuged.

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98 OKASINSKI AND VELICER

This was repeated one additional time. The final precipitate was solubilized for SDS-PAGE as

de-scribed below, or trichloroacetic acid-precipitable

radioactivity was assayed aspreviously described

(17).

SDS-PAGE.Electrophoresisinthepresence of 1% SDS wasdone using a 9%polyacrylamide gelsimilar

tothatdescribedbyFairbanksetal. (11). Samples

were solubilized in sample buffer (0.01 M Tris-hy-drochloride-5 mM EDTA-1% SDS-2% mercaptoeth-anol) and heated for 3 min at 100°C. Electrophoresis was performed at 70 V for 3 h. The gels were

frac-tionated and assayedforradioactivityaspreviously

described, (17), using 3a70B scintillation cocktail

(Research Products International Corp., Elk Grove Village, Ill.).

Tryptic peptide analysis. Immune precipitates fromcells pulse labeled with 3H-amino acidswere

electrophoresedinthe presence of 1% SDS.Thegels

were fractionated into 2-mm slices as described

above,andthepolypeptides, were eluted with 0.4 ml of 0.1% SDS at37°Cfor 24 h.Small portions of each fraction wereassayedforradioactivity to locate

la-beled polypeptides.3H-amino acid-labeledFeLV was

preparedandelectrophoresed,andp30waselutedin

asimilarmanner.

The eluted polypeptides plus 1 mg of BSA as carrier were precipitated with 15% trichloroacetic

acid and1 volumeofethanol. Theprecipitated

pro-teinwascentrifugedandthe pellet waswashedfour timeswithethanol andoncewithether. The final

pelletwasdriedunder a stream of nitrogen.

The precipitated protein was oxidized as

de-scribedbyHirs(19) with 1 ml ofperformicacid (4.5 ml of formicacidplus 0.5 mlof 30%hydrogen perox-ide kept at250Cfor 1.5h) for 1 h at40C.Atotal of 15 ml ofdistilledwater wasadded, followedby lyophili-zation.Thelyophilized proteinswereresuspendedin

15mlofdistilledwaterandlyophilizedagain.

Theoxidizedproteins wereresuspendedin 3ml of

0.15MNH4HCO3containing 300 ,ug oftolylsulfonyl

phenylalanyl chloromethyl ketone (TPCK)-treated

trypsin (WorthingtonBiochemicalCorp.)and 10,ul

oftoluene and thenincubatedfor 4 hat 370C. An

additional 300 ,ug of TPCK-treated trypsin was

added anddigestionwascontinuedfor 15 hat370C.

The digested polypeptides were lyophilized,

resus-pendedin 3 ml ofdistilled water, and lyophilized

again. Thedigestedpeptideswerestoredat -76°C.

Cation-exchange chromatography ofthe tryptic peptides was done by a modification of the technique ofSchroeder (28), using a high-pressure column of type P chromobeads (Technicon) maintained at 52.5°C. The tryptic peptides were suspendedin 1.5 mlofpH 3.1 buffer (16 ml ofpyridine and 278 ml of acetic acidperliter) and thencentrifuged at1,000 rpm for 5 min to remove insoluble cores.The pep-tides were loaded onto the column under pressure developed from a 30-mldisposablesyringeand tight-fitting tygon tubing. Thepeptideswereeluted with alineargradientof 300ml ofpH3.1bufferand300 mlof pH 5.0 buffer (161 ml ofpyridineand143ml of aceticacid perliter)ataflowrateof30ml/h. Frac-tions(3ml)werecollected,evaporatedat600C, and

radioactivelyassayedwith10ml of3a70B

scintilla-tioncocktail.

RESULTS

Immune precipitation of FeLV p30 from disrupted virus. [35S]methionine-labeled FeLV wasprepared and electrophoresed inthe pres-ence of1% SDS. The polypeptide profile (Fig.

10

A 35S labeled FeLV

p30 p15

8-

p70

6-

4-0

B Immune precipitated

p30

E 8N F p35

0.

