Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus.

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0022-538X/89/062657-10$02.00/0

Society

forMicrobiology

Multiple

EBNA1-Binding

Sites

Are

Required

To Form

an

EBNA1-Dependent Enhancer

and

To

Activate

a

Minimal

Replicative

Origin

within

oriP of

Epstein-Barr Virus

DOREEN A. WYSOKENSKI ANDJOHN L. YATES*

Departmentof Human Genetics, Roswell Park Memorial Instituite, Buffalo, New York 14263

Received 1 December1988/Accepted 8March 1989

EBNAl activatesthe EBV plasmid maintenance sequenceoriP by bindingtoitstwoessential regions. One

region isafamily of 30-base-pair (bp)repeatsandis activated by EBNA1to actas atranscriptionalenhancer. The other region contains a 65-bp dyad symmetry and lacks enhancer function. To explore the functional

differencesbetween thetworegions,wedeterminedoriPactivitiesasfunctions of thenumberof30-bprepeats

andcompared them withactivities determined when tandem copies ofthe dyadsymmetryregionwereusedto

replace the 30-bp repeats.Three conclusions have been drawn. (i) Activation of the 30-bprepeatsby EBNA1

toenhancetranscriptionortopermit plasmid maintenance isahighly cooperativeprocessinvolvingatleastsix or seven30-bprepeatsforfullactivity. (ii) Tandemcopiesofthe dyadsymmetryregion cooperativelyenhance

transcription butarelesseffectivethan 30-bprepeatsprovidingasimilar number ofEBNAl-bindingsites.(iii)

Tandemcopies ofthe dyadsymmetryregion alone cooperatively activate replication, suggesting thatthe region

contains theactual origin ofreplication. We alsoreport thatwhile rodent-derived cell lines do not support

replication of EBV-derived plasmids they do permit EBNAl-dependent enhancer activity. EBV plasmid

replication thus requires the interaction of EBNA1 or oriP with a host factor that is not required for

enhancement of transcription.

The170-kilobase-pair (kbp)genomeof the human

herpes-virusEpstein-Barr virus (EBV) is maintained extrachromo-somallyinlatentlyinfected cells (24)as acovalently closed,

circularplasmid (18)thatisreplicated duringSphase ofthe cellcycle (13). The EBV genomeencodes two components

known to function in plasmid maintenance: an 1,800-bp

cis-acting region, oriP,thatsupportsthestablemaintenance ofrecombinant plasmids carrying it (37), and the nuclear antigen, EBNA1, that is required for oriP to function in permissive cells (19, 38). Since EBNA1 is the only EBV-encoded protein requiredforplasmid maintenance, plasmid replication is presumedto be performed largely by thecell DNAreplication machinery, directed by EBNA1 to initiate replicationwithinoriP(see reference 21forareview).

Two salient characteristics of oriP and its activation by EBNA1 that are common features of the control of DNA replication in both procaryotes and eucaryotes are the

involvement of multiple initiator protein-DNA interactions (8)and oftranscriptional control elements (5). oriP is

com-posedoftwoessentialregions(31), eachcontaining multiple EBNA1-binding sites(28) (Fig. 1A). One essential regionis composed ofapproximately 20 tandem copies of a 30-bp sequence, each copy containing an EBNA1-binding site.

This family of 30-bp repeats acts as a transcriptional

en-hancer inastrictlyEBNA1-dependent manner(30, 31).The other essential component oforiP, located almost 1,000 bp from the 30-bp repeats, contains two symmetrically posi-tionedEBNA1-binding sites withina 65-bp dyadsymmetry

and two additional EBNA1-binding sites flanking the

sym-metrical sequence. This region, referred to as the dyad symmetry region, lacks significant enhancer activity. By analogy to known replicative origins, particularly those of

simian virus 40 and polyoma virus, for which bidirectional

DNA synthesis begins at a region ofdyad symmetry

con-*Correspondingauthor.

taining symmetrically positioned binding sites for the

initia-torprotein large Tantigen, it was suggested that the dyad symmetry region oforiPcontains theactual origin of repli-cation(31). The 30-bp repeatscan thus be viewed as

acti-vating this origin from a distance, perhaps through an

enhancerlike mechanism. The 30-bp repeatsand the region of dyad symmetry of oriP, in fact, exhibit the spatial independencecharacteristicofenhancer-promoter combina-tions;whilebothcomponentsoforiPmustbepresentonthe sameDNAmoleculetopermitDNAreplication,the relative orientationofthetwocomponents and thedistance between them, when varied fromzerotoseveralthousand basepairs,

areof littleimportance (31;D. Reisman, Ph.D. dissertation, University ofWisconsin, Madison, 1986).

Wesoughttoascertain howmultiple EBNA1-bindingsites in the two components of oriP are involved in apparently

distinct functions. First, the minimal numbers of 30-bp

repeats needed for full enhanceractivity and for oriP repli-cation were determined. Next, to determine whether the

functional differences between thetworegionsoforiP result

from the different numbers ofEBNA1-binding sites in the

two regions or from functional differences between these

binding sites and theirneighboring sequences, tandem

cop-iesof thedyadsymmetryregionweretested fortheactivities

of the 30-bp repeats. The results imply that efficient

en-hancerfunction and activation of oriPreplication minimally involve the cooperation ofseven, or possibly six, EBNA1

dimers. EBNA1-binding sites within the 30-bprepeats were

found to activate replication and transcription more

effi-ciently than those within the dyad symmetry region, yet tandem copies ofthe dyad symmetry regionexhibited

lim-ited EBNA1-dependent enhancer activity and could

repli-catein the absence ofthe30-bprepeats. The resultsprovide experimental evidence that the dyad symmetry region

con-tains thereplicative originof oriP and thatthe 30-bp repeats

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A

30-bp Repeats Dyod Symmetry

ao b a c'b a c a da 'hb""' " b a c' b a c doeA

III I I I

E Ns N N N N Bx N N N Bx 4 960bp EV S H

a . a% -'

0x30 2x30

B.

