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JOURNAL OFVIROLOGY,JUIY 1989, p.3190-3194 Vol. 63, No.7 0022-538X/89/073190-05$02.00/0

Copyright© 1989, American Society for Microbiology

Negative

Modulation of Epstein-Barr Virus

Episomes

by

a

Human

B-Cell

Growth Factor

JOHN MORGAN, AARON ROOMEt ABBY MAIZEL, AND SURENDRA SHARMA*

Deparutnent ofPathology, Roger Williams GeneralHospital-Brown University, 825Chalkstone Avenue, Providence, Rhode Island 02908

Received 27October1988/Accepted 31 March 1989

Tostudy the effectof T-cell-derivedBCGF-12kDonhuman B-cellautocrinegrowth-associatedfunctions,we cultured Epstein Barrvirus (EBV)-transformed normal B cells(LCL 72285) and Burkitt's lymphoma cells (Raji)in the presence or absenceof BCGF-12kD. When cultured inmedia supplementedonly withfetalcalf serum, the LCL andRaji cell lines maintained relatively highlevels ofepisomes. Althoughasimilar levelof proliferation couldbe maintained under defined culture conditions in mediasupplementedwithBCGF-12kD, these conditions resulted in a time-dependent reduction of EBV sequences, as detected with EBV nuclear antigen (EBNA-1 and EBNA-2) gene probes. These results suggest that stimulation with T-cell-derived BCGF-12kD can alter a regulatory stepwhich may be involved in the EBVtransformation of B cells. It is invariably found that human Bcellsimmortalized by

eithermalignantorEpstein-Barr virus (EBV)transformation

proliferatecontinuously in culture in the apparent absence of exogenous growth factor (14). This immortalized state may bemaintained byanautocrine loop mechanism (4, 5, 11). In contrast, normal antigen-dependent B-cell maturation,

which includes activation, proliferation, anddifferentiation, is thought to be a highly regulated event controlled by distinct T-cell- and monocyte-derived cytokines. Insupport of this notion,a humanT-cell-derived B-cell growth factor, BCGF-12kD, has been shown to act as a costimulant with antibodies toRchains in inducing B-cellproliferation (22, 25, 30). Inaddition,this growth factor has been showntoinduce S-phase entry of malignant Bcells from hairy cell leukemia, B-cell chronic lymphocytic leukemia, non-Hodgkin's lym-phoma (9), and B-cell precursor acutelymphoblastic leuke-mia (36). These observations suggest an important role for BCGF-12kD in the regulation of human B-cell proliferation and are further supported by the recent observations that other described B-cell lymphokines, such as interleukin-2 (IL-2), IL-4, IL-5, and IL-6, are multilineage specific and mayinduce both proliferation and terminal differentiation.

EBV is a human lymphotropic herpesvirus that usually showsamarkedpropensity to infect B cells. The majority of the infected B cells grow in an autocrine manner and maintain the virus latently in the form of multiple nuclear

episomes (2, 20). Events contributing to autocrine growth may include amplification of EBV episomes, induction of autocrine B-cell growth factors, and up-regulation of the B-cellactivation antigen CD23 (Blast-2) (16). However, little is known regarding the immunoregulatory role of T-cell-derived B-cell-specific lymphokines in the maintenance of latently infected B cells. Can BCGF-12kD exert controlled growthby modulating autocrine growth-regulated functions, e.g., a negative modulation of EBV functions that may be

involved in the pathogenesis of B-cell neoplasia?

A critical question is whether B-cell growth mediated by anautocrine loop mechanism represents a unique biological

situation in which T-cell-derived

BCGF-l2kD-regulated

B-* Correspondingauthor.

tPresent address: Department of Microbiology, University of Connecticut HealthCenter, Farmington, CT 06062.

cell growth isundermined. Acorollarytothisquestionisthe putative existence of distinct autocrine mechanisms in EBV-positive andEBV-negative immunoblastoid cells. To resolve this question, we have investigated the influence of T-cell-derived BCGF-12kD, a B-cell-specific lymphokine, on several EBV-transformed B-cell lines. Our results suggest that BCGF-12kDnegatively modulates the episomalstateof the EBVgenomeand that thisphenomenon is not theresult of selective growth ofa B-cell subpopulation(s).

