Integrated viral DNA sequences in Epstein-Barr virus-converted human lymphoma lines.

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JOURNALOFVIROLOGY, Mar.1978,p.710-718 0022-538X/78/0025-0710$02.00/0

Copyright0 1978 AmericanSocietyforMicrobiology

Vol.25, No.3 Printed inU.S.A.

Integrated

Viral

DNA

Sequences

in

Epstein-Barr

Virus-Converted

Human

Lymphoma

Lines

MARIAANDERSSON-ANVRET* ANDTOMASLINDAHLt

Departments ofChemistyandTumorBiology,KarolinskaInstitute,10401Stockholm,Sweden

Receivedfor publication 19September 1977

Most humanlymphoid celllines containmultiplecopies ofcircular, noninte-grated Epstein-Barr virus (EBV) DNA molecules as wellasviral DNA sequences

withproperties ofintegrated DNA. The

physical

state ofthe EBV DNA ina humanlymphoma line that only containsonevirusgenomeequivalentper cell has nowbeen studiedbythree differentmethods,neutralCsCldensity gradient centrifugation, actinomycinD-CsClgradient

centrifugation,

and Hirt fractiona-tion. This cellline,AW-Ramos,has beenobtainedbyEBVinfection invitro of theapparently EBV-negative Ramoslymphoma line. Theresults indicate that the EBV DNA in AW-Ramos is presentexclusivelyinalinearly integratedform. Similar data were obtained with two otherEBV-converted sublines of Ramos cells.

Human lymphoid cells cannot be grown in long-term tissue culture, as a rule, unless the cells have been transformed by Epstein-Barr

virus (EBV;forreviews, see27, 38). Lymphoid celllines derived from blood ofhealthy donors

alwaysareof the B (bone marrow-derived)-cell

type,invariablycontainmultiplegenomecopies

of EBV DNA, and express an EBV-associated nuclear antigen, EBNA. Similar EBV-trans-formed B-cell lines can be obtained at a high frequency fromblood of infectious mononucle-osispatientsorfromBurkittlymphoma biopsies. Although the establishment of EBV-negative

humanlymphoid cell lines from nonmalignant

sourceshasneverbeenreported, biopsiesfrom

afew

undifferentiated,

highly malignanthuman

B-cell lymphomas have yielded cell lines that appeartobe free fromEBV,asjudged byEBNA

negativity (22) and by absence of detectable

amountsof EBV DNA (3). Two such

EBV-neg-ative lymphoma lines, Ramos and BJAB, are

of particular interest because they can be in-fected with EBV in vitro and converted to

EBNA-positivesublines(7, 13,21). These

EBV-converted lines contain small but detectable

amountsof viralDNA,andonelineofthistype,

AW-Ramos, only has about one EBV genome

equivalentper cell (3).AW-Ramoscellsare

sta-bly convertedby EBV,andEBNA-negativecells

do not appear in cultures at a detectable

fre-quency.

The latent EBV DNA in typical human lymphoid celllinesismainly presentascircular

tPresentaddress:DepartmentofMedicalChemistry, Uni-versity ofG6teborg,400 33Gi6teborg,Sweden.

DNAmolecules of viral genome length (17, 23), butintegrated viral DNA sequences also seem

tobepresent (1, 2, 17). In this work, we have extended the studies on the physical state of intracellular EBVDNAtoEBV-converted sub-linesof Ramos that carrysmall amounts of viral DNA. We have used three different methods suitable for the analysis ofintegration of

high-molecular-weightviralDNA and show that such lines contain EBV DNA sequences with the expected properties ofintegrated DNA, whereas circular,nonintegrated viral DNA molecules ap-peartobeabsent.

MATERIALS AND METHODS

Cell lines.The humanlymphoma-derived B-cell

lines Raji (10), Ramos, AW-Ramos clone 2,

EHRA-Ramos (21), Ramos/B95-8 (13), and U-698 M (22) were obtained from G.Klein,Department of Tumor

Biology, Karolinska Institute, Stockholm. The cells

were grown at 370C in suspension culture in RPMI 1640medium (Grand Island Biological Co.) supple-mented with 15% fetalbovineserum,100U of penicil-lin perml,and100ug ofstreptomycin perml.Actively growing cell cultures, containing5 x 106 to 1 x 106

cells/mlandmorethan 85%livingcellsaccording to

the trypan blueexclusion test,wereused in all exper-iments. The cells wereharvested by low-speed

cen-trifugation and washed twice in0.01 Msodium

phos-phate, pH 7.4,immediately before use.