- 7x30

5x30

pFR-TKCAT

20x30bp cat 5, amp

I B So '0' II

vdeletions-- pTKCAT-9x30topTKCAT-Ox30

cat pTKCAT amp

I

BSo IAI1

5I1010 pTKCAT-3xDS

BS S S

C.

orip

B So

pHEBo

hph *a

I

pHEBo&FR

I I

B So

Ito=

pHEBo&FR-2x30

B/Ns So

_ 7/pHEBo&FR-3xDS

B So

Ikbp

FIG. 1. Structure of oriPof EBV strain B95-8(1)andplasmidsusedtostudyitscomponents.Restriction endonuclease sites areshown for BamHI(B), BstXI (Bx), EcoRI (E),EcoRV(EV), HpaI (H),NdeI(N),NsiI(Ns), Sall (Sa),andSmaI(S).(A)Structureof oriP.Segments ofthefamily of 30-bprepeatsandthedyadsymmetryregion (DS)thatweresubclonedfor functional tests(BandC)areindicatedbybrackets.

Open boxes in the30-bprepeats and within thedyad symmetry region representthe18-bp, symmetricEBNA1recognition sequences(28). Closedboxesrepresentthe12-bpspacersthat alternate withEBNA1-bindingsitestoformthe30-bp repeats. Convergingarrowsindicatethe

65-bpdyadsymmetry.For the30-bprepeats,theletterarepresents theconsensus18-bp symmetricsequence;b,c,and drepresentsequences

that differfrom theconsensus sequence atoneposition; anderepresentsasequencethat differsat twopositions. Each superscript prime symbolrepresents onebasechange fromtheconsensus sequence inthe 12-bpspacer, whileeachsuperscriptdeltarepresentsashortened

spacer.(B and C) Plasmids usedin thisstudy,shown linearizedatthepBR322-derivedEcoRI site.Thin linesrepresentE.coli-derivedDNA

(amp, ampicillin resistancegene;hph,hygromycinBphosphotransferasegene;cat,CATgene).Closedboxesrepresentthepromoter(-197

to+56[arrow]) and 3-flankingsequences(626 bp from SmaItoPvuII)of the HSV type 1 TKgene. Openboxesrepresent oriPDNA,with

vertical linesindicating the 30-bprepeatsandacircleindicating the dyadsymmetryregion.

supportits activation throughacooperativeaction of

multi-ple EBNA1 molecules.

MATERIALS ANDMETHODS

Cells and media. The EBV-positive Burkitt's lymphoma cell lineRajiandtheEBV-negativeBurkitt's lymphomacell lineWilsonwere growninRPMI 1640 medium supplemented

with 7% fetal bovine serum plus 7%Nu-serum

(Collabora-tiveResearch, Inc.). LMTK- cellsweregrowninminimum

essential medium alpha with the same serum supplement.

Wilson(p174) is a derivative of Wilson carrying multiple

copies ofthe oriP-based, hygromycin B resistance plasmid p174, whichexpressesEBNA1 (38).Wilson(p174) cellswere

maintained in medium containing 300 FLg of hygromycin B

per ml, except for a few generations before and during

chloramphenicol acetyltransferase (CAT) gene expression

studies.

Recombinantplasmids. Plasmids were constructed by

us-ing standard methods (20). pFR-TKCAT carries the family of 30-bp repeats on a fragment of approximately 900 bp

extendingfrom an EcoRI site to aBaI31 deletion endpoint

positioned between the EcoRI and BamHI sites of pBR322-derived DNA and located 2.1 kbp 3' or 2.5 kbp 5' to the thymidine kinase (TK) promoter (Fig. 1B). It was

con-structedfrompABal4(37) by replacing theneogene,located

between the promoter and polyadenylation signals of the herpessimplexvirus TKgene,with the bacterialCATgene.

pTKCATwasgeneratedfrompFR-TKCATby replacingthe

DNA between an EBV SphI site and the SphI site in

pBR322-derivedDNA withaBamHI linker, leavingonly 15 bpof EBV DNA between theEcoRI andBamHIsites (Fig. 1B). pTKCAT-9x30 was generated by deleting DNA

be-tweenthe BstXI sitenearthecenterof the30-bprepeatsand the flanking BamHI site ofpFR-TKCAT. pTKCAT-9x30 through pTKCAT-Ox30 were constructed by partially di-gesting pFR-TKCATwith NdeI,followedby digestion with BamHI, repair with the Klenow large fragment of DNA polymerase I, ligation, and a final digestion with BstXI to remove unwantedproducts of NdeI partialdigestion.

The dyad symmetry region of oriP was excised from

pHEBo (34) as a 139-bp fragment by using endonucleases

EcoRV andHpaIandwassubcloned inpUC12attheHindll site.Next,aBglIIlinkerwasintroducedintotheneighboring

PstI site of the pUC12 polylinker, generating p400. The 159-bp BamHI-to-BglII fragment containing the dyad sym-metryregionand20bpof linker DNAwasexcisedfrom p400 andinserted atthe BamHI siteofpTKCATin one,two, or

three tandemcopies, all in theorientation shown inFig. 1B (indicated by the SmaI site), generating pTKCAT-lxDS through pTKCAT-3xDS.

DS

amp

..V..

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pHEBoA&FR was created by deleting DNA between the unique Sall site of pHEBo and an NcoI site immediately flanking the 30-bp repeats, regenerating the Sall site (Fig.

1C).

The shortened 30-bp repeat arrays and tandem copies of the dyad symmetry region were excised from the pTKCAT vector with

NsiI

plus

SalJ

and with EcoRI plus

SalI,

respectively, and inserted into pHEBoAFR between its filled

BamHI

site and itsSall site.

p294, obtained from Bill Sugden, is a 10.4-kbp, oriP-carrying plasmid derived from pHEBo that expresses the

EBNA1

gene from the immediate early promoter of human cytomegalovirus.