Several malignant and normal established B-cell lines wereanalyzed for theirinteraction with BCGF-12kD. Raji is anonpermissive cell line derived fromaBurkitt'slymphoma (27) expressing approximately 50 to 60 extrachromosomal copies of the EBVgenome percell (20). LCL 72285 isan in vitro-transformed cell line derived from normal peripheral blood B cells and expressing a relatively high number of EBV copies (28). The B9 cell line was obtained from a normaldonorbychronicexposureof anti-pL-chain antibody-activatedperipheralblood B cells toBCGF-12kD(21).Allof these cell lines demonstrate exquisite sensitivity to B-cell growth factorpreparations (data notshown). To investigate theeffectoflymphokine interaction upon the maintenanceof the immortalized state, oursubsequent experimentsfocused on the immunomodulation of the EBV genome. Raji and LCL 72285 were chosen as representative of high-EBV-copy-number-bearing B-cell lines, whereasB9representeda low-EBV-copy-number-bearing cell line. Additionally, the Namalwacell linewasutilizedas acontrol cell line express-ing two EBV genomic sequences (18, 19, 32).

All cell cultures were routinely maintained in media sup-plemented either with only 5 to20% (vol/vol) fetal calf serum (FCS) or with BCGF-12kD and 5% FCS. The BCGF-12kD utilized for these initial experiments was a partially purified preparation obtained commercially (Cellular Products, Inc.) and willbe referred to as cBCGF. These cell cultures were analyzed for their relative numbers of EBV genomic se-quences via the slot blot technique (in accordance with manufacturer directions; Schleicher & Schuell, Inc., publi-cation371).Theslotblottechnique provides a rapid analysis of relative amounts of EBV genomic sequences, including both integrated (1, 13, 24) and extrachromosomal (20, 26) EBV copies. At intervals ofculturin.g, cell aliquots (1.5 x

106) were removed, totalcellular DNA was bound to

nitro-3190

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A

z

.CM

:

w

z

w

control

14

days

, 28

days

=14

days

28

days

mm :==- control

C a

14 days

28

days

B c n .QCIv

CM

5kb

*-

m

a b c d

B9

C) CM qq

e f g

LCL

FIG. 1. (A) Slot blot analysis ofnegative modulation of EBV

sequences by cBCGF. The in vitro-transformed B-cell line LCL

72285 was maintained in either 10% FCSonly or10% cBCGF-5%

FCSfor4weeks. DNAwasextractedfrom 1.5 x 106viable cellsin

the logarithmic growth phase and vacuum aspirated onto

nitrocel-lulose viathe slot blot technique. The filters were hybridized with 32P-labeled DNA probes specificfor EBNA-1. EBNA-2, and IL-2.

The exposure time of slots hybridized with the 32P-labeled IL-2 probe wasapproximately 10 timesthat of slotshybridized with the EBNA-specific probes. (B)Southern analysis representing chrono-logical reduction of EBV genomic sequences. DNA was isolated

from celllines cultured inthepresenceorabsence of cBCGFvia the guanidinium isothiocyanate-cesiumchloridecentrifugation method. BimiHI restriction enzyme-digested DNA (30 ,ug per lane) was

electrophoresed onan 0.8% agarosegel, transferred to

nitrocellu-lose, and hybridized with a 32P-labeled DNA probe specific for

EBNA-1. Lanes:a, B9cells maintained in 10% FCSonly: b,c,and d, B9 cells maintained in 10% cBCGF-5% FCS for 1. 2. and 4 weeks, respectively; e, LCL 72285 cells maintained in 10% FCS only;f and g, LCL 72285 cellsmaintained in10% cBCGF-5% FCS

for 2 and 4 weeks, respectively. The hybridizing 5-kilobase band

representstheBacmHI Kfragment (12).

