Viral Glycoproteins Destined for Apical or Basolateral Plasma Membrane Domains Traverse the ...

16 Pages·2002·2.27 MB·English

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Viral Glycoproteins Destined for Apical or
Basolateral Plasma Membrane Domains Traverse the Same
Golgi Apparatus during Their Intracellular Transport in
Doubly Infected Madin-Darby Canine Kidney Cells
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MICHAEL J. RINDLER, IVAN EMANUILOV IVANOV, HEIDE PLESKEN, de
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ENRIQUE RODRIGUEZ-BOULAN,* and DAVID D. SABATINI fro
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DepartmentofCellBiology, NewYork UniversityMedical Center, NewYork 10016; and *Department of http
Pathology, Downstate Medical Center, Brooklyn, NewYork 11203 ://ru
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ABSTRACT Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs /jcb
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of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus rticle
[VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins -p
d
ofthe two viruses are synthesized simultaneously and assembled into virions at their charac- f/98
/4
teristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane ofthe /1
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cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic 4/1
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examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) 77
5
5
and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi 5/1
3
cisternae. This indicates that the pathways ofthe two proteins towardsthe plasma membrane 04
.p
do not diverge before passage through the Golgi apparatus and therefore that critical sorting df b
steps musttake placeduring or after passage ofthe glycoproteins throughthis organelle. After y g
u
e
its passage through the Golgi, the HA accumulated primarily atthe apical membrane, where st o
influenza virion assemblyoccurred. Asmall fraction ofHAdid, however, appear on the lateral n 1
8
surface and was incorporated into the envelope of budding VSV virions. Although predomi- M
a
nantly found on the basolateral surface, significant amounts ofG protein were observed on rch
2
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the apical plasma membrane well before disruption of the tight junctions was detectable. 23
Nevertheless, assembly ofVSVvirions was restricted to the basolateral domain and in doubly
infected cells the G protein was only infrequently incorporated into the envelope of budding
influenza virions. These observations indicate that the site ofVSV budding is not determined
exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV,
othercellular or viral components are responsible forthe selection ofthe appropriate budding
domain.
Transportingepithelialserveasselective permeabilitybarriers cells. In lightofcurrent models for organelle andmembrane
between different physiological compartments. The func- biogenesis (cf. 44), it is reasonable to propose that these
tional polarization ofcellswith suchepithelia is reflected in proteinspossessaddressingsignalsthatdetermine theirintra-
theexistenceofmorphologically distinct apical andbasolat- cellular itinerary and ultimate destination. The capacity of
eral plasma membrane domains that contain characteristic plasmamembraneproteinsofepithelialcellstoreturn totheir
enzymaticandtransportactivities(cf. 20, 25).Littleisknown correct siteoffunction in the course ofmembranerecycling
about the biogenesic mechanisms that ensure that newly (26)suggests thatsuchsignals are not transientfeaturesofthe
synthesized proteins specific for these domains achieve the polypeptides but are retainedin matureproteins.
specific distribution necessary for the function ofpolarized Virally infected cultured epithelial cells provide aconven-
THE JOURNAL OF CELL BIOLOGY - VOLUME 98 April 1984 1304-1319
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(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)
ient model system to study sorting-out processes that effect onMDBKorMDCKcells,andinfluenzaWSN(providedbyDr.A.Gregoriades
[PublicHealth Research InsitituteoftheCityofNewYork])onMDCK.VSV
the distribution ofplasma membrane proteins to apical or
(Indianaserotype,from J.Vilcek [NewYorkUniversityMedicalCenter])was
basolateral domainsinpolarizedcells(38).Whenmonolayers
plaqued and grownonVerocellsand titeredonMDCK.Themethods used
ofMadin-Darby canine kidney (MDCK)' or Madin-Darby have alreadybeendescribed(38).
bovine kidney (MDBK) cells are infected with enveloped Monolayerswereinfectedby incubationwith thevirusinasmallvolumeof
viruses, virions assemble only on specific domains ofthe MEMfor1 hat 37°Cwith frequentshaking.WhenVSVwas used,75 ug/ml
ofDEAEdextran (Pharmacia Fine Chemicals, Piscataway, NJ) was alsoin-
plasmamembrane.Influenza,sendai, orsimianvirus5(SV5)
cluded. After incubation the medium was removed and replaced with an
bud exclusively from the apical surface ofthecells, whereas appropriate volume ofMEM(containing 2% fetalbovine serumfortheSV5
vesicular stomatitis virus (VSV) particles assemble only on infection).
basolateral domains. Because polarized budding ispreceded SuccessfuldoubleinfectionsofMDCK monolayersrequiredadherencetoa
by an accumulation ofviral envelope proteins in the corre- strict protocol. For SV5 and VSV, itwas foundthat theoptimal time for
superinfectionwith VSVwas 24 to42 hafterSV5inoculation.Atthattime,
sponding plasma membrane domains, it hasbeen suggested
themedium wasremoved and atypical infection with VSV was conducted.