DNA preparations and hybridization proce-dures.High-molecular-weight cellular DNA for gra-dientcentrifugation experimentswasobtainedby lysis

ofacellsuspensioncontaining107cells/mlin0.01M

sodiumphosphate (pH 7.4) byaddition of0.5volume of 0.075 M Tris-hydrochloride (pH 8.5), 0.025 M

EDTA,and 1.5%sodiumdodecylsulfate. After20min

at 20°C, 0.2 volume of 0.5% Pronase (Calbiochem;

710

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self-digestedin 0.05 MTrs-hydrochloride[pH7.5]at

46°C for45minbeforeuse)wasadded,and thelysate

wasincubatedat370Cfor 8 h(25).The viscous

solu-tionwasthenbroughtto200C, diluted withanequal

volume of 0.1 M NaCl-0.075 M Tris-hydrochloride

(pH 8.0), and extracted with 1 volume offreshly redis-tilledphenol equilibratedwith the samebuffer. The phenol extraction was performed by slowly pouring

the mixtureback and forthbetween twoglassbeakers.

The mixture was then chilled to 8°C, and the two phases wereseparatedbycentrifugation.The aqueous phase wasextracted once more with phenol in the same fashion and then dialyzed against 0.1 M

NaCl-0.01 M Tris-hydrochloride (pH 8.0)-0.001 M

EDTA for 48 h at20C,followedby dialysisfor 4 h

againstthesamebuffer withoutNaCl.

Nonradioactive and 3H-labeled (105cpm/pg)EBV

DNA preparations from virus particles released by

the P3HR-1celllineweregiftsfrom A. Adams. The viral DNApreparations had more than 8.5% of the DNA in intact, 59S form. KlebsielIa pneumoniae

[3H]DNA (10' cpm/,ug, p = 1.717 g/cm3, molecular

weight=3x 107) wasprepared by standard methods.

The preparation of3P-labeled EBV

complemen-taryRNA(cRNA)and the conditions for DNA-cRNA

hybridizationwereasdescribed (23).

COsCldensity gradient centrifugation.ForCsCl

gradient centrifugation, DNA solutions were diluted

tolowconcentrations,2to6

jg/ml,

andsupplemented

with solidCsCl (MerckSuprapur) to afinaldensity

of 1.710 to 1.714 g/cm3 and a trace amount of K.

pneumoniae[3H]DNA.The DNAsolutionswere

cen-trifugedin18.5-mlsamplesinaSpinco6OTirotor at

33,000 rpmand210Cfor65h.Fractions(0.4ml)were

collected through alargehole in the bottom of the

tube with the aid ofaclosed-systemcollection device

and analyzedfor refractive index, radioactivity,

ab-sorbancy at 260 nm, and hybridizability with EBV

[32P]cRNA

asdescribed(23).FurtheranalysesofCsCl

gradientfractionsbyneutralglycerol gradient

centrif-ugationwerealsoperformedasdescribed(23).

Actinomycin D-CsCl gradient centrifugation.

Theexperimentswereperformedessentially according

toBinstieletal.(5).DNAsolutionswereextensively

dialyzed against0.05M sodium tetraborate (pH 9.0)

at30Cand dilutedto aDNA concentration of2

i&g/ml.

Fourteenmiflilitersof each DNAsolutionwas

supple-mented with a trace amount of K. pneumoniae

[3H]DNA, 0.2 ml ofa 0.2% actinomycin D (Sigma)

solution,andsolidCsClto arefractive index of 1.3885

at30C.Thesolutionswerethenoverlaidwithparaffin

oil andcentrifuged ina Spinco 6WTi rotor at33,000

rpm and 30C for 96 h. Collection and analysis of fractionswere asforCsCl gradientswithout

actino-mycin D,except that eachfractionwasdilutedwith

an equalvolume of0.05 M sodium tetraborate (pH

9.0) before denaturationforhybridizationanalysis.

