EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication.

0022-538X/92/010489-07$02.00/0

CopyrightC 1992, American Society for Microbiology

EBNA1 Can Link the Enhancer

Element

to

the

Initiator

Element of

the

Epstein-Barr Virus Plasmid

Origin of

DNA

Replication

TIM MIDDLETON* AND BILL SUGDEN

McArdleLaboratory forCancer Research, UniversityofWisconsin,

1400 UniversityAvenue, Madison, Wisconsin53706 Received29July1991/Accepted 8 October1991

The plasmid origin of DNA replication ofEpstein-Barr virus, oriP, is replicated once per cell division, employing cellularreplication machinery and onlyoneviral protein.Tounderstandhow replicationfrom this

origin is initiated andregulated,wepurified this viral protein, EBNA1. EBNA1wasexpressed inCV-lpcells

by using aninfectious simian virus 40 vectorcontaining the EBNA1 gene. Itwas purified in two chromato-graphic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferasegenefromareporterplasmid carrying the FR enhancerelementtowhich EBNA1 binds.EBNAldoes nothave oriP-dependentATPase activity, indicating that it doesnotcarryoutanenergy-dependentstepin the initiation of DNA replication.However,EBNAldoes mediateanassociationbetween thetwoelementsoforiP. We measured thisassociation by bindingone of the elements, the enhancer element, to a solid matrix and

measuring retention by thiselement of the otherone,the initiator element, in the presenceofEBNAl. This retention is specificforDNA fragments containing EBNAl-binding sites.EBNA1 thuscanlink thetwoelements of theorigin, providingalocally highconcentration of EBNA1atthesiteof initiation of DNA replication. We proposethat thisassociationis important either (i)toaffect DNAstructure toallowacellular helicasetoinitiate DNA strand separationor(ii)tobindreplication proteinstobring themtothe origin of replication.

DNAreplication requires careful coordination of the

en-zymatic steps of replication with other aspects of cell

divi-sion. Studies usingprokaryotic replicons have identified the

primary enzymatic activities required for DNA replication

and, in some cases, mechanisms by which replication is

regulated (21). More recently, genetic studies using herpes

simplex virus and reconstructions of replication activity in

vitro using simian virus 40 (SV40) and adenoviruses have

demonstrated that the enzymology of DNA replication is

well conserved from bacteriato mammals (6, 31). However, the regulation ofDNA replication within the cell cycle in

mammalian cells isnot well understood.

In mammalian cells, replicons must be copied only once per cell cycle. There have been two impediments to deter-mining how this regulation occurs. (i)Those viralorigins of replicationused toidentifythe

required

enzymatic functions

are derived from

lytic

viruses and are, by their nature, not

limitedto oneroundofreplicationpercellcycle.

(ii)

Eukary-otic chromosomal origins of

replication

have notbeenmade tofunction invitro; incontrastto

origins

from

Saccharomy-cescerevisiae, mammalian chromosomal

origins

have been studiedasplasmids in vivo with

only

limited success. Until the features of these chromosomal

origins

that limit their study in isolationare

identified,

these

replicons

will

provide

cumbersome models both forthe

study

ofthe initiation of

DNAreplication and for itscellcyclecontrol.

Latently replicating viruses

bridge

the gap between

lytic

viruses and chromosomal

origins

ofDNA

replication.

The

best studied of these virusesare

papillomaviruses

and

lym-photrophic herpesviruses.

Epstein-Barr

virus

(EBV)

is a

lymphotropic

herpesvirusthatimmortalizesBcellson

infec-tion.

Replication

of the viral DNA is

subject

to the same

constraintsaschromosomalDNA

during

one

phase

of its life cycle. EBVis

distinguished

from chromosomal

replicons

in

*Corresponding author.

that its origin has been identified andfunctions in plasmids

(34). EBV has a linear genome ofapproximately 172 kbp which iscircularizedafter infection and is maintained in the nucleus as aplasmid at a copy number of between 5 and 500

(30). While there is considerable variation incopy number betweenclonesof infectedBcells, oncethe copy numberis reached at some early stage afterinfection, itis stable for many generations thereafter. Furthermore, density shift experimentshave demonstrated that EBV DNAisreplicated

once per cell cycle (1, 36).

Genetic analyses of the plasmid origin of EBV DNA

replication (oriP)havedemonstratedthat less than 800bp of

EBVDNAisneeded toformafunctionalreplication origin. ThisDNA is divided intotwoelements, which inthe virus are separated by about 1,000bp of

intervening

DNA (27).

Both elements, the family of repeats (FR) and the dyad

symmetry region (DS), consist of multiple

copies

of a

recognition site for viralprotein EBNA1 (25), which is the

only viral protein

required

for DNA

synthesis

from oriP

(Fig. 1; 16,37). DNA

replication

initiatesat or nearthe DS element(10), while FRisanaccessory element

required

for DNAreplication (27).

