An Epstein-Barr

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Proc.NatiAcad.Sci. USA

Vol.78, No. 11, pp. 7162-7165,November1981 Microbiology

An Epstein-Barr virus early protein induces cell fusion

(nasopharyngealcarcinoma/biologicalpropertiesofEpstein-Barrvirus) GARY JAMES BAYLISSAND HANSWOLF

MaxvonPettenkoferInstitute,Pettenkoferstrasse9a, 8000Munich2,Federal Republic ofGermany

Communicated byWernerHenle, July1, 1981

ABSTRACT Superinfection of Raji cells with Epstein-Barr virus(EBV) leads to syncytium formation. Studies using metabolic inhibitors and amino acid analogues suggest that the fusion-inducing factor belongs to the early group of virus-specified proteins. Induction ofearly EBV protein synthesis in Raji cells by using various chemicals also leadsto syncytiumformation, indi- cating that the fusion process is not causedby a virion membrane proteinintroduced into the cells upon infection. The relevance of these findings to the association of EBV with carcinoma of the nasopharynx is discussed.

Previous studies(1) indicated thatsuperinfection ofRajicells withEpstein-Barr virus(EBV) derivedfromP3HR1cells leads tothe formationofpolykaryocytes. Moreover, itwasdemon- strated thatsuperinfected Raji cells could fuse to cellslacking receptors for the virus. Virus treated with neutralizinganti- serum orinactivated with UVfailedto induce cellfusion, in- dicating thatviable virusisrequiredfor this process. Further- more treatment of infected cells with sodium azide, which prevents virus penetration, orcycloheximide, which prevents all protein synthesis, precluded the formation ofpolykaryocytes. These data

suggested

thataviral protein synthesized aftersuperinfectionwasresponsiblefor the fusionpotential of EBV. However, theseexperiments didnotexclude the possi-

bility

thatavirus membrane protein, which couldhave been integratedintothecellularmembraneduringpenetrationofthe virus, caused fusion and thatthe various treatments weused preventedthe necessary processingofthis protein (e.g., pro- teolytic cleavage) that would enableittoinduce cellfusion. In the seriesofexperimentsdescribedinthis communicationwe show that thesynthesis ofviral proteins insuperinfected cells isessential for fusion and thatthe viral genes,inducibleinRaji cells

by

treatmentwith chemicals suchas

iododeoxyuridine

(2) or

phorbol

ester(3), code for thenecessaryfunctionsthatlead tosyncytiaformation.

MATERIALS AND METHODS

TissueCulture and VirusPreparation. Raji cellsweregrown in plastictissue cultureflasks inRPMI 1640culture medium supplementedwith 10% heat-inactivated fetal calf serum. Cul- tures weresplit1:4when the celldensityreached 106 cells per ml. P3HR1cells,which release infectious EBV, weregrown as 200-ml cultures in32-oz(950-ml)glass prescriptionbottlesin RPMI 1640 medium. After subculture the cellswere allowed tostandat

34°C

for10days, the culture supernatantwasclar- ifiedby centrifugationat4000 x g for15min,and thevirus was sedimentedby centrifugationat27,000Xgfor2hrat4°C. The virus pellet was resuspended in tissue culture medium and storedoverliquidnitrogen.

Preparation of Lymphoblastoid Cell Monolayers. Plastic plates (FlowLaboratories)35 mm indiameterwereincubated with2.5%

(vol/vol)

glutaraldehydeatroomtemperature for 2 hr and thenthoroughlywashed with sterilephosphate-buffered saline; 1mlofdiluted anti-lymphocyte globulin (ALG)(16pug/

ml)wasadded and theplateswereincubated overnightat40C.

TheunadsorbedALG wasremovedwith a pipetteandtheplates werethen rinsed withphosphate-bufferedsaline several times to removeremainingunadsorbedALG.Cellswereaddedtothe coatedplates and after 1-2 hr at 370C the nonadherent cells wereremoved byrinsingtheplateswith tissue culture medium.

Afterrefeeding the cultureswereready foruse.

FluorescentAntibodyStainingof FixedCells. Smearsofsus- pension cultures or monolayers ofadherent cells were dried underastreamofcoldairand thenfixedfor10min at -200C inacetonewhenglass surfaces wereused. Forplasticsurfaces weused petroleumbenzene(bp

50-75QC)

(4).The smears were stained either directly byusingserum conjugated with fluorescein isothiocyanate or indirectly by using unconjugated serumfollowedby fluorescein isothiocyanate-conjugatedanti- humanIgG. Stainedcells were embedded insemipermanent mountingmedium (5) and examinedin a Zeissepi-illuminated fluorescencemicroscope.