(37)thatthepolarizeddistribution ofenvelopeproteinsisthe For influenza and VSV double infections, it was determined that unless
maindeterminant ofasymmetricbudding. influenza was addedatleast 3 h before VSV little production ofinfluenza
It is widely presumed that viral glycoproteins reach their proteins couldbe detected by immunofluorescence orbypulselabeling. If,
however,the influenza infection was allowed toproceed for6hormore the
destinationvia thesamebiogeneticpathwaysusedbycellular
subsequent infection by VSVwas severely curtailed. Optimal results were
membrane proteins. These glycoproteins are known to be obtained when VSV infection was carried out 4.5 h after inoculation by
synthesized onmembrane-bound ribosomes and inserted co- influenza.SimilarobservationsweremadebyRoth andCompans(41)intheir
translationally into the endoplasmic reticulum (ER) mem- studiesofphenotypicmixingindoublyinfectedMDCK.
D
branes(12, 18, 19, 22).During theirsynthesis in the ERand Antibody Preparations: Rabbit anti-SV5 antiserum was obtained ow
subsequenttransferthrough theGolgiapparatustotheplasma from Microbiological Associates. It was tested by immunofluorescence and nloa
showntoreactonly with the apical membrane ofSV5-infected MDCK and d
mtreamnbslraatnioen,altmhoedipfoilcyapteipotnisd,essuucnhdaesrgprootseeovleyrtailcccol-eaavnagdep(o4s7t)-, nagoatinwsittheggu-nginrfoewcntevdircuesllasn.dAtnhisanatniti-bWoSdNyawnatsibsopdecyifwicasfoprrienpfalrueednzianinrfaebcbtietds ed from
glycosylation (1, 16, 31, 32),andadditionoffatty acids(46), cellsasassayedby immunofluorescence microscopy.Mousemonoclonalanti- http
which are alsocharacteristic ofcellularproteins. Gbo.diWeesbsatgeari,nstStt.heJuhdeemaCghgilludtreinn'isn (HoHsAp)itparlot(eMienmopfhiWsS,NTNw)er.eEaacghiftofofthDer.twRo. ://rup
Inthisstudywecompared, inMDCKcellsdoublyinfected re
with viruses ofopposite budding polarity, the intracellular dmiefmfebrrenatnemoonfocilnfolnuaelnzaantiinbfoedciteesdceelmlps,loayseddetreeracmtiendedstbryongilmymuwniotfhlutohreepslcaesnmcae ss.org
pathways followed bythe different viral glycoproteins, from microscopy and ultrathin frozen sectionimmunocytochemistry, but did not /jcb
tchheemEicRaltodettheectaipoincaolfotrhebavsiorlalatearnatligseunrsfaicnesu.ltIrmamthuinnocfryotzoe-n wraeasctprweiptahruendiansfedecstcerdicbeeldlseolrseVwShVe-rien(f3e7c)t.eTdhicesllnse.uAtrnatlii-zVinSgVaGntipbroodteyisnpeacnitfiicbaoldlyy /article
immunoprecipitated only the G protein from radiolabeled VSV-infected -p
sections demonstratedthat proteins destined tothe different MDCKcellsand showed no reactionwithany otherviralorcellularproteins. df/9
plasma membrane domains share a common pathway that Immunofluorescenceandimmunocytochemicalcriteriademonstrateditsspec- 8/4
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includespassagethroughthesameGolgiapparatus.Thus,the ificity for VSV-infected cells. 3
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necessarysorting stepscannot take placebefore traversal of All otherantibody preparations were obtained from commercial sources. 4/1
this organelle. Preliminary accounts ofthis work have been CRahpopdealmiLnaeb-ocroantjoruigeaste(dCocghoraatnvainltlie-,raPbbAi)t aIngdGaaffnidnigtoy-apturainfiteid-maonutsiebodIigeGs ffrroomm 07755
presented at meetings(34, 35). Tago Scientific(Burlingame, CA).Goat serumwaspurchased from Pocono 5/1
3
RabbitFarmandan IgG fractionwaspreparedbyammoniumsulfate fraction- 04
ationfollowedby DEAE-cellulosecolumn chromatography. .pd
MATERIALS AND METHODS [35S]Methionine Pulse Labeling: MDCK monolayers infected f by g
withtheappropriatevirusesweretransferredtoandmaintainedfor30 minin ue
a 10:1 mixture ofmethionine-free MEM(made from aGibco kit) before a st o
Cell Culture: Cultures of MDCK (originally obtained from Dr. J. 30-min incubationwith 0.5 mlofthesame medium containing-25 ACi of n 1
8
Leighton [MedicalCollege ofPennsylvania]), MDBK (obtained from Drs.R. ['SS]methionine(NewEnglandNuclear, Boston, MA).Afterlabelingcellswere M
KVreurgoa(nAdTCPC.)R.cEeltlksinwder[beotmhaionftMaeimnoerdiainlpSllaosatincKettitsseureincgulCtaunrceerflCaesknste(rF]a)lacnodn rdeeomxoyvcehdolabtyesicnraapihnygp,otsoonliubcilTirziesd-HiCnIdbeutfefregre)ntan(d0.p5r%oceNsosnedidaestdPe-s4c0r,ibe0d.5b%y arch 2
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Labware, Oxnard, CA) using minimum essential medium (MEM) supple- Greenet al (11).Samples were analyzed by PAGE on 10% gelsfollowed by 2
3
mentedwithfetalbovineserum(GibcoLaboratories, Inc.,GrandIsland,NY), fluorography(3) usingKodakXR-5orDupontCronex film.