Transient expression and replication assays. DNA was introduced into 6 x 106to 1 x

107

Raji, Wilson, or Wilson (p174) cells by electroporation with a ZA 1000 instrument (Promega) having a 0.5-cm-wide chamber of 0.5-ml volume at 1,500 V (3,000 V/cm) with 5

pug

of DNA per ml of RPMI 1640 medium on ice. (Calculated resistance x

capacitance,

approximately 1 ms.)

MouseLMTK-cells were transfected with 1.5

jig

of CAT expression plasmid plus 5

p,g

of

EBNA1

expression plasmid per 6-cm dish by using the calcium phosphate method (37) and were shocked 5 h later with 20% glycerol for 2min.After 24 h, the cells were washed with phosphate-buffered saline containing 1 mM EDTA, trypsinized, and split into two 10-cm dishes so that one dish could be harvested for CAT and the other for DNA, as described below. Cells were harvested after 72 h in culture, lysed by three freeze-thaw cycles, and assayed for CAT levels as previously described (12), with the following modifications. Reaction mixtures contained 10 nmol of

chloramphenicol

at a specific activity of5 mCi per mmol in a volume of 70

,ul.

Acetylation rates were linear with time up to 7 nmol of acetylation (70%) or for at least 4 h and were linear with respect to added cell extract up to 100

,ug

of protein. For experiments with Raji cells, 50 to 100

,ug

of protein, measured by the Lowry method, was assayed for 25

min

to 3 h, as appropriate for the CAT activities present.

104, 103,

or102 cells perwell. Cells werefed atleast onceper week by replacing part of the mediumin each well. Wells with proliferatingdrug-resistant cells were scored after3 to 4 weeks in culture. The frequency of outgrowth of drug-resistant cloneswas then calculated byassumingaPoisson distribution involving the fraction of negative wells at the dilution for eachthatgavesomepositive andsomenegative wells.

DNAs from Hirt supernatants ofexpanded hygromycin B-resistant clones or pools of clones were analyzed on Southern blots of agarosegelsasdescribed above. Plasmids wererecoveredfromHirtsupernatantsbytransformation of Escherichia

coli

as described previously (37).

RESULTS

The experiments weredesignedtodetermine

(i)

the activ-ities of the 30-bp repeats of oriP asfunctions of the number oftandem30-bpcopiesand(ii) whether tandem

copies

ofthe dyad symmetryregionoforiPcontaining similar numbers

of

EBNA1-binding sites function similarly. As measures of 30-bp repeat activities, both

EBNA1-dependent

enhance-ment oftranscription and cis-complementation ofthe

dyad

symmetry region oforiP to allow EBNA1-dependent DNA replication weredetermined.

Enhancer activity as function of numberof

EBNAl-binding

sites. Deletions were introduced into the

family

of

30-bp

repeatsbyusingthe plasmidpFR-TKCAT, which carriesall 20 copiesof30-bprepeats oforiPandthebacterial CATgene expressed from the rather weak TK promoter of

herpes

simplex virus. Expression of the CAT gene from the TK promoterisenhancedup to200-fold in

EBNAl-positive

cells by the presence of the 30-bp repeats on

pFR-TKCAT

(30).

Thenumber of30-bp repeats

carrying

intact

EBNA1-binding

sites onpFR-TKCAT was reducedfromthefull20

copies

to

9, 7, 5, 2, and 0 copies (Fig. 1A and B).

Next,

the

dyad

symmetry region of oriP, defined here as a

139-bp

DNA segment containing the 65-bp

dyad

symmetry with its two

EBNAl-binding sites and the two additional

flanking

EBNAl-binding sites, was inserted into

pTKCAT

in one, two, or three tandem copies (Fig. 1A and

B).

pTKCAT

is identical to pFR-TKCAT except that it lacks the

30-bp

repeats. The enhancer activities of these constructs were

then compared by using

electroporation

to introduce the plasmids into Raji cells and by

measuring

CAT levels 72 h later.BecauseColEl-relatedplasmids

(such

as

pBR322)

can

dimerize during propagation in Escherichia

coli

and the 30-bprepeatscan enhance

transcription

from

long distances,

we took care that only plasmid

preparations

with less than 5%dimeric molecules were usedforenhancer assays.

The relative CAT activities are shown in

Fig.

2. Nine intact 30-bp repeats were

fully

active,

enhancing

CAT expression 70% more than didall 20

30-bp

repeats, while 7 copies were two-thirds asactive asall 20. This isconsistent with the observation by Reisman

and

Sugden

of full

en-hancer activity with 12 30-bp repeats

(30).

With five intact 30-bp repeats,

activity

dropped

to

11%;

two

30-bp

repeats

gave only 1%of fullactivity,

raising

CAT

expression

to

only

twice the unenhanced level. These results

suggest

that a

minimum of six or seven

30-bp

repeats are

required

for efficient enhanceractivityand that

activity

drops

rapidly

as

the numberof30-bp repeats are reduced below this. Because there are sequence differences among

30-bp

repeats, we do not know whetherindividual

repeats

within thesegments tested contribute

equally

to enhancer

activity.

It is conceivable, for

example,

that

30-bp

repeats

6 and 7

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200r

> too

O

50-0 2 4 5 7 8 9 12 20

EBNA-1-Binding

Sites

FIG. 2. Enhanceractivitywithvarious numbers of30-bprepeats

(closedbars) or copies of thedyad symmetry region(open bars).

