Sequence complexity of circular Epstein-Bar virus DNA in transformed cells.

0022-538X/81/100011-09$02.00/0

Sequence

Complexity of Circular Epstein-Barr

Virus DNA

in

Transformed Cells

BEVERLY E. GRIFFIN,t EVA BJORCK, GUNNAR BJURSELL, AND TOMAS LINDAHL*

Department of MedicalBiochemistry, Gothenburg University, S-400 33 Gothenburg, Sweden

Received11February 1981/Accepted 3 June 1981

A simplified procedure, based on several methodspreviously used to isolate

circular DNAmolecules from bacteria,was devisedforthe preparationof

cova-lently closed circularviral DNA molecules from large quantities oflymphocytes

transformed by Epstein-Barr virus. The protocol can be applied both to virus

nonproducer lines and to lines containing cells activated to virus production.

Sufficient amounts of highly purified viral DNA of intracellular origin were

obtainedfrom B95-8 and Raji cellstoallow direct visual analysis of theirsequence

complexities after cleavage with EcoRI and separation of fragments by gel

electrophoresis. No major differences in complexity were observed between

circular DNA and linear virion DNA from B95-8 cells. The fragmentpatterns

observedinthis fashionagreewell withthosedetectedbyconventionalblotting

and hybridization methods. The procedure can also be used as an analytical

methodtoassayfor smallamountsof circular Epstein-Barr virusDNAmolecules

inothertransformedcells. In this connection,nocircular Epstein-Barr virus DNA

wasdetected inNamalva cells.

TheEpstein-Barr virus(EBV) transforms

hu-manBlymphocytes effectivelyto acellular form

thatgrowswell in tissueculture,andthe

trans-formed cellsexpress avirus-determined nuclear

antigen,EBNA (17, 27). Inmanysuchcells, the

viral genomes are present mainly as

non-inte-grated, covalently closed circular DNA

mole-cules (21, 23). Small amounts of EBV DNA

circles, suitable for characterizationby electron

microscopy, have been isolated previously in

more than 90%pureform(21).However, larger

quantities of circular EBV DNA formore

de-tailed studies ofsequenceorganization and

com-plexity havenotbeenavailable,because

stand-ardmethods usedtopreparesmall circular DNA

molecules from varioustypes ofcells have not

been applicable to EBV-transforned

lympho-cytes. Comparisons among viral DNA circles

presentin differenttypesof transformed cellsby

analysisof restrictionenzymecleavagepatterns

have therefore only been perforned with

unpu-rified (32)orpartly purified (16,29) EBVDNA

preparations, using selective hybridization

pro-cedures to detect the EBV DNAsequences. In

the present.study, wedescribe the purification

ofrelatively largeamountsofcircular EBV DNA

fromRajiand B95-8cells,free from host nuclear

DNA. The availabilityof such material has

al-t Presenal-t address: Imperial Cancer Research Fund,

Lin-coln's InnFields,London WC2A3PX,England.

11

lowed an analysis of restriction enzyme digests

of circular intracellular EBV DNA by optical

methods.

MATERIALS AND METHODS

Cefls. The EBV-transformed lymphoid cell lines

Raji, Namalva, and B95-8 were propagated without

agitationassuspensioncultures in2-liter volumes in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100U ofpenicillinperml,and100,ugof streptomycin per ml. Raji and Namalvaare Burkitt

lymphoma-derived lines (11, 27), whereas B95-8 isa

marmosetlymphocytelineimmortalized with human EBVfromamononucleosispatient (22). Best results wereobtained with actively growing cells, harvested

at adensityof106cells per ml andcontainingahigh

proportion ofliving cells (>85%) as judged by the

trypanblueexclusiontest.

Viral DNA and hybridization probes. Linear EBV DNAwasprepared from virus particles released by the B95-8 cell line (21). The EcoRI fragmentof circular EBV DNA from Raji cells, which contains sequencescorrespondingtothe endfragmentsof the linear DNA from virus particles, wascloned in the cosmid pHC79 (2a). These DNA preparations were radioactively labeledinvitrobynicktranslationinthe presenceof[a-32P]dCTP(28).Thespecific activityof thelabeled DNAwas1 x 108to2x108cpm

Lg-'.