~6-

4-0 20 40 60 80

FRACTION NUMBER

FIG. 1. SDS-PAGE ofimmune precipitated p30 fr-omNP-40-disrupted FeLV. -"S-labeled FeLVwas preparedfrom100x1O6cellsincubated with100uCi

of[3S]methioninefor 24 h in 50 ml of growth

me-dium. The virus was purified as described in Materi-alsand Methods.(A)FeLV(20,000 cpm) was resus-pended in sample buffer and electrophoresed; (B) FeLV(30,000 cpm) wasresuspended inlysisbuffer, incubatedfor0.5h at370C,and thenrapidly freeze-thawed 15 times. The disrupted virus was incubated with 200plofanti-p30,and an immune precipitate was collected, resuspended in sample buffer, and electrophoresed,asdescribedinMaterials and Meth-ods.

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1A) obtained was similartothat seenwith 3H-aminoacid-labeled FeLV (17; see Fig. 4). There was,however,little methionine label in the plO and pll position of the profile. A shoulderon thehigh-molecular-weightside of thep15peak wasroutinely seen and may correspondtothe previouslyreportedp21 of FeLV (17). The ma-jorityof labelwas distributed amongp15, p30, and p70.

To demonstrate the specificity of anti-p30,

[35S]methionine-labeled FeLV was disrupted with 0.5% NP-40 and 15 rapidfreeze-thaw

cy-cles and then immune precipitated. This dis-ruption procedure solubilizes all of the major

structural proteins, except p70 (Okasinskiand Velicer, manuscript in preparation). SDS-PAGE of the immune precipitate (Fig.1B)

dem-onstratedasingle polypeptide,whichmigrated atthe position of FeLVp30. Immune precipita-tion of '4C-amino acid-labeled FeLV yielded similar results(datanotshown). The data indi-cated that antiserum to p30 was monospecific

with respect to FeLVstructural proteins. Con-trol experiments using 5 ,ug of BSA and200 ,IA

of anti-BSA showed virtually no precipitation of labeled viral proteins, indicating little or no nonspecific trapping.

Immunodiffusion ofintracellular proteins. ACEanda PF wereprepared and examinedfor the presence ofp30 by immunodiffusion with

anti-p30.Boththe CE and thePFwerepositive forp30, asjudged bythe presence ofaline of identity with disrupted FeLV (Fig. 2A). This antiserum had previously been shown to be monospecific with respect to FeLV proteins in both immune precipitation (Fig. 1B)and

immu-nodiffusion(17). Antiserum to BSA wasusedin similarimmunodiffusion experiments, withno precipitin linesevident (datanotshown).

Estimation of the level ofintracellular p30 synthesis. To estimate the percentage of host

cellproteinsynthesisdirected toward synthesis

ofp30, aPF and aCE from long-term labeled cells (200 x 106) wereeach divided intoequal

portions (counts per minute per portion) and

incubated with increasing amounts of anti-p30.

Theimmuneprecipitable counts per minute in each portion of the CEandPF areexpressedas the percentage of total counts per minute (counts per minute in the CE portion and

countsperminute inthe PF portion). The CE

contained90%ofthe total counts perminute in this experiment (data notshown). Maximal im-muneprecipitation occurred with 50 ,ul of

anti-serum(Fig. 3), which wasequivalent to 400 ,ul

of anti-p30 to maximally immune precipitate intracellularp30from the CE or PF of 100x 106 cells.ThedatainFig. 3indicated that approxi-mately 0.8% ofthe total hostcell protein

syn-FIG. 2.Immunodiffusion oftheCE,PF,and

NP-40-disruptedFeLV with anti-p30. Wells A, B, and C

contained CE,NP-40-disrupted FeLV, and PF,

re-spectively. Well D contained anti-p30. The CE and

PF wereprepared from 100 x 106 cells disrupted with 0.6ml oflysisbuffer, asdescribedinMaterialsand

Methods. NP40-disruptedFeLV waspreparedfrom

unlabeled virus as described in the legend to Fig. 1.

