Effect of interferon on the replication of mink cell focus-inducing virus in murine cells: synthesis, processing, assembly, and release of viral proteins.

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0022-538XI82/070213-10$02.00/0

Effect

of Interferon on the Replication of Mink Cell

Focus-Inducing

Virus in Murine Cells: Synthesis, Processing,

Assembly, and Release of Viral Proteins

JOHN A. BILELLO,1*NELSON A. WIVEL,2AND PAULA M. PITHA1

Biochemical Virology Laboratory, Johns Hopkins Oncology Center, Baltimore, Maryland 21205,1 and

Laboratory

of

CellBiology,National CancerInstitute,Bethesda,

Maryland

202052

Received 14 December 1981/Accepted 15 March 1982

Treatmentof mink cell focus-inducing (MCF) virus (isolateAK-13)producing SC-1 cells with mouse fibroblast interferon (150 to 600 U/ml) led to a 100-fold

decrease in the release of infectious virus, whereas there was a 2.5- to 10-fold

decrease in variousparameters of virus particle release. Analysis of labeled virion

proteins indicatedthat a temporal change in virion protein composition occurred

after interferontreatment. After a 24-h exposure of chronically infected cells to

interferon,thevirionsproduced contained a85,000-dalton glycoprotein

(apparent-ly of nonviral origin) whichwas in excess of the virusenvelope glycoproteingp7O. Particles produced fromcells treated with interferon for 32 to 48 h were nearly

devoidofgp7Oand contained substantially lower quantities ofp30. Intracellular

processing of viralprecursor polyproteins to the mature virion structural proteins was not altered inthe presence of interferon. However, an accumulation of the

viral p30and pl2Eproteins was observed ininterferon-treated cells, consistent

withan increase incell-associated virions. Immunoprecipitation analysis of the

tissue culturefluids from

[35S]methionine-labeled

control and interferon-treated cells revealedamarked decrease inp30andpl5E/pl2E releasedafter interferon treatment. In contrast, gp7O did not accumulate in interferon-treated cells, but wasreleased into the culture medium in a form that was neither pelletable nor

associatedwithpl5E/pl2E.

Interferons are afamily of cellular

glycopro-teins synthesized in response to viral infection

or nonviral inducers such as double-stranded

polyribonucleotides and certain halogenated

pyrimidols

(for review,seereference 16).

Expo-sureofcellstointerferonresults in the

establish-mentofan"antiviral state"asmanifestedbythe

inhibitionof thereplication ofawide variety of DNA and RNA viruses. Although the

mecha-nism bywhich interferon inhibits viral

replica-tion isnotfullydefined, themajority ofviruses

are inhibited at the level ofmRNA translation

(16, 21, 22, 28). However, our own laboratory

and others have demonstrated that interferon

inhibits thereplication ofmurineretrovirusesin bothchronicallyandacutelyinfected cells, and this inhibitionoccursafter the

synthesis

ofviral RNAand proteins (4, 15, 32-35). Anumber of studies have suggestedthatinterferon interferes with retrovirus assembly and maturation, lead-ing to the "trapping" of the virions atthe cell surface(5,10, 34,36). Insystems wherevirions

were released frominterferon-treated cells, the

released particles had reduced

infectivity.

Ex-amination of the structural

proteins

ofvirions

released from interferon-treated cells had

indi-catedthatnewproteins, presumably incorrectly processed viral precursor polyproteins, were

incorporatedinto virions(9, 33).

Our previous results had indicated an

alter-ationin assemblyastheprimarydefect in

retro-virus production after interferon treatment of both chronicallyand denovo-infectedcells (32,

33, 36). Retrovirus assembly is a complicated

processrequiring the propertemporal and

spa-tialcleavage of threemajorprecursorproteins:

the gag precursor

(Pr67sar)

is cleaved to the virion core proteins (plO, p12, p15, and p30), gag-pol

(Pr1809a59Pol)

is cleaved to the virion

RNA-dependentDNApolymerase(and

presum-ablygag geneproducts),and the envprecursor

(gPr8Senv)

givesriseto the virionenvelope

gly-coprotein gp7O and the virion matrix

protein

pl5E/pl2E (for review, seereference

40).

Viral

polyproteins function directly in the

assembly

process; for example, Pr675ag contains both

RNA-binding

proteins

which bind the

genomic

RNA and proteins which

specifically

interact with the virion transmembrane

protein p15E,

affecting attachment of the viral core to the membrane. Theenvprecursor

gPr85nv

mustbe further

glycosylated

and in aproper

configura-213

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214 BILELLO, WIVEL, AND PITHA

tion in the membrane topermit both the interac-tion of plSE withPr679'9 and theformation ofa

stable disulfide linkage between pl5E andgp7O before proteolytic cleavage (7, 29, 31).