10%forMDCKandMDBK and5%forVero.For experimental work,cells Immunofluorescence Microscopy: InfectedMDCK monolayers
(1.5 x 105) were plated on 35-mmdishes (Falcon Labware). Infectionwith on 13-mm coverslips were fixed for 30 minwith 4% paraformaldehyde in
viruseswas always performed at least 3 d pastconfluence, at which point phosphate-bufferedsaline (PBS)plusCa`*(0.1 mM), washed andstainedfor
monolayerswere more resistanttothecytopathiceffectsofviralinfectionthan immunofluorescence asdescribed elsewhere (1l). Observations were carried
thosewhichhadjustreachedconfluence.Forstudiesinvolvingultrathinfrozen out withaLeitzOrthoplan immunofluorescence microscopeequipped with a
sectioning, MDCK cells(1.5 x 106)were platedin35-mmdishescontaining Wildcamera.
25-mmglasscoverslips (FisherScientificCo., Pittsburgh, PA)coatedwith rat- Preparation ofColloidal Gold: Colloidalgold particleswere pre-
tail collagen. For immunofluorescent studies, cellswere plated onto 13-mm paredbythecitrate method (18-nm particles) ofHorisberger and Rosset (14)
coverslips(SGA Scientific, Inc., Bloomfield, NJ)ina24-well dish (4 x 10° andbythewhitephosphorustechnique(5-nmparticles)ofFrens(8),asoutlined
cells/well).Electricalresistance measurementswere performed on monolayers Geuzeetal.(9)usingtetrachloroauricacid(Kodak).Threetimestheminimal
ofcellsthatwereplatedoncollagencoatednylondisks(5). amountofantibody(storedin aborate buffer, pH 8.0) necessary tostabilize a
Virus Stocks andInfection Protocols: SV5(originally obtained givenamountofcolloidal gold was dilutedin2 ml ofdistilled HZOandthe
fromDr.P.Choppin [RockefellerUniversity])wasplaqued,grown, andtitered goldsolution(firstbroughttopH7.8 usingK2CO3)wasaddedoveraperiodof
1min.Afteradditionofpolyethylene glycol 20,000 (FisherScientificCo.) the
solutionswerecentrifuged eitherat12,000 gfor60mintosediment 18nmor
at50,000gfor120minfor5-nmparticles.Sedimentswereresuspendedgently
'Abbreviations usedinthispaper: ER, endoplasmic reticulum;HA,
inPBS(butatpH 7.8) with0.02% polyethyleneglycol andtheprocedure was
hemagglutinin; MDBK, Madin-Darby bovine kidney;MDCK, Ma- repeated.Thematerialwasstoredinthesamebufferwithsodiumazide(0.02%)
din-Darbycanine kidney; MEM, minimal essential medium; PBS, at4°C.Before use, goldpreparations dilutedforimmunolabelingwerecentri-
phosphate-buffered saline;SV5, simian virus5;VSV, vesicular sto- fugedin an Eppendorfcentrifugefor4mintoremoveanylargeaggregates.
matitisvirus. For immunocytochemical studiesrequiring only a single label, affmity-
RINDLERETAL. Epithelial Cell Polarity 1305
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purifiedgoatanti-mouseandgoatanti-rabbitantibodiescoupledto l8-nmgold RESULTS
particleswereemployed.For double labeling,goat anti-rabbitantibodies cou-
pledto5-nmgold particles were used,although nosignificant differences in
theamountordistribution oflabelwas observed whentheconversecombina- Double Infection ofMDCK Monolayers with
tionofantibody-goldcomplexeswas used.