Isolation of circular EBV DNA.Lymphoidcells

(2 x 109 to 4 x 109) were collected by low-speed

centrifugationandwashedoncewith0.14MNaCl-0.01

M sodium phosphate (pH 7.4). The cells were

sus-pendedinasmall amountof this saline solution by

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12 GRIFFIN ET AL.

brief blending inaVortex mixertogenerate athick but homogeneous cellsuspension of about109 cells per ml. Analkaline sodium dodecyl sulfate buffer was then immediately added to lyse thecells (1 ml per 107 cells). This buffer, which contained 50 mM NaCl-2 mM EDTA-1% sodiumdodecyl sulfate, broughttopH12.4 with 2 MNaOH, wasfreshly made and adjusted to thecorrectpH usinganalkali-resistant electrode,or by correcting for the reading error of conventional combination electrodes(by calibration withapH12.4 standard buffer that contains25ml of0.2MKCI,16.2 mlof 0.2 MNaOH,and58.8ml ofwater). A10- to 15-mlportionof the very viscous cell lysate was poured into a50-mlconical centrifuge tube (Falcon Plastics, Oxnard,Calif.),withsample separation being achieved by cutting through theviscouslysate with a pair of scissorsduring pouring.The tubewascapped, and the sample of the lysatewassheared for2min on aregular laboratory Vortex apparatus operating atmaximum speed (e.g.,aSupermixer, Lab-Line Instruments, Inc., Melrose Park, Ill.). The lysate should form a thin liquid film coveringmostof thewall of the tube during the Vortextreatment.The processwasrepeated until all of the DNA had been sheared. The combined, slightly viscous lysatewasthen incubatedat300Cfor 30mintoallowcomplete alkali-induced strand sepa-rationof linear DNAmolecules. The final lysatewas supplemented with 0.05 volume of 1 M Tris-hydro-chloride (pH7.1), toobtain apHvalue of 8.5to 9.0, and then with 0.2volume of3MNaCl. These additions weremadeslowly withgentle swirling. Proteinase K (E. Merck AG,Darmstadt, Germany), 0.02 volume of a0.5%solution,wasthenadded, and the lysate was incubatedat37°Cfor30mintoallowpartial degra-dation of cellular proteins. This protease digestion

appeared necessary forobtaining goodyieldsof EBV

DNAduring subsequentsteps.Thelysatewaschilled

to200Candmixed with one-third volume of redistilled phenol, previously saturated with0.2MNaCl-0.2M

Tris-hydrochloride (pH 8.0). The phenol extraction

was performed byslowly pouring the mixture back

and forth betweentwoglassbeakers. Afterchillingto

8to100C,thelysatewascentrifugedfor 20 min in

250-ml Corex bottlesat6,000 rpm inaSorvalllaboratory

centrifuge. A clear aqueous layer, containing RNA

fragments and covalently closed circular DNA, was observed aboveawhiteinterphaseofasingle-stranded

DNA andremainingprotein.The aqueous, nonviscous phasewasrecovered with awide-mouth pipette and

gentlyextracted with1volume ofchloroform-isoamyl

alcohol(24:1)toremove mostof thedissolved phenol. The phasesseparated quickly onstanding, and the bottom phase was removed and discarded. The aqueousphasewasmixed with2volumes of cold 95% ethanol and leftovernightat-20°C.Asmall precipi-tate developed, containing RNA and circular DNA. The RNA apparently servedascarrier and ensured effectiveprecipitation of the very dilute DNA in this step. The precipitate was recoveredby centrifuging portions of themixture for10mineachat6,000 rpm in thesame250-ml Corex tube. The tubewasdrained upside down for15 to 20min,and thenaCsCl(Merck, Suprapur) solution (6 g of CsCl in6ml of10mM Tris-hydrochloride-1 mM EDTA [pH 7.5]) was added to

