DNA Epstein-Barr

Proc.Nati.Acad.Sci. USA

Vol.77, No. 1, pp. 433-436,January1980

CellBiology

Expression of Epstein-Barr virus genes in different cell types after microinjection of viral DNA

(early antigen/viral capsid antigen/thymidine kinase)

A. GRAESSMANN*, H. WOLFt, ANDG. W. BORNKAMMt

*InstitutfurBiochemieundMolekularbiologie derFreienUniversitAtBerlin,Arnimallee 22,1Berlin33,Germany; Max-von-Pettenkofer-Institutder UniversitatMfinchen, Pettenkoferstr.9a,8Muinchen,FederalRepublicofGermany;andtInstitutfurVirologie,Zentrum furHygiene, UniversititFreiburg, Hermann-Herderstr.11, 78Freiburg, FederalRepublicof Germany

CommunicatedbyWernerHenle, October 22,1979

ABSTRACr Gene expression of Epstein-Barrvirus(EBV) was studied aftermicroinjection of viralDNAintodifferent types of cells. Raji TK- cells, known to express viral gene functions aftersuperinfectionwith theEBV-P3HR-1 virusstrain, wereattachedtoplasticdishesby usinganti-lymphocyte IgG, phytohemagglutinin,orconcanavalinAasaligand.Itwasdif- ficult to injectDNA intothe smallandfragile Rajicells. After formation of polykaryonsby cellfusion, microinjectionbecame moreefficient.At 24hr afterinjectionof P3HR-1virusDNA, 90-100% of the injected cells expressed the early antigen complexasobservedby immunofluorescence staining 70-80%

ofthe cellssimultaneously incorporated

[3H]thymidine,

indi- catingthatthymidinekinase isexpressedafterinjectionof viral DNA. Additionally, synthesisof the viruscapsid antigenwas demonstrated in20-30% of therecipient Raji cells. Human diploidfibroblasts,Africangreenmonkeykidney cells,and rat fibroblasts, which do not represent natural target cells forEBV, could also be induced tosynthesisofearly antigen complexby injectionofP3HR-1virus DNA.

Epstein-Barrvirus(EBV)has been foundregularlyassociated withtwohumanmalignancies,Burkitt'slymphomaandnaso- pharyngeal carcinoma (for review, see refs.1and 2). Thevirus transforms human lymphocytes into permanently growing lymphoblasts(3-6)and induces a malignantlymphoprolifer- ativediseaseuponinoculation into NewWorld primates (7-10).

It isofparticularinteresttostudythetransforming capacity of EBVbecause of a possible causative role of the virus in the developmentofmalignanciesinman.

Oneapproach to thestudy ofthe EBVgenome has been comparisonoftheDNAofviralstrainswith differentbiological properties(11-13). The majority of EBV isolates, regardless of whether derived from patients with infectious mononucleosis, Burkitt'slymphoma,or otherdiseases, are transforming viruses and donotinduce theearlyantigen complex (EA) upon su- perinfectionof Raji cells (a nonproducer cell line of Burkitt origin). In contrast, the strain P3HR-1 (originating from a Burkitt tumor) is a nontransforming virus with the unique ca- pacitytoinduce theEA inRajicells (14, 15).

Descriptive comparativestudies have been published which identified sequencesdeleted from or inserted into different viral strainsby partial denaturation mapping (16) and by analysis oftherestrictionenzymecleavage patterns (17, 18). Although substantialprogresshas been made in the physical character- izationof theEBVgenome by Givenand Kieff (18), attempts tomapbiological functions on the genome have not been re- ported.Considerable effort was made in different laboratories toadapt transfection techniques that appeared to be useful in thestudyof other herpes viruses (19-21) to EBV; however, so farthey have beenwithout success.

Here we report thatbiological functions of EBV can be

studiedby microinjectionof EBV DNA into different typesof cells. Rajicells were selected formicroinjectionof EBVDNA because theycan beabortively infected with P3HR-1 virus, resulting eveninvirusparticleproduction. Inaddition, viral gene expressionwasstudied afterinjection of P3HR-1 virus DNA intohumanand rat fibroblasts and into Africangreen monkey kidney cells,which are notnaturally susceptibletoviral infection.