Wehaveattemptedto comparethesynthesis, processing, glycosylation, and release of viral proteins in thepresenceand absence of

interfer-on in order to determine whether interferon-induced alterations in these processes are

re-sponsible for the defective assembly. Our studies have concentrated on gp7O expression, since the envelope glycoprotein determines

vi-rus infectivity, neutralization, interference,and hostrangeproperties(reviewedinreference40). Additionally, the malignancy of murine

lympho-sarcomacell lines appears tocorrelate with the cell surface display of gp7O (37). Interferon-sensitive SC-1 cells infected with the envelope

gene recombinant mink cell focus-inducing

(MCF) virus isolated from AKR mice(19) were

used in this study. This virus-cell system has severaluniqueproperties relevant for studies of theeffect ofinterferononretrovirusreplication.

MCF viruses canbe titrated byfocus assayon mink cells whichareinsensitiveto mouse

inter-feron; thus, theinfectivity assayis notaffected

bythepossiblepresenceof residualmouse inter-feron in the system. Since earlier studies had indicated that interferonmayaffect viral precur-sorproteinprocessing,theuseof theSC-1/MCF

cell line with asubstantially slowerrate ofenv

geneprocessing (12) could amplifyminor inter-feron-induced differences in thispathway.

Last-ly, the in vivo administration of interferon has been showntoprolong thesurvival of AKR mice (18). Since this MCF virus is highly leukemo-genic and implicated in the appearanceof

s'pon-taneous T-cell lymphomas in AKR mice (19),

information gathered from this in vitro study

may be relevant to the previously observed antitumor effect of interferon in this mouse

strain.

MATERIALSAND METHODS

Cells and viruses. Interferon-sensitive SC-1 cells chronically infected with the AK-13 isolate of AKR-MCF virus (19) were used. Virus was titered by enumerating cytopathic foci on mink lung cells (19).

Interferon.Experiments were performed with a

sin-glelot ofpurified mouse interferon induced in L cells with Newcastle disease virus (specific activity, 1.1 x 107reference units per mg of protein). The interferon was assayed in L929 cells by the cytopathic assay of Finter (13), using vesicular stomatitis virus as the

challenge virus. Interferon titers are expressed in internationalreference units; 1 U of theinternational standard titrated as 1 U in our assay. Subconfluent monolayers of chronically infected SC-1 cells were treated, 24 h after trypsinization and passage, with the indicated amount of interferon for the indicated length oftime.

Reversetranscriptase assay. The virus was pelleted

from thesupernatant fluid at100,000 x g in anSW40

rotor for 90 min, and the virion-associated reverse transcriptase was assayed as described previously

(34).

Electronmicroscopy. We prepared monolayers for transmission electron microscopy as described previ-ously (36). Thin sections were examined in a Hitachi HU-11Emicroscope withanacceleratingvoltage of 75 kV. Pellets of the fixedmonolayersweredividedinto multiple fractions, and sections from the separate fractions were used for countingtominimizesampling error and avoidrepetitive counting of a few cells.

Radiolabeling. In experiments where metabolically radiolabeledvirus or virion proteinsweretobe charac-terized, cells were labeled fora16-h interval either 8 or 32 h after interferon treatment (24- or 48-h virions, respectively). Labeling with [35S]methioninewaswith 50 plCi/ml in medium containing 5% of the normal level of methionine. Tritiated monosaccharides (AmershamCorp.,Arlington Heights, Ill.)wereused at 20,uCi/ml inamediumcontaining 10% the normal concentration of glucose.

Pulse-chaseexperimentswereperformedat24or48 hafter interferon treatment. Cellswere preincubated for1 hin mediumlacking methionine before labeling for 30 min with 125 ,uCi of [35S]methionine per ml (Amersham;specific activity, 1,100to1,300 Ci/mmol). Chase wasincomplete mediumcontaininga100-fold excessof unlabeled L-methionine. Cellswerewashed twice with phosphate-buffered saline before prepara-tion of cellextracts.

We performed lactoperoxidase radioiodination of cell surface proteins after washing the monolayers twice with buffer(phosphate-buffered saline[pH 7.4]

or 50 mM

N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid [HEPES; pH 7.4]-buffered saline as indicated). Cells were labeledat roomtemperature for 10 min in 1 mlof the indicated saline solution contain-ing 5 p.goflactoperoxidase (Calbiochem-Behring, B grade) 0.5 mCi of1251,and 10 pl1 of 0.03%H202. Ifno chasewasdesired,monolayerswerewashed andlysed

in extraction buffer. Chase was performed after the reaction was stopped byaddition of cold wash buffer

containing0.5 mMtyrosine.Cells were washed twice and transferred to fresh medium. After the indicated length of chase, the cells were washed and superna-tantsandcells were analyzed as described above.