J. VIROL.

thestill-moist precipitate. The latter dissolvedreadily,

and the solutionwastransferredto anultracentrifuge

tube. After the addition of 0.1 ml of 1% ethidium bromide andoverlayingwithparaffinoil,thesolution wascentrifuged for40hinaBeckman 50-Tirotor at 40,000 rpmand200C.At the endof the run, the tube wasexamined underlong-wave UVlight.Apelletof RNA and two DNA bands of approximately equal

intensitywere seenintypicalexperiments. The lower band, containingcovalently closed circular DNA,was collected through alargehole in the bottom of the tube with the aid ofaclosed-systemcollectiondevice,

andethidiumbromidewasremoved from this solution by fourextractions withanequalvolume of

isopropa-nol, presaturatedwithasimilarly concentratedCsCl

solution. The DNA solutionwasthensupplemented

withadditionalsolidCsCl (0.25 g/ml) and dilutedto

avolume of 6 mlbythe addition ofmoreCsClsolution.

The densityof this solutionwasadjustedto1.716g/

cm3 withthe aid ofarefractometer,and thesolution wascentrifuged for48h ina50-Tirotor at34,000 rpm

and200C.Atthe end of the run, 0.2-ml fractionswere

collected throughalarge hole in the bottom of the tube. Each fractionwasdiluted with3volumes of10

mM Tris-hydrochloride-1 mM EDTA (pH 8.0), and

theabsorbancy at260 nmwasdetermined.

Low-mo-lecular-weight RNA mainly resided in the bottom

fractions, and the peaks ofcircular EBV DNA and

lighter mitochondrial DNA wereseparated by2to 3 fractions. The EBV DNA (usually intwo fractions)

waspooledintoasiliconized Corex glass tube, mixed

with 2volumes of cold ethanol, and kept at -20°C

overnight. After centrifugation, the DNA was

dis-solved in50plof10mM Tris-hydrochloride-1 mM EDTA(pH8.0) and storedat40C.Long-term storage of thecovalentlyclosed circular EBV DNA in concen-tratedCsClsolution should beavoided, because inour

experiencenicksslowlyappear.

If theyieldof circular EBV DNA is low, the position of thelighterband of mitochondrial DNA may be used as reference to localize the virus DNA. A sensitive fluorescence method (9)orhybridization of portions of eachgradientfraction with32P-labeledEBV cRNA has also beenemployedtolocalize the circular EBV DNAafterCsCl density gradient centrifugation. As an alternative tothe second CsCl fractionation step, in

someexperiments glycerol gradient centrifugationof

thecovalentlyclosed circular DNA from the ethidium

bromide-CsCl gradient was performed as described (21), and the materialsedimentingat 90 to115Swas recovered.

Inanalytical experimentstosearchfor the presence of circular EBV DNA in cells, fractions from the ethidiumbromide/CsCl gradientwereextractedwith isopropanol, boundtomembraneifiters,anddirectly hybridized withEBV32P-cRNA(21).

Electronmicroscopy. DNA preparationswere ex-amined by the formamide modification of the Kleinschmidt technique, and length measurements wereperformedasdescribed (21).BacteriophagePM2 DNAwasusedastheinternal size marker.

Gelelectrophoresisof EBV DNAfragments.A

2-pg amount of EBV DNA (circular or linear) was incubated with 20 U of EcoRI restriction enzyme

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CIRCULAR EBV DNA 13

(Boehringer, WestGermany) in 40 ,ul of 0.1 M NaCl-50mMTris-hydrochloride (pH7.5)-10mMMgC12-2

mMdithiothreitol at370Cfor 2 h. The reaction was stopped by the addition of 10

pd

of 40% Ficoll-0.1 M Tris-hydrochloride (pH 7.5)-50 mM EDTA-0.075% bromophenol blue, and 0.03- to0.6-jIgportions of the cleaved DNA wereapplied to an agarose slab gel (15 by 20 cm). Both 0.35% and 0.8% gels were used. Elec-trophoresis wasperformedfor 20 h and 1.5 V/cm in 40 mM Tris-acetate (pH 8.0)-5 mM sodium acetate-i

mMEDTA-0.01% ethidium bromide. Gels were pho-tographed under short-wave UV light.Southern blot-tingof the gels (31), hybridization with EBV DNA probes,and fluorography were performed as described

(29).