Hirtfractionationprocedure.The fractionation

wasperformedafter Pronasetreatment(16)and at a

relatively low cell concentration (4). Cells (2 x 107

living cells) were suspended in 8 ml of 0.13 M

NaCl-0.01 M sodium phosphate (pH 7.4). An equal

volume of 1.2% sodium dodecylsulfate-0.01 M

Tris-hydrochloride (pH 8.0)-0.01 MEDTA wasadded at

200C, and thesolutions were gently mixed. After 60

min, 4 mlofpreincubated 0.5% Pronase was slowly

added, followedby incubation at370C for 4 h. The

solution wasthen chilled to 0°C and gently mixed

with 6.7 ml of4 M NaCl-0.4 M Tris-hydrochloride

(pH 7.6) (at00C).After 12 h at00C,themixturewas

centrifugedat24,000xg for 45 min. Thesupernatant

wasrecovered anddialyzedagainst 0.1 M

Tris-hydro-chloride (pH8.0)-0.001 MEDTA, and the precipitate

wasdissolvedinthe same buffer at200C.The solutions

wereseparatelyextracted with 1 volume of phenol at

200C and freed from phenol by centrifugation and

dialysisof the aqueousphaseagainst 1 MNaCl-0.05

MTris-hydrochloride(pH 8.0)-0.001 M EDTA for 48

h,followed bydialysis againstthe same buffer without

NaCl. Severalsamples (10to 12,ugeach)of the DNA

solutions were thendenatured byincubation with an

equal volumeof0.5MNaOH at800Cfor 10min,and

thisprocedure also served to cleave large, covalently

closed circular DNA molecules and to degrade

con-taminatingRNA. The DNA was subsequentlyfixed

to membrane filters and analyzed by hybridization with EBV [uP]cRNA. The DNA-containing filters wererecovered after theradioactivitymeasurements, and the DNA on eachfilterwasacidhydrolyzedand

quantitated by the diphenylamine reaction as

de-scribed (23). The latter values were used to correct

thehybridizationdata to 10.0

itg

of DNA per filter. RESULTS

Distribution of integratedviral DNA se-quences after CsCl

density

gradient

cen-trifugation. DNA molecules of different base

compositioncanbefractionated byCsCldensity

gradient centrifugation, and this method has often been usedtoseparatefree viral DNA and host DNA andtostudytheintegrationofviral DNA (for reviews, see 9, 24). In its simplest

form, the method can be used to measure the

integration ofasmall viral genome, or a

frag-ment of a virus DNA molecule, into a large

piece of host DNA. In this case, theintegrated viral DNA sequenceswillexhibit a density very close to that of the cellular DNA, and it has been shown in this fashion that fragments of adenovirus 12DNAareintegratedinto

cellular

DNA after abortive infection ofbaby hamster

kidney cellsgrown inthe presence of bromode-oxyuridine (8), and that the DNA provirus of

spleen necrosis virus is integrated into the chicken cell genome (14). When the virus

ge-nome is large, as in the case of a herpesvirus DNA molecule, the analysis becomes slightly more complicated, because standard methods

for the preparation of high-molecular-weight

DNA from

mammalian

cellsusing Pronase and

phenol treatments (33) yield shear-produced

large fragments of the host chromosome that

are of amolecularweightcloseto

10',

i.e.,similar

in sizetothe viralgenome under

study.

Inthis case,integratedviralDNAsequences should be found atdensities intermediate between those

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712 ANDERSSON-ANVRET AND LINDAHL

offree viral DNA and host DNA afterdensity

gradient centrifugation ifentire virus genomes are integrated, because random fragmentation

of hostchromosomescontainingintegratedvirus

genomes will result in the formation of "joint

molecules"comprisedofvarying proportionsof

viral and cellular DNA sequences With the assumption that the joints between viral and

cellular DNA sequences are not anomalously

shear sensitive, a typical jointmolecule might

then be comprisedofapproximately equalparts

ofviral and cellular DNA.

There isnomethod availabletoequally mea-sureallsuch joint molecules.Instead, viralDNA sequencesinjoint moleculescanbe determined

by nucleic acid hybridization, usingaradioactive

virusnucleic acid probe. Thismeansthatajoint

molecule comprised ofalongsequence of viral

DNAandashortsequenceofcellular DNA will

provide more sequencescomplementaryto the

radioactive probe than a joint molecule

com-prised ofashortsequence ofviral DNA and a

long sequence ofcellular DNA. Consequently, eveniffree virusDNAmolecules,cellular DNA

"molecules," and viral-cellular DNA joint mol-ecules are all of the same size, the profile

ob-tainedbyhybridizationwithaviral nucleic acid

probe over aCsClgradient containing cellular

DNA with integrated virus genomes will not

yield a symmetric radioactivity peak at equal

distance between viral and cellular DNA. In-stead, a skewed peak localized closer to the

positionof free viral DNAthantocellular DNA

would beexpected.