.@ 0.5

0

-O.

' 0.4 . 0

a 0.3

E 0.2 0.

Ile0.1 I.

F~

F

Cytoplasmic Extract

YO,~~~

- --

-%-, 0

//

Particulate

Fraction

1,

50 100 150

anti-p30 (1l)

200

FIG. 3. Maximal immune precipitation of intra-cellularp30.Atotalof 250 x 106 cells were labeled for3h with 250,uCiof 3H-amino acid mixture in 125 ml of growth medium, and a CE and a PF were prepared as described in Materials and Methods. Each subcellular fraction was divided into six ali-quots (counts per minute per aliquot), and 5 pgof

unlabeled NP-40-disrupted virus (prepared as

de-scribed in the legend to Fig. 1) was added. The aliquots were incubated with either 5, 10, 25, 50, 100, or 200

pl

ofanti-p30,and immune precipitates werecollected as described in Materials and Meth-ods. The immune precipitates were resuspended in 1% SDS and the radioactivity was assayed. The countsper minuteof immune precipitates from each

aliquotareexpressedas the percentage oftotal counts

per minute (counts per minute of aCE aliquot plus countsper minuteof a PF aliquot).

thesis wasdirected toward production of FeLV

p30.Thedatafurther suggested that intracellu-larp30was equally distributed between the CE (anNP4O-solubleform) and the PF (an NP-40-insoluble form) and indicated a 10-fold enrich-ment ofintracellularp30 in the PF relative to thetotal counts per minute present in this

frac-tion. Nonspecific precipitation was determined

from a parallel experiment employing 5 ,ug of BSA per portion and increasing anti-BSA.

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talnonspecific precipitation was less than 5% of the anti-p30 immune precipitable counts per minute. The total counts per minute (counts per minute in CEplus counts per minuteinPF) in this experiment represented greater than 95% ofthe total trichloroaceticacid-precipitable counts per minute incorporated during a 3-h labelingperiod (data not shown) and indicated that the cell fractionation procedure allowed

examination of greater than 95% of the total proteincontent of these cells.

SDS-PAGE of intracellular p30 immune precipitates from long-term labeled cells. SDS-PAGE of immune precipitates from the CE andPFoflong-termlabeledcellsroutinely

yielded alabeled polypeptide that comigrated

with FeLVp30(Fig. 4AandC). Inaddition,two verysmallpeaks (a andb)areconsistentlyseen inbothexperimental andcontrol (Fig. 4Band D) profiles. Two polypeptides that migrate slower than polypeptide a were consistently

seen inp30 immuneprecipitates from the CE.

The nature of these polypeptides isunknown,

buttheymay represent hostpolypeptides

non-3.0

[

.5

[

A

Anti-p

30 p30 Cytoplasmic

I

Extroct i p15

I. and

p-A

p1

Is 4: *.

b

covalently linked to intracellular viral p30. Noncovalent association of host polypeptides with intracellular oncornavirus polypeptides has been reported in Roussarcoma virus trans-formed hamster cells (15).

Immune precipitation and SDS-PAGE of intracellular p30 from pulse-chase-labeled cells. Pulse-chase labeling inconcert with SDS-PAGE of immune precipitates was done to de-termine whether a high-molecular-weight pre-cursor ofp30exists and tomonitor the release of intracellular labeled p30 into extracellular vi-rus. Cytoplasmic extracts andparticulate frac-tions were prepared from pulse-labeled and

pulse-chase-labeled cells followed by immune precipitation withanti-p30. Phenyl methyl sul-fonyl fluoride (Sigma Chemical Co.) was added tothe lysis buffer to prevent proteolytic cleav-ageduring preparation of subcellular fractions. When cells were pulse labeled for 2.5 min, the immune precipitate from the PF (Fig. 5A) contained a single polypeptide with a molecular weight of 60,000 (Pp6O), whereas the immune precipitate from the CE (Fig. 6A) contained a

