Rescue of Epstein-Barr Virus from Somatic Cell Hybrids of Burkitt Lymphoblastoid Cells

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JOURNALOF VIROLOGY, Aug. 1972, p. 288--296

Copyright ©T 1972 AmiiericanSocietyforMicrobiology

Vol. 10, No. 2 Printted ill U.S.A.



Epstein-Barr Virus from Somatic Cell


of Burkitt

Lymphoblastoid Cells


Department of Microbiology, College of Medicinie, Miltont S. Hershey Medical Celnter, Penllnsylvaniia State

Untiversity, Hershey, Pennsylvania17033

Receivedforpublication 23 May 1972

A Burkitt lymphoblastoid cell line, P31-HR-I, was fused and hybridized to a

human sternal marrow cell line. The somatic cellhybridswere negativewhen

ex-amined forEpstein-Barrvirus(EBV)markers. When thehybridcellswereexposedto

5-iododeoxyuridine, both EBV-specific antigensand virusparticleswereinducedas

determined bythe immunofluorescence testand byelectron microscopy. The data

presented suggestthat the EBVgenome canbe transferred froma lymphoblastoid

cellto anothercelltypeduringcellhybridization, that the EBVgenome canpersist in the hybrid cells forlong periods oftime, andthat synthesis ofthe viruscan be

induced in the heterokaryons.

Inactivated Sendai virus has been used to

in-ducecell fusion inseveral laboratories(7, 12, 21). Theprocedurehasbeen appliedtocancerresearch where somatic cell hybridization (3, 15) has

proventobeafruitfultechniquein manystudies. The rescue ofsimian virus 40 from transformed cells after they were fused to potentially suscep-tible cells (6, 16) hasgreatly stimulateduseof the method invirus laboratories. The method, when applied to papovavirus systems, has already al-lowed observations that suggest that, in somatic

cell hybrids of normal and virus-transformed

cells, virus-specifictumorandtransplantation

an-tigens can be transferred intothe hybridandcan

persistinthehybridcells forlongperiodsof time



Theassociation of a herpesvirus designated the

Epstein-Barr virus (EBV) with many human

lymphoblastoid cell lines has been reported (8).

A small percentage of the cells containthe EBV

(9) as wellasantigens which canbe detected by

the immunofluorescence test (13). There is also

some evidence that theEBVgenomeisintegrated

inthe host cell genome (19, 27).

Recent findings have indicated that Burkitt lymphoblastoid cells can be fused and hybridized

to both mouse and human cells (R. Glaser and

F. J. O'Neill, Science, in press). In this report, further data is presented concerning the proper-ties of the Burkitt lymphoblastoid somatic cell hybrids and the induction of synthesis of EBV-specificantigens and virus particles using the drug 5-iododeoxyuridine (IUDR).


Cells. The human sternal marrow cell line, D98'

AH-2, (D98) (25) was maintained in Eagle medium

supplemented with


fetalcalfserum, 100units of

penicillin/ml, 100 ,ug of streptomycin/ml, 1 jAg of

fungizone,/ml, 10unitsof mycostatin/ml, and 0.075%

NaHCO3 in 8-oz (ca. 0.24 liter) glass prescription

bottlesat37C. The Burkittlymphoblastoidcell line,

P3J-HR-1, (HR-1) was maintained in RPM1 1640

mediumsupplemented with


fetal calfserum, 100

units ofpenicillin/ml, 100,ugofstreptomycin/ml, 1Mg

of fungizone/ml, 10 units of mycostatin/nd, and

0.075c% NaHCO3 in 8-oz (ca. 0.24 liter) glass pre-scriptionbottles at35C.

Fusion andhybridization. Themethod forfusion of

D98cellstoHR-1 cellshasbeenpreviously described

(R. Glaser and F. J. O'Neill, Science, inipress). The

procedure was as follows: 2 X 106 D98 cells were

seededinto tissueculture platesand grownfor 24 hr

at37C.HR-1 cellswereprepared at aconcentration

of 107 cells in0.2ml inEagle medium without serum.