Immunoprecipitation analysis of labeled proteins. Preparation of cell extracts and immunoprecipitation with monospecific goat antisera to purified retrovirus proteins was described previously (3). To compensate fordifferences in protein synthesis, each immunopre-cipitation was done with approximately 108 dpm of cell extract; the volume of each samplewas adjusted to 1 mlwith extraction buffer. Analysis of viral pro-teins released into the medium was done with 1.0 ml of clarifiedsupernatant(4,000x g, 5min) adjusted to 1% Triton X-100 and0.5%deoxycholate. Cell extracts and

supernatants were precipitated with normal goat se-rumbefore specific immunoprecipitationwith a previ-ously determined amount of antiserum. Staphylococ-cus aureus immunoabsorbant was used to collect antigen-antibody complexes by centrifugation. Pellets were washed twice with 20 mM Tris (pH7.4), 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40 and once with the same mixture containing 2.5 MKCl.

Particles were analyzed aftercentrifugation through

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EFFECT OF INTERFERON ON MCF VIRUS REPLICATION

a 2%o sucrose cushion. Seven milliliters of radiola-beled tissue culturesupernatantwaslayeredover5 ml

of 20% sucrosein Tris-buffered saline (TBS; 20 mM Tris,pH 7.4,and 100 mMNaCI) and centrifugedfor 90

minat35,000rpminanSW41rotor at4°C.Tubeswere

fractionated fromthetop,and theupper4mlwasused

asapost-centrifugation sample.Justbeforeremoval of thesucroselayer, the sides ofthe tubeswerewashed

threetimes with TBS.Atapointwhere 1 ml ofsucrose

remained above thepellet, the sides of the tubeswere

again washed three times with TBS. The remaining

sucrose was removed, and the pellet was gently

washed twice with TBS.Pelletsweresuspended in the extraction buffer used for immunoprecipitation and analyzed either directly on polyacrylamide gels or

afterimmunoprecipitation with specific antisera. Anal-ysis of virus prepared by bandingonsucrosegradients

gaveidentical results.

Polyacrylamide gelelectrophoresis and quantitation of labeled protein bands. Samples were analyzed on

12.5 or 15% sodium dodecyl sulfate-polyacrylamide

slabgels, using the discontinuoussystemdescribed by Laemmli (24). Fluorographywasperformed according

tothe procedure ofBonnerand Laskey (8), and the dried gelswereexposedtopreflashed Chronex4X-ray filmat-70°C.

Autoradiographs of the dried gelswerequantitated

by theuseofanintegratingspectrophotometer (Ortec model 4310) whenanappropriateexposuretimewas

available in which the film gave a linear response.

Routinely, labeled bandswereexcised fromthedried

gels, dissolved in 0.3 ml of 15% hydrogen peroxide, and counted in Aquasol II liquid scintillation fluid (NewEngland Nuclear Corp.). Thetwomethodsgave

comparable results.

RESULTS

Interferon treatmentof MCF virus-producing SC-1cellsleadstotheproduction of noninfectious

virus. SC-1 cells chronically infected with the AK-13 isolate of MCF viruswere treated with

150 to600 U ofpurified mouse fibroblast

inter-feron for 24 or 48 h. The levels of infectious

viruspresentinthemedium ofinterferon-treated

cultures were reducedby 100-fold, but reverse

transcriptase assay of the same supematants

indicated onlya10-foldreduction in virus

parti-cles. Metabolic labeling of virions with [35S]methionineindicateda2.5-to10-fold

reduc-tion in virusparticles(Table 1). Theseresultsare

consistent with earlier studies with

AKR-L1-and Rauscher virus-infected cells which had

indicated that noninfectiousvirusparticleswere

formed inthepresenceof interferon.

Analysis of the numberofcell-associated

vi-rusparticles produced by interferon-treated

SC-1/MCFcellswasperformed byelectron

micros-copy.Transmissionelectronmicroscopy(TEM) of representative sections of SC-1/MCF cells

indicatedthat the block in virus release occurred at astep afterbudding. The numberofplasma membrane-associated virus particles visible by TEMofthinsectionswasincreasedby

approxi-TABLE 1. Effect ofinterferononMCF virus release

Re-Infec- verse Plasma

Exp

Inerfrontious

vi- tran- mem-

[S35]methionine-Expt Interferon rus scrip- brane-as- labeled virions no. treatment (FFU/ tase sociated

ml) activity virions

1 None 4 x104

ioo'

1,983b

300U/ml,3 x 102 11 4,894 48 h

2 None lOOc 194,660d

600 U/ml, 23 42,310

24 h

300U/ml, 14 31,453

48 h

600U/ml, 15 29,694

48 h

a 100% = 120

pmol

of

[3H]dTTPincorporated into DNA in response to poly(rAdT)12_18 per 20)

RI

of pelleted virus obtained from 40 ml of culture fluid.

bTEM, 600 cellscounted.

c

100o

= 223,000 cpmincorporated.

dTotal hot trichloroacetic acid-precipitable cpm in

samples of[35S]methionine-labeledvirusparticles

pre-paredasdescribed in Materials and Methods.

mately 2.5-fold in interferon-treated cells when

compared with untreated SC-1/MCFcells(Table

1).We havepreviouslyshown that theestimates of virus particlesonthe cell surface quantitated by scanning electron microscopy and computer-generated image analysisarein complete agree-mentwithTEM estimates(36). Ultrastructural-ly, there was no detectable difference between virions assembled in the presence and absence of interferon.