C

Anti-p

30

Particulote Froction

p30

I

p70

1

..0

I a

l*,

MOs"

A

pl5

plO

and

I

1 1:

4

2

~~~~~MM a

-3

6

2 p30 D

Control

P30

B

Control

,

op15

.

p70:: p11 p70 ~~~~~0 and

I 11

p11~~~~~~70I

em

0 r

0 20 40 60

I

CM

I

x

4-c

0

N.

E

0

to

FRACTION NUMBER

FIG. 4. SDS-PAGEofimmuneprecipitates fromthe CEand the PFof long-term-labeledcells.A CE and PFwereprepared from100 x 106cellslabeledfor3 h with100 pCiof[35S]methionine in50 mlof growth

medium, asdescribed in Materials and Methods. The CE and the PF were each divided into two equal portions (countsperminuteperportion)andincubatedwith either 200plofanti-p30orwith5pgofBSA and 200 of anti-BSA. The immuneprecipitates were collected and coelectrophoresed with 3H-amino acid-labeledFeLVasdescribedin Materials and Methods.(A)CEandanti-p30;(B)PF andanti-p30;(C)CE with

5pgofBSA andanti-BSA;(D)PF with 5pgofBSA andanti-BSA.

C-I

0

x

4-c 0

L..

N%..

E

U) In) I')

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FeLV p30 PRECURSORPOLYPEPTIDE 101

20 4C)

6v a a

FRACTION NUMBER

bU 0vu

FIG. 5. SDS-PAGEof immune precipitates fromPFsofpulse-chase-labeledcells.Atotalof700 x106cells

werepulselabeled for 2.5 min with1 mCiof[35S]methionineand then divided intosevenaliquots,oneof

whichwaslysedimmediately, whereas sixwerechasedfor periodsof0.5,1, 2, 3, 6,and24h(thecellsin each

chasealiquotwerecounted andadjustedtocontain 100 x100cells).A PFfractionwasprepared fromboth

pulse-labeled and pulse-chase-labeled cells, using lysis buffer containing300pgofphenyl methyl sulfonyl fluorideperml,asdescribed inMaterials and Methods.NP-40-disruptedFeLV(5pg)wasaddedtoeach PF

followed by 500p1ofanti-p30. Immune precipitateswerecollectedandcoelectrophoresedwith 3H-amino acid-labeled FeLVasdescribed in Materials and Methods. Arrows indicate positionsof nonglycosylated3H-amino

acid-labeled FeLVpolypeptides. (A) PF from cells pulse labeled for2.5min; (BtoF) PFs fromcells chasedfor 0.5to6 h.

singlepolypeptidethatcomigratedwith FeLV

p30. Control experiments employingBSA and

anti-BSAyieldedapolypeptideprofile (datanot

shown) identical to that seenwith

long-term-labeled subcellular fractions (Fig. 4B and D).

SDS-PAGE of immuneprecipitates from cells

chased for 0.5 hyielded quite different profiles.

The PFcontainedonlylow levels ofa

polypep-tide thatcomigratedwithFeLVp30 (Fig. 5B),

whereastheCE containeda30,000-dalton

poly-peptideinquantities much greater thanseenat

the end ofthepulse (Fig. 6B).

The PF and CE from cells chased for 1 h

contained immune precipitable p30 (Fig. 5C

and 6C).The lossof labeled p30seenintheCE

duringthe 0.5- to 1-h chase interval could be

accountedforbytheappearanceof labeledp30

present at 1 h in the PF (Fig. 5C) andbythe

labeled p30recoveredasextracellular FeLV at

1 h (Fig. 7).

Immune precipitatesofcellschased for 1, 2,

and 3 hcontainedp30inboththe PF(Fig.5C to

E) and the CE (Fig. 6C to E). The level of

immune precipitable p30 inthe PF decreased

duringthis 2-h chaseperiod, whereas the level

ofp30 in the CE remained relativelyconstant

(several experiments indicateda slight loss of immuneprecipitablep30inthe CE).The level

ofimmune precipitablep30 inthe PF andthe

CE at the 6-h interval (Fig. 5F and 6F) was

relativelyequal andbothwerereducedas

com-pared with the 3-h chase interval. No labeled

I

N C

6

0

E

Co

In

;R'

A 2.5 min D 2hchase

pulse Pp6o p70 p30 p1SplO

a-;"

a1I

Pl

St

!n

I

ndd

4-

p70

p30

p5

plIO

Krp

2 lilT"~~~~~~~i

B Q5h chase E 3 h chase