The D98 cells were washed with phosphate-buffered

saline (PBS). Sendai virus (400-1,000

hemagglutinat-ing units in0.2mlof PBS) wasinactivated with

ultra-violet light andadded to the center of the cell sheet and to the HR-1 cell suspension. Both theHR-1 and

D98 cellswerecooled onice for 5min. Then thecell

suspensionwasplacedon themonolayer culturesand

cooled for5 min onice, and thecultureswereplaced

at 37 C for3 to4 hr in an atmosphereof



Threedays afterfusion,thenormalEagle mediumwas

replacedwith the selective HAT medium containing

aminopterin and thymidine (17), supplemented with

10%c fetal calfserum, 100 units of penicillin/ml, 100

lAg of streptomycin/ml, 1 ,Ag of fungizone/ml, 10

units of mycostatin/ml, and


NaHCO3. The

cells were maintained in HAT medium in 100-mm


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tissue culture plates inanatmosphereof5%OCO2 at


Somaticcell hybrids. Afterthe D98 cells haddied

and colonies of hybrid cells (D98/HR-1) were

ob-served, thecellsweretrypsinizedandclonedin 35-mm

tissue culture plates. The cells weremaintained

con-tinuouslyin HAT selective mediumat37C in 250-ml


Treatment withIUDR. The D98/HR-1 cells were

trypsinized and grownon18-mmglasscoverslipsin

60-mmplastictissueculture plates.Whencell

mono-layerswereobtained, the HATmediumwasreplaced

with 5 ml of Eagle medium containing 40 ,g of

IUDR/ml. The cells were incubated at 37 C for 3

days,atwhich time theEaglemediumcontainingthe drugwasreplacedwith normalEaglemedium foran additional3days.

Immunofluorescencetechniques.Both the direct and


EBV-specific antigens.Coverslipswithmonolayersof

IUDR-treated cells were washed with

tris(hydroxy-methyl)aminomethane (Tris)-buffered saline, pH7.4, air dried, and fixed in acetone for 3 min. For the

directtest, the cellswere rehydratedin Tris and

ad-sorbed with humanserumpositivefor EBV antigens

(by immunofluorescence) previously conjugated to

fluorescein isothiocyanate (FITC) (obtained from

CharlesPfizer &Co.)for 30min,washed three times with Tris, two times with distilled water, air dried,

and mounted in elvanolonglassslides.

Fortheindirecttest,acetone-fixed cells were

rehy-drated in Tris, adsorbed with human EBV-positive

serum (tested on HR-1 cells) for 30 min, washed

three times with Tris, and readsorbed with rabbit


for 30 min. The cells were washed as before and

mounted. Preparations were examined for

EBV-spe-cific fluorescence with a Zeiss microscope with an



mon-olayer D98/HR-1 cell cultures that had been treated

with IUDRwereprepared, washed,and fixedas

pre-viously described.The cellswereadsorbed with

EBV-specificantiserum for 30min, washedasbefore, and


FITC, washed, dried, and mounted. The cells were

thenexaminedforEBV-specific antigensby usingthe


Electron microscopy. Cultures ofD98/HR-1 cells

weregrownin 250-mlplastictissue culture flasks and

treatedwith IUDRaspreviouslydescribed. At3, 5,7

and 10 daysafter removal ofIUDR, the cell

mono-layers were washed with


7.3, and scraped

off the glass with arubber policeman. Any floating

cellsin theculturewerecombined with the cells from

themonolayer andfixedinKamovsky'sfixative (14)

overnight at 4 C. Thespecimens were postfixed for 2 hr with Dalton's chrome-osmium (4) (containing 2%osmiumtetroxide), dehydrated, andembedded in

Spurr's low-viscosityplastic (24).Thin sections were

prepared by using an LKB ultratome; the sections

were stainedwith 0.5% uranylacetatein


meth-anolandReynold's lead (22), placed on naked copper

grids, and examined with an Hitachi HU-12 electron microscope at 75 kv.