Viruses ofOpposite Polarity
Preparation ofUltrathin Frozen Sections: InfectedMDCKcell
monolayerson collagencoatedcoverslipswere washed in PBSwith Ca'*,fixed To compare the intracellular pathways that glycoproteins
for 30 min at room temperaturewith 2% glutaraldehyde in thesame buffer of enveloped viruses with opposite budding polarity follow
washed threetimes in PBSandstoredat 4°C untilfurtheruse.Frozensections from their site of synthesis to the eventual site of virion
were obtainedby amodificationofthetechniqueofTokuyasuandSinger(48,
assembly, wedeveloped conditions toallowthe double infec-
49).Briefly fragments ofthefixed monolayer, infusedwith 0.6 Msucrosefor
at least2h, were cryosectioned at -80°to90°Cusing aMT-2B LKB Sorvall tionofMDCKmonolayers (see Materials and Methods).
microtome(LKBInstruments, Inc.,Gaithersburg, MD) equippedwith afreez- The simultaneous synthesis of enveloped glycoproteins of
ingattachment (LTC-2).Sections werepicked up oncarbon-Formvarcoated both viruses was demonstrated in cultures pulse-labeled with
coppergrids, washedinPBScontaining 10mMglycineandtreatedwith a2%
['SS]methionine atvarious timesafter VSVaddition (Fig. I).
gelatin (300 bloom, Sigma Chemical Co., St. Louis, MO) solution in PBS-
Synthesis ofSV5 orinfluenza proteins was clearly detectable
glycine.Sectionswereincubated for 30minon topof10,al dropsofantibody
solutions(a 1:10 dilution ofacombination ofthetwoascitesfluidscontaining during the first 7.5-8 h following superinfection with VSV,
monoclonalantibodiestotheHAofinfluenzaora 1 mg/mlsolutionofrabbit whenthe rateofsynthesis ofVSVproteins increased dramat-
IgGagainstVSV Gproteineach containing2mg/mlofgoatIgG).Gridswere ically. In fact,the rateofsynthesis ofSV5 proteins(Fig. 1 a),
then washed with PBS-glycine and incubated for 30 minwith the secondary
appeared to remain relatively constant during the first 7.5 h
goatIgGantibodycomplexedtocolloidalgoldparticles,whichhadbeendiluted
with PBS-glycine untiljustbarely pink.After several washes thegridswere of superinfection; on the other hand, the synthesis of WSN
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fixedin glutaraldehyde and stainedfor 20 minwith 0.3% OS04 in cacodylate proteinsreachedapeak at5 h, decreasingsomewhatthereafter ow
buffer, washed incacodylatebufferand H20, and placed for 20-30minover (Fig. I b). nlo
an embedding mixture containing 0.8% methyl cellulose (Fisher Scientific, ad
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blotted away toleaveafilmthatafterdryinghad asilver-golden interference http
ocpoelroar.teTdheaste60sakmVpl.es were viewed ina Phillips EM 301 electron microscope ://rup
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monolayersincubatedwith theinappropriateantibody ornonimmuneserum. /jcb
Backgroundlabeling in allcaseswas verylowand,inparticular, labeloverthe /a
Golgi apparatus wasnegligible. rticle
Conventional Electron Microscope Studies: Monolayers -pd
growneitheron35-mm dishesoron collagen-coated coverslips were fixedin f/9
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glutaraldehyde for 20 min, scraped, sedimented inanEppendorfcentrifuge, /4
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washedinPBS,and processedasdescribed (38). 30
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IodinationExperiments: Atvarioustimes afterinfectionwith VSV, /1
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MDCK monolayersgrownon 35-mm disheswere washed threetimesinice- 77
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coldPBSplusCa",placedon ametal blockimmersediniceand iodinatedat 55
4°Caspreviouslydescribed (l5,39).0.5mlofiodination mixturecontaining 1 /13
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IU/mllactoperoxidase-coupled Sepharosebeads (P-LBiochemicals, Inc., Mil- 4.p
waukee, WI), 10 mMglucose (Aldrich Chemical Co., Milwaukee,WI), 0.14 df b
mU/mlglucoseoxidase(SigmaChemicalCo.),2.5nmol/mlNaI,and0.3 mCi/ y g
ml['2'I]iodine (New England Nuclear) was addedtoeach platefor30 min. ue
Beforeusetheenzyme-coupledbeadswere sedimentedbycentrifugationfor20 st o
n
sinan EppendorfcentrifugeandwashedfourtimeswithPBS.Afteriodination, 1
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monolayers were washed threetimes at4°Cina1:1 mixture ofPBSwith Ca- M
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and 150 mM Nal containing 10'Na2S203, and two more times inice-cold rch
PBS. Samples were then processed asdescribed above for ["S]methionine 2