cellulose filters,and filterswere hybridized with32P-labeled

probes encoding genomic sequencesfor either EBNA-1 (12)

or EBNA-2 (15, 35). Hybridization ofan adjacent slot blot

lane with a radiolabeled cDNA for IL-2 (6) was used to

detect variations in the quantity ofDNAadded. In a

repre-sentativeexperiment (Fig. IA) the lymphoblastoid cell line

LCL 72285 exhibitedatime-dependentdiminution of

hybrid-izing genomicsequenceswithcontinued exposureof the cell

culture to cBCGF. This decrease ofhybridizable genomic

copieswasdetected with either the EBNA-1orthe EBNA-2 DNA probe. Densitometric scans of the autoradiograms

were performed. Integration of the peak area revealed a

7-foldreduction ofhybridizationto the EBNA-1 probeanda

10-fold reduction ofhybridization to the EBNA-2 probe. A

densitometric scanofan adjacent series of slots hybridized

with a cDNA for IL-2 revealed less than a 12% (1.3-fold)

variation between slots, confirming that approximately

equivalent amounts of DNA were added to each slot. Similar results were obtained with the Burkitt's lymphomacell line Raji; culture maintenance in cBCGF resulted in a chrono-logical reduction of EBV-related sequences. Furthermore, when anegatively modulated Raji culture (i.e., maintained in the presence ofcBCGF) wasreturned to conditions equiva-lent to those of the control culture (20%c FCS only), a reduced level of EBV genomic sequences was maintained and restoration of EBV copy number to control levels was not observed (data not shown). These results suggest that cBCGFmodulation of EBV-related genomic sequences is a permanent rather than a transitory event.

Additional confirmation of the apparent modulating effect ofcBCGF on the number of EBV genomic sequences was obtained viaelectrophoreticseparationofBawinHIrestriction enzyme-digested total cellular DNA (20 p.g) obtained fi-om cells maintained in the presence of either FCS or cBCGF-FCS (23). The nonpermissive B-cell lines B9 and LCL72285 exhibited significant and specific hybridization to the

-3P-labeled EBNA-1 probe at a region corresponding to 5 kilobases (Fig. iB). Analogous to results obtained with the slot blot technique, the Southern blot revealed pronounced diminution ofhybridizing sequences in chronological DNA samples from LCL 72285; however, no pattern of modula-tion wasobservedfor DNAfromthe B9 cell line. When this filter was stripped of radiolabeled probe and rehybridized with the IL-2 cDNA probe, no significant variation of hybridization was detected, indicating that approximately equivalent amounts of digested DNA were added to each lane(datanotshown). In contrast tothe modulation ofEBV sequencesobservedintheLCL 72285 and Raji cell lines,cell lines bearing low EBV copy numbers exhibited no detect-able alteration of the quantity of EBV-hybridizing se-quences. It is interesting to note that the low-EBV-copy-number-bearing cell lines did notexhibitanincrease ofEBV copynumbers,suggestingthatBCGF-12kDmay not support the selective proliferation of either low- or high-EBV-copy-number-bearing cells.

Todetermine whether this observed negative modulation ofEBV copies was related toreduced FCS concentrations, culturesof theRaji cell line were maintainedfor4weeks in various concentrations of FCS (5 to 20%). At intervals of culturing, cell aliquots were removed and analyzed for EBV-hybridizing sequences via the slot blot technique. No

significant modulation of EBV copy number was detected throughout the culture period (data not shown). These results suggest that the chronological reduction of EBV copies detected in cultures maintained in the presence of cBCGF is not a result of culture maintenance in reduced serumconcentrationsbut ratherisadirect effectof cBCGF. As mentioned above, cBCGFis a partially purified prep-aration enriched for B-cell mitogenic activities (25). A sig-nificantconcernintheinterpretationof results obtainedwith thispartially purified material isthe presenceof contaminat-ing factors which may mimic the effects of BCGF-12kD. Indeed, it has recently been reported that lymphotoxin, contained within the cBCGF preparation, may provide a mitogenic signal(s) forStaphylococciusaureius-activated hu-manB cells(17).Toconfirmourdata obtained withcBCGF, we cultured the Raji cell line in the presence of purified

preparations of 10%1 (vol/vol) recombinant BCGF-12kD (rBCGF; 30; A. Vazquez, S. Mills, S. Sharma, and A. Maizel. Eur. J. Immunol., in press) and 5% FCS. The rBCGF used represented a purified cell extract obtained fromEsclherichia coliB-cellgrowth factor cDNAwhich has been shown to function as a hybrid protein capable of