Generalpropertiesofsomatic cell hybrids. When D98 cells were grown in selective HAT medium, the cells died within 2 to 3 weeks. Briefly, D98

cells do notcontain the enzyme IMP

pyrophos-phorylase and are not able toutilizehypoxanthine in the "scavenger" pathway of deoxyribonucleic

acid (DNA) synthesis. The HAT medium

con-tains aminopterinand thymidine which selectively

inhibits de novo purine synthesis but allows cells

capableofsynthesizingDNA, utilizing preformed

purines, to grow (25). However, after fusing the

D98 cells to the Burkitt lymphoblastoid cell line

HR-1 (acellline which only grows insuspension

invitro),theresultingheterokaryons were able to

growinthe selectivemedium, due to the synthesis

ofthe enzyme IMP pyrophosphorylase mediated

by the genome of the HR-1 parent. The fused

cells developed into somaticcell hybrids and were

cloned. Detailed chromosome studies were

per-formed. The modal numbers of the D98/HR-1

clonesexaminedwere approximately equal to the

sumof the modal numbers of the D98 andHR-1

parental cell lines (R. Glaser and F. J. O'Neill,

Science, inpress).

Themorphology of the D98/HR-1 cells, which growasmonolayers, was unlike the morphology

of theD98 orthe HR-1 cells, presumably due to

thecomplement of chromosomessupplied by both

parental lines. The hybrids were larger in size,

and thegrowthpatterns on aglass or plastic

sur-face weredifferent (Fig. 1 and 2). The D98 cells

were contact inhibited (Fig. 3). In contrast, the D98/HR-1 cells were observed to grow frequently

inmultilayered foci (Fig. 4).

EBV specific antigens. When cover


of D98/HR-1 cells were fixed in acetone and tested

for EBV-specific antigens by the direct and

in-direct immunofluorescence test, no evidence of

virus-specific antigenswasfound. These tests were

performed at2, 3,4and 8monthsafter fusion.

Induction ofEBV-specific antigens after treat-ment with IUDR. Coverslip cultures of D98,

hu-manembryoniclung(HEL),and D98/HR-1 cells

clone (cl) 1, 2, 3,and 8 were prepared. Attempts

were made to induce EBV-specific markers by

using 40 ,ug of IUDR/ml in Eagle medium. The

cells weretreated for 3 days after which the

me-diumcontainingIUDR wasreplaced with normal medium for an additional 3 days. Acetone-fixed D98, HEL, and D98/HR-1 cells were tested for EBV-specific antigens by using the direct and

in-direct immunofluorescence tests. The results are

VOL. 10, 1972 289

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.:S ",

.-r. y


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#. w *i

_-t d: ..

.w: ^

FIG. 1. Photornicrograplhof D98 cellsstainied with hematoxylini antd eosini. X527.

FIG. 2. Pliotomnicrograph ofD98/HR-1 cellsstainied with hematoxylintandeosini. X527. 290

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''9~~~~~~~~~~~~~O 1

Va JL~~~~~~~~~~~~~~~

IO ~ %* p

* ,.. ; * 4


Sw~~b~~: 4P~~~







~ ' -~.