p70 p30 p15 plO p70 p30 p15plO

o

I

I I

and

C Ihchase p30 F 6h chase

p70 p15 plO p70 p30 p15 plO

4p

I°P

IF

1

and

o-

A

_

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A 2.5min pulse D 2h chase

p70 p30 p15 plO p70 p30 p15 plO

4 aond I and

l

I

II

B

C I h chase E 36h chase

p70 p3OpISpI p70 p30 p15plO

4 and ond

CL

0

N

C Ihcoef 6h chase

p70 p30 p15 plO p70 p30 p15plO

1 'If~~an

IaInI

4

plAi

20 40 60 80 20

FRACTION NUMBER

40 60 80

FIG. 6. SDS-PAGE of immune precipitatesfrom CEs ofpulse-chase-labeledcells. CEswereprepared from

thesame700 x106cells labeledasdescribed in the legendtoFig. 5, using lysis buffer containing300 Pgof phenyl methyl sulfonyl fluorideperml,asdescribed in Materialsand Methods.Deoxycholatewasaddedtoa

final concentration of 02%.NP40-disrupted FeLV (5pg)wasaddedtoeach CEfollowed by 500pi of anti-p30.Immune precipitates were collected andcoelectrophoresed with 3H-amino acid-labeledFeLV, as

de-scribed inMaterials and Methods. Arrows indicate positionsof nonglycosylated3H-amino acid-labeled FeLV polypeptides. (A) CE from cells pulse labeled for2.5min; (BtoF)CEsfromcells chasedfor0.5to0.6 h.

p30could be recovered at the 24-h chase time

(data not shown). Deoxycholate was added to

the cytoplasmic extracts (NP-40-soluble

frac-tion)inthisexperiment(Fig. 6)todemonstrate

thatPp6Ois foundonlyinthePFunder

identi-cal detergent conditions. Identical results to

thosepresented in Fig. 6wereobtained in the

absence ofdeoxycholate (datanotshown).

To determine whether labeled extracellular

FeLV p30 was contaminating the subcellular

fractions, a mixing experiment was done

em-ploying purified labeled FeLV and unlabeled

cells. When 50,000 cpm of purified

[35S]-methionine-labeled FeLV was incubated with

100 x 106 cells prior to fractionation, no 35S

label could be recovered in the subcellular

frac-tions (data not shown). The absence of

[35S]methionine-labeled intracellularp30inthe

subcellar fractions after a 24-h chase also

ar-gued against extracellular contaminationof the

CEand PF. The influence of labeledp30

associ-ated withnewlyassembled FeLVintheprocess

ofbuddingfrom the surfaceof these cells could

notbe determined.

Analysis of intracellular andextracellular

p30 levelsduring pulse-chase labelingof cells.

The levels of intracellular andextracellularp30

weremonitored duringa0.5- to 24-h chase

pe-riod aftera2.5-minpulse(Fig. 7)tofollow the

incorporationof intracellularp30into

extracel-lular FeLV. Extracellular p30 levelswere

de-termined by collecting FeLV from the chase

media in the previousexperiment (Fig. 5 and

6). Unlabeled carriervirus (100 ug)wasadded

toeach chaseportion (0.5,1, 2, 3, 6, and 24h),

and the viruswaspurifiedandelectrophoresed

in the presence of SDS. The amount of

[35S]-methionine-labeled p30 was determined from

J. VIROL.

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[image:7.508.96.404.70.394.2]

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E

C)

0

c0 to

-5

0

2 4

[image:8.508.55.245.53.196.2]

HOURS FIG. 7. Analysis of intracellular and p30 levels duringa0.5-to24-hchase 2.5-min pulse. The levels ofp30areexi

percentageof total intracellularorextri

Totalintracellular p30 is the level of both the CE and PF of cellschased for mined from Fig. 5 and 6). Totalextrac

the entireamountofvirion-associated leased duringa 0.5- to24-h chase afi

pulse (determined from SDS-PAGE beledFeLV released duringthisexper

bols: 0,percentageof totallabeledimi

