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
*-
ma 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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[image:2.612.83.272.82.348.2]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 variationwithin 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 extrachromosomalepisomes.
A direct enumeration ofepisomes
wouldprovide
themostmeaningful analysis
ofthis inducedmodulation; however,
thevast excess ofhost chro-mosomalDNAprecludesanyquantitation techniquewithoutprior partial
purification
of the EBV-relatedplasmids.
To circumvent thispotential
dilemma yet attempt toquantitate
therelative numbers ofextrachromosomalcopies,
weusedamodification of the
techniques
of Eckhardt(8)
andGardellaetal.
(10).
Thistechnique
offersexquisite sensitivity
in thataslittle as0.25 EBVgenome canbedetected percell. Cells
(103
to104)
weresuspended
in 20[L1
ofbufferA,
consistingof 15 mM Trishydrochloride (pH 7.6),
60 mMKCI,
15 mMNaCl,
and 340mMsucrose. Toeffect cell lysis,weadded 20 pA of bufferB,
consisting
of 150 mM NaCI, 15 mM EDTA(pH 8.0), 50 mM Tris hydrochloride (pH 7.6), and 0.3% sodium
dodecyl
sulfate. Afterbeing gently mixed,
the solu-tionwas incubated for10to15minatroomtemperature and addeddirectly
tothe well ofa0.5%agarosegel.Thegelwaselectrophoresed
for 5 hat 5.5V/cm and then for 15 h at2.8 V/cm. To inhibitnonspecific binding
to the 32P-labeled EBNA-1probe,
wehybridized
the filters overnight at 65°C underhigh-stringency
conditionsin theabsence of formam-ide. The filters were washed in 2x SSC(lx SSC is 0.15 MNaCl
plus
0.015 M sodiumcitrate)-0.1%
sodiumdodecyl
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 mediasupplemented 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 theorigin
andwaspresumablycellular DNA anddegraded EBVepisomal 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 demonstratedby
Gardella et al. (10), thisregion
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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[image:3.612.86.277.79.162.2]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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