Induction of EarlyAntigen(EA)SynthesisinRaji Cells. Raji cells weresynchronized byusing adoublethymidine blockand EA synthesis was induced according to the procedures described (2)by addingiododeoxyuridine (20

jig/ml),

hypoxan- thine(14 ,ug/ml),and aminopterin(1

,ug/ml)

1hrafter the onset ofSphase. Otherinducingagents-sodium butyrateat3mM (6),phorbolester(3), orthemethylationinhibitorcycloleucine at 2mM-wereaddeddirectly afterthe releaseofthethymidine block. Inexperimentsinwhichinducedcells were treated with inhibitorsor aminoacid analoguesthedrugswereaddedatthe sametime asthe EA-inducingreagents.

RESULTS

Superinfection of Raji cells with P3HR1 EBVleads to alytic cycle of viral expression. In a number of studies we (7-9) and others (10)have described an ordered synthesis of virally induced proteins withinsuperinfected Rajicells. Furthermore, we wereable to divide the replicativecycleof EBV into several phasesbythe use ofanumber ofmetabolic inhibitors, these data aresummarizedinFig. 1. Fig. 2 shows theappearance ofRaji cellssuperinfected andimmobilizedinthe presence ofphos- phonoaceticacid (Lower

Left)

and canavanineinthe presence of arginine(LowerRight).ReferencetoFig. 1shows thatinthe presenceoftheseinhibitors a limited expression ofthe spectrum ofEBV-induced proteins occurs and these cells arecapableof undergoingfusion. In aprevious

study

wehaveshown(1)that additionofinactivatedvirus toRajicells failedtoinducefusion.

Abbreviations:EBV, Epstein-Barrvirus;ALG,anti-lymphocyte globulin;EA,earlyantigen.

Thepublicationcostsofthisarticle weredefrayedinpartbypagecharge payment. This article musttherefore be hereby marked "advertise- ment"inaccordancewith18 U. S. C.§1734solelytoindicate this fact.

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Proc.NatL Acad. Sci. USA 78

(1981)

7163

Paa AztCan+ My 2 4 6 10 16 24 Total HU Azet Arg Can

_ 15C0_v.

-. ~~~138143_

SL.- ~~~120 110

* age-i *.- * 105 102 -_ -_ A _._ 90

83-80 S.

..b

-4f- MM 63

54

-5 >-5>- . 549 -

==* .' 40

41ON

aW_*6

_35 - I

33 -

FIG. 1. Synthesis of proteinsinEBV-superinfectedRajicells.Raji

cells weresuperinfectedwithEBVderived from P3HR1 culturesand pulse labeledwith[35S]methionineatvarioustimesafter infection(the starting times,inhr,forthe 30-minpulsesaregiven above thetracks;

MIistheprofile of proteins observedinuninfectedRajicells).The approximatemolecularmasses aregiveninkilodaltonsattheside of thegel. Theinfected labeled cellsweredisruptedinsamplebuffer[50 mMTris-HCl (pH7.0)/2%sodiumdodecyl sulfate/5%(vol/vol)2-mer-

captoethanol/30%sucrose] and heatedto1000Cfor5min.Thesamples wereresolvedon 10%polyacrylamidegelscrosslinked with0.26%diallyltartardiamide, using a stacking gel containing 3% acrylamide, 0.08%diallyltartardiamide,andadiscontinuous buffersystem.The resolved stainedgelsweredried andautoradiogramswereprepared, usingLKB 2208Ultrofilm.In a seriesof similarexperimentsvarious metabolic inhibitorswereaddedatthetimeof infection and the cells werelabeledat 12-16hrafter infection.Solid linesontherightin- dicate proteinsynthesisinthepresenceoftheinhibitor namedinthe columnheading. PAA, phosphonoacetic acidat200

pug/ml;

HU, hy- droxyureaat4mg/ml;araC, cytosine arabinonucleosideat50,tg/ml;

Azet,azetidineat500/Ag/ml;Can+ Arg,canavanineat500

Ag/ml

innormalarginine-containingmedium;andCan,canavanineat500 ,ug/mlinarginine-freemedium.Thecolumn headed"Total" indicates thespectrumofproteinsseeninuninhibited infectedRajicells. The 150-kilodaltonprotein issynthesized inculturestreatedwithphos- phonoacetic acidorhydroxyurea butnotincultures treated withcy- tosinearabinonucleoside;.the 80-and63-kilodalton proteinscanbe identifiedinextractsofcanavanine-treated cellsonlyafterimmuno- precipitation because they are relatively poorly synthesized under theseconditions.