When DNA molecules isolated from

trans-formedcellsand free viralgenomes areof similar

size, the determination ofthe expected distri-bution of integrated viral DNA after density gradient centrifugation becomes formally equiv-alenttothe theoretical analysis of the density

distribution of bacteriophage T4 recombinant

DNA molecules performed by Tomizawa and Anraku(34). These authors infected cells simul-taneously with bromouracil-containing,

nonra-dioactiveT4particlesofhigh density andasmall

amountof32P-labeledT4phageofnormal

den-sity,andregistered joiningofparentalT4DNA

molecules bymeasuring the amounts of

radio-active T4 DNA foundatanomalousdensities in

CsClgradientsafter extractionandfractionation

of high-molecular-weight DNA from infected

cells. In thisconnection,anequationwasderived

toshow theexpected distribution of radioactive

residuesinjoint molecules (equation2in

refer-ence34). Here,wehaveapplied the

Tomizawa-Anrakuequationtoestimatethe expected

den-sity distribution in neutral CsCl gradients of

EBV DNA integrated into human DNA. EBV

DNA sequences can be separated from host

cellularDNA inCsClgradientsbecausealarge,

natural density difference exists between the virus DNA (p = 1.718 g/cm3) and mammalian

DNA (p = 1.700 g/cm3), whereas EBV DNA

has little internal density heterogeneity (30). Figure 1 shows the experimentally determined density values for free EBV DNA from virus particles and for host DNA, aswell as the

ex-pectedtheoretical distribution ofintegrated

vi-usDNA sequencesfor thecase ofintegration

of entire EBV genomes as single entities.

Whereasthe freevirus DNA is foundas a narrow peakatadensityof 1.718g/cm3,theintegrated viral DNA would be detected as a broader,

slightly skewed profile with a peak density of

1.713g/cm3 by hybridizationwithaviral nucleic

acid probe. There are no detectable free viral

DNAsequencesatthedensityof cellularDNA, whereas a signiicant tail of integrated viral

DNAsequenceswould be found at thisdensity.

Thelatter material wouldrepresent

viral-cellu-lar DNA joint molecules mainly consistng of

cellular DNA. Several ssumptions have been

made inthisanalysis.

(i)All cellularDNAmoleculeshave thesame

density.This is notentirelycorrect,but

moder-ate density heterogeneity within the peak of

cellular DNAwould not markedly change the

theoretical distributionprofileofintegratedviral

DNA (34). It should be noted, however, that

E

z

I

ro m m

UJ

wp

V _1.740 ₁₇₂₀ ₁₇₀₀ _1.680 V

x(density,gcm-3)

FIG. 1. Estimateofthedensitydistribution of in-tegratedEBVDNA inaneutralCsClgradient. The dashed line shows experimental data for EBV

PHIDNA,isolatedfromvirusparticles, after centrif-ugationat33,00()rpminaSpinco6i rotorfor65 hat20°Cinan185-mlneutralCsClgradient. The datacanbeapproximated byaGaussiandistribution with apeak density of1.718 g/cm3 anda = 0.004

g/cm3.Thearrowshows thepeak position ofhuman ceUDNA (1.700g/cm3)asdetermined inaseparate experiment. The solid line shows the theoretically expected distribution ofEBV DNA in theform of viral-cellularjointmolecules, c(x), as afunction of

density,calculatedaccordingtoTomizawa and

An-raku(34).Forfurther details,seetext. ' 4

I

4 I I

I

1~~~~~~1

2 /

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the presentestimate concerns viral DNA inte-grated into cellular DNA of typical base

com-position (p = 1.700 g/cm3). It is obvious that differentdistributionswould be expected for the unlikely cases of integration of EBV DNA into guanine-cytosine-rich DNA such as nucleolar DNA, or into satellite DNA of unusual base composition.

(ii) All DNA molecules are of the same length. Thisrequirement isessentially followed in the presentwork, as the isolated cellular DNA was ofsimilar orslightlylargersizethan intact EBV DNA (seebelow).

(iii) Fragmentation of cellular chromosomes containingintegrated viral DNA occurs by ran-dom shear-induced breakage, so that ratios of viral to cellular DNA in joint molecules range from zero to infinity, andall values occur with

equal probability.

(iv) Viral DNA is integrated in the form of entire butsingle genomes. Clearly, ifthe virus DNA isintegratedasmany smallfragments of viral DNA at multiple positions, the integrated virus DNA sequences should be found at the

density positionofcellularDNA. On the other hand, if several EBV genomes are tandemly

integrated, theywouldbe,difficulttodistinguish fromnonintegratedvirus DNAby CsCl density

gradient centrifugation.

(v) Differences in DNAmethylationbetween EBV DNA from virusparticlesand intracellular EBVDNA, ifthey exist, would notbe of such amagnitude as to cause marked differencesin

buoyantdensity(20). In support of this

assump-tion,it is noted that intracellularnonintegrated

EBV DNAmolecules,isolatedmainly as nicked circular DNA molecules, have densities very closetothat of EBV DNA from virusparticles

(17).