MATERIALS AND METHODS

Cells andVirus. Humanembryonicfibroblasts(strainWI-38 and our ownisolates),secondary green monkey kidneycells(TC 7and our ownisolates),and rat fibroblasts(rat 1)were grown onglass slidesin aminimal essential mediumsupplemented with10% fetal calfserum. P3HR-1cells were obtainedfrom W.Henle; RajiTK-cellswerefrom M.Nonoyama.These cells weregrownin RPMI-1640(GIBCO)with 10%fetal calf serum.

SpontaneousreversionofRajiTK-cells to theTK+ phenotype was notobserved, eventhoughthe cells werekeptinnonse- lective medium for many months.

EBVwaspurifiedfromP3HR-1cells.Virus wasinducedby addition of12-O-tetradecanoylphorbol-13-acetate (20 ng/ml;

Sigma) 1 day after the cellsweresplit (22).Virus waspurified from cells and supernatantsasdescribed(23),startingfrom1.2 liters ofculture fluid, except that dextran T-10 (Pharmacia)was used forgradient centrifugation instead of sucrose (24). The visible virus bands were collected, and the virus was pelleted at 20,000 rpmfor 1 hrat40Cina SW 27 rotor(Beckman), resuspended in virus suspension buffer (VSB; 0.01 M Tris- HCI/0.01 M KCI/0.005 M EDTA, pH 7.4), and stored at -70'C. Oneof fiveviruspreparationswaslabeled with[3H]- thymidine(1

ACi/ml;

1Ci=3.7X1010 becquerels) 1 day after addition of thephorbol.

Purification ofP3HR-1VirusDNA. Virusisolatedfrom a total of 12liters ofP3HR-1 cell cultures waslysed with 1%

sarcosyl/0.025MTris-HCI/0.01 MEDTA, pH 8.5, and incu- bated with proteinaseK(100,ug/ml;Merck) at370C for2hr.

Then,0.01 MTris-HCI/1mMEDTA, pH 7.5,and solid CsCl wereaddedto afinal volume of 10mland a final density of 1.705g/ml. Thelysate was centrifuged for 72 hr at 30,000 rpm ina 50 Tianglehead rotor (Beckman) at 20'C. Fractions (four drops) werecollected fromthe bottom of the tube through a syringe needle(1.25-mm innerdiameter) at a restricted flow rate. Aliquots (3Ml) were assayed for radioactivity, and the DNA concentration was determined in the fractions neigh- boringthepeak fraction by measuring the optical density at 260 nm. TheDNAconcentration in the peak fraction estimated fromthe specific activity was 140Ag/ml.A portion (100Ml)of thepeak fraction was loaded onto a Sephadex G-50 column (5.0

Abbreviations: EBV, Epstein-Barrvirus;EA,earlyantigencomplex;

VCA,viruscapsidantigen.

433

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434 Cell Biology: Graessmann et al.

X 0.35 cm) and elutedwith 0.01 MTris-HCI/1mM EDTA,pH 7.5,in

25-,Ml

fractions.The two peak fractions of the Sephadex column withDNAconcentrations of100,g/mlwere usedfor microinjection experiments.

Attachmentof Raji Cells to Plastic Petri Dishes. Plastic 60-mm petridishes containing2.5ml of2.5%glutaraldehyde wereincubated for2hrat room temperature. The plates were washedfour times with sterilewaterandcovered with

200,gl

of anti-lymphocyte IgG (1.3 mg/ml) (kindly provided by H.