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3192 NOTES

a control

b cBCGF

=== control

d

rBCGF

FIG. 2. Modulationof EBVsequencesdetectedin cultures

main-tained in purifiedrBCGF. Raji cellsweremaintained inthepresence

ofFCS only, control bacterialextract,cBCGF,orrBCGF. After 10

days of culturing, DNAwas extracted and boundto nitrocellulose

filtersvia the slot blottechnique, asdescribedin thelegendtoFig.

1,andhybridizedwitha32P-labeled DNAprobespecific for

EBNA-1.Raji cellswereculturedin thepresenceof10% FCS only(a),10%

cBCGF-5%FCS(b), 10%control bacterial extract-5% FCS(c),or

10%rBCGF-5% FCS (d).

modulating those affects associated with BCGF-12kD (30). After only 10daysin culturein the presenceof rBCGF, total

cellular DNAobtained from this Raji cellcultureexhibited a

considerable reduction of hybridization to the EBNA-1 probe, analogousto the negative modulation observed with cBCGF (Fig. 2). Densitometric scans of the autoradiogram

for the culture maintainedoncBCGF-FCSexhibited

approx-imately a 3.8-fold reduction ofhybridizable EBVsequences

when compared with the control culture maintained in the

presenceof FCS only. AparallelRaji cell culturemaintained

in thepresenceofrBCGF-FCSexhibiteda4.4-fold reduction

of hybridizable sequences when compared with the same

control culture (FCS only). An additional Raji cell culture

was maintained in the presence ofcontrol bacterial extract

and FCS; no significant difference in EBNA-1 hybridization was detected between blotted DNA of this control culture

and that of Raji cellsmaintained in the presenceofFCS only

(1.5-fold variation).

Although our results (Fig. 1 and 2) suggest that the BCGF-12kD-induced negative modulation of EBV copiesin bothlymphoblastoid andmalignant B cellsisauniqueevent,

it is possible that certain selective culture conditions may

induce an unstable maintenance of EBV genome copy

number (3, 31, 32). To ascertain ifBCGF-12kD provided a

selective advantage for the clonalproliferation of those cells possessing relativelyfew EBVgenomic copies, we analyzed

20 pigof DNA from cells maintainedin thepresenceofeither

FCS orcBCGF-FCS for variations ofimmunoglobulin

rear-rangement by using a probe specific for the heavy-chain

joining region

(Jl),

a precise marker for clonal variation

within a polyclonal population ofB cells (7). Although this

technique is capable of detecting B-cell clones accounting for as little as 1% of the total population, no detectable

variation of the hybridization pattern was observed for the

LCL 72285, Raji,orB9cell lines maintained inthe presence or absence of cBCGF (data not shown). These results confirm thatthesubstantial diminutionofEBNA-hybridizing

sequences in those cultures maintained in BCGF-12kD

can-not be attributed to clonal selection of a population(s)

characteristically exhibiting low EBV copy numbers.

The Rajicell linehas been showntocontain EBV genomic

sequences as extrachromosomal covalently closed plasmids (20). As demonstrated by the slot blot technique, DNA derived from Raji cells (as well as from the lymphoblastoid

cell line) maintained in the presence of BCGF-12kD

exhib-itedasignificant reduction of EBNA-1- and

EBNA-2-hybrid-izing sequences; therefore, it is reasonable to hypothesize

thatBCGF-12kD

negatively

modulates the relative numbers of extrachromosomal

episomes.