(vi) The radioactive viral nucleic acid probe

usedtodetect viral DNA sequencesby

hybridi-zation experiments isequally representative of

allsequences inthe EBV genome. In the present work we have used 32P-labeled EBV cRNA, made with Escherichia coli RNA polymerase (containingsigma factor),and such cRNA

prep-arationshave been showntorepresentmost or

all of the EBV genome (3). However, it is

pos-siblethatabundancedifferences between

differ-entsequencesoccurinthe cRNApreparations.

As the aboveassumptions maynotbestrictly

fulfilledinthepresent case, the theoretical

dis-tribution shown inFig.1shouldonlyberegarded

as an approximation. Nevertheless, it seems

likelythatthisestimateis aconsiderablybetter representation of the expected location oflong integrated sequences of EBV DNA than the

simple assumption that all integrated

herpes-INTEGRATED

virus DNA sequences might be found at the density of cellular DNA.

Fractionation of lymphoma cell DNA by neutral CsCl density gradient centrifuga-tion. Three different EBNA-positive sublines of the EBV-negative Ramos cell lines were used. Two of these lines, AW-Ramos and EHRA-Ra-mos, were obtainedby infection of Ramos cells with the P3HR-1 strain of EBV (21), whereas the third line, Ramos/B95-8, was obtained by infection with the B95-8 strain of EBV (13). Inagreement with previous results (3, 12), the AW-Ramos cells were found to contain ap-proximately one EBV genome equivalent per cell, as determined by DNA-cRNA hybridiza-tion, whereas EHRA-Ramos had four and Ra-mos/B95-8 had two EBV genome equivalents per cell. High-molecular-weight DNAwas pre-pared from these linesby lysisof thecells with

sodiumdodecylsulfateand

EDTA,

followedby prolongedtreatmentwithahigh concentration ofPronase, and finallytwogentle phenol

extrac-tions followedby dialysis.The DNAwas

subse-quentlymixed witha traceamountofadensity marker,K.pneumoniae[3H]DNA,fractionated

byneutral CsCldensitygradient centrifugation,

and collected under conditions minimizing

shearingforces(23). Such DNA hada sedimen-tation coefficient of62S,correspondingtoa

mo-lecular weight of 1.1 x 108 (reference 11), as

determined by cosedimentation of [3H]DNA

from AW-Ramos, recovered from a CsCl

gra-dient, with phage T4 [14C]DNA in a neutral glycerol gradient.

As a control, DNA was isolated from the

extensively studied Raji cell line by the same

procedures,and EBV DNA sequences were lo-calizedby hybridization ofindividualCsCl gra-dient fractions. This Burkittlymphoma-derived

linecontains 50 to 60EBVgenomeequivalents per cell (28), and most of this virus DNA is presentascircular, nonintegratedDNA,but vi-rusDNAsequences withproperties of integrated DNA arealso present (1). The data are shown in Fig. 2. The peak of EBV DNA sequences from Raji cells is found at a density of 1.716 g/cm3, i.e., at adensity lowerthan thatof free viralDNA(1.718

g/cm3)

butat adensity higher than that expected for singly integrated viral genomes (1.713 g/cm3). Furthermore, the peak

isslightlyskewed inshapeandextendsinto the

densityregionofcellular DNA. These observa-tions areconsistent with the presence ofboth

nonintegrated and integrated EBV DNA se-quencesinthesecells. Thehybridizationprofile obtained isrepresentativeof theintrinsic density of the intracellular EBV DNA, because it has

been shown (2) that the radioactiveviralDNA

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o

0L' " &

10 20 30

Fraction number

T

x

E

0X

N

15.

10z lx

uJ

0I

FIG. 2. Fractionation of high-molecular-weight

DNAfromRajicellsbyneutralCsCIdensitygradient

centrifugation.DNAwasextractedfromthe cellsby

lysis with sodium dodecyl sulfate and EDTA,

fol-lowedbyextensivetreatmentwith Pronase. The DNA wasthen extracted twice withphenol, dialyzed,mixed

witha trace amountofK.pneumoniae13HJDNAas densitymarker, diluted,andsupplementedwith solid

CsCIto afinaldensityof1.710to1.714g/cm3.Each 18.5-mlgradientcontained the DNAfrom4 x106to

6x 106cellsin 0.01 MTris-hydrochloride-0.001 M

EDTA

LpH

8.0)andwascentrifugedinaSpinco6OTi rotor at 33,000 rpm and 200Cfor 65 h. Fractions

were slowly collected through a large hole in the

bottom ofthetube, and thecellular DNA and the

densitymarker were localizedby absorbancyat260

nm

(A200

andradioactivitymeasurements.TheDNA

in theindividualfractionswasthenalkali denatured and immobilized on membrane filters. Each filter

washybridizedwith6ngofEBV[2PJcRNA(8.8x

I&.cpmafter correctionfor32Pdecay),and the

back-groundof cRNAnonspecificallyboundtofilters

con-tainingheterologousDNA(80() cpm)wassubtracted.