RESULTS

Isolationof circular EBV DNA. The

stand-ard methods usedfor the enrichment of circular

viralDNAmolecules frommammaliancells,e.g.,

by Hirt precipitation of the host DNA (18),

causeconsiderable losses whenappliedtoDNA

moleculesaslarge asthe EBV genome (2).

Re-cently, several methods involving alkali

treat-ment as an initial step to denature selectively

hostlinear DNA, butnotcovalently closed

cir-cular DNA,were described for the preparation

ofplasmidsfrombacteria, includingDNA

mol-ecules as large as Ti plasmids, F factors, and

cosmidscontaining recombinant DNA (3, 5, 8).

In ourhands,noneofthese methodswere

suc-cessful when directly applied to

EBV-trans-formed lymphocytes. The study of Currier and

Nester (8) on the preparation of Ti plasmids

contains several potentially useful purification

procedures,includinglimited shear treatment of

neutralcelllysatestoallow efficient strand

sep-aration of the host DNA onsubsequent alkali

denaturation, andphenolextractionathigh salt

concentrationto removethe denatured DNA. In

apparent contrast totheresults of thoseauthors,

weobserved that thenecessarysheartreatment

maybeperformedathigh pH withoutcausing

damagetoDNAcircles of the size of

10'

daltons,

and this allows cell lysis and shearing to be

carried out directly in an alkaline buffer.

Fur-ther,after neutralization of such lysates,abrief

proteasedegradationstepappears toberequired

before phenol extraction. The procedure

adopted here avoidsasmuchaspossible lengthy

incubations under conditions that may cause

slowbutsignificantintroduction ofsingle-strand

breaks inlargeDNAcircles.

In ourpenultimate purificationstep, circular

DNA was isolated by banding in an ethidium

bromide-CsCl gradient.The band ofcovalently

closedcircularDNA,which isclearlydetectable

by illuminationwithlong-wave UVlightifmore

than109cellsareusedasstarting material, would

beexpectedtocontain all covalently closed

cir-cular DNAmoleculespresentin the cells,

inde-pendent of their size (upto atleast 105 daltons)

and base composition. Examination of such

DNA preparations from several lymphoid cell

linesby electronmicroscopyindicated thatthey

contain a mixture of mitochondrial DNA

(mtDNA) and EBV DNA, but no other circular

DNA molecules. In a typical experiment with

the Raji line, which carries multiple copies of

circular EBV DNA, the relevant ethidium

bro-mide-CsCl fraction contained about 30

mole-cules of mtDNA (average contour length 5.07

,pm)

permolecule of EBVDNA (average contour

length 51.5 ,um). Small amounts of catenated

circular DNA ofcontourlengths of10and15,um

were also observed, which may be ascribed to

thedimers and trimers of mtDNA knowntobe

present (6), and occasional linear DNA

mole-culeswerefound insomepreparations.As afinal

purification step, we employed either density

gradientcentrifugation in CsCl (without

ethid-ium bromide,toseparatethe circular molecules

accordingtobase composition) orglycerol

gra-dientcentrifugation (toseparate onthe basis of

size).Both methods reduced the level of

contam-ination ofthe circular EBV DNAwith mtDNA

toabout onemtDNA molecule perEBV DNA

molecules (Fig. 1). (With the glycerol gradient

centrifugation method, the EBV DNA fractions contain mtDNA enriched for the faster

sedi-menting dimer and trimerforms.) However,we

mainly used centrifugationin CsCl asthe final

purificationstep;since itavoidsanintermittent

dialysis step, the EBV DNA is obtained in a

smallervolume, and circular viral DNA

inad-vertently nicked during and after collection of

therelevant ethidiumbromide-CsClfraction is

recovered. Byacombination ofCsCl and

glyc-erolgradientpurifications, itshould be feasible

toreduce the level of contaminationtoless than

0.05mtDNAmoleculeperEBV DNAmolecule.