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labeling.Before SDSelectrophoresis, sampleswere precipitated withtrichloro- 23
acetic acid, etherextracted,andresuspendedingel sample buffer.Theincor-
poration of12'Iintocell protein wasnot due toleakage ofenzyme from the
beads during theincubation period. This was demonstrated by the fact that
incubation mixturesthathadbeen previously used forlabelingcellsbut from
FIGURE 1 Synthesis of viral proteins in doubly infected MDCK
whichthebeadshad beenremoved bycentrifugationfor20 sinthe Eppendorf
cells. Cell monolayers on 35-mm dishes were infected and incu-
centrifuge, were nolongercapable oflabelinguntreated monolayers duringa
second incubation. batedwith [35S]met(50,uCi/ml) for20minas described below.The
Labeledvirionsreleasedintothemedium wererecoveredbysucrosedensity cells were solubilized with detergent (11) and processed for SDS
gradientcentrifugation(11)afteradding 60kg ofpurified unlabeled virionsas electrophoresisin a 10%polyacrylamide gel followed by fluorog-
a carrier.Aftersedimentation(100,000gfor60 minin SW56 Beckmanrotor raphy. (A) Cells were infected with SV5 (5 pfu/cell) and superin-
[BeckmanInstruments, Inc.,PaloAlto,CA])andresuspensionin10mMTris- fected with VSV (10 pfu/cell) 24 h later, Total cell extracts (3-d
HCI, pH 7.6, the samples were loaded onto a 7-52% K*-tartrate gradient exposure) were analyzed from cultures which were radiolabeled
containingthesamebufferandcentrifugedovernightat 100,000g.Material in before each infection (a and c) or at the times indicated after
avisiblebandwasrecoveredbycentrifugation,solubilizedingelsample buffer addition of VSV (e, g, i, k, m, and o). The correspondingimmuno-
and analyzed by electrophoresis.Thedata have been corrected for recoveries
precipitates (6-d exposure) obtained with anti-SV5 antiserum from
whichtypicallyranged from 60% to80%.
the cell extracts were also analyzed (b, d, f, h, i, 1, n, and p). (t3)
Electrical Resistance Measurements: For each time point four
Cells were infected with influenza WSN (10 pfu/cell) and superin-
disksofcells infectedby thestandard protocolwere used.Measurementswere
carried out in a modified Ussing chamber as previously described (5). To fected with VSV (15 pfu/cell) 4.5 hlater. Infections were allowed to
ascertain thattheinfectionwasproceedingnormally,wepulse-labeled,5hafter proceed for the times indicated before radiolabeling. Total cell
infection, one infectedand onecontrol diskwith ["S]methionine for30min extracts were analyzed from cultures infected with WSN alone(a-
and analyzedby SDSelectrophoresis. f),with both viruses (g-k) and with VSV alone(I-p).
1306 THE JOURNAL OF CELL BIOLOGY " VOLUME 98, 1984
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FasIGdUeRsEcr2ibedPofloarriFziegd. 1bAu.dSdaimnpgloefsStVa5keannd11VhSVafvteirrisounpsefrrinofmecditfifoenrewnitthsuVrSfaVcewserofetphreocseasmseedMfDorCcKoncveellnt.iInofneacltieolnesctwreornemciacrrroisedcoopuyt. f by gu
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FbaisloalmaetnetraolusdoSmVa5ivnir(ieoln)so(fatrhreowchelela.dB)arb,ud1 gfrmo.mxth2e2,a0p0ic0a.l surface (Ap)whilebullet-shaped VSV particles (arrows)assemble onthe st on 1
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Electron microscopyrevealedthatviralassemblyproceeded Intracellular Distribution of Viral Glycoproteins in 23
in doubly infected monolayerswith the polaritythat is char-
Doubly Infected Cells
acteristic of each virus in singly infected cells. Filamentous
SV5 virions, or spherical influenza particles assembled atthe The localization of HA of influenza or G of VSV was
apical surfaces of cells thatat the same time manifested the determined by immunolabeling of ultrathin frozen sections
bullet-shaped VSV particles budding from lateral and basal usingspecific antibodies toeither viral glycoprotein andcor-
domains (Figs. 2 and3, a, b, andc). With theprogression of responding affinity-purified second antibodies complexed to
the double infections budding of VSV predominated and colloidal gold particles. Intracellularly, the G protein was
fewerSV5 or influenza virions were produced. After several preferentially localized intheGolgiapparatus (Fig. 4a) where
hours of VSV infection many cells became rounded, tight it first appeared 3 h after infection, a time when there was
junctions weredisrupted and bullet-shaped VSVvirions were practically nolabelingonthe cell surface. Goldparticleswere