A direct enumeration of

episomes

would

provide

themost

meaningful analysis

ofthis induced

modulation; however,

thevast excess ofhost chro-mosomalDNAprecludesanyquantitation techniquewithout

prior partial

purification

of the EBV-related

plasmids.

To circumvent this

potential

dilemma yet attempt to

quantitate

therelative numbers ofextrachromosomal

copies,

weuseda

modification of the

techniques

of Eckhardt

(8)

andGardella

etal.

(10).

This

technique

offers

exquisite sensitivity

in that

aslittle as0.25 EBVgenome canbedetected percell. Cells

(103

to

104)

were

suspended

in 20

[L1

ofbuffer

A,

consistingof 15 mM Tris

hydrochloride (pH 7.6),

60 mM

KCI,

15 mM

NaCl,

and 340mMsucrose. Toeffect cell lysis,weadded 20 pA of buffer

B,

consisting

of 150 mM NaCI, 15 mM EDTA

(pH 8.0), 50 mM Tris hydrochloride (pH 7.6), and 0.3% sodium

dodecyl

sulfate. After

being gently mixed,

the solu-tionwas incubated for10to15minatroomtemperature and added

directly

tothe well ofa0.5%agarosegel.Thegelwas

electrophoresed

for 5 hat 5.5V/cm and then for 15 h at2.8 V/cm. To inhibit

nonspecific binding

to the 32P-labeled EBNA-1

probe,

we

hybridized

the filters overnight at 65°C under

high-stringency

conditionsin theabsence of formam-ide. The filters were washed in 2x SSC(lx SSC is 0.15 M

NaCl

plus

0.015 M sodium

citrate)-0.1%

sodium

dodecyl

sulfate at roomtemperature and in0.lx SSC-0.1% sodium

dodecyl

sulfate at 65°C. The filters were subsequently air dried and exposed toXAR and XRPfilms at -80°C.

Raji

cells were maintained in the presence of media

supplemented either with FCS only or with cBCGF and FCS. After culture maintenance for 4 weeks, lysed cells were directly electrophoresed on a 0.5% agarose gel. As detected by ethidium bromide staining (Fig. 3A), the

major-ity

of DNAmigratedto aregionapproximately 7cmfrom the

origin

andwaspresumablycellular DNA anddegraded EBV

episomal DNA(10). Minimal DNAwasdetectable via ethid-ium bromidestaining throughoutthe intermediategel

region.

In contrast, theautoradiogramof thetransferred and

hybrid-izedgelshowed adistinct reaction patternwith the EBNA-1

probe (Fig. 3B). Significanthybridization wasobserved ina region approximately 3.5 cm from the origin (denoted as

"E");

as demonstrated

by

Gardella et al. (10), this

region

corresponds to the migration zone of EBV episomal DNA. Minimal hybridization was detected in the area associated with cellular DNA andlinear viral DNA (approximately7cm from the origin; denoted as "C"). Although equivalent

numbers of cells were added to each corresponding pair of

lanes, those lanes loaded with Raji cells cultured in cBCGF

consistently exhibited less EBNA-1-hybridizing DNA. This

autoradiogram confirms a reduction ofEBNA-1-hybridizing

DNA (episomes) from cells cultured in the presence of cBCGF for each of the three cell dilutions (Fig. 3B, lanes2, 4, and 6). This observation supports results obtained with the slot blot and Southern techniques; furthermore, the data suggest that the observed negative modulation of EBV-related sequences is pronounced for the covalently closed EBV episomes.

Ourdataclearly showed that transformedB-cell linesfrom both normal (LCL 72285) and malignant (Raji) sources exhibited a time-dependent diminution of EBV-related se-quences in the presence of BCGF-12kD. This observed reduction of EBV-related sequences was substantiated and further characterized by electrophoretic separation of viral integrated and extrachromosomal copies; asignificant reduc-tion of EBV plasmid copies was observed. These results J. VIROL.