Tests were performed in Raji cells as described in Materials and Methods. Mean CAT activities per microgram of extract were obtained fromfourelectroporations. Relative valuesareindicated

along with the number-corrected standard errors. The activity obtained withpFR-TKCAT,set at100%,wastypically2.5 nmol of

acetylationperhper100 ,ugofprotein.Theunenhanced CAT level

obtained with pTKCAT, typically 0.012 nmol/h per 100 p.g of

protein,wasthreetofivetimes thebackground valueobtained when an extract of mock-electroporated cells was tested. The values shownhavebeen corrected forbackground.

from the leftarealone responsible formostof theenhancer activity of the seven leftmost repeats rather than acting cooperatively with the other repeats. However, consider-ationof thesequencedifferencesamongtheserepeatsmakes this seem very unlikely. In Fig. 1A each 30-bp repeat is depicted as an open box representing the 18-bp symmetric

region containing the EBNA1 recognition sequence and a

closedboxrepresenting the 12-bpspacersequence. Repeats

1, 6,and 8containtheconsensussymmetric region (Fig. 1A,

a), while repeats 2, 4, 5, and 7 differ from the consensus sequenceinthisregion by only 1 bp (b and c).Thusrepeats

6 and 7are identical to repeats 3 and 4 exceptfora single

changeinthespacerofrepeat4(denoted by '). Similarly,we

do not knowwhether the EBNA1-binding sites in the dyad

symmetryregioncontribute equallytoenhanceractivity. We

note,however, thatmostofthenonlinearincrease in activity

was observed when going from four to eight binding sites from thedyad symmetryregion or whengoing from five to seven ornine 30-bprepeats,implyingthatsimilar numbersof

binding sites fromtworegionsarerequired for cooperativity.

While one copy of the dyad symmetry region had very

littleenhancer activity(30, 31)(Fig. 2),twoorthree tandem

copies exhibited significant activity, being 25 and 35% as

activeastheintactfamily of 30-bprepeats,respectively.The increase in enhancer activity from3.5%withone copyof the

TABLE 1.

EBNA1-dependent

enhancer activity in human andmousecells

CATactivityafor cell line:

LMTK- cotransfected Testplasmid Wilson Wilson(p174) with:

(EBNA1-) (EBNA1+) p392 p294

(EBNAl-) (EBNA1+)

pTKCAT 0.17 0.2 1.0 1.0

pFR-TKCAT 0.11 42 1.9 13

pTKCAT-3xDS 0.14 7.4 1.4 13

a Valuesareaveragesofduplicatedeterminations andareexpressedasthe percentageofchloramphenicol(total,10nmol)acetylated per hourof

reac-tion. Assayed were 100 pLg ofprotein extracted from approximately 10'

WilsonorWilson(p174)cellsand1.5,ug of protein extracted from approxi-mately 5 x 103LMTK- cells.

dyad symmetry region to 25% with two copies clearly exceededalinearincrease,asobserved for enhanceractivity

asa function of low numbers of30-bp repeats (Fig. 2). As with the family of 30-bp repeats (30, 31), the enhancer activity of three tandem copies of the dyad symmetryregion wasEBNA1dependent; itwasobserved inEBNAl-positive Wilson(p174) cells butnotinEBNA1-negative Wilson cells (Table 1).

As it will be shown below, tandem copies of the dyad symmetry region allow DNA replication in Raji cells. It is therefore possible that some ofthe increase in CAT gene expression observed withtwoorthree tandem copies of the dyadsymmetryregionwasduetoanincrease in the number oftranscriptionally active plasmids because ofreplication. We can roughly estimate the extent of replication if we

assume thatoriP-carrying plasmids replicatean average of once percelldivision cycle during the transient expression assay, asthey dowhentheyhave beenstably introducedby genetic selection. Recent experiments indicate that this is thecase with EBV-derived plasmids introducedintohuman 293 cells(J. L. Yates, unpublished results). Following elec-troporation, the surviving Rajicells appear tolagfor24 hand then to undergo two population doublings before being harvested 72 h after electroporation. If an oriP-carrying plasmid is assumed to replicate twice under these condi-tions, then pTKCAT carrying three copies of the dyad symmetryregion that replicates 40%asefficientlyasdoes a plasmid carrying oriP (see Fig. 4) would be expected to increase in number by less than twofold. In addition, as shownbelow, the enhanceractivity of tandemcopies of the dyad symmetry region or of the 30-bp repeats can be demonstrated by using rodent cell lines, which are nonper-missivefororiP-specificreplication.

In conclusion, a sufficient number of EBNA1-binding sites, 8 or 12, from the dyad symmetry region can act to enhance transcription, although not as efficiently in the human cell lines tested as a similar number of EBNA1-binding sites in thecontextofthe 30-bp repeats. Because the 30-bp repeats and the region of dyad symmetry have no common sequences other than the EBNA1-binding sites, and because these DNA segmentshave no enhancer activity in the absence of EBNA1, we conclude that multiple EBNA1-bindingsites,minimallysix or seven, are necessary and sufficient forenhancer activity.

EBNA1-dependent enhancer activity in cells nonpermissive forEBNA1-specific replication. Totesttheenhanceractivity of tandem copiesof thedyadsymmetryregionin the absence of DNA replication, we took advantage of the fact that

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rodent cell lines are nonpermissive for oriP-specific replica-tion but support the EBNA1-dependentenhancer activity of the 30-bp repeats. pTKCAT, pFR-TKCAT, or

pTKCAT-3xDS

DNA was cotransfected into mouse LMTK- cells along with a plasmid that expresses EBNA1 from the cyto-megalovirus immediate early promoter or with a control plasmid lacking the

EBNA1

gene. Three tandem copies of the dyad symmetry region enhanced CAT gene expression in an

EBNA1-dependent

manner to the same extent as did the family of 30-bp repeats (Table 1).