-FIG. 3.Photomicrographof D98 cellsgrowingincell culture. X280.

shown in Table 1. All4clonesofD98/HR-1 cells containedEBV-specificantigens bythe direct and indirect immunofluorescencetestwithavarietyof human sera (pretested on HR-1 cells). The sera

werefrompatientswith infectiousmononucleosis

or with nasopharyngeal carcinoma. Cytoplasmic

and nuclear fluorescence was observed (Fig. 5



infrom1 to20% of thecells. Normal and


D98 and HEL cells under thesame

test conditions were


Serumblockingtests.Toobtain additional data



of the reactions observed,serum

blocking tests were performed. D98/HR-1 cells,

grown on cover slips and treated with IUDR,

were exposed to serum containing antibodies to

EBV. This was followed by exposure to serum




(used in the direct immunofluorescence


Re-actions wereobserved incellswhere the



serum was used at adilution of 1:10,

but no EBV-specific antigens were observed


fluorescence when undiluted EBV antiserum was used to block thereaction.

Todetermine if the EBV antiserum used in the serum blocking experimentswould block the im-munologic reaction in a nonspecific manner, it was tested on rabbit kidney (RK) cells infected with herpes simplex virus type 2 (HSV-2). The EBV-positive serum was adsorbed on acetone-fixed monolayercell cultures ofRKcells infected with HSV-2 (pretested with rabbit anti HSV-2 antiserum and found positive for HSV-2-specific antigens by immunofluorescence). The cells were then adsorbed with rabbit anti-HSV-2 antiserum followed by horse anti-rabbit antiserum conju-gated to FITC. The EI3V-specific antiserum did

not interfere with the detection of the

HSV-2-specific antigens.

Synthesis of virus particles. When cultures of D98/HR-1 cells were examined at 3, 5, 7and 10

days afterseedingin tissue cultureflasks, novirus

particles were observed. However, when D98/


10, 1972 291

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FIG.4.Potom aWh o growti-ng in ce re. X280n,

FIG.4.Photomicrograph of


cellsgrowiniginl cell culture. X280.

HR-1 cells previously treated with IUDR were


nu-clei ofdegeneratingcellsondays7and 10(Fig.7).

Both enveloped (inthe cytoplasm) and

unenvel-opedparticleswerefound in many cells examined.

In asecondsetofexperiments, D98/HR-1 cells


intotissue culture flasksorgrownoncoverslips.

One set offlasks and cover slip monolayer cell

cultureswastreated withIUDR; the otherset was

notexposedtothedrug. Seven days after removal of IUDR from the treatedcultures, both sets of cell cultures were fixed for electron microscopy

examination. Thecoverslip monolayercellswere

fixed and examined by the direct

immunofluores-cencetest,by the methodalready described.

The D98/HR-1 cells treated with IUDR

con-tainedvirusparticlesin the nucleiof about


of the cells examined and some cells exhibited

particlesin thecytoplasm;however,novirus

par-ticleswereobserved in the untreatedcells. When

the specimens prepared for immunofluorescence

were examined, the cells treated with IUDR con-tained EBV antigens as described previously. No EBV antigens or particles were detected in the untreated cells (Table2).


The results presented in this report and in a preliminary report (R. Glaser and F. J. O'Neill, Science, in press) suggest that Burkitt lympho-blastoid cells have been fused andhybridized with

both mouse and human cells. The chromosome

data, along with the alteration in cell morphology and ability to grow ina selective medium, were the criteria used to support evidence of somatic


The data obtained from chromosome analysis

from earlierwork suggested that most, ifnotall,

the HR-1 chromosomes were in the nuclei of the D98/HR-1 cells. In addition, EBV-specific

anti-292 J. VIROL.

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FIG. 5. Inmunofiluorescencephotomicrograph cf EBVantigenislocalized in the cytoplasm inD98/HR-J cells

treated with40 ,ug ofS-IUDR/ml


r3 daysand thenexaminted3dcays afterthe cells weregr.wninnormal

me-diumn. Many oftilecellscontainin1gEBVantigeniswereroun1ded. X)527.

FIG. 6. Immunofluorescence photomicrograph cfEBVantigens localizedin the nucleuisofa D98/HR-I cell

treated with40,ugcfS-IUDR mlfor3daysandthen examined3days afterthe cellsweregrowninnormal

me-dium. Note theevendistributionicfEB Vantigensthroughoutthenuclelus. X 840.