Thegradients werelinear within the entiredensity

rangeofinterest(1.68to1.75g/cm3) asdetermined

byrefractiveindexmeasurements.Symbols:Celular

DNA of buoyant density 1.700g/cm3 (0); K.

pneu-moniae pHlDNA of buoyant density 1.717 g/cm3

(a); EBV DNA sequences(A).

component inanartifical mixture of3H-labeled EBVDNAfromvirusparticles and

high-molec-ular-weight cellularDNAbands at the density of free viralDNA inneutral CsClgradients, that theintracellular EBV DNA sequencesfromRaji

cells found at an anomalously low density re-main atthe same density inrebanding

experi-ments at low DNA concentrations, and that

suchlow-densityEBVDNAisalsofound after CsClgradientcentrifugation ofPronase-treated

celllysatesnotextractedwithphenol.

Figure3showsaneutral CsCl density gradient

fractionation experiment with high-molecular-weightDNAfromAW-Ramoscells,which

con-tam 50timesless EBV DNA than Raji cells. In

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this case, the EBV DNA sequences are found at apeak density of 1.713g/cm3,and the hybrid-ization profileis similar to the theoretical esti-mate for an integrated EBV DNA molecule in Fig. 1. These data indicate that the viral DNA sequences in AW-Ramos are present in inte-grated form. The results do not seem compatible with a situation in which AW-Ramoscells con-tain onenonintegratedEBV DNAcircle percell

of density of 1.718

g/cm3.

In agreement with

thisnotion, no viralDNAwith the sedimenta-tion properties of covalently closed circular DNA of viral genome size was detected when the AW-Ramos DNA banding at a density of 1.710 to 1.718

g/cm3

inCsClgradients was fur-ther fractionated by neutral glycerol gradient

centrifugation and analyzed by hybridization

(datanot shown). Moreover, the viral DNA in AW-Ramos cells is notpresent in the form of many smallfragments integrated at several lo-cations,as thereare few EBV DNA sequences

foundatthedensityofcellularDNA.The data donotdistinguishbetween thepossibilitiesthat AW-Ramos contains one integrated complete EBVgenome or a couple oflargefragments of

viralDNA.AW-Ramos cells, incontrast to Raji

cells,cannotbeinduced to express EBV antigens associated with an abortivelyticvirus cycle by

iododeoxyuridine treatment (21), and it is not knownifallsequences of the EBV genome are present in AW-Ramos.

When DNA from AW-Ramos cells was re-duced in sizeby shear treatment to a molecular weight of 6 x 106 before CsCl density gradient

centrifugation, the EBV DNA sequences were instead foundasapeakat1.718

g/cm3,

i.e., the

densityof freeviral DNA (3). This showsthat

0

0.6-6 0

N

0

0--0.~~~~~~~~~1

--10 20 30

Fraction number

FIG. 3. FractionationofA W-Ramos DNA by neu-tral CsCl density gradient centrifugation. Experi-mental conditions andsymbolsasinFig.2.

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715

the low density observed for the intracellular

EBVDNA after fractionation of high-molecular-weightDNA from AW-Ramos cells is notdue toanunusualbase

composition

of the viral DNA sequences presentinthecells.

Results similar to those obtained with AW-Ramos were also observed on fractionation of

high-molecular-weightDNAfrom the two other EBV-convertedRamossublines,EHRA-Ramos andRamos/B95-8,by CsCl density gradient

cen-trifugation. In both cases, the EBV DNA se-quences were detected as broad hybridization profiles at densities markedly lower (1.710 to 1.714g/cm3) than that offreeEBVDNA (data notshown).

Actinomycin D-CsCl gradient centrifu-gation. InCsClgradientscontaining actinomy-cin D, guanine-cytosine-rich DNA bands at a lower density thanadenine-thymine-rich DNA (5, 18). Consequently, integrated EBV DNA

would be expected to band at a higher density than free EBV DNA insuchgradients, and the

distribution ofintegratedviral DNAsequences would be approximated by a mirror image of Fig. 1.