The size distributions of circular EBV DNA

from B95-8 andRajicells, isolated onthe basis

of its covalently closedstructure and

guanine-cytosinecontent,weredetermined. Inagreement

withprevious results onglycerol

gradient-frac-tionated material (21), the EBV DNA circles

fromRajicellsappearedtobe ofuniforn length.

Ten molecules were measured, with contour

lengths of 50.5 to 52.2 ym, andanaveragelength

of 51.4,tmwith a standard deviationof1.2%was

observed. In contrast,theB95-8 EBV DNA

cir-cles showed afinite but distinct size

heteroge-neity,althoughtheiraveragelengthwassimilar

to that of Raji EBV DNA. In 11 molecules

measured, contourlengths variedbetween47.6

and 57.7,tm, andan average lengthof52.4,um

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FIG. 1. Electronmicroscopiccharacterization ofpreparations of circular EBV DNA. In a typical field of

apreparation fromRajicells,employingamodified Kleinschmidt technique forspreading,only covalently

closedcircular DNA molecules were observed. Two large circles that correspond to EBV DNA and three small circles that correspondtohuman mtDNA could be seen. The bar in the figure represents 1,um.

with a standard deviation of 5.6% was found. (Raji),sincelinear viral DNA molecules present

Although the Raji and B95-8 circles are of the incells activated to virus production in the

for-samelengthwithinexperimental error,the size mer lines areremoved together with the linear

variation in the B95-8material is significant (P host DNA during the initial stages of

purifica-<0.01) incomparisonwith Raji. This variation tion. Theexistence of circular EBV DNA

mole-is due presumably to the presence of differing cules in the cells of producer lines has been

amounts ofrepeatedsequences atthe site cor- demonstrated previously by treatment of

cul-responding to the ends of the linear molecule tures with inhibitors of viral DNA replication

(seebelow, Fig.2c) and/or at the internal repeat before preparation of DNA circles (7, 14). No

region (4, 12, 29). It is noteworthy that the such pretreatment with inhibitors is necessary

procedure for isolation of circular EBV DNA in thepresentprocedure.

devised here appears to work aswell for virus TheEBV DNA in Rajicells, which is present

producer lines(B95-8) asfor nonproducerlines as 50 to 60copies ofcircular DNA molecules and

14 GRIFFIN ET AL. J. VIROL.

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[image:4.496.69.446.61.464.2]

CIRCULAR EBV DNA 15

amuchsmaller amountofintegrated viral DNA

sequences, amounts to about 0.1% of the total

DNAis eachcell(21). Weobtained6to

9,ug

of

circularE5BVDNAfrom2x 109to3 x109Raji

cells; since each cell contains about6pgofDNA,

thisrepresents ayield of circular EBV DNA of

about 50%.

EcoRI cleavage patterns of DNA circles.

TheintracellularcircularEBVDNApresentin

virus-transforned cells is of similar lengthtothe

linear virion DNA (21), and reassociation

kinet-icsdata (19) andanalysis of restrictionenzyme

fragments by Southernblotting followed by

hy-bridization with radioactively labeled virion

DNA (16, 29, 32) have suggested that the

se-quencecomplexity of the circular DNA is similar

tothat ofvirion DNA.Nevertheless, it hasnot

1 2 3 4

been established whether some of the EBV DNA

circles in a cell are nonidentical, or whether

defectivemoleculescontainingcellular DNA

se-quences(notdetectedby hybridizationwith an

EBV DNA probe) do not also occur. These

possibilities havenowbeenrendered much less

likely by the direct visualization ofthe DNA

fragments obtained by cleavage of circularDNA

preparationswith theEcoRIenzymeand

sepa-ration byagarosegelelectrophoresis. The

frag-ment pattern obtained by EcoRI digestion of

twodifferent preparationsofcircular EBVDNA

from the marmoset B95-8 lymphocyte line is

shown inFig. 2a, together with fragments

ob-tained from linear B95-8-derived virion DNA

and from hostmarmosetmtDNA.Although

cir-cularEBV DNA preparations used stillcontain

1 2 3 4 1 2 _{3 4}

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FIG. 2. EcoRIcleavagepatternof circular EBVDNAfromB95-8cells,asdeterminedbyelectrophoresis in a0.8% agarosegeL Thecapital letters in theleftmarginrefertotheEcoRIfragments of B95-8 linear EB virion DNA(12). Lane1containsmarmosetmtDNAfromB95-8cells; lanes2and3contain two preparations