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TABLE 1. Iniductioni of EBV-specific antigens in

D98/HR-J cells treated withIUDR asmeasured

by the immuniofluorescenice test

Immunofluorescence-Avg percent ofpositivecells Cells




D98 + IUDR


Clone I

Clone 2 Clone 3 Clone 8

D98/HR-l + IUDR

CloneI Clone 2 Clone 3 Clone 8 Directa test 0 0 0 0 0 0 10-20 10-20 10-20 10-20


la 2b 3'

0 0 0 0 0 0 0 0 0 0 0 0 0 0 5-15 5-15 5-15 5-15 0 0 0 0 1-2 1-2 1-2 1-2

a Serums(diluted1:10)frompatientswith

naso-pharyngeal carcinoma.

bSerum (diluted 1:5) from a patient with in-fectious mononucleosis.

cSerum (diluted 1:2) from a normal patient knowntobenegativeforantibodies againstEBV.

dHumanembryonic lungcells.

e 40jig/ml.

gens, as determined by the immunofluorescence

tests, have beeninduced in thehybrid cellswhen

the cells weretreated with IUDR. Thereare

pre-liminary results (Glaser, unpublished observations)

that induction can also be accomplished with

5-bromodeoxyuridine (BUDR). The data suggest

thatatleastpart ofthe EBVgenome was

trans-ferred from the HR-1 cells to the hybrids and

has persisted for 10 months without expression

of EBV markers, as determined by electron

mi-croscopy and by immunofluorescence. These

re-sults support current concepts on the intgerated

state of the EBVgenome (11). Cloning and

nu-cleic acid hybridizationdatasupportthe concept

thatthe EBVgenomecanpersistwithout

expres-sion for long periods oftime in what had been

presumed to be EBV-negative cells (19, 20). To

furthersupport thisconcept,preliminary data

in-dicate that thereareseveral EBVgenomesineach

D98/HR-1 cell,asdetermined by nucleic acid

hy-bridization experiments now being performed

(M. Nonoyama, personal communication).

An important conclusion can be drawn from

thedatapresented. Namely,the EBVgenomecan

persist and can be activated to synthesize virus

in a cell type other than a lymphoblastoid cell. The possibility exists that the lymphoblastoid cells, in which the EBV is found invivo, maynot

be the sole site of infectious virus replication, an

observation already made in the case of the Marek's disease herpesvirus (2).

Otherinvestigators have reported the induction of EBVantigens in "normal" lymphoblastoid cell lines with BUDR (10). In thepresenceofBUDR, both nuclear and cytoplasmic antigens were de-tected. In addition, ribonucleic acid (RNA)

vi-ruses have also been induced in cell lines with

BUDR or IUDR (1, 18). Thus, the results

pre-sented here concerning induction of EBV

anti-gensin somatic cellhybrids reveal that induction of virusin hybrids mayfollowa pathway similar

to that invirus-transformed cells.

The sera used in the indirect and direct im-munofluorescence tests were known to be highly specific. They had been tested against cells in-fected with other herpesviruses and RNA tumor

viruses as well as avariety of celllines in this

lab-oratory and at Pfizer & Co. (K. Traul,personal

communication). Thedata fromthe serum-block-ing experiments and the induction experiments with D98 and HEL cells also support the

speci-ficity of the antigens observed in the D98/HR-1


There was no emperipolesis of the HR-1 cells by theD98 cells asdetermined bybrightfield and phase-contrast microscopy, discounting the possi-bility ofcarry-over oflymphoblastoid cells. To further support this, no evidence of the

persist-ence ofHR-1 cells was found by visual

observa-tions and bychromosome analysis when the cells

were cloned. Furthermore, the cells that are posi-tive in theimmunofluorescence test and by

elec-tronmicroscopy areepithelial-like in morphology. Whether the D98/HR-1 cells are transformed

isstill not clear. Thehybrid cells grow in

multi-layers, indicating a loss of contactinhibition. The D98 cells have never been observed to do this. However, further work is necessary to clarify this point.