The cotransfected plasmids p294, which expresses the

EBNA1

gene, and p392, which lacks the EBNA1 gene, both carry oriP. To test for replication of these DNAs in the transfected LMTK- cells, low-molecular-mass DNA was isolated 72 h after transfection froma portion of the LMTK-cells that had also been transfected with pTKCAT-3xDS. The DNA was digested withBamHIto linearize the plasmids and with

DpnI

to degrade unreplicated DNA. (DpnI cuts only DNA containing methylated adenines on both strands of its recognition sequence, GATC. This modification is present on plasmids propagated in dam' E. coli; it is lost if the plasmids are replicated after being introduced into mam-malian cells.) Results of Southern analysis of the DNA are shown in Fig. 3. The same low levels of DpnI-resistant plasmids were detected whether the cotransfecting plasmid carried the EBNA1 gene (p294; Fig. 3, lanes 1 and 2) or lacked it (p392; lanes 3 and 4). In similar studies of transient replication with several human-derived cell lines, EBNA1 stimulated replication of oriP-carrying plasmids 10- to 50-fold (36; Yates, unpublished results). To verify that theDpnI cut DNA to completion, an E. coli-prepared (dam-methy-lated) plasmid DNA, pHEBo, intermediate in size between the two introduced plasmids, was mixed with a portion of the DNA isolated from the transfected cells and then digested and analyzed in parallel (Fig. 3, lane co.); noDpnI-resistant pHEBo DNA was detected. The low-level, apparently non-specific replication of transiently introduced plasmids is not peculiar to LMTK- cells; we have consistently observed it with each of several mammalian cell lines tested, including human 143 cells (36) and Raji cells (see below).

We conclude that any EBNA1 oriP-specific replication that may occur in LMTK- cells is less efficient than the EBNA1-independent, apparently template-nonspecific con-version of input DNA to a DpnI-resistant form and is therefore undetectable. This is consistent with the inability of EBV-derived plasmids to be maintained as plasmids under selection in any of several tested cell lines derived from rodents (38). Yet theEBNA1-dependent enhancer activity of the 30-bp repeats is readily observed in LMTK- cells. Transactivation of the 30-bp repeats by EBNA1 and the inability of EBNA1 to activate oriP-specific replication in other mouse-derived cell lines has been independently ob-served (J. Mecsas and B. Sugden, personal communication). Multiple

EBNAI-binding

sites activate replication of dyad symmetry region. Both the 30-bp repeats and the dyad symmetry region oforiP must be present on the same DNA molecule to allow efficient EBNA1-dependent DNA replica-tion (19, 31). The shortened arrays of the 30-bprepeats were tested for the ability to cis-complement the dyad symmetry region of oriP by substituting them for the 30-bp repeats of theoriP-containing hygromycinB resistance vector pHEBo. The family of repeats was deleted from pHEBo, generating pHEBoAFR,to which was added the shortened 30-bprepeat arrays (Fig.1C). The tandem copies of the dyad symmetry region were also moved topHEBoAFRinorder totestthem for the ability to functionally replace the 30-bp repeatswithin

FIG. 3. Resultsof assays for replication ofplasmids transiently introduced into LMTK-cells.Seventy-two hours aftertransfection, low-molecular-mass DNAs were isolated from portions (4 x 106 cells) of the transfected cells used in CAT expression assays, digested with BamHI (to linearizetheplasmids)and withDpnI, and electrophoresed on 0.7% agarose gels. An autoradiograph from a

Southern analysisisshown. Lanes 1 and 2, Samplesfromduplicate transfections with pTKCAT-3xDS plus p294 (EBNA1+); lanes 3 and 4. samples from duplicate transfections with pTKCAT-3xDS plus p392 (EBNAl-). Arrowheads mark the positions ofthe full-length, linear, DpnI-resistant plasmids: pTKCAT-3xDS, 4.7 kbp; p392, 8.4 kbp; and p294, 10.4 kbp. For lane co., 5 ng of dam-methylated pHEBo(7.2 kbp) wasmixed withequalportions ofDNA

from samples represented inlanes 1 and 2 andanalyzed inparallel. Some of the p294 DNA molecules carried a spontaneous, 700-bp deletion of the IR3 repeated sequences within the EBNA1 gene.

Such deletionshave nodeleteriouseffectsonEBNA1 functions(36,

38).

oriP. The plasmids were tested for the ability to replicate transiently following electroporation of Raji cells and to be maintained as plasmids in drug-selected cells.

To measure transient DNA replication, low-molecular-mass DNA was isolated from Raji cells 96 h

following

electroporation, digested with Salltolinearizethe

plasmids,

and analyzed for the presence ofDpnI-resistant molecules. Low-level replication of the vectorspTKCATand

pHyg

was

observed (Fig. 4B). No DpnI-resistant DNA could be de-tectedwhen dam-methylated plasmid DNAwasaddedtothe DNA extracted from mock-transfected cells

(Fig.

4,

lanes Co). TheoriP-carrying plasmid pHEBo replicated at a level 50 times more efficient than the

background,

nonspecific

level. At most, pHEBoAFR carrying all of oriP except the 30-bp repeats replicated at a level twice that ofbackground (Fig. 4B).

Replication activity as a function of the number of

30-bp

repeats washighly nonlinear(Fig. 4A). Twocopies of

30-bp

repeats stimulated replication to onlytwice the

background

level, at most, while five 30-bp repeats supported transient replicationabouthalf asefficientlyasdid all 20

copies.

Seven 30-bp repeats were fully effective. The steeprise in

replica-tion activity as afunction of the number of

30-bp

repeats is thus similar to the enhancer activity curve but is shifted to

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A

A .:o c^0 t t

7

10 3.5 54 137 5-6 27 70 100

i--.

so

B

0,6

4 *)

l: q , ,

2.2 6.1 4.7 12 42 25 2.7 3.2 28 70

FIG. 4. Assays for transient replication of plasmids in Raji cells.

DNAs were isolated from cells 96 h (A) or120 h(B) after

electro-poration, digested with Salland Dpnl, and subjected to Southern

analysis. Hybridization signals representing Dpnl-resistant,

full-length plasmidswerequantified 'bylaserdensitometryof

autoradio-graphs exposed for appropriate periods without intensifying

screens. Relative values, averaged forduplicates, are indicated at

thetops of theblots,withpHEBogivenavalue of100inpanelAand

pHEBoAFR-3XDSgiven avalue of 70 inpanel B. FortwoDNAs,

duplicatetransfectionswerenotavailable foranalysis. For lanesCo.