Because addition of inhibitors touninfected cultures did not result inthe formation ofpolykaryocytes, it can be concluded that at leastapartialexpressionofEBV isrequiredforfusion to occur. Although it cannot be said that the fusion-inducing proteinis oneof those visualizedinthegel profiles presented inFig. 1(because.theproteinmightcomigratewithahostpro- tein orbe presentinminor amountsandnotbevisualized), it maybe concludedthat the protein has thesameproperties as the proteins seen in the early profiles and in the inhibitor- treatedsamples.

Fluorescentantibody stainingof the cultures revealed that thecellswere

synthesizing

EAbutnotviral

capsid

antigen. It has beenshown that chemicals(2, 3,6)andotheragents (11, 12)canbeusedtoinduceEAsynthesisinRajicells, andit was necessaryto testwhether.theproteins

responsible

forfusionare synthesizedininducedoronlyin

superinfected

Raji cells. Pre- viousexperiments(11)haveshown thatRajicellsimmobilized inconfluentmonolayerscan nolongerbe inducedtosynthesize EA.Becauseconfluencyisnecessaryfor theobservationof cell fusionwe overcamethis

problem by synchronizing

theRaji cells

1-4 ...n.....;

w ~

**:Fi)o *

as~~~~0

t j w

4._;im

J :3. A-.;~~~~~~~~~~~~~~~~~~...:..

%4 ,I -

'K>

^x

2 t

$ *

i H e at .S.,

jm

a

f

FIG. 2. Formationofpolykaryocytes incultures of immobilized superinfected Raji cells. Raji cellsweresuperinfectedormockinfected.

Totheculture showninLowerLeft, phosphonoacetic acidwasadded atthe timeofinfection;canavanine wasaddedtothecultureshown LowerRightatinfection. After thevirushadabsorbedtothecells,the cellswereappliedtothe ALG-coatedplatesandallowedtoformmon-

olayers. Uninfected cells do not form polykaryocytes (UpperLeft), whereas after4hrthe superinfected culturescontained fused cells (UpperRight). Thetreatmentwith eitherphosphonoacetic acidor canavanine (Lower Left and Right) did not prevent the formation of polykaryocytes.(x650.)

in suspension; thesefree-floating cells were then induced to synthesizeEA bytheaddition of various inducingagents, in-

cluding iododeoxyuridine (2), phorbolester(3), sodiumbutyr- ate(6),orthemethylation inhibitorcycloleucine.After theRaji cells had entered into a cycle of EAsynthesistheywereapplied toALG-coatedplates and allowed to formpartially confluent monolayers. Inmanyplacesthe cellshad settledout asislands andthus thefusionprocesscouldoccurif the

required

proteins werepresent. Twelve hours after the cells were immobilized theywereprepared for fluorescent staining orexamined and photographedwith aphase-contrast photomicroscope. Under conditions inwhich thecells synthesize EAthe cellswereca-

pableofundergoing fusion (representativeculturesareshown inFig.3UpperRight and Lower

Left

andRight).

Immobilized,

synchronizedRajicells athigh density (when they donotsyn- thesizeEAinthe absence ofinducers) didnotleadto

polykar-

yocyteformation (Fig.3Upper

Left).

Treatmentof theinduced cells with the variousinhibitorsdescribedinFig. 1(we didnot testcytosinearabinonucleosideorhydroxyurea)didnotprevent theformation ofpolykaryocytes, once again suggestingthat a subset of very early proteins may be involved in the fusion process.

0*so*

Microbiology: Bayliss

andWolf

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7164 Microbiology: BaylissandWolf

CH TREATED FOR HRS

FTTTFTI';

Is~

I

Vw .b .

-"..-

-49m-~~~Z.

v1..ar,,400 ft is

FIG. 3. Formation ofpolykaryocytes incultures of induced immobilized Rajicells.Rajicellswereinduced tosynthesizeEAasde- scribedinthetext,the cellswereappliedtoALG-coatedPetriplates andallowedtoattach,and 12 hrlater the cultureswerephotographed inphasecontrast. (Upper Left)uninducedcells;(UpperRight) cyclo- leucinepresent;(LowerLeft)sodiumbutyratepresent;(LowerRight) 12-O-tetradecanoylphorbol 13-acetate present.(x650.)