ofcircular EBV DNAfrom different batchesofB95-8 cells, carriedoutatdifferent times; lane4contains linear EBV DNAfrom virusparticles produced byB95-8 cells. (a) Direct visualization of fragments by

ethidium bromidefluorescence;(b) detectionofEBV DNA sequencesbySouthernblottingandhybridization

withnick-translated32P-labeledvirionDNA; (c)same as(b), but thenick-translated32P-labeledprobe used was acloned EBV DNAfragmentthat containedthe terminal repeat sequences rather thanwhole virion DNA.

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16 GRIFFIN ET AL.

about 1 mtDNA per EBV DNA molecule, in

other respectsthe fragments from circular

ver-sus linear B95-8 EBV DNAs appear virtually

indistinguishable. Possibly, theD,Efragment is

broader and ofslightly higher molecular weight

in thecircular material.Itisclear, however, that

extra bandscorresponding todefective viralor

other DNAsare notseen, evenwhen the method

ofmeasurementdoes notrelyonhybridization

with aselected probe. Moreover, it canbe

as-sumed thatfragments observed in the

prepara-tionsof circular EBVDNA are notdue to

con-taminating virionDNA, sincemorethan98% of

thestarting materialwascircularasdetermined

byelectron microscopy. Inaddition,

hybridiza-tionexperimentswithradioactively labeled EBV

virion DNA after Southern blotting ofthe gel

showed similar patterns for both circular and

linear B95-8 EBVDNAs(Fig. 2b).Asexpected,

thelow-molecular-weight bands in thecircular

DNAswhose positionscoincided withfragments

ofmtDNAdidnothybridize with the probe.

After EcoRI cleavage of linear virion DNA,

oneendof the molecule is knowntobecontained

inthe D fragment. AnEcoRI cleavage site

oc-cursclosetothe otherend, and it is observedas

aseries of smallfragmentsof different sizes since

the terminalsequenceiscomprised ofavariable

numberofcopies ofa 500base-pairrepeat(12).

Ourresults with linear virus DNA (Fig. 2c)are

inagreementwith those data. However,aclear

difference between linear and circular B95-8

DNAs isrevealed byhybridization withacloned

EBV DNAfragment that contains the terminal

sequencesof thelinearmolecules.Inthecircular

EBV DNAmolecules,sequencesrepresentedby

thisprobe occur at theposition of the D

frag-ment, which hybridizes strongly, whereas the

small fragments are not found (Fig. 2c). This

would be theexpected result if thetermini of a

linear molecule werejoined during

circulariza-tionby recombination betweenrepeat sequences

present atboth ends (13, 20,21).Inaddition,a

bandslightly largerthan theDband isobserved

inthecleavedDNAcirclesby hybridization with

the sameprobe (Fig. 2c). Thisrepresents a join

fragment ofthe D and Ibands, present in

sub-molar amounts due to partial resistance to

EcoRIat acleavagesite closetothe leftendof

the linear molecule (L. Rymo, T. Lindahl, S.

Povey,andG.Klein, Virology,inpress).

Similarexperiments with circular EBV DNA

fromRajicellsareshown inFig.3togetherwith

data for the circular andlinear forms ofB95-8

EBV DNA. To obtainbetter resolutionoflarge

fragments,a0.35% agarosegelwasusedinstead

of a 0.8%gel.The data showthat,inthe case of

Raji EBV DNAalso, thefragment patternsas

observedbyethidium bromidefluorescenceare

closelysimilar to thoserevealed byhybridization

withnick-translated 32P-labeledEBVDNAfrom

B95-8 virus particles. In contrast to a recent

report (16), several types ofdefective or

rear-rangedEBV DNA moleculeswere not seen in

thepurifiedcircular viral DNA fromRajicells.