Viral protein synthesis and processing in con-trol and interferon-treated cells. Viral protein

synthesis and processingwerestudiedby pulse-chase labeling and immunoprecipitation

analy-sis. Since other authors (27) have indicated that interferon may exert its inhibitory effect on

membranebudding viruses by virtue ofa

tunica-mycin-like activity associated with or induced

by interferon,

tunicamycin-treated

cells were

also included for comparison. Figure 1 shows the synthesis and processing of

gp7O

in SC-1

cells infected with MCF virus as well as in

infected cells treated with interferon or tunica-mycin. Unlike tunicamycin treatment, which leadstothe accumulationofa

nonglycosylated

precursor to the virion envelope

glycoprotein

(Pr69enlV),

treatment with interferon didnotlead

to aninhibition of

glycosylation

or

processing.

The majordifference observed inthe

synthesis

and processing of the

envelope

glycoprotein

precursor related to the accumulation of the

nonglycosylated virion matrix

protein

pl5E/

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L

FIG. 1. Anti-gp7. immunoprecipitates of cell

ex-tractsfrom SC-1/MCF cells treated with interferonor

tunicamycin.SC-1/MCF cellsweretreatedwith 600 U ofpurified mouseinterferon per ml for 48 h before a

30-mm pulse-labeling with [35S]methionine as de-scribed in the text. Parallel control cultures were

eitherlabeledatthesametimeortreatedfor1hbefore

labelingwith 1 pLgoftunicamycinperml. Cellextracts

werepreparedeitheratthe end of the pulseoraftera

90-min chase in medium containinga100-foldexcess ofunlabeled L-methionine. Immunoprecipitation with anti-gp7Owasasdescribed in thetext.

theimmunoprecipitation shown,where thesame number ofcounts was used for each

precipita-tion.The observed lack of effect oftunicamycin

onprocessingofPr67919andrelease ofp30into the supernatants of tunicamycin-treated SC-1

cells infected with MCF virus correlated with the resultsfound in othersystems(Bilello,

War-necke, Koch,andOstertag, submitted for publi-cation). Chase of the reverse transcriptase

pre-cursor,

PMWag-PO',

also appeared to proceed

normally in interferon-treated cells. The

-195,000-molecular-weight protein which ran above PMWag-Po' and was found in anti-gp70,

-p30,and-pl5E immunoprecipitateswas an SC-1 cytoskeletal protein which contaminated

im-munoprecipitates made from interferon-treated

cells. Both thecharacterization andsignificance

of the interferon-induced increase in the 195-kilodalton proteinareunderinvestigation.

Release of viralproteinsfromcontrol and

inter-feron-treated cells. Our data indicated that the effect ofinterferon on MCF viralprotein

synthe-sis and processing was minimal. However, an

intracellularaccumulationofp30andpl5E/pl2E

wasobserved,whereas no increase in the

intra-cellular level of gp7O occurred. Since other studies have indicated thatgp7Ocanbe released

EISERASE

NTLERFERO - - _

-TLUrICvli CN

pl2E in interferon-treated cells. When cell ex-tracts wereimmunoprecipitated with antiserum

to the virion plSE protein (Fig. 2), there was

little difference in the amount plSE and a

marked increase in pl2E in interferon-treated

cells, whereas in control cells only plSE was

detected. Previous studies (23; Bilello,

unpub-lished data) as well as those described below

indicated thatpl5E isprimarily cell associated and pl2E islocalized in virions, where it is the

major p1SE-related protein. It was recently

shownthatpl5Eis aprecursorofpl2Eand that apeptide from the carboxyterminusofpl5E is removed during itsprocessing topl2E(17).

Analysis of the virion p30-related proteins

indicated no major differences in the cleavage

patternofPr67gag (Fig. 3). Ininterferon-treated cells, a small but reproducible accumulation of

p30 (three- to fivefold increase) was measured

by quantitation of the autoradiograph ordirect

determination of label in the p30 band. The

increasein cell-associatedp30isaccentuated in

GpR85EN

GP/uO

-.- .sm fa ins

p[E,9ENf

-FIG. 2. Anti-pl5E immunoprecipitates ofcell ex-tractsfrom SC-1/MCF cells treated with interferon or

tunicamycin.Anti-pl5E immunoprecipitates were pre-pared as described in the legend to Fig. 1 and analyzed on a 15% acrylamide-0.2% bisacrylamide slab gel to enhance the separation between pl5E and pl2E.