Theproduction of EBV particles in the IUDR-treated cells clearly shows that the EBV genome maintained in the hybrid cells are complete enough to code for EBV DNA and capsid pro-teins, since complete virus particles with dense cores were found. Whether enough information is presenttocode for the synthesis of "infectious"

EBV with a higher oncogenic potential than the

EBV presently obtained from lymphoblastoid cells remains to be determined. If infectious, the

propertiesofthis virus and its ability to replicate

or transform various cell types will represent an important extension of this work.

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FIG. 7.Electronz micrograph of D98/HR-1cells treated with 60 jig of


7days after replacemenzt of medium conitaining IUDR with ntormalmedium. Noteparticles with densecoresin the niucleus and cytoplasm (arrows). Barrepresenits I,um.

TABLE 2. Iniductionz of EBV-specific anttigenis antd

virus particlesinD98/HR-1 cells treated with

5-iododeoxyuridinie (IUDR)

CellsCells Presence of virus


Presenceparticlesof virus

D98/HR-1 None detected None detected

D98/HR-1 + Present Present


Serum(diluted 1:10)fromapatientwith


bAt 60


for 3 days; cells were fixed for

electron microscopy 7 days after the removal of

IUDR (performedat37C).


We are grateful to Patricia Nelson and Ross Farrugia for excellenttechnicalassistance. Wethank Karl Traul andGuyde Thefortheseraused in thestudy.

Thisstudy wasconducted under Public Health Service con-tract 70-2024 within the Special Virus Cancer Program of the Nationial Cancer Institute.


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FIG. 1. Photornicrograplh of D98 cells stainied with hematoxylini antd eosini. X527.
FIG. 1. Photornicrograplh of D98 cells stainied with hematoxylini antd eosini. X527. p.3
FIG. 2. Pliotomnicrograph ofD98/HR-1 cells stainied with hematoxylint and eosini. X527.
FIG. 2. Pliotomnicrograph ofD98/HR-1 cells stainied with hematoxylint and eosini. X527. p.3
FIG. 3. Photomicrograph ofD98 cells growing in cell culture. X280.
FIG. 3. Photomicrograph ofD98 cells growing in cell culture. X280. p.4
FIG.4.Potom Photomicrograph
FIG.4.Potom Photomicrograph p.5
FIG. 5.diumn.treated Inmunofiluorescence photomicrograph cf EBV antigenis localized in the cytoplasm in D98/HR-J cells with 40 ,ug of S-IUDR/ml f
FIG. 5.diumn.treated Inmunofiluorescence photomicrograph cf EBV antigenis localized in the cytoplasm in D98/HR-J cells with 40 ,ug of S-IUDR/ml f p.6
FIG. 6.dium.treated Immunofluorescence photomicrograph cf EBV antigens localized in the nucleuis of a D98/HR-I cell with 40 ,ug cf S-IUDR ml for 3 days and then examined 3 days after the cells were grown in normal me- Note the even distributioni cf EB V an
FIG. 6.dium.treated Immunofluorescence photomicrograph cf EBV antigens localized in the nucleuis of a D98/HR-I cell with 40 ,ug cf S-IUDR ml for 3 days and then examined 3 days after the cells were grown in normal me- Note the even distributioni cf EB V an p.6
TABLE 2.virus particles

TABLE 2.virus

particles p.8
FIG. 7.of(arrows). medium Electronz micrograph of D98/HR-1 cells treated with 60jig of S-IUDRI'ml 7 days after replacemenzt conitaining IUDR withntormal medium
FIG. 7.of(arrows). medium Electronz micrograph of D98/HR-1 cells treated with 60jig of S-IUDRI'ml 7 days after replacemenzt conitaining IUDR withntormal medium p.8