The fragment patterns obtained here are in

agreement with recent mapping data and

hy-bridization studies (4, 16, 26; Rymo et al., in

press), which show that the EcoRI C and D

fragments ofRaji EBV DNA are larger than

those of B95-8 EBV DNA. Thus, the Raji C

band occurs immediately below the B band,

whereas the D band is foundat aposition similar

tothat of the B95-8C band.Hybridization with

the cloned EBV DNA probe containing

se-quences present atthe termini of linear B95-8

DNA yielded hybridization with the Raji D

band, butnot with the A, B, C, or any of the

smaller bands. Asdiscussed elsewhere(Rymoet

al., inpress), partial resistance toEcoRI

cleav-age at two sites generated two submolar

frag-ments. One ofthese, which iscomprised of the

G1 and Lfragments, comigrated with the largest

mtDNA fragment and was only detected by

hybridization (Fig. 3b). The other, which

mi-grated between bands B and C (Fig. 3b), was

comprised of the D and I fragments, since it

hybridized both with a proberepresenting

ter-minalsequences anda cloned EcoRI fragment

Iprobe. Additional bands visually observed in

Raji circular DNA correspond to EcoRI

frag-ments from human mitochondrial DNA. The

latter yields three fragments on cleavage with

EcoRI (10); two of these are present between

the EBV DNA F and G fragments (Fig. 3),

whereas the third is smaller thanthe EBV DNA

Jfragment.

Search for smallamountsofcircular EBV

DNA. The procedurewe employed for

prepa-ration ofcircularEBVDNAdependsonefficient

removal oflinear DNA beforedensity gradient

centrifugation,sothe material used for ethidium bromide-CsCl fractionation is about 200-fold

purifiedwithrespect to circular EBV DNAas

comparedtowholecelllysates.Hybridization of

suchmaterial,orofgradientfractionscontaining

covalently closed circularDNAmolecules,with

an EBVprobeprovides asensitive method for

detecting smallamounts ofcircular viral DNA

molecules in varioustypes ofcells. Control

ex-perimentswithmixturesofRajicells anda

100-foldexcessof Africangreenmonkeykidneycells

indicatedthat onecovalently closedEBV DNA

circle per 5 to 10 cellscouldbe detected (data

not shown). We have used this approach to

search forcircular EBV DNA in the Namalva

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CIRCULAR EBV DNA 17

1 2 3

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FIG. 3. EcoRIcleavagepatternsofcircular EBV DNAfromRajicellsand B95-8 cells,asdetermined by

electrophoresisina0.35%agarosegel.Letterscorrespondtobandsobtained withreference B95-8 virionDNA (lane 4).Lanes1, 2, and3 contain cleavedRajicircular EBVDNA, 0.6, 0.3,and0.03pg,respectively. Lane4 contains cleavedB95-8 virionDNA,0.4 pg.Lanes5, 6,and 7contain cleavedcircularEB VDNAfrom B95-8

cells, 0.6, 0.3,and 0.03 pg,respectively. (a)Directvisualization offragmentsby ethidium bromide fluorescence; (b)oneofseveralexposurestodetect EBV DNAsequencesbySouthernblotting and hybridization,asin Fig.

2b. Thephotographshown isalongexposuretoreveal thesmall DNA fragments.

line, an EBV-transformed lymphoid cell line

that contains much less EBV DNA thanmost

other such lines, i.e., only 2 to 3 viral genome

equivalents per cell (1, 25). No sequences

hy-bridizingtoEBV DNAweredetected in circular

DNAfrom Namalvacells, indicatingthat EBV

DNA is not present as covalentlyclosed DNA

circles in this line. Similarly, nocircular EBV

DNAhas beendetected inAW-Ramos, another

EBNA-positive lymphoid cell line that contains

only small amounts of EBV DNA (E. Bjorck,

unpublishedobservations). AW-Ramos has been

shown previously to carry its EBV DNA in

integrated form (2). It now appearslikelythat

thisis also the case forthe Namalva cell line.