seen budding over the entire cell surface. However, even in found on the different Golgi cisternae as well as adjacent
these cases, when virions from the first infection (SV5 or Golgivesiclesandvacuoles.Thereseemedtobeno preference
influenza)were still assembling, thisoccurredinareasdistinct for the rims of the individual cisternae over the middle
fromthose producing VSV particles (Fig. 3d). In SV5/VSV regions, and no marked accumulation towards one side or
doubly infected cells, SV5 virions tended to cluster towards theother sideofthe Golgistacks.In contrasttothe prominent
the apical pole ofthe cell, away from areas in the same free labeling ofthe Golgi apparatus, which persisted even when
surface wherepatches ofVSVbudding virions appeared (not the G protein became more abundant on the plasma mem-
shown). brane, littleaccumulation ofG protein waseverdetected over
RINDLER ETAL. EpithelialCellPolarity 1307
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FIGURE 3 Polarized budding of influenza and VSV virions from doubly infected MDCK cells. Monolayers were infected with
influenzaand 4.5h latersuperinfected withVSVand incubated for5(a, b,andc)or9h(d) before fixation forelectron microscopy.
(a) Numerous influenza virions (arrowhead) averaging 90nm indiameter are present ontheapical surface (Ap) of the cell,while
bullet shaped VSV particles of 65-nmdiameter (arrow) budding fromthe basolateral plasma membrane domains accumulate in
the intercellular spaces (BI). (band c) Higher magnification views ofsegments of the apical (band lateral (c) surfaces.(d)Acell
late ininfection, with tight junctions no longer intact. Bothtypesof viruses can be seen budding from nonadherent regions of
the plasmamembrane. Budding virions of each type, however, do not intermingle completely on the surface. Distinct regions
are observed where influenza (arrowhead) or VSV (arrow) particlesassemble. Bar, 1 gm.x26,000.
308 THE JOURNAL OF CELL BIOLOGY - VOLUME 98, 1984
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FIGURE 4 Localization ofviral glycoproteins inMDCKcells infected with influenza and VSV. (a) Thin frozen sections of doubly
infected cells were processed for immunoelectron microscopy using rabbit antibodies to G or mouse monoclonal to HA as
described in Materials and Methods. 3 h after superinfection with VSV, before significant amounts of G are detected in the
plasma membrane, the G protein is found throughoutcisternae of the Golgiapparatus (arrow). (b) 5 h after superinfection with
VSV the HA of influenza isfound in the Golgiapparatus (GA, arrows) as well astheapical plasmamembrane (arrowhead). Bar,
0.25 Pm.x60,000.
RINDLER ET AL. EpithelialCellPolarity 1309
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the rough endoplasmic reticulum, where the polypeptide is label found on the apical membrane. The segregation ofG
synthesized.Thus,traversal oftheGolgiapparatus appears to was even lessstrict;the densityofanti-Gprotein labelonthe
beamuchslowerprocessthanexitoftheGproteinfrom the apical membraneranged widely from-20%ofthatfoundon
ER. basolateral surfaces to almost 100% in several cells in ad-
HAwas alsolocalizedthroughouttheGolgiapparatus (Fig. vancedstatesofVSVinfection.Thepresence ofGprotein on
46), although in this case when labeling ofthe cell surface theapical surface, which wasalso observedinsingly infected
was intense (5 or more hoursafter VSV infection), labeling cells(Fig. 9), was only prominent after mostofthe infected
over the Golgi elements decreased. Notably, the remaining cells had Gonthe basolateral surface. Thisobservation is in
few gold particles observedovertheorganelle appeared to be agreement withpreliminary results fromworkinthislabora-
preferentially located towards therim oftheGolgicisternae. tory(manuscript in preparation)usingtheVSVtemperature-
Theseimmunoelectron microscopic observations are consist- sensitive045 mutant thatpermits abetter synchronization of
entwith thechanges intherate ofsynthesis oftherespective the intracellular traffic ofG proteins from the Golgi to the
viralproteinsdetermined from radioactive pulse-labeling ex- plasma membrane. Itmustbestressed, however,thatevenin
periments (Fig. 1). those cases where the density oflabeling with anti-G anti-
Todetermine whether theGand HA glycoproteins simul- bodies was equally intense on both surfaces, VSV virions
taneously traverse thesameGolgiapparatus, wetreated thin budded exclusively fromlateral and basalplasma membrane
frozen sectionsofdoublyinfectedcellswithantibodiesagainst domains.