Control experiments with the EBV-negative cell line BJA showed that the addedchemicals didnotcausefusionbythem- selves. The morphologyof immobilized cells seemstobede- pendent^uponthelengthoftimefor whichthe cells have been attachedto the surface. A few hours after immobilizationthe cellsare rounded andappear asnormal lymphoblasts (Fig. 2 UpperLeft); atlatertimesthecellsspreadoutandpresentan

epithelioid-orfibroblastoidmorphology (Fig.3UpperLeft).The infectedorinducedcells^arealsocapableofadoptingaflattened morphology ^even under conditions when fusion takes place.

Certain metabolic inhibitors (e.g., canavanine; Fig. 2 Lower Right) ^or inducers (e.g., phorbol ester; Fig. 3 Lower Right)

seem topreventthe adoptionof^aflattened morphology; this islikelytobeduetointerference with normal cellularprocesses

inaddition tothe effectswhich theseagentshaveonviralan-

tigensynthesis. The^reasonsfor theadoptionof thisflattened

appearanceandtheformationof the fineprocesses(a good^ex- amplecanbeseeninFig.3UpperRight)arenotclear,but this morphologycould be due tothe interactionofthe cell mem-

branewith theantibodies usedtoimmobilize the cells.

Previousstudiesbyus(9) havesuggestedthattreatmentof uninfected Rajicells withcycloheximideforseveralhours fol- lowedbyitsremoval leadstoaverylimitedexpression of the endogenous EBVgenome. For this reasonwe decidedtosee

ifsuch atreatmentwould leadtopolykaryocyte formation. As showninFig. 4, treatmentofRajicells withcycloheximidefor

102jjjo S e' j

* & s

FIG. 4. Induction ofpolykaryocyte formation inRajicellsbycy- cloheximide(CH)treatment. RaJicells were treated withcyclohexi- mide for0, 2,or 4hr,thedrugwasthenwashedfrom the cells, andthe cells werelabeled for30 minwith

[3'Slmethionine.

Cell extracts were preparedandanalyzedasdescribedinthe legendtoFig.1.Thetwo cycloheximide-inducible proteins (whicharealsoinducedbysuperin- fection;seeFig. 1) are marked with open circles. Adjacent to thecell profilesarephase-contrastphotomicrographsofcultures of immobi- lizedRajicells that were exposed tocycloheximidefor 3hr;after this exposurethedrugwaswashedawayandthe cultures were incubated for 2hr(Upper)or18hr(Lower).Notethe formation ofpolykaryocytes andtheincreasein sizeafterlongerincubation.(x650.)

2-4hrfollowedbyitsremovalleadstothe synthesis oftwonew proteinswithmolecularmassesof

120,000

and 102,000daltons, respectively. Afterimmobilization of such cultures. localpoly- karyocyteformationcan,be observed. The number andsizeof thepolykaryocytes, incomparisontothose seen in Figs. 2 and 3, is..small andmayindicate that occasionally cells express ad- ditionalproteinswhich inducefusion. The amounts of thetwo cycloheximide-inducible proteinsseenonthegelssuggest that mostofthe cellsinthe treated cultures mustbe synthesizing them. Iftheywere responsible for the fusion, thenpolykary- ocyteformation should be observedmoreofteninthetreated cultures. Staining of these cultures with antisera against EA failedtorevealthepresenceof thisantigenwithinlargenum- bers ofcells; however, afewweakly positive cellswere seen.

Further studies usingimmunoprecipitation should be carried out toinvestigate thispoint.

DISCUSSION

The data presented indicate that theEBV-mediated cell fusion occursfrom within andisnotduetoavirionmembraneprotein as is thecase with theparamyxovirus group. Further studies will be necessarytoidentify the protein responsiblefor thefu- sionprocess, althoughtheresultsofthe experiments within- hibitorsnarrowthenumber ofpossible candidates toagroup ofproteinssynthesizedveryearlyinthe viralreplicativecycle.

Thisobservationisof importance for processesinvivo,where antibodies in the serum would probably attack and destroy EBV-carrying lymphocytes before they entered into the later phases of the viral replicative cycle.