Rodt). Alternatively, other ligandsknown tobind tocellsur- faces,such as phytohemagglutininP(Serva), concanavalinA (Serva),pokeweed mitogen (Serva), and poly(l-lysine) (Sigma) wereused ina concentration of 100

gg/ml

for coating the plates.Afterincubation for 1-2 hr at 370C in a humidified at- mosphere,the fluid was once more homogeneously distributed onthe surface of the plates and allowedtodry. Afterdrying, theplates were washed twice with phosphate-buffered saline without Mg2+andCa2+andfilled with3mlof RPMI-1640 mediumwithout serum. Raji cells were washed four times in serum-free RPMI-1640 medium.About107 cellswereseeded per60-mmplateand wereallowedtosettle for2-3hrat370C inanatmosphereof 5% CO2inair.

Fusionof Raji Cells. Cell fusion was induced by polyeth- ylene glycol 1000 (Sigma) as described byNorwoodetal. (25).

Plates with attached Raji cells were washed three times with phosphate-bufferedsaline(free of Mg2+ and Ca2+)inorderto removelooselybound cells. The saline was aspirated and 1.5-2.5 ml of 45-47% (wt/wt) polyethylene glycol in serum-free RPMI-1640medium,preincubatedat 370C, wasadded to the celllayer.Theglycolsolutionwasleftonthe cells for60-70sec;

then it was removed by three successive washes with phos- phate-buffered saline. The cells wereincubatedinserum-free RPMI-1640medium at 37°C in ahumidified CO2 incubator.

Cell fusion started immediately after polyethylene glycol treatmentandproceeded for several hours.

Immunofluorescence Staining. Fluorescence staining for EPV EAand viruscapsid antigen(VCA) was by standard pro- cedures using indirect immunofluoreseence techniques (14, 26).

Microinjection. Microinjection has beendescribedindetail (27).

Autoradiography. Kodak nuclear track emulsion (NTB) was usedat adilutionof 1:10 (inH20). After exposure for3days at4°C, autoradiogramsweredevelopedwith KodakD-19de- veloper for 10 min atroomtemperature. Afterwashingfor5 min indistilled water, autoradiogramswerefixed with Kodak Unifix for 5 min, washed again for 10 min indistilled water, andairdried.

RESULTS

Expression ofEBVDNAinRajiCells AfterMicroinjec- tion. Largecells firmly adherentto asolid supportarewell suited for microinjection experiments. ThelymphoidRajicells, however, growinsuspensionand aresignificantly smaller than mostmononuclearcells (e.g., human fibroblasts). Raji cellscan beattached efficientlytothe surface ofcoatedplasticdishes (Fig. lb). Coating the dishes with anti-lymphocyte IgG, phy- tohemagglutinin, orconcanavalin A as ligands gave results about equally good. In the case of concanavalin A, serum- containingmediumcould be used. Plates coated withpokeweed mitogenorpoly(l-lysine)had lessbindingcapacityorappeared

I':

I

FIG. 1. (a)Suspension cultureofRajicells. (b) Adherent and spread Raji cells, 5 hr after plating onto an anti-lymphocyte IgG-coated plastic dish;uncoatedregionsarefreeofcells.(c)Fused Rajicells,10hr afteraddition ofpolyethylene glycol. (d) Phase-contrast picture of fused cells.

(X 120.)

Proc.Natl.Acad.Sci. USA 77

(1980)

Proc. Natl.Acad.Sci. USA 77(1980) 435 to be more toxic to the cells. Pretreatment of the cells with

glutaraldehyde prior coating was not essential but seemed to givesomewhat better reproducible attachment.

Single mononucleated Raji cells tolerated injection of only about 5 X 10-12 ml. This volume could be increased about 10-fold when polyethylene glycol-fused Raji cells containing 5-10 nucleiwereusedas recipientcellsfor microinjection (Fig.

1c and d). No spontaneous EBV antigen synthesis wasobserved incellsmade adherent and fused.

After injection of EBV DNA (100 ig/ml) into fused Raji cells, the following virus induced functions were tested:(i)synthesis ofEA'(ii) expression of thymidine kinase; and (iii) synthesis of VCA. EAand VCA were detected by indirect immunoflu- orescence; thymidine kinase was studied by uptake of3H-la- beled thymidine into RajiTK-cells. To studyEAand thymi- dinekinase expression, microinjected cells were incubated for 24hrinserum-free medium with [3H]thymidine (0.1 ACi/ml), fixed with methanol, stained for EA, and subjected to autora- diography. Of the injected cells, 90-100% stained forEAand 70-80%of these incorporated

[3H]thymidine

(Fig. 2 aand b).