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EFFECT OF INTERFERON ON MCF VIRUS REPLICATION 217

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em~~~~~~~~~~~~~~~~~~~~~~~..

FIG. 3. Anti-p30 immunoprecipitates of cell

ex-tractsfrom SC-1/MCFcelltreated with interferon or tunicamycin. Anti-p30 immunoprecipitates were

pre-pared from extracts of control, tunicamycin-treated, andinterferon-treatedcells as described in the legend toFig. 1.

either freeorassociated with virus particles (6;

Bilello,unpublisheddata), wedecided to

exam-inetheeffectof interferon on the release of viral proteins as well as the incorporation of these

proteins into virus particles. Control and

inter-feron-treated cells(600U/ml;48h)werelabeled for 30 minwith[35S]methionineand thenchased for2, 4, and 24 h in medium containing a 100-foldexcessofunlabeled L-methionine. Analysis

of gp70 in thetissue culture fluidatvarious time

points duringthe 24-h chase indicated that there

was nodifference in the release of labeled gp7O into the medium in the presence andabsence of interferon (Fig. 4a). Similar analysis of p30

re-leaseindicatedamarked decrease (73 to87%)in the release of p30intothe supernatant(Fig.

4b)

ofinterferon-treated cells.

Figure5showsanimmunoprecipitation

analy-sis oftissue culture fluidsfrom cells labeled with [35S]methionine before and after centrifugation

toremove virusparticles. In lanes A throughD,

equal volumes (1 ml) of labeled tissue culture fluid were immunoprecipitated with either anti-p30 (panel a) or anti-gp7O (panel b) serum. A comparison of the levels of p30 present in the supernatant from untreated MCF

virus-produc-ing SC-1 cellsbefore (lane A) and after (laneC)

centrifugation indicated that nearly all (88 to

94%) of thep30 in the supernatantwas associat-ed with particles. Tissue culture fluids from interferon-treated SC-1/MCF cells contained verylittle p30, and nearly all of it appeared tobe sedimentable. Similar analysis using anti-gp7O

serumindicated that although gp7O was present inthe supematants of both control and interfer-on-treated cells, areduction (38 to 43%) in the amount of gp7O present after pelleting of the virus was observed only in supernatants from

untreated cells (Fig. Sb).

b

a

N _Iso

gas

FIG. 4. Immunoprecipitationofsupernatant fluids from[35S]methionine-pulse-labeledcontrol and interferon-treatedcellsatvarious times aftertransfertomediumcontaininga100-foldexcessof unlabeled L-methionine. (a)

Anti-gp7O immunoprecipitates; (b)anti-p30 immunoprecipitates.

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218 BILELLO, WIVEL, AND PITHA

a~

a _fVkI _Dr,

IrMT'EFs,Jj _

-4.

FIG. 5. (a) Anti-p30 [35S]methionine-labeled cell from MCF virus-infected S fromuntreated cells;B,sup(

with 600 U of interferon p supernatant fluid as in lar

200,000 x gfor 90minover removevirusparticles; D,s

fugedasdescribedabove.(b

pitates.

Analysis of virion strui formed on particles rele

interferon-treated cells

either [35S]methionine o

rides. A study of virion interferontreatment rev( inp30releasedescribed

al in virions labeled bel interferon treatment (da

er, the amount of viric

substantially lower,and

new glycoprotein ofapj tons which was the ma

labeledwith[3H]glucosa

kilodalton glycoprotein

pitated with antiserum virusparticles, butnotN viralproteins (33; Fig.6

data), and represented presentin controlvirion

Figure 6b shows the

[35S]methionine-labeled

48 h of treatment with inhibitoryeffectonvirus

production was maxima virionsreleasedfromun

virion structural proteii

protein component, wh

werealsopresent in a ra foundinothervirusprep in the middle ofa groul proteins. The identity of

confirmed

by

immunoprecipitations

of the virion

.

....preparations

...

with antisera to the

purified

pro-teins

p30

(lane C) and

gp70

(lane E).

When

particles frominterferon-treated cells were

ana-lyzed, amarked difference in the virion compo-sitionwasfound. Verylittlep30or

lower-molec-ular-weight virion proteins were sedimentable

from the supematants from interferon-treated

cells

(Fig.

6b,lanes BandD).

Immunoprecipita-tion of MCF

particles

from untreated

virus-in-fectedSC-1 cells with

anti-gp70

sera

precipitated

gp7O as well as the virion matrix

protein

pl2E

(and asmallamount of

unprocessed

pl5E)

(Fig.

6b, lane E). In contrast,

only

asmall amountof

gp70andnopl2Ewas

immunoprecipitated

from

particle preparations from interferon-treated

MCF-infected SC-1 cells (lane F).