1 ng ofaplasmid DNA was added to DNAextracted from

mock-electroporatedcells and analyzed inparallel.

fewer copies of30-bp repeats, with five copies supporting

justover50% ofreplication activity.Thereasonfor thismay

be that in the replication assay, a vectorcarrying the dyad

symmetry region (with its four EBNAl-binding sites) is by

necessity used in addition to the 30-bp repeats, and these distant sites are likely to cooperate.

followed

by

the

dyad

symmetry

region

in its normallocation.

Multiple

copies

of the

dyad

symmetry

region

act

cooper-atively

to

permit replication

rather than

acting independently

(Fig.

4). Ifindividual

copies

of the

dyad

symmetry

region

acted

independently

when present on a

plasmid

in

multiple

copies,

the

probability

of such a

plasmid being replicated

would be

approximately

thesumofthe low

probabilities

that each

dyad

symmetry copy,and thevectorsequences,would be

replicated.

For

example,

with

pHEBoAFR-3xDS,

which carries atotal of four

copies

of the

dyad

symmetry

region,

each copy and the vectorwouldcontribute about 0.025 and 0.02 unit ofreplication activity, respectively (relative tothe

valueof 1.0 forpHEBo),to

yield

avalue of0.12.

Instead,

a

valueof0.70 wasobserved

(Fig.

4).

Theratherefficient

replication

obtained withtandem cop-ies of the

dyad

symmetry

region

suggests that this

140-bp

segment contains the actual

origin

of

replication

of oriP. Activation ofthis

origin

appears to

require multiple (five

to

seven)

EBNA1-binding

sites in cis, either from the

dyad

symmetry

region

itself when

repeated

tandemly

orfromthe

30-bp

repeats. As with enhancer

activity,

this activation process is

accomplished

more

effectively by

the

30-bp

re-peatsthan

by

asimilar numberof

EBNA1-binding

sites from the

dyad

symmetry

region

(Fig. 4).

Numberof30-bprepeats

required

for

plasmid maintenance;

inefficient functional substitution by tandem copies ofdyad symmetry region. The

oriP-carrying, hygromycin

B

resis-tance

plasmid

pHEBocanbe

efficiently

introduced into

Raji

cells,

where it is carried under selection as a

stable,

multi-copy

plasmid (34).

Removalof the

30-bp

repeatsoforiPfrom pHEBo decreased the

frequency

at which

hygromycin

B-resistant

Raji

clonescould be obtained

by

almost

1,000-fold,

from 1 to 2% of

Raji

cells electroporated with pHEBo to

0.005% of cells

electroporated

with pHEBoLFR, and pre-vented the plasmids from being maintained

extrachromo-somally (Table

2and

Fig.

5).

Adding

back230-bprepeatsto pHEBoAFRdid notchangeitsproperties,whileaddingback

7, 9,

orall 20

30-bp

repeats

fully

restored efficient

plasmid

maintenance. As with the tests for enhancer activity and transient replication, an intermediate result was obtained when five 30-bp repeats were tested for plasmid

mainte-nance. Although pHEBoAFR carrying five 30-bp repeats

wasabletoconferhygromycinBresistancetoRajicells with

only

1% of the normal

efficiency,

the

drug-resistant

clones carried the

plasmids

asextrachromosomal, circular, unrear-ranged molecules (Table 2 and Fig. 5). In two clones

analyzed,

1 and 6 molecules of

pHEBol\FR-5

x30 were recovered per cell, compared with 12 to 20 copies per cell recoveredwithplasmids carrying fullyfunctionalconstructs oforiP

(Table

2).

Addingone or twotandem copies of the dyad symmetry

region

to

pHEBoAFR

hadnoapparenteffects.Threetandem

'g,sc.,. "Y',,O 0 (i Ili CO

A..-Q * 4z) 0 'Q"

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TABLE 2. Plasmidmaintenancein Raji cells under selection as a

functionof the number of 30-bp repeats

Frequencyof No. ofplasmid Plasmid hygromycin molecules recovered

resistance" percell"

pHEBo 1 x 10-2 20, 12

pHEBo/FR 4 x 10-5 0, 0

pHEBozFR-2x30 2 x 10-5 0, 0

pHEBoAFR-5x30 2 x 10-4 1, 6

pHEBoAFR-7x30 2 x 10-2 12, 15

pHEBoAFR-9x30 2 x 10-2 20, 12

pHEBoAFR-20x30 2 x 10-2 ND"

pHEBoAFR-lxDS 2 x 10-5 0, 0

pHEBoAFR-2xDS 4x 10-5 0, 0

pHEBoAFR-3xDS 3 x 10-4 20, 5, 7, 5

aFrequencies were determined by limiting dilution in hygromycin

B-containing medium as described in Materials and Methods. Values are

averaged frequenciesfor duplicateelectroporations,whichgenerally differed

fromeach other by afactorof 2 or less.

6Values wereobtainedby Southern analysisof DNA insupernatantsof

Hirtextracts(Fig.3) andrepresent individual hygromycin B-resistantclones, exceptforthelasttwo values forpHEBoAFR-3xDS, whichrepresentspools

of100to 200clones.Limit ofdetection:0.05molecule recoveredper cell. C ND, Notdone.

A

pHEBo

sthd 45

r3-3-0,0,cQ-,00Q0ve0- ;

B pHEEo

std. 45.

N 0

'5,0, e0 e 0

0 q k1- Ai A2a2'2oR Z

pHEBoAFR-3xDS

std

1- '' I ---I I

pHEBo std.

C, Co CoXe

'21 A21 q-lI~ O "

copies ofthe dyad symmetry region restored plasmid main-tenance to about 1% of normal efficiency, similar to the result obtained with five 30-bp repeats. Most hygromycin B-resistant clones obtained with this construct, pHEBoAFR-3xDS, carried fewer copies of the plasmid per cell than resultedwithpHEBo. However, one clone carried about 20 copies ofthe plasmidpercell (Table 2 and Fig. 5).