When high-molecular-weight DNA from an EBV-negative humanlymphoma linewasmixed

with small amounts of EBV DNA from virus

particles andK.pneumoniae[3H]DNA,the

lat-terDNAserving asdensity marker,theresults shown in Fig.4were obtained by actinomycin

D-CsCl gradient centrifugation. Although K.

pneumoniae DNA and EBV DNA have very similar base compositions, the EBV DNA banded at a slightly higher density than the

bacterialDNA,presumably becauseof sequence effects. As expected, the human cellDNA was foundat amarkedly higherdensity than either of the two more guanine-cytosine-rich DNAs. The properties of high-molecular-weight AW-RamosDNA,centrifuged togetherwith K.

pneu-moniaeDNA in thesamefashion,areshownin

Fig. 5. The separation between the bacterial DNA and the human cell DNA is similar to that observed in the controlexperiment. How-ever, theintrinsicEBVDNA sequences of AW-Ramos arefound at asignificantly higher

den-sitythan that of free viral DNA in this type of gradient. These data are in agreement with those

showninFig.3andprovide additional evidence

for the presence ofintegrated EBV DNA

se-quencesinAW-Ramos cells.

Hirt fractionation procedure. DNA from

smalltumorvirusescanbeefficiently separated fromhigh-molecular-weight cellular DNA in cell lysates by coprecipitation of the latter DNA

withsodiumdodecyl sulfate in cold 1 M NaCl. The relative sizes of the DNA molecules are clearlyimportant forthefractionation, and the

0I

4

n

Q

E

a-C-)

0)

N

.n

10

->

z

5_DL

N

m

uJ

0

1

10 20

[image:6.501.253.444.58.260.2]

Fraction number

FIG. 4. Actinomycin D-CsClgradient centrifuga-tion ofhuman DNA from an EBV-negative

lym-phoma line, U-69a, mixed with trace amountsof

EBV DNAfromvirusparticksandK. pneumoniae

pHJDNA. A 30-pg amount ofthe

high-molecular-weight DNA was dialyzed against 0.05M sodium

tetraborate(pH9.0),suppkmentedwithactinomycin

D andCsCl,andcentrifugedin afinal volumeof19 ml in aSpinco6OTi rotorfor4days at33,000rpm and3°C. Fractions (0.4ml) were coUectedfromthe bottom of the tube, followed by measurements on

individualfractionsof refractive index, absorbancy

at 260nm(A2,3Hradioactivity,andhybridizability

withEBVf2PJcRNA.SymbolsasinFig.2.

method is also applicable to Pronase-treated

lysates (16). It has recently been found that

molecules as large as intact herpesvirus DNA

can be separated from cellular DNA in this fashion (4, 29).Here, we have used this proce-dure to obtain partial separation of

noninte-grated EBV DNA from human cellular DNA. In control experiments with phage T4

[14C]DNA,which issimilar in sizetoherpesvirus DNA, added in trace amounts to Pronase-treatedlysates ofhumanlymphomacells, con-siderabletrappingofthe T4 DNAwasobserved.

Using a

relatively

low cell concentration

(106

cells/ml),which has beenreported tofacilitate

the separation of herpesvirus DNA from cell DNA (4),weobtained 13 to15% of the cellular

DNAand50 to60% of the T4 DNA in the "Hirt

supernatant." It is clear that the presence of

viral DNAin the "Hirt precipitate" cannot be takenasevidence forintegrationwhenthevirus genome hasa molecularweightof108, but it is alsoclear that theT4DNAwasenriched in the Hirtsupernatant. Table1shows that whenRaji

cellDNA wasfractionatedinthesamefashion,

theHirtsupernatant wasthreefold enrichedin

EBV DNAsequences. These data indicate that

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o 0. N

q 0.

0

10 20

[image:7.501.65.254.54.260.2]

Fraction number

FIG. 5. Analysis of intrinsic EBV DNAsequences

in AW-Ramos cells byactinomycin D-CsCl gradient centrifugation. ExperimentalconditionsasinFig.4

andsymbolsasinFig.2.

some nonintegrated EBV DNA is present in

Raji cells. There is much better evidence for

this notion obtained by othertechniques (23),

and the EBV DNA present in the Hirt

super-natantfromRajicellshas beenfurtherpurified

and shown to consist largely of circular viral

DNA molecules (data not shown). Incontrast,

therewas nodifference between theproportion

ofEBV DNAtocellular DNA betweenthe Hirt

supernatant andthe corresponding precipitate

fromAW-Ramoscells(Table 1). These dataare

again consistent with the absence of

noninte-gratedEBV DNA molecules inAW-Ramoscells.

Furthermore, the results indicatethat the EBV

DNAsequencesinAW-Ramoscellsarenot

pres-ent in the forn of circular DNA molecules of

viralgenomesizecomprisedof bothcellularand

viral DNAsequences.