Proc.Nad Acad.Sci. USA 78

(1981)

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Microbiology: Bayliss andWolf

EBV genomes have been found in epithelialcells ofnaso- pharyngealcarcinomabyseveral unrelatedtechniques:in situ hybridization (13, 14), cell separation (15), passage of cells through nude mice (16), and identification of EBV-specified nuclearantigen intouch preparations of tumor tissue (13, 17).

These observationspresent an enigmabecauseattempts to in- fecthuman nontumorouscellsotherthan Blymphocytesin vitro have failed (18). Also, negative results from binding studies usingradiolabeled EBV(unpublished data)do not support the presence of EBV receptors on human untransformed nasopharyngeal epithelial cells. One report (19) indicated that explants of normal human nasopharyngeal tissuecouldbe stim- ulatedintorapid outgrowth by additionof EBV and could be cultured for periods ofup to 1 year; however, no EBV "foot- prints" could be foundintheinfected cells. It isof interest to note that theinfected explants, in contrast totheuninfected ones,contained multinucleate giant cells and cells with abnor- malnuclei that could havearisenfromafusionprocess. Ourdata describing EBV-induced cell fusion provide apossible expla- nationfor thepresenceofEBV in EBVreceptor-negativecells.

Itcannotbe ruledoutthatlymphocytespresent inthe spec- imens werethe primary targets forEBVand afterentering a lytic cycle theytransferred their viral genomes totheepithelial cells via apartialorcomplete fusionprocess.Theexceptionally closecontactbetween the immobilized cellswithinthemono- layersprepared for these studies allowedustodetectthefusion- inducing ability ofRaji cells expressing early viral functions.

Onemight speculate thatsimilareventscould takeplacein vivo.

Theexceptionally closecontactbetween lymphocytes andep- ithelialcellsfoundintheWaldeyer'sringof the throat(20)may providethe necessaryconditionsofintimatecell-to-cellcontact for cell fusion. A

lymphocyte

carryingEBVandexpressing the fusion-inducingproteins inclosecontactwithanepithelialcell couldfuseto itandso transferitsloadofEBVgenomesalong with other cellularcomponents. Theinfectability of epithelial cells from tumors(21)mayreflectanalterationinthe

biological

properties ofthesecells; itis conceivable thatamongthe ma- terialstransferred from thelymphocytes duringafusionevent maybenot onlyEBVgenomesbut alsosomethingconferring theabilitytocarrytheEBVreceptorsonthe cell surface. Under normal conditionsonewouldexpectthat fusionis not afrequent eventbecause the induction ofEBVinlymphocytesseems to bea rare event[aconsequenceof thisisthat antibodiesto EA disappear rather rapidly after acute disease (22)], thus one would expect that EBV-containing epithelial cells would be present in verysmall numbersand would finally, after yearsof latency, give rise toclinicallymanifestedmalignant growth.

The geographical distribution ofEBV-associated nasophar- yngealcarcinomacould reflect thedistributionofvariousstrains ofEBV

differing

intheir

ability

toinducecell fusion. Alterna- tively, itmay reflect the presence ofagents within

high-risk

areaswith thepotentialtoinduce thesynthesis ofearly EBV proteins inlatently infected lymphocytes andso increase the probabilityof fusion events. Indeed, recent studies (23)dem- onstrate that thehigh incidenceareaofnasopharyngeal carcinoma insouthern China follows almostexactlythegeographical distribution of Croton tiglium Linnaeus, a plant containing phorbolesters,whichisusedintraditional Chinese herbalrem- edies. The apparent correlation between the presence ofphor- bolesters intheenvironmentand theoccurrenceofnasophar- yngeal carcinomamight atfirstseemratherpuzzlingbecause these substances lead to expression of viral antigens and hence cell death. Synthesis of early membrane antigens may be

Proc.NatL Acad. Sci. USA 78 (1981) 7165 enough for the immune system toeliminate these lymphocytes.

However, iflatently infectedlymphocyteswereinduced to syn- thesizeimmediateearly EBV proteinsthat were nonantigenic (9), they could then fuse to the epithelial cells in Waldeyer's ring, transferring their EBV genomes.

Previousstudies (24)have shown that the inducibility of EBV ishighlydependentuponthe type ofcellwithin which it resides and, furthermore (25), it was shown thatahuman epithelioid cellline(D 98 variant ofHeLa)containedafactor that repressed theexpression of EBV in superinfected nonproducer cells (F 256).