Properties of pHEBoAFR-5x30 and pHEBoAFR-3xDS maintained as plasmids in Raji cells. Because hygromycin-resistant clones are selected to carrypHEBoAFR-5x30and pHEBoAFR-3xDS at low frequencies, it seemed possible that genetically altered forms of these DNAs had been selected. To test this possibility, Hirt extracts from two hygromycin B-resistant clones carrying pHEBoAFR-5x30 and from four different populations carrying pHEBoAFR-3xDS were used to reintroduce the plasmids into E. coli. Mostlymonomericclones but alsoafewdimeric cloneswere obtained fromeach. In every case, analysiswith restriction enzymesthat would have revealed differencesas smallas30 bp in theoriPregion showed the plasmids tobe identicalto theoriginal constructs. Next, therecovered plasmids, both monomeric anddimeric, were testedfor efficiency ofstable introduction into Raji cells. The recoveredplasmids (mono-mers) were stably introduced into Raji cells at frequencies notsignificantlydifferent from thefrequencies obtainedwith the original plasmidDNAs and about 1% of thefrequency observed with pHEBo(Table 3).

The dimer of

pHEBoAFR-3

xDS did not produce signifi-cantly more hygromycin B-resistant clones than did the monomer, while the dimer ofpHEBo/FR-5x30 yielded 10 timesasmanydrug-resistant clonesasdiditsmonomer. We didnotdeterminewhether the increasedefficiency observed with thedimerofpHEBoAFR-5x30resultedfrom its

being

a dimerorfrom amutation in one of its monomericunits. In any event, thedimeric forms of theseplasmidswereaminor fraction of the plasmid molecules maintained in Raji cells selectedto carry the original

plasmids (Fig.

5A).

From these results we conclude that the stable mainte-nanceof these DNAsas replicating plasmidsdoesnot arise throughthe selectionofmutantforms. Itmightalsobenoted thatthefrequencywith whichthese

plasmids

are selectedto

be stably maintainedin Raji cells, 1%relativeto

pHEBo,

is

..p mI_

FIG. 5. Southern analysis of Hirt supernatants of Raji cells selectedtocarry theindicatedplasmids.DNAobtainedfrom 3x 106 cells was analyzed uncut(A) or wascut with EcoRI (B). Forthe

standards,20 pgcorrespondsto oneplasmid molecule recoveredper

cell.ForuncutDNAs,the bandseentomigrateaheadoftherelaxed

circularform in the standards and samples represents the

super-coiled dimeric form. The band seen in one sample ofundigested

DNAfromcellscarrying pHEBoAFR-2x30islocatedattheposition ofundigested, residual cell DNA in the Hirt supernatants. DNA from oneclone carryingpHEBoAFR-9x30 contains alarger,

rear-rangedbutnonintegrated formof theplasmidatless thanonecopy percellinadditiontomultiplecopiesof theintact,originalplasmid.

PartofpanelAisfromablotofasimilargelbecauseofdistortionin thisregion oftheoriginalgel. SizesofEcoRIdigestionproductsare

4,500bp (pHEBo), 2,300bp(allplasmids),and 400bp(notseen).In

one sample of pHEBoAFR-3xDS, the partial digestion product (2,300plus400bp) ispresent.

more than 100-fold greater than theobserved mutation rate

of the TK gene carried on an oriP-based vectorand intro-duced intoan EBV-transformed

lymphoblastoid

cell line

by

electroporation (7).

Given the rather efficient

replication

of

pHEBoAFR-5

x30 and

pHEBoAFR-3xDS

in the first few celldivisions follow-ing electroporation, their low

efficiency

of

being

stably

maintained-1%comparedwith

pHEBo-may

seem

surpris-ing. We suggest two

explanations. First,

efficient

plasmid

maintenance of oriP-based vectors such as

pHEBo

under selection is likelyto

depend

onthe enhancer

activity

ofthe 30-bp repeats in addition to the efficient

replication

of oriP

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TABLE 3. Frequencieswith whichpHEBozFR-5x30

andpHEBo/FR-3xDS isolatedfrom Rajicells

canbestably reintroduced intoRajicells

Plasmid Frequencyof

hygromycinresistance'

pHEBo 2 x 10-2

pHEBoA\FR 1 x 10-5

pHEBoAFR-5x30 2x 10-4

pHEBoLFR-5x30, recovered 2x 10-4

pHEBoAFR-5x30, recovered dimer 2x10-x

pHEBoAFR-3xDS 1 x 10-4

pHEBoAFR-3xDS, recovered 1 x 10-4 pHEBoAFR-3xDS,recovered dimer 2 x 10-4

"Averagevaluesfor duplicatedeterminations (see Table 1.footnote).

(19, 31). Thus the weak enhancer activities of 5X30 and 3XDS may lead to insufficient expression of the hph gene from the TK promoter and resultin lowcloning efficiencies inhygromycin B-containing medium. Second, the molecular requirements for transient replication may be less than what is required for plasmid maintenance. If each molecule of pHEBoAFRcarrying 5X30 or 3xDS were tocontinue rep-licatingindefinitely, witha50 to70%probability ofdoubling in each S phase, we would expectto obtain manycolonies that would grow slowly under selection and then lose the plasmid rapidly when grown without selection, as is ob-served with "weak" ars elements in Saccharomyces cere-v'isiae (14, 17). This was not observed.

sequence differences among members of the EBV 30-bp repeats suggest that a duplication event of the 30-bp repeats(orageneconversion of 10membersbytheother10) occurred in the recent evolutionary pastofstrain B95-8 (1) (Fig. 1A).