DISCUSSION

Several differentmethodshave been usedto

study the integration oftumor virus DNA

se-quences in virus-transformed host cells, and

thereare nowmanyexcellenttechniques

avail-abletoconvincinglydemonstrate theintegration

ofsmall tumor virus genomes or fragments of

virus DNA (6, 8, 15, 31,36,37). Unfortunately,

most ofthese methods depend on the smaller

size of the tumor virus genomes under study

than ofgentlyisolatedcellular DNA, and they

arenotreadilyapplicabletothe characterization

of intracellular forms ofhigh-molecular-weight

viral DNA. In that case, nonintegrated viral

genomesmaybe foundas amixture of covalently

closedcircular DNA and nicked circular DNA, and one orthe other of these forms islikelyto havefractionation propertiessimilartothose of

thehost DNA in different types ofcentrifugation

experiments. Inaddition, methodsthat involve

precipitationstepstoseparate viral and cellular

DNA are marred by trapping artifactsthat do

notoccurtoasignificantextentwith small DNA

molecules. When alargenaturaldensity

differ-ence between the viral DNA and host DNA

exists, asinthepresentcase,neutral CsCl

den-sity gradient centrifugation is a useful method

forsearchingforlarge viral DNA molecules in

integratedform. Alkaline CsClgradientsareless

satisfactoryfor suchstudieson

high-molecular-weightDNA because of theslow,salt-promoted hydrolysisof DNA thattakesplace in that sol-vent (19, 35). Inthe presentwork,the analysis of EBV-converted lymphoma lines that carry

relativelysmall amountsof EBVDNAby

neu-tral CsClgradient centrifugation yieldedresults

that are strongly indicative of the presence of

integrated viral DNA sequences in the cells,

whereasthe data donotseemcompatiblewith

the occurrence of intracellular nonintegrated viral DNA. Similar results were obtained by

centrifugation of AW-Ramos DNA in

actino-mycin D-CsCl gradients. Although the latter

methoddoesnotseemtoofferanygainin

sen-sitivityoverregularCsClgradient centrifugation

inthepresentcase(Fig.3and5), the dataserve

to confirm that the anomalousdensity

proper-tiesof theintracellular viral DNAsequencesare

due to an association with DNA of relatively

lowerguanine-cytosinecontent.

The EBV-converted sublines of the Ramos

lymphomaline havedifferentgrowth

character-istics andsurvive much better in crowded

sus-pensioncultures than the parent line (32), and

theyalsoexpressEBNAincontrast tothe

par-TABLE 1. AmountsofEBV DNA sequences in DNA

fromhumanlymphomalinesafterHirt fractionation

EBV[32P]cRNAbound/10jigof

DNA(cpm)a

Source of DNA

Hirtsuperna- Hirt

precipi-tantb tate

Ramos ... 497 532

AW-Ramos ... 908 944

Raji... 28,790 9,&36

aAverage values of

triplicate

determinations. Each

0.3-mlreaction mixture contained2.5ng(370,000 cpm) of EBV[3P]cRNA.

bThe Hirtsupernatantfractions contained14± 2% of the total cellular DNA, using a low initial cell concentration andPronase treatmentbefore fraction-ation.

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25,1978

entline,sofrom thispointof viewlines suchas

AW-Ramos may be regarded as

"EBV-trans-forned." On the otherhand, the parental,

ap-parently EBV-negative Ramoslymphoma cells havealready beentransformed in an unknown event, since they grow indefinitely in culture and show malignant potentialoninjectioninto nude mice (21).Toavoidanomenclature prob-lem,the term "EBV conversion" has been used inthe present work todescribe the stable alter-ation ofpropertiesobserved after EBVinfection ofanEBV-negative lymphoma line.

Theintegration of EBV DNAinto the DNA of human lymphocytes does not appear to be

sufficient by itself for transformation to

malig-nancy, since small amountsofintegratedEBV DNA sequenceshave been found innewly estab-lished human lymphoblastoid cell lines of dip-loidkaryotypethatdonot cause tumorsinadult nudemice(17, 26).It may be necessarytostudy

the sites of viral DNA integration to evaluate the transforming potential of EBV, and such

measurements will have to await the

develop-ment of suitable

technology

fordetailed

char-acterizationof

integrated

sequences of

large

viral DNAmolecules.

ACKNOWLEDGMENTS

We thankBirgittaMoller andUlfBrodin oftheKarolinaka InstituteComputerCenterforwritingaFortran program of theTomizawa-Anrakuequation.

This work was supported bygrants from the Swedish

CancerSocietyand theSwedish Natural ScienceResearch

CouncilandbyPublicHealthServicecontractsNO1CP33316 and NOI CP81020within the Virus CancerProgramof the

U.S.National CancerInsttute.

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