table intracellularp30foundinbothti CE;0,percentageoftotal labeledextrac

p30.

the resultingelectropherograms (

gramsnotshown). The 24-h chase

vided the total extracellular FeLl whereasallremainingvalueswere

as apercentageofthetotalextrac

p30 (Fig. 7). All values for extrace

were corrected for virus released

first 0.5h ofeach chase period by

theamountoflabeledp30appearin

the 0.5-h time point. This analysi

examined theappearanceoflabele

lar FeLVp30, using the 0.5-h cha starting point and the 24-h chase total.

Intracellularp30levels during th chase period were obtained froi

shown inFig. 5B to F and 6B to

chasetimeprovided the total (1009

larlevel ofp30 inthis analysis. Th

minute comigrating with viral p3

theCEandPFwereusedtodeterm of intracellular p30 at each chas levelof p30ateach chase time isre

thepercentageof totalintracellull An analysis of the data present indicated thatat atime (approxin

when 50% of the total extracellul

appeared,approximately50%of th

cellular p30was nolonger detectab

suggested that the intracellularp34

these experiments was indeed a precursor of o / extracellular FeLV.

o.I Tryptic peptide analysis. High-pressure,

cation-exchange chromatography of tryptic

peptides was employed to determine whether

labeledpeptidesof FeLV p30 were also found in Pp60. 3H-amino acid-labeled Pp6O was used for thisanalysis to insure labeling of all peptides. The tryptic peptide map (Fig. 8) contained 25 3H-labeled peptides present in Pp60, whereas 6

224

3H-labeled

p30

contained15

peptides.

Of the15

peptidespresentin3H-labeled p30, 13 are also extracellular present in

Pp60.

These chromatograms of la-periodaftera beled

tryptic

peptides

indicated

that

p30

is pressed as the found within the

Pp6O

molecule.

azcellularp30. DSUSO

p30found in DISCUSSION

r0.5 h (deter- Evidence from tryptic peptide analysis and

ellular

p30

Is pulse-chase labeling demonstratesthat Pp60 is FeLV

p3O

re- a rapidly cleaved precursor ofFeLV

p30.

An

tera 2.5-mmn examination ofintracellular and extracellular

iment)

Sym-

p30 levels during pulse-chase labeling

experi-mune

precipi- ments suggests that newly synthesized p30 is

wePFandthe subsequentlydistributed intoanNP-40-soluble cellularFeLV and -insoluble form and indicates that most of

the intracellularp30isassembled into extracel-lular virus.

Results of SDS-PAGE of immune precipitates

electrophero- from FeLV-infected cells demonstrated the !portionpro- presenceofintracellular p30 in both an NP-40-V p30 value, soluble (CE)and -insoluble (PF) form (Fig. 4).

represented Solubilization of p30 from the PFbytheuseof

ellularFeLV 0.2%deoxycholatesuggestsamembrane

associ-allularFeLV ation;however, furtherexperimentationwillbe I during the requiredtoconfirm this point. Asimilar distri-subtracting bution of intracellular oncornavirus proteins Lg in FeLV at between amembrane fraction anda

cytoplas-is, therefore, mic extracthas been reportedfor murine

sar-d extracellu- coma-leukemia virus-infected rat cells (29).

Lsetime asa Pp60appearstobelimitedtothe PFandisonly

time as the observable upon double-detergent treatment (0.5% NP-40 and 0.2% deoxycholate). This

in-ie0.5- to24-h ferred membrane associationof an

oncornavi-m the data rusprecursorpolypeptidehas beenobserved by

F. The 0.5-h other workers. AMV-infected primary chick

fi-9o)

intracellu- broblasts synthesize a 76,000-dalton precursor

le countsper polypeptide, whose cleavage in vitro can be

30 from both inhibited by membrane-dissociating agents

mine