both types ofviral glycoproteins. In this case, the primary Thepresence ofGprotein ontheapicalmembraneofsingly
anti-HA mousemonoclonalandanti-Grabbitantibodieswere infected cells was also determined by selectively iodinating D
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localized by secondary goat anti-mouse or anti-rabbit anti- theexposed surface ofVSV infected monolayerswith lacto- wn
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bodiescoupled togoldparticlesofdifferentsizes(seeMaterials peroxidase coupled to large (60 um) Sepharose beads. Only ad
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and Methods). In cellsinwhich both HA and G were found complete opening oftight junctions could allow access of d fro
intracellularly, theGolgiapparatus,regardlessoftheirlocation these beadstothe basolateral domains. However, asearly as m
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within the cell or their proximity to either ofthe plasma 4 h after VSV infection (Fig. 10), soon afterthe G protein ttp
membrane domains, were always labeledwith both typesof was first detectable on the plasma membrane byimmunoe- ://ru
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the same cisternae (Fig. 8, a, b, and c). These clusters were intheapical membrane. Thisapical polypeptidewas indistin- /jcb
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ofseveral viralglycoproteins, whereas the irregularly shaped cipitationwithanti-G antiserum (notshown)intotalinfected f/9
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clumps couldalso bethe resultofanartifactual aggregation cell extracts and purified virions. When cells were labeled /4/1
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oflabel around a single antigen. The G protein showed no with"'Iafter5hofinfection andsubsequently incubated at 0
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preferential distribution, but HA was more frequently found 37°C for 1.5 and 3 h, the amount of labeled G protein /10
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over peripheral regions ofthe cisternae, especially in cases recovered in cell extracts diminished to 60% and 40%, re- 5
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when high concentrations ofG were present in the Golgi spectively. This probably reflects degradation oftheapically /1
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(Figs. 5 and 8c). These observations clearly indicate that exposed G protein rather than itsincorporation into virions 4.p
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envelopeglycoproteins ofviruses withopposite budding po- and subsequent release from the monolayer, since virions f b
larity traversethesameorganelle. collected and purified from the medium accounted for only y g
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Anti-viral glycoprotein antibodies were also localized on a small fraction(--5%) ofthedecreasein radioactivity. st o
vesicular elements located inthe Golgiareaandin thecyto- Theintegrityofthejunctions attimeswhenGprotein was n 1
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plasm between the Golgi apparatus and the plasma mem- already detected onthe apical plasma membrane was moni- M
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brane. In most instances these vesicles contained only gold tored by measuring the electrical resistance (5) ofinfected rch
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particlesofonesize(Fig.6),butoccasionally, andinparticular monolayers(Fig. 11a).Despite anactive VSVinfection (Fig. 0
2
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insomeofthelargervesicles, bothtypesofgoldparticles were 11 b), thisbegan todecrease only 6.5hafteraddition ofVSV
present.Itisworthnoting thatdue tothelow level oflabeling but remainedsignificant even 2 h later. Thus,theappearance
and the small size ofmost vesicles it cannot be concluded of VSV G protein on the apical membrane is unlikely to
thatthosevesicles labeled withonlyone typeofantibodydid result from a redistribution ofsurface proteinfollowing dis-
not carrysomeoftheotherantigenaswell. ruptionofthetightjunctions during VSVinfection.
Distribution of Viral Glycoproteins in the Formation of Mixed Virions in Doubly
Cell Surface Infected Cells
As double infection proceeded, both viral glycoproteins Cultured cells infected with two different viruses can pro-
were observed in the same Golgi, while HA accumulated duceaberrantparticles inwhichthenucleocapsidofone virus
predominantlyontheapicalandGonthebasolateraldomain issurroundedby anenvelope composed partially (phenotyp-
oftheplasma membrane.However, segregation oftheglyco- ically mixedvirions)ortotally (pseudotypes) ofglycoproteins
proteins toeach domainwas not as strict as suggestedprevi- from the other virus (28). A recent study (41) measured
ouslyinstudiesconductedwithsinglyinfectedcells(37).Even pseudotypeand mixed virion formation inpolarizedMDCK
beforeviralbuddingbegan,somelabelingofHAwasobserved monolayers infectedwith influenza andVSV bytheneutral-
in the basolateral domains and this persisted when viral ization of VSV plaque forming units with anti-influenza
production was intense(4.5-11.5 h afterinfluenza infection, antibodies. This indicated that the production of aberrant
see Fig. 12), but this rarely exceeded 10% ofthe density of virions containingaVSVnucleocapsidincreaseddramatically
310 THE JOURNAL OF CELL BIOLOGY - VOLUME 98, 1984
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FIGURE 5 Simultaneous localization of VSV and influenzaglycoproteins inadoublyinfected MDCK cell. Frozen thin sections of
doubly infected cells(4 h after superinfection with VSV)were incubated with mousemonoclonal antibodies against the HA of
influenza and rabbit anti-VSV G protein antibodies, followed bythe appropriate second antibodies complexed to colloidal gold
particles of two different sizes. Largegold particles, correspondingto the HA of influenza (arrowhead), and small gold particles
localizing the Gof VSV (arrow), are found in the same Golgiapparatus (GA).The apical surface (Ap) contains predominantly HA
butalso some small amount of G. Nosignificant label isobserved overthe nucleus (N) or the cytoplasm. Bar, 0.2 arm.x 57,000.