Analysis

of immunoprecipitates of virion phosphoproteins labeled with32P,failedto 1-free tissue culture fluids indicate any alteration of the

32P-labeled

species C-i cells. A, Supernatant

(Pr67ag p12,

and other minor species) present enatantfrom cellstreated in the

virion;

however, the approximate level of

er ml for 48 h; C, same

32P-labeled

p12

protein

was

reduced fivefold

in

a20%

sucrosencushion

to MCF virions released from interferon-treated

ame as lane B, butcentri- SC-i cells (data notshown).

)Anti-gp7Oimmunopreci- Interferon-mediated inhibition of murine

leu-kemia virus releasepersists aslongasinterferon is present in the culture medium, and virus production resumes within 16 to 24 h after

cturalproteins was per- interferon removal (34). A reversal

experiment

-ased from control and was performed as follows. Cells were treated

labeled for 16 h with with interferon for 48 h and then washed and

r tritiated monosaccha- refed with medium without interferon. At 24 h

is produced early after after removal of interferon, the cells were la-ealed that thereduction beled for 16 h with [35S]methionine, and the

above was only margin- labeled supernatants were analyzed for virion tween 8 and 24 h after proteins. Supernatant fluids from parallel cul-ta not shown). Howev- tures labeled between 32 and 48 h after

interfer-in-associated gp70 was on treatment were also immunoprecipitated to

the virions contained a indicate the extent of the block in retrovirus proximately 85,000 dal- release before reversal. Interferon treatment

re-jor virion

glycoprotein

sulted in a marked decrease in

pl5E/pl2E

re-Lmine (Fig. 6a). The 85- lease into the supernatant as well as a lack of could be immunopreci- association of gp7O with either pl2E or

pl5E

prepared against total (Fig. 7). Transfer of the interferon-treated cells with antiserato purified to medium without interferon resulted in the and Bilello,unpublished increased release ofp30 (data not shown) and

a cellular glycoprotein the association ofgp7O with

pl5E

and pl2Ein

ks as a minor species. immunoprecipitates ofsupernatants (Fig. 7) or

protein composition of virions (data not shown).

virions harvested after Glycosylation of gp7O after interferon

treat-i interferon, when the ment of AKR/MCF-infected SC-1 cells. One

pos-sinfectivityandparticle siblemechanism for inhibition of the infectivity d. Lane A shows MCF of membrane budding viruses was the proposed itreated SC-1 cells. The tunicamycin-like activity induced by interferon n, p30, was the major (26). Although the experiments described above

iereas pl5E and pl2E indicated thatgp7Owas glycosylated, processed,

ttioconsistent with that and shed,we were interested to ascertain wheth-arations. gp7Oisvisible er there were any detectable alterations in the

p ofnon-virion-specific carbohydrate moiety of

gp7O

which would lead

f thelabeled bands was to its inability to be assembled into virions.

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EFFECT OF INTERFERON ON MCF VIRUS REPLICATION 219

a

m

..::x

FIG. 6. (a)Glycoprotein composition of virus particles produced by control and interferon-treated cells.

SC-1/MCFcells werelabeled for16hwith tritiated glucosamineandpelleted througha20% sucrosecushionas

described in thetext.Thefirsttwolanes show the glycoproteincomposition of particlesproduced by control and

interferon-treated cells. The last two lanes indicate anti-gp7O immunoprecipitates from the two particle

preparations. (b) [35S]methionine-labeledparticles released from controlcells and cells treated for 48hwith600

Uof interferonperml.(A)Controlparticles; (B) particlesfrom interferon-treated cells; (C)anti-p30 precipitation

ofA; (D) anti-p30precipitation ofB; (E) anti-gp7O precipitationof A; (F) anti-gp70 precipitation ofB.

When gp70 labeled with

[3H]glucosamine,

[3H]mannose, or [3H]fucose was analyzed in

virionsor intissueculturefluids before and after

centrifugation, apatternsimilar to thatobserved for [35S]methionine-labeled gp7O was found

(datanotshown). Although we observed minor

differences in labeling with monosaccharides,

wefound noevidencefor an overt alteration in

the glycosylation of free or virus-associated

gp70. Differences in the amount of carbohy-drate-labeled MCF virusgp7O released by

con-trolandinterferon-treated SC-1/MCF cellswas

generally proportional to the extent of overall inhibition of protein synthesisseenin interferon-treated cells (30to50% of control).