the level (34). Van Zaane etal. (31) have alsodescribed

e time. The two membrane-associated precursor polypep-,presented as tidesinJLS-V9cells infected with RLV.

ir

p30. The results of pulse-chase labeling experi-ted in Fig. 7 ments (Fig. 5 and 6) indicate that Pp60 is rap-nately2.0h) idly cleaved to form a large pool of NP-40-solu-lar viral p30 blep30 (CE), which subsequently becomes dis-etotalintra- tributed between the CEand thePF.The pres-)le. Thedata ence ofPp60 in the PF andp30inthe CE at the 0obtainedin end ofa 2.5-minpulse could also suggest that

%of Total Intracellulor p30

Remaining---VOL. 20, 1976

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c~'lV~

VVVXI

L J L

4

E viralp30

to

'Lli~~~~~~~'

2

0 P

2

5 ItiJ5

In'0

20 40 60 80 100 120 140 160 180

[image:9.508.67.461.60.359.2]

FRACTION NUMBER

FIG. 8. Tryptic peptide analysis ofPp60and FeLVp30 eluted from SDS-polyacrylamide gels. Labeled Pp6Owasimmunoprecipitatedfromthe PF of 500 x 106 cells pulselabeled for 2.5minwith 500 XACiof 3H-labeled amino acid mixture. The immune precipitate was electrophoresed in parallel with 3H-amino

acid-labeled FeLV,asdescribedinMaterials and Methods. The labeled polypeptides were elutedfromthe gelsand

trypticpeptideswereprepared as described in Materials and Methods. The recovery for the entire procedure was 70 to 80%.Variouspeptide peaksareidentifiedby pH ofelutiondeterminedfromreading pH values with aPHM 26 expanded-scalepH meter(Radiometer, Copenhagen,Denmark). O, Elution pH values ofp30 trypticpeptides absentinPp6O.

p30 issynthesizedbothaspartofalarge

precur-sorandas amaturevirionpolypeptide.

Prelim-inary pulse-chase labeling experiments

em-ploying a general protease inhibitor (manu-scriptinpreparation)suggestthat inhibition of

Pp60 cleavage isassociated witha decrease of

labeledimmuneprecipitable p30attheendofa 2.5-minpulse, which wouldargue against syn-thesisofp30as a mature virionpolypeptide.

Therelativelyrapid loss of intracellular p30

from the PFduring the 1- to 3-hchase period,

combined with aslow loss ofp30from the CE during this interval, suggeststhat

extracellu-larFeLVp30may arise from an

NP-40-insolu-ble form of intracellularp30. The data (Fig. 5

and 6), however, donotruleoutthepossibility

that NP40-soluble p30 is directly assembled

intoextracellular FeLV. Thewell-documented

cell surface assembly of the oncornaviruses

seemstofavoramembraneassociationof viral

proteinsduringassembly and could be takenas

supportive ofour interpretation of these re-sults.

The existence of two intracellular pools of

p30,differinginsolubilityinNP-40and

appar-entlyinthekinetics ofincorporationinto

extra-cellular virus, raises several possibilities. For

example,theappearanceofaninitiallarge pool oflabeled p30 in the CE after pulse labeling

(Fig. 6) maysuggest furtherposttranslational

processingofintracellular p30 undetectablein

theseexperiments. The slow release ofp30from the CE into extracellular FeLV may suggest some fimctional role forp30 within these cells or mayreflect host regulation ofintracellular p30levels.

The analysisofintracellular and extracellu-larp30levelspresentedinFig. 7indicates that

most ofthe immune precipitable p30

synthe-sized andprocessed duringa2.5-minpulseand

30-min chase is subsequently assembled into

extracellularvirus. The30-minchasetimewas J. VIROL.

http://jvi.asm.org/

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chosen asthe starting point for this analysis becauseitprovided thehighestlevel of labeled