Moreover, these data indicate that thepresence

ofcircular EBV DNAmolecules,although

usu-ally found in Burkitt lymphoma lines, is not

obligatory for cellular transformationtooccur.

DISCUSSION

The circular EBVDNAmoleculespresentin

virus-transformed humancells occurclosely

as-sociated with the host DNA. Thus, such EBV

DNAisorganized in nucleosomes (30) andonly

replicates during the S phase (15). In these as well asotherregards, it has properties similarto

anotherplasmid found in eucaryotic cells, the

2-ttmDNAfrom

yeast (24).

The

large

size of EBV

DNA and its association with host DNAhave

made itdifficulttopreparecircular EBVDNA

exceptby laborious procedures which in the end

yield only minute amounts of material (21).

Thus,manystudiesoncircular DNAhave used

crudepreparations of this material (7, 14, 16, 29,

32). The procedure described here, which is

adapted with important modifications from

sev-eral methodspreviouslyemployed in other

sys-b

40,1981

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18 GRIFFIN ET AL.

temsfor isolation of circularDNA, allows

con-venient isolation of EBV DNA circles in

amountsreadily detected byconventional

opti-cal methods. The only contaminating material

appears to be host mtDNA. Such DNA

prepa-rationsmaybe used forgenetic mapping studies

(Rymo et al., in press) and other experiments

that require access torelatively large amounts

of circular EBV DNA. The procedure should

alsobe well suitedtosearch for smallamounts

ofcircular herpesvirus DNA molecules in

var-iouscell lines and tissue biopsies.

In our experiments, we have used circular

EBV DNAtostudy itscomplexity andcompare

sequenceswith thosefound in virion DNA. The

sequence complexityofsuchintracellular EBV

DNA, as estimated by direct visualization of

restriction enzyme fragments after gel

electro-phoresis,appears tobesimilartothat previously

determined by nucleic acid hybridization (29).

Ouranalysis of the EcoRIfragmentsof circular

EBV DNA indicatesthe virtual absence ofany

marked heterogeneity or defectiveness in the

viral DNA preparationsinvestigated. In this

re-gard, it isinteresting to notethatcircular EBV

DNA fromRaji cells kept in acell bank since

1967 and recently thawed shows an identical

EcoRIcleavagepattem toEBVDNA prepared

from Raji cells grown continuously for many

yearsintissueculture,asestimatedbySouthern

blotting and hybridization (Rymo et al., in

press).Althoughasurveyof theEcoRI fragment

patterns of circular EBV DNA from several

differentsourcesrevealed detectabledifferences

between almost all natural isolates (29), their

overall similarity and the clean fragment

pat-terns observed here (Fig. 2and 3) indicate that

the sequences of intracellular circular EBV

DNAdonotrearrange to amarkedextentduring

continuedproliferation of the hostcells. These

resultsareinapparentconflict withareportby

Helleretal. (16) ofrearrangementand

hetero-geneity within EBV DNA from Rajicells.Those

authors,however, employedcrudepreparations

of viralDNAcontaininganexcessof host DNA,

and the occurrence ofpartialrestrictionenzyme

cleavage productswasnotassessed.

The relationship between the linear EBV

DNA in virus particles and the intracellular

circularandintegratedviral DNA formsremains

unclear. It seems likely, however, that the

cir-cular form is generated by recombination

be-tweenthe terminiof incomingvirion DNA (13,

20, 21). It is noteworthy that the size

heteroge-neity present in B95-8virionDNA (12) also is

observed in B95-8circular DNA. Theabilityof

the hostcelltoreproducefaithfullythe circular

EBV DNA,without generation of shortened or

defective molecules, may be an obligatory

re-quirement forthepreservation of this commonly

occurringlatent form of the virus.

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This work was supported by research grants from the Swedish and Danish Cancer Societies andby Public Health Service contract1CP8-1020 within the Virus CancerProgram

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