Expressionofgp7Oonthesurfaceof interferon-treatedSC-1/MCF cells.Our studies of

metaboli-cally labeled viral proteins did not exclude the

possibility that interferon treatment leads to a redistribution of viral glycoproteins in the cell membranewhichmayleadtoinefficient

assem-bly. It was previously reported that unlike in other retrovirus systems, cell surface iodination ofMCF-infected SC-1 cells leads tothe

detec-tion of both labeled gp7O and gPr85env after

immunoprecipitation (12).We

performed

similar

experiments to determine whether we could

determineaninterferon-induced alterationinthe

labeling of gp7O, its precursor, or both. Our results indicated that detection of

gPr85eenV

on

the cell surface appearedto beprimarily due to

the low viability of the cells in phosphate-buff-ered saline. Labeling in HEPES-bufferedsaline,

which maintains -100% cell viability and a

minimum of cell breakage (as indicated by a

failure to label the intracellular protein actin), resulted in labeling ofgp7O exclusively. Under theseconditions, nodifferencewasobserved in the accessibility ofgp7O to lactoperoxidase io-dination between control andinterferon-treated

cells(datanotshown).

Theturnoverofgp7Oonthe surfaceof control and interferon-treated cells was examined by following the fate ofiodinated

gp7O

at various times of chase. Control and interferon-treated

cells were iodinated and chased for 0.5or 3 h.

Theamountof labeled

gp7O

associated with the cellorpresentin virionswasthendetermined

by

immunoprecipitation

as described above. No

difference was observed in the levels of

gp7O

associated with control and interferon-treated

cells. However, theamount of125I-labeled

gp7O

present in virions released at 3 h after

labeling

wassignificantly lowerininterferon-treated

cul-tures(Fig. 8), whichcorrelates withourresults

with metaboliclabeling.

DISCUSSION

The resultspresentedhereindicatethat

inter-ferontreatmentof SC-1 cells

chronically

infect-"AM

ia

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[image:7.504.122.403.83.284.2]

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.Pi P15E

a BC D

GP70

--P15E

-

P12E---"'IS'.

_4

St

..

FIG. 7. Effectofthe transfer of interferon-treated cellstonormal mediumonthe release of viralproteins

intothetissue culturemedium.Anti-pl5E immunopre-cipitates of[35S]methionine-labeledvirionproteins in the supernatants of control, interferon-treated, and interferon-treated cells shifted for 24 h to normal growth medium for24hbefore labeling.(A)

Superna-tants from control cells labeled with [35S]methionine from32to48 hasdetailed in thetext.(B)Supernatants fromSC-1/MCFcellslabeledbetween 32 and 48 h after

treatmentwith 600 U ofinterferonperml.(C)

Super-natants from a culture of untreated cells grown in

parallel to those described in A, but grown for an

additional 24h infresh normalgrowthmedium before

a16-hlabeling with[35S]methionine.(D)Supernatants from an interferon-treated culture grown for48 h in paralleltotheinterferon-treatedcells analyzed in lane B, buttransferredtofresh mediumwithoutinterferon for 24 h before a 16-h labeling in the absence of

interferon.

ed with MCF virus leads to the formation of noninfectious virions which are deficient in

gp70. No evidence was obtained for the

inhibi-tion of viral protein synthesis or processing. Similarly, wecould notdetect apreferential or

increased incorporationof uncleaved virion

pre-cursorproteins into virionsreleased from

inter-feron-treated cells. Intracellular levels of the

fully processed p30orpl5E/pl2E proteinswere

elevated ininterferon-treated cells.Precipitation

of the cell-free supernatants from interferon-treatedcells withanti-pl5Eserafailed todetect

significantlevels of eitherpl5Eorpl2E (Fig.7). Althoughtheamountofp30released from

inter-feron-treatedcellswasmarkedly reduced, near-lyall of thep30 released from control or interfer-on-treated cells was in a particulateform. The reduction in the release ofp30 and pl5E/pl2E

proteins from interferon-treated cells was

ac-companied by an increased intracellular

accu-mulationof the fully processed virion structural proteins. Incontrast,the levels ofgp7Oreleased into the medium werethe samein the presence

andabsence of interferon. In control cells,gp7O

can be either assembled into particles or

re-leased free into the cell culture fluid. However,

gp7Opresentin the medium ofinterferon-treated

cells was primarily nonpelletable, and little if

anyof thegp7Owasfound associated withvirus

particles.

The second major difference found was that

no gp7O/pl2E complex was detected in either virions or tissue culture fluids from interferon-treated cells. The gp7O/pl2E complex can be readily demonstrated by reciprocal co-immuno-precipitation of untreated SC-1/MCF cells, since disulfide bonds between gp7O and pl2E are

maintainedthroughout theimmunoprecipitation

procedure, and the individual proteinsare

sepa-rated by reduction of the complex with 13-mer-captoethanol before electrophoresis. There are two major interpretations of these data: first,

FIG. 8. Anti-gp7O immunoprecipitates of cell

sur-face-iodinatedcellextracts and particles released from control and interferon-treated cells. (1) Cell extract preparedimmediately after iodination; (2) cell extracts aftera30-minchase; (3)particles after a30-minchase; (4) cell extract after a 3-h chase; (5) particles after a 3-h chase.