Grains were observed over EA-positive cells but not over mock-injected oruninjectedcells. Also autoradiography of cells immediately afterDNAinjectiondid not reveal any grains.

In order to test, whether thymidine incorporation was a consequenceofEAinduction,EAsynthesiswasinducedinRaji TK-cells by the diterpene ester 12-O-tetradecanoylphorbol- 13-acetateTPA.About 10% of the Raji cells expressed EA after 4daysbut did not reveal any[3H]thymidineuptake.

VCA expression was studied 48 hr after injection ofviral DNAinto Rajicells; 20-30% of the recipient cells stained for VCA. The VCA-positive cells were always found to incorporate

[3H]thymidine

(Fig. 2 c andd).

The induction ofEAandthymidine kinase was specific for intactviral DNA; EcoRIdigestionof the DNA abolished both effects.

_P

FIG. 3. EA-positive rat fibroblasts, fixed and stained 48 hrafter injection of EBV DNA. (X450.)

The presence of a vast majority of uninjected cells sur- rounding adefined number ofinjectedcells withinadefined areaof theplate servedas aninternal control for antigenex- pressionandthymidine kinase activity.-

Expression ofEBVDNAinHumanFibroblasts, Monkey KidneyCells, and Rat Fibroblasts. Expression ofEAandVCA wasalso studiedinhumanfibroblasts,secondary African green monkey kidneycells, andratfibroblasts after microinjection ofEBVDNA. These cells donotrepresentnatural targetcells forEBVandcannotbeinfected by thevirus.

At 48hrafter injection, about one-fourth humanfibroblasts, monkeykidney cells, andratfibroblastsrevealedEAstaining (Fig. 3). Fig. 4 shows the time courseof EA appearance in WI-38cells injected with viralDNA.EA-positivecellswerefirst observedat 48hrafter injection.Atthis time,25-30% of the cells exhibitedEAfluorescence. Withprolongedincubation the number of EA-positivecells decreased slightly. After8days, 15% of theinjected cellswerestill^EApositive.NoVCAsyn- thesis could beobservedincells studied 48, 72,or96hr after injectionof viralDNA.

DigestionofP3HR-1 virus DNAwithEcoRIdestroyedthe EA-inducing activity.

b 4

2

u030

0._

20

10_

.~~~~~~~~~~~~~~~~~~~*.... d

FIG. 2. Expressionof viralantigensandthymidinekinase inRaji TK-cells injectedwithEBV DNA.(a) EA-positivemultinucleated Raji cell,24 hr afterinjection. (b) Autoradiogramofsectionina.(c) VCA-positivemononucleatedRaji cell,fixed and stained48 hr after injection. (d) Autoradiogramofareainc.(X450.)

0 48 96

Time,hr

144 192

FIG. 4. TimecourseofEAsynthesisinhuman fibroblasts. Each timepointis basedon100injectedcells.

40H

- _.o

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Cell

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Graessmannetal.

436 CellBiology: Graessmann etal.

DISCUSSION

Microinjectionallows the transferofabout10-11mlintoeach mononucleatedtissueculture cell (27).ForaDNAwithamo-

lecular weight of100X106theDNAconcentrationhastobe atleast17

;g/ml

inordertotransfer statisticallyoneviralge- nomeperinjectedcell. Obviously, higherDNAconcentrations

should be usedtoobtain^anefficienttransfer ofintactDNAinto

the cells, becausesomeof theDNAmight be sheared during theinjectionstep.InductionofEBVsynthesisin carriercelllines byditerpeneestersincreasesthe yield ofvirusDNAconsider- ably (22, 28). This enabled^ustoprepareviralDNAinacon-

centrationof100jig/mlfrom CsCl density gradients without

anyotherconcentrationprocedure, whichmaylead^tofrag- mentationofthe high molecularweightDNA. Freezingofthe DNA,required for lyophilization, destroysatleastpartofthe biologicalactivityof^EBVDNAandwastherefore avoided.