intracellular p30 among the times examined. Anearlier starting time was notpossibledue to the simultaneouscleavage of Pp6O andthe ap-pearanceofp30.Approximately5%of the total

extracellularviralp30 is releasedduringa 30-min chase of pulse-labeled cells (data not

shown),whichindicatesthat theinitial levelof intracellularp30is atleast 5%higherthanwas

determinedinthisanalysis.

Trypticpeptide analysisofPp6Oandp30 dem-onstratesthatPp6Ocontainsp30(Fig. 8).Using a similar experimental approach,

AMV-in-fected cells have been shown to contain a

76,000-daltonprecursorpolypeptide(10,33, 34).

Precursorpolypeptideshave also beenreported inRLV-infected cellsbytwodifferentgroups of

workers (23, 31). One group (23) originally

re-ported the existence of 140,000-, 65,000-, and

50,000-daltonprecursorsofRLVp30inJLS-V16

cells. More recently the same group detected

the existence of anapproximately 200,000-, as

well as80,000-and65,000-, daltonprecursorof RLVp30inthe samecellsystem,usinga differ-ent SDS-PAGE system and the analysis ofa

limited number ofmethionine-labeled

tryptic

peptides (1). A second group, using JLS-V9

cells, couldonly detect 72,000- and

65,000-dal-ton precursors of RLV p30. Although a final

judgmentmust awaittrypticpeptide

analysis

of RLV precursorsidentifiedby bothgroups, pref-erably with larger numbers of

peptides

than canbe labeled with methionine as

pointed

out

by Arcementetal. (1),their resultsmayreflect host cell influenceonprecursorprocessing.

Re-cently, Oskarsson et al. (26) have suggested

that a 60,000-dalton polypeptide at the FeLV

pseudotype

ofMaloney sarcoma virus may be anuncleaved precursor ofMaloneysarcoma vi-rus FeLV p30. Although the evidence from avian, murine, andfelineoncornavirusis con-sistent in demonstrating precursor polypep-tides within infected cells, the processing of these precursors ranges from the very rapid

rate reported here to anaberrantcleavage

re-sultingintheincorporation oflargeamountsof a possible uncleavedprecursor into assembled

virions (26).Thesedifferencesinprocessing in-dicate that it may be essential to investigate notonly both avian andmammalian

oncornavi-rusprecursor polypeptides but also the effects of varioushostcells upon precursor processing. IfoncornavirusmRNA is equivalent to the 3 x 106-dalton viral genome subunits (12, 16, 31) andtranslated from asingle initiation site,one would expect a precursor polypeptide with a molecular weight of approximately 300,000.

The failure to detect such a giant precursor

polypeptidein our experiments may be dueto several factors: (i) the p30 antigenic determi-nantof suchalarge polypeptide may be inac-cessibletoantibody;(ii) Pp6O may be acleavage productofanascentpolypeptide; (iii)

antibody

prepared against p30 purified by gel filtration in guanidine hydrochloride may not contain

antibody binding sites to all native p30

anti-genic determinants, thus lowering the effi-ciencyofimmuneprecipitation; (iv)the

hypoth-esized 300,000-dalton precursor may notexist.

ACKNOWLEDGMENTS

We thank Alice Swanson and Catherine Sekerke for

technical assistanceandDorisMunro fortyping.

Thisresearch was supportedby Public Health Service grantCA-12101from theNational Cancer Institute, grant

DRG-1230 from the Damon Runyon Memorial Fund for

Cancer Research, Inc., and a grant from the Elsa U. Pardee

Foundation.L. V. isarecipientofPublicHealth Service ResearchCareerDevelopment Award CA-70808 fromthe NationalCancer Institute.

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