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NOTES 3193

A

B

B1;2 3 4 b 6

:

*

*4v*%N

.}

zz ^ ^

_w

_F

w_

_

_

1 2 3 4

..W .4

...-..NV

E

o

I

CW~

FIG. 3. Gelelectrophoretic analysis of integrated and extrachromosomal EBV sequences. Raji cellsweremaintained in the presenceof

either10% FCSor10%cBCGF-5% FCS for 4 weeks. Various numbers of cellswerelysed and loaded directlyontoa0.5%agarosegel.(A)

Ethidiumbromide-stained gel after electrophor-esis for 5 h at5.5 V/cmand then for 15 hat2.8V/cm. Lanes 1. 3. and5wereloaded with2

X 104, 3 x 104, and4 x 104cellsperwell, respectively, ofRajicellsmaintained inthe presenceof10%)g FCS only. Lanes2. 4. and6 were

loaded with 2 x 104, 3 x 104,and 4 x 10'cellsperwell, respectively, of Raji cells maintainedinthepresenceof 10% cBCGF-5% FCS. (B)

Thegelwastransferredtoanitrocellulosefilter, hybridized witha32P-labeled EBNA-1probe, and autoradiographed. Episomal EBVcopies

arerepresented by the region of the gel denoted as E';degraded plasmids and integrated EBV copiesare represented in thegel region

denotedas "C."

may have implications for the maintenance ofan

immortal-ized state and EBV-associated autocrine B-cell growth.

As apossible mechanism for the reported negative mod-ulation of EBV episomes, we propose that BCGF-12kD

interacts with asignal(s) controlling plasmid replication. As has been suggested, EBV transformation of B cells may

involve activation and immortalization processes. Many

cellular and viral proteins, including the B-cell activation

antigen CD23 (Blast-2), EBNA-1, and EBNA-2, have been implicated as necessary components in the initiation and maintenance of EBV immortalization of B cells. Most

im-portantly, up-regulation of CD23duringthe initial phases of EBV infection has been associated with amplification of circular EBV episomes (16). It is therefore possible that BCGF-12kD modulates the expression of some of these

functions, thus disrupting the constitutive presence of a

threshold amount of theseproteins.

Is modulation of plasmid copy number reflected in an

alteration ofvirus-related functions, and ifso, what are the biological consequences to the host cell'? Pertinent to this questionis theobservation that when T cellswere addedto

cultures ofin vitro-infected B cells within a few hours of

their exposure to EBV, a significant delay occurred in the

detection of proliferating colonies (34). This phenomenon

wasthoughttobethe result of B-cellinteractionwith T-cell-derived soluble mediators, such as gamma interferon (33).

However, it is reasonable to hypothesize that BCGF-12kD

may impose controlled growth by down-regulating signals whichpromoteautocrinegrowth (29). Furthermore,wehave

observed that transformed B-cell lines derived via chronic

exposure of activated B cells to BCGF-1'kD

characteristi-cally exhibit very low EBV copy numbers (Fig. 1). Our

studies will, therefore, provide a basis for experiments on

the possible inhibition of virus replication by biological

response modifers.

We express our appreciation to Aime Vazquez and Marshall

Posner forhelpful discussions. We thank Fred Wang and Elliot Kieff

for the EBNA-2 probe. Janet Hearing for the EBNA-1 probe. and

Suresh Arya for the IL-2 probe. Thanks are also due to Deborah

Morgain for performing fluorescence-activated cell sortinganalysis.

This work was supported by Public Health Service grants

CA46959 (to S.S.) and CA45148 (to A.M.) from the National

InstitutesofHealth.

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J. VIROL.

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Figure

FIG.1.BimiHIThefromguanidiniumd,electrophoresedtheonly;forprobelose,weeks,logicallulose72285sequencesrepresentsEBNA-1.32EBNA-specificFCS B9 P-labeled (A) Slot blot analysis of negative modulation of EBV by cBCGF
FIG.2.cBCGF-5%tainedoffiltersdays1,1.10% Raji and FCS Modulation of EBV sequences detected in cultures main- in purified rBCGF

References

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