Thus EBV genomes from induced lymphoid cells may be transferredbyfusiontoepithelialcellsdevoid ofEBV receptors, where cellspecific factors could repress the further expression of the viralgenomes,allowing the manifestation of the immor- talizing potential of EBV.

The authors thankDr. H.Rodt forsupplyingALGandMrs. G. Deby forexcellent technicalassistance. The workwassupported bya grant from theDeutscheForschungsgemeinschaft (DFG 227/2) and fromthe Sonderforschungsbereich51.

1. Bayliss, G. J. &Wolf, H.(1980)Nature(London) 287, 164-165.

2. Hampar, B., Derge,J. G., Nonoyama, M.,Chang, S.-Y.,Taga- metes, M. A. &Showalter, S. D. (1974) Virology 62,71-89.

3. zur Hausen, H., O'Neill, F. J., Freese, U. K. & Hecker, E.

(1978)Nature(London) 272,373-375.

4. Bayliss, G.J. &Wolf,H. (1981)J.Gen.Virol,54, 397-401.

5. Rodriguez, J. &Deinhardt, F.(1960)Virology12, 316-317.

6. Luka, J., Kallin,B. &Klein,G. (1979) Virology94,228-232.

7. Bayliss, G. J. &Nonoyama,M. (1978)Virology 87,204-207.

8. Wolf,H. &Bayliss,G. J. (1979)inAntiviralMechanismsinthe ControlofNeoplasia, ed. Chandra,P.(Plenum, NewYork),pp.

315-329.

9. Bayliss, G.J. &Wolf,H. (1981)J.Gen.ViroL,inpress.

10. Feighny,R.J.,Farrell, M. P.&Pagano, J. S.(1980)J.Virot87, 204-207.

11. Bayliss,G.J.&Wolf,H.(1979)inAdvancesinComparativeLou- kemiaResearch, eds. Yohn, D., Lapin, B. &Blakeslee, J. (El- sevier, NewYork),pp.381-382.

12. Tovey, M. G., Lenoir,G. &Begon-Lours, J. (1978)Nature(Lon- don) 267, 270-272.

13. Wolf, H., zurHausen, H. &Becker,V. (1973)Nature(London) NewBiol244,245-247.

14. Wolf,H.,Werner,J. & zur Hausen, H.(1975) Cold SpringHar- borSymp. Quant.BioL39,791-796.

15. Desgranges, C., Wolf, H., de The, G., Shanmugarantom, K., Cammoun, N., Ellouz, R., Klein, G., Lennert, K., Munoz, N.

& zurHausen, H.(1975)Int.J.Cancer16,7-15.

16. Klein, G., Giovanella,B., Lindahl,T., Fialkow, P.,Singh, S. &

Sehlin,J. (1974)^Proc.Natl, Acad.Sci. USA71,4747-4751.

17. Huang, D. P., Ho, J. H.,Henle, W. &Henle,G. (1974)Int.1.

Cancer14, 580-588.

18. Glaser,R., Lang,C. M.,Lee, K. J., Schuller,D. E.,Jacobs,D.

&McQuattie,C.J. (1980)NatICancer Inst.64,1085-1090.

19. Huang, D. P., Ho, H.C., Mun, H. N.-G. &Lui, M. (1977)Br.

J.Cancer35,630-634.

20. D6hnert, G. (1977)in Sitzungsberichtder Heidelberger Akade- miederWissenschaften (Springer, Berlin),Vol. 3,pp.96-168.

21. Glaser,R., deThe, G., Lenoir,G.&Ho, J.H.(1976)Proc.NatL Acad.Sci.USA73,960-963.

22. Wolf, H., Bayliss, G. J. &Wilmes, E. (1981) in Cancer Cam- paign, NasopharyngealCarcinoma, ed. Grundman, E. (Gustav Fischer, Stuttgart),Vol. 5,inpress.

23. Ito, Y.,Kishishita,M., Morigaki,T., Yanase, S.&Hiragama,T.

(1981) in CancerCampaign, Nasopharyngeal Carcinoma, ed.

Grundman,E. (GustavFischer,Stuttgart),Vol.5, inpress.

24. Glaser, R., Farrugia, R. & Brown, N. (1976) Virology 69, 132-142.

25. Glaser, R., Zimmermann,J., St. Jeor, S. &Rapp, F. (1975)Vi- rology64,289-292.