RINDLER ETAL. EpithelialCell Polarity 131 1
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FIGURE 6 AppearanceofVSVand influenza glycoproteins in multiple Golgistacksofthe same doublyinfected cells.Frozen thin
sections were prepared and labeled as described in Fig. 5. Large gold particles (arrowheads) representing the HA of influenza,
aredistributed in eachGolgiapparatus (GA) depicted, as well ason the apical surface (Ap).Smallgold particles (long arrows) are
found within the same Golgi stacks, showing that the VSV G protein and the HA of influenza are sharing the same organelle.
Most cytoplasmic vesicles (shortarrows) are labeled with only one type ofantibody. Bar,0.25 lAm.x45,000.
with the course ofinfection and with theonset ofcytopathic The formation ofmixed virions was demonstrated by im-
effects, suggesting that opening of the tight junctions pro- munolabeling in frozen sections of doubly infected MDCK
moted the intermingling of viral glycoproteins in the cell cells (Figs. 12 and 13). Bullet-shaped virions (i.e., with VSV
membrane. nucleocapsids) containing varying amounts of label corre-
312 THE JOURNAL OF CELL BIOLOGY " VOLUME 98, 1984
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FIGURE 7 Distribution of HA andGwithin the same Golgiapparatus ofadoubly infected MDCKcell. Infected monolayerswere st o
n
prepared and processed as described in Fig. 5. Each Golgi apparatus (GA) contains both large gold particles (arrowheads) 1
8
representing HA of influenza and smallones (arrows) corresponding to the G ofVSV. The two typesof glycoproteins are found M
a
inthe same cisternae and inassociatedvesicular structures, some of which aredilated. Bar, 0.2jum.x63,000. rch
2
0
2
3
sponding to the HA glycoprotein were commonly seen both HA ofinfluenza. We observed thatincellslabeled duringthe
free in the intercellular spaces and budding from the lateral productivestages ofinfection, HA ofinfluenzaandGprotein
membrane, wellbeforethere was anyevidence for the lossof of VSV could be detected throughout all cisternae within a
junctional integrity (Figs. 12 and 13a). Mixed-type virions Golgi stack. This most likely represents the distribution of
with a rounded influenza nucleocapsid but labeled with anti molecules inpassagethroughthe organelle from the cistothe
G antibodies were rarely observed (Fig. 13b), despite the trans face, since it is now clear that the Golgi apparatus is a
relative abundance of G protein on the apical membrane, polarized structurein whichdifferentenzymatic activities are
whereinfluenza virions assembled. confined tospecialized regions (cf. 7, 40).
Even though the viral glycoproteins are synthesized in the
DISCUSSION ER, we detected little labeling of this organelle. This may
The realization that many plasma membranes proteins are reflect the low concentration of newly synthesized glycopro-
glycoproteins bearing complex oligosaccharides (cf. 45) tein in ER membranes, resulting from their rapid transit to
known to be processed by enzymes located in Golgi mem- the Golgi apparatus. Somewhat higher levelsof ER labeling
branes (4, 13) suggested that these proteins musttraversethe for the glycoproteins of SFV (10) and VSV (2) have been
Golgiapparatusontheirwaytothe cellsurface. Thishasonly observed by otherinvestigators in nonpolarized cells.
recently been demonstrated directly by immunoelectron mi- In many secretory cell types the Golgi apparatus has a
croscopy for the viral envelope glycoproteins ofVSV (2) and characteristic disposition andorientation,whichappearswell
SFV (10), and asa resultofthe work presented here, for the suited for the directional transfer of its contents to the cell
RINDLER ETAL. EpithelialCellPolarity 131 3