Enhancer functionofEBNA1 and30-bp repeats. Originally it was hypothesized that a role of the 30-bp repeats in plasmid maintenance could be that of relocalizing the plas-mid to the reforming nucleus at the end of mitosis or of targeting the plasmid to a nuclear subcompartment where DNA replication occurs (31). Although there is no real evidence for such a function of the 30-bp repeats, it is conceivable that the function exists and is at least part of what is measured as enhancer activity in the CAT gene transientexpressionassaysdescribed here. In any event, the 30-bp repeats constitute an EBNA1-dependent enhanceras defined operationally. Previous observations that the mea-sured enhanceractivity of the 30-bp repeats depends on the particular promoter and on the cell line tested argue for a rather direct effect ofthe30-bprepeatsontranscription (30, 31), since all cell lines used in the studies supported main-tenanceoforiP-carrying plasmids and therefore were ableto localize the introduced DNAs to the nucleus. In addition, the 30-bp repeats are likely to play an important role in the control of EBV gene expressionduring latency. A promoter located 2,200 bp from oriP which directs synthesis of the EBNA family of spliced mRNAs in some latently infected cell lines (2) has been shown todepend on EBNA1 and the 30-bp repeats for its activity (35). In the present studies, the demonstration of EBNA1-dependent enhancer activity by tandem copies of the dyad symmetry region implies that multiple EBNA-1 binding sites are necessary and sufficient for this activity, since the 30-bp repeats and the dyad symmetry region share no other sequence.

It is not known to what extent, if any, oriP replication depends on the 30-bp repeat enhancer function per se. However, the requirements for EBNA1-binding sites for enhancerfunction parallel the requirements for activation of replication: seven to nine 30-bp repeats are required for full activity; tandem copies of the dyad symmetry region en-hance transcription, but less effectively than the 30-bp repeats; and the nonlinear dependence of activity on the number of EBNA1-binding sites suggests cooperativity. These similarities suggest that transcriptional enhancement and activation of replication by the 30-bp repeats share certain steps. A recent deletion analysis of the EBNA1 gene supports this notion (36). Deletions within the EBNA1 open reading frame that did not alter DNA binding were found to reduce or eliminate enhancement and transient replication functions similarly or to affect neither, implying that the enhancer-activatingproperty of EBNA1 is an essential com-ponent ofreplication activation by EBNAl.

Host-specific factor required forEBNAI-dependent replica-tionbut not for activation of transcription. In light of several recent experiments demonstrating that activators of tran-scription can function in cells of the most distantly related eucaryotic organisms (27), the finding that EBNA1 can activate the 30-bp repeat enhancer in mouse cells is not surprising. The fact that EBV plasmid replication does not

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cross species lines from cells of primates to cells of rodents indicates that activation of DNA replication requires an activity ofEBNA1(or of oriP) in addition to what is needed for enhancer activation, such as an interaction with a host protein involved in DNA replication. The results suggest thatEBNA1 may interact with separate cell factorsinvolved in the control of transcription and of DNA replication.

Like EBV plasmid replication, simian virus 40 DNA replication occurs in monkey or human cells but not in rodent cells. The host range for simian virus 40 replication has been explained by the ability of simian virus 40 Tantigen to bind to DNA polymerase-alpha or an associated factor from human or monkey cells but not from mouse cells (23, 33). It should be possible to test for a comparableassociation of EBNA1 with DNA polymerase-alpha from permissive cells.

Cooperativity of EBNA1. The cooperative actions of EBNA1 molecules that lead to activation of transcription and DNA replication could occur, presumably, at the stage of DNA binding or subsequent to DNA binding. Recent studies in vitro withEBNA1 partially purified from Raji cells revealed very little cooperativity in the binding ofEBNA1 to the 30-bp repeats (16). Similarly, studies with the DNA-binding domain of EBNA1, a carboxyl-terminal fragment synthesized in E. coli, failed to show cooperativity of binding to adjacent sites, although binding to a single site was very concentration dependent, suggesting multimeriza-tion of theEBNA1 fragment (22). The studies of Jones et al. indicated that the 30-bp repeat sequence has an intrinsic affinity forEBNA1 that is severalfold higher than the affin-ities of the binding sites within the dyad symmetry region (16). Features of the 30-bp repeats that allow them to act cooperatively to enhance transcription and to activate repli-cation more efficiently than tandem copies of the dyad symmetry region could be this greater binding affinity for EBNA1, a more favorable spacing of the binding sites, or structural features that could impart DNA flexibility, such as the A+T-rich (85%) spacer that alternates with the 18-bp EBNA1 recognition sequence.

The lack of a high degree ofcooperativity in binding of EBNA1 to the 30-bp repeats in vitro leads us to speculate that the cooperativity of seven or more EBNA1 dimers observed for the functions of the 30-bp repeats in vivo reflects events that occur subsequent to DNA binding. Since EBNA1 is likely to activate DNA replication and transcrip-tion through an interactranscrip-tion with cellular factors, it ispossible that it is the binding of EBNA1 tothese factors, rather than to DNA, that is highly cooperative in cells. A requirement for cooperative action of multiple DNA-bound transcription-activating proteins in directing transcription initiation by polymeraseII has been inferred from several studies and is presumably an importantfeature in preventingsequestration of the transcription machinery by transcription activators bound at nonspecific sites (10, 25, 27).

It is also possible that a multi-EBNA1 protein complex, formed through binding to multiple DNA sites, isrequiredto destabilize the DNA double helix in leading to initiation of DNA replication, analogous to the actions of the multipro-tein complexes of E. coli dnaA protein at oriC and of bacteriophage AO protein at A ori (3, 6, 11, 32).

ACKNOWLEDGMENTS

We thank Sarah Camiolo for performing some of the plasmid

constructions, plasmid purifications, and CAT assays; Lynne Ma-quat, Tim Middleton, and Bill Sugdenforcomments on the

manu-script; several colleagues at Roswell Park Memorial Institute for

discussions and support; and Nancy Framefor preparingthe manu-script.

Thiswork was supported by aPublic HealthServicegrantfrom

the National Cancer Institute.

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