(P3HR-1 virusDNAataconcentrationof25,ug/ml,which^was usedinthe initialexperiments,lostitsEA-inducingactivityafter

twocycles of freezing and thawing.)

Itwasnotpossibletousethe small andnotadherent lymphoid Rajicells directly formicroinjection. But^{we were}able^toanchor these cells byusing plates coated withanti-lymphocyteIgG,

phytohemagglutinin, orconcanavalin A,andtheproblemof the small cellsizeofthe lymphoblastswassolved by cell fusion of the attached cells^topolykaryons.

BecauseRajicellswereattachedtoplasticpetridishes,^ace-

tonecouldnotbe used for fixation, and the cells hadtobefixed withmethanol, whichinactivatestheRcomponentoftheEA (29).Thus, only theexpressionof theEADcomponentcould bestudied.

Wehave shown that P3HR-1 virusDNAiscapable ofin-

ducingEA,VCA,andthymidine kinase afterinjectionof viral DNA into Raji cells. From these experiments, however, no

conclusion^canbedrawn^astowhether thegenesexpressed after viralDNAinjectionareencodedby theinputviralDNAorby endogenous EBV genomes. Itevencannotbeexcluded that thymidine kinaseexpression isduetotheactivationofapre-

viously silent cellulargene.Itwas ourmainobjectivetodem-

onstrate that injected viral DNA shows thesame biological activityastheinfectingP3HR-1 virus.

ThehighrateofconversionofRajicellstoEA expressionafter injection of P3HR-1 virus DNA indicates that the injection

processishighly efficient. Thisisnotsurprisingifoneconsiders that,inplaque-purifiedsimianvirus40preparations, about100 particles^arerequiredtoyield1plaque-formingunit intissue cultureinfection,whereas 1 virusparticleisinfectiousupon

injection intothe cell(30).

EAcanalsobe induced invirus-nonsusceptible human fi- broblasts, African^greenmonkey kidney cells,andratfibroblasts afterinjection of P3HR-1virusDNA.This demonstrates that the natural barrieragainstanEBVinfection of these cellsmust

be locatedinoneof theearlyevents-adsorption, penetration,

oruncoating.Virusadsorptionstudies indicate that these cells donotpossessreceptorsfor EBV(unpublished observation).^A similar mode ofhostrangerestriction, which couldbeby-passed byinjectionofvirusparticles^orviralDNA,has been demon- strated forsimianvirus40andpolyomaviruses(for review,see

ref.30). The factthat humanfibroblasts, monkey kidney cells, andratfibroblastsdidnotsynthesizeviralcapsidproteinsmay

pointtoanadditionalregulationmechanism fortheexpression oflate viralgenesinthese cells.

Untilnow,noEBVnuclearantigenexpressioncould be de- tectedafterinjectionof viral DNAintohumanandratfibro- blasts.Thereasonforthismaybetechnicalandrequiresfurther investigation.

Theexpression ofEBVgenesaftermicroinjectionofviral DNAobviously providesanapproachto mappingbiological functionsonthe viralgenome. Wehave been abletoinduceEA byinjectionofDNAfragments, which will be reportedindetail elsewhere. It will be ofspecialinteresttostudythetransforming potential of EBVDNAand of EBVDNAfragments and to learn if transformationcanbeinitiatedbyinjection ofviral DNA intohumanfibroblasts.

The excellent technical assistance of E. Guhl, M. Haus, and U.

Zimberisgratefullyacknowledged.Wethank Dr. H. zur Hausenand Dr.H.Deliusfor helpful discussions andcritically reading theman- uscript. This workwassupportedby Die DeutscheForschungsge- meinschaft(Gr. 384/5,Ha449/12,Wo227/2, and SFB51).

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