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[image:8.504.73.221.71.394.2] [image:8.504.258.447.449.612.2]

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221

that gp7O is particle associated in interferon-treated cells, but because of the lack of

associa-tionwithpl5E/pl2Eisverysusceptible to shear forces generated during centrifugation; and sec-ond, that gp7O is not assembled into particles and isreleased from pl5E/pl2E after proteolytic cleavage ofgPr85e"v. Several lines of evidence favor the latter hypothesis: (i) virus particles associated with thecells have a decreased

infec-tivity (36); (ii) the amount of gp7O detected in virions isindependent of virion preparation and purification; (iii) failure to detect a gp70/pl2E

complex in the medium before centrifugation;

and (iv) the startling difference in the kineticsof release for gp7O compared with p30 and pl5E/ pl2E.

Themolecular basis for thefailure of gp7O to be incorporated into retroviruses produced by interferon-treated cells is yet unknown. Mahesh-wari et al. (26) observed that interferon treat-mentof L-cells infected with vesicular stomatitis virus resulted in the production of vesicular

stomatitis virus particles which had markedly reducedlevels of theglycoprotein (G) and mem-brane protein (M). They also reported that

[3H]glucosamine

incorporation into the gp7O of

Moloney leukemia virus particles was reduced (12). Maheshwari et al. later related these find-ings to an inhibition of the formation of

aspara-gine-linked oligosaccharide chains, an effect similar to that induced upon exposure of cells to

tunicamycin(6). We would like to suggest that

since they analyzed only virus particles, the apparentlack of[3H]glucosamine-labeled

enve-lopeglycoproteinmayhave been due to afailure to incorporate viral glycoproteins into virions. In the SC-1/MCF system, as well as in other

cellswehave studied,noinhibitionofN-linked

glycosylation in the presence of interferon has beenobserved (J. Bilello and P. M.Pitha,

manu-script in preparation). The envelope

glycopro-tein both from control and interferon-treated

cells contains mannose, fucose, and glucos-amine. Aboud et al. (1) recently reported an

early decrease in the rate of [3H]glucosamine incorporation into the envelope

glycoprotein

precursor in interferon-treated cells.

Although

wehavenotbeenabletoconfirmthese

observa-tions, there may be differences in

methodology

andthecellsystem studied. Onepossible mech-anism for the failure to incorporate

gp7O

into

virus particlescomesfromnucleotideandamino acid sequenceanalysisof

gp7O (39;

S.

Oroszlan,

personalcommunication),where itappears that the majorityof the

carbohydrate

acceptor sites

are at the

carboxy-terminal

end of

gp7O.

It is

conceivable that an altered

carbohydrate

side

chain could interfere with the formation orthe

stability of disulfide bonds between

gp7O

and

pl5E/pl2E, allowing the release of

gp7O

upon

proteolytic cleavage ofgPr85e"v. Alternatively, conformation changes at the membrane either

duetointerferon-induced alterations in gp7O or membrane stiffening (11, 30) may prevent the stable association between gp7O and pl5E/12E

orlead to anincreased release of gp7O at points distal to viralmaturation sites. Experiments are inprogress to distinguishbetween these

alterna-tives.

Apart from their effect on virus replication,

interferons affect a number ofcellular functions and processes. Although we have found no

interferon-induced alteration in the expression

ofgp7Oat the cellsurface, a number of studies have indicated thatinterferon alters the expres-sion ofhistocompatibilityand otherantigens (for review, see reference 41). We also have found noalteration in the total amount of gp7Oreleased orin thekineticsof this process, whereas others havereported changes in the release of plasmin-ogen activator (20, 38) and carcinoembryonic

antigen (2). We would like to stress that our

observations oncell surface expression and re-lease of gp7O apply only to this system. The studies ofplasminogen activator releasealluded

to above have indicated that interferon can

eitherpotentiate or inhibit the release of a single protein, depending on the cell type or system studied.

Studies of themembrane budding virus vesic-ularstomatitis virus have indicated that specific host cell surface proteins can be incorporated into virions even in the absence of the viral envelope (G) glycoprotein (25). We have noted thatafter treatment ofSC-1/MCF-infected

cells

withinterferon,there is atemporal difference in theaccumulation of a cellular85-kilodalton

gly-coprotein into virions. This furtherstrengthens

our suggestion that the restriction in virus

as-sembly is not selectively localized at the virus structuralproteins,but maybeaconsequence of

an interferon-induced change in plasma mem-brane ofthe infected cells, e.g., a decrease in

fluidity.

ACKNOWLEDGMENTS

We thank S. Oroszlanfor his generous giftofanti-pl5E

serumand David Carbone for assistance withradioiodination studies.

Thisstudywassupported byPublic HealthServicegrant 5ROI-CA19061-06(to P.M.P. from the National Institutes of Health) and by American CancerSociety grantIN-11R (to

J.A.B.). P.M.P. isaLeukemiaSocietyscholar.

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