Two features of

particular

interestin

understanding

oriP

function are the interactions ofEBNA1 with other

replica-tion proteins and the mechanism

by

which the FR and DS elementsinteracttoformafunctional

origin.

To

study

these

features,

we

purified

EBNA1 from cells in which EBNA1

and oriPfunction. We demonstrated that

purified

EBNA1 is

biologically

active

by

measuring

its

activity

in vivo after

introduction of the

protein

back into mammalian cells.

Purified, functional EBNA1 does not have detectable ATPase

activity,

indicating

that it isnot

likely

to

perform

the

energy-dependent

strand

separation

thatrepresents an

early

enzymatic

activity

in DNA

replication.

It

does,

however,

promoteanassociationbetween FR andDS that is

likely

to

be

required

for initiation of DNA

replication.

489

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FR

-1p

I*-*A

FIG. 1. Structure of oriP. FR and DS are indicated as boxes correspondingto therepeat units. Thepositionof oriPontheEBV

genomeisgiven bythenucleotide numbersof thesequenceofBaer

et al. (4). The arrows indicate inverted repeats. The lower part showsa consensusrepeatsequence.The asterisksmarkmismatches

intheinvertedrepeattowhichEBNA1binds(3, 13).

MATERIALS AND METHODS

Plasmids. The basepairnumbersgivenfor EBVarethose describedbyBaer et al.(4).p1010 andp304contain the DS

(EBV bp 8992 to 9132) and FR (EBV bp 7315 to 8191)

elementsinserted intotheBamHIsites ofpBKSandpSP65, respectively. p972 contains oriP (EBV bp 7333 to9257) in the

HincII

and SphI sites ofpUC19. p896isa derivativeof pBluescript

KS'

with a duplex oligonucleotide with the sequence AGCTTATCTATATCTGGGTAGCATATGCTA TCCTAATCTAGGATCCAinserted at theHindIll site. The binding siteforEBNA1 isunderlined.

p1013 (tk-luciferase) was constructed by removing from

pA2(-260/-87)tkCAT8+

(14) the HindIII-to-BamHI

frag-ment containing DNAfrom thepromoter region ofa vitel-logeningeneandreplacingthechloramphenicol

acetyltrans-ferase gene on a BgIII-to-HpaI fragment with the firefly

luciferase gene on a HindIII-to-HpaI fragment from pSVL (8). p985 (FR-tk-luciferase) contains the FR element on a BamHIfragment fromp304 inserted into theBamHI siteof

p1013.

The SV40-EBNA1 expression vector has been described previously (11). Briefly, aBamHI K fragment containinga

deletionof mostof theglycine-glycine-alanine repeat region

of EBNA1 was inserted into the XhoI site of plasmid pSVEpR4. The deleted region is not essentialfor EBNA1-mediatedactivationoftranscriptionorreplication (35).EBV

bp 109,982 to 112,065 were replaced by aBamHI linker to give p398Y. Digestion of this plasmid with BamHI gave a fragmentofpackageablesizecontainingtheSV40 originand

early region,withtheEBNA1 genereplacingthelateregion.

The EBVsequencesincludedarefrombp107,567to109,982

and 112,065to 112,622.

The plasmid used to complement the deletion in p398Y

contained SV40 DNA with a deletion ofbp 4974 to 5046,

whichcreatesanout-of-framedeletion intheT-antigengene

(provided by JanetMertz).

Infectionand harvestingofCV-lpcells. Stocksofviruses

were obtained by introducing the complementing viral

DNAsintoCV-lpcells byDEAEtransfection(17). At 96 h later, the cells were harvested and virus was recoveredas

described in reference 18. These virus stocks were used to infect CV-lp cells for purification of EBNA1. The ratio of

virustocells touseforinfectionwasdetermined by titrating

thevirus onCV-lp cells, measuring the amountof EBNA1 produced by an enzyme-linked immunosorbent assay (see

below),andusingthattiterwhich maximizedtheamountof

EBNA1detected.

Infections for isolation of EBNA1 were done on

15-cm-diametertissueculturedishes.CV-lpcellsweregrownto50

to 80%confluence inDulbecco'smediumplus 10% neonatal

calfserum. Medium was

removed,

and viruswas incubated

with the cellsin 3 mlof mediumfor1 h.

Thirty

milliliters of medium was

added,

and dishes were incubated until about

10% ofthe cells started to detach. Medium was

aspirated

fromthe

dishes,

and cellswere

scraped

offthe

plates

intwo

volumes of buffer D

(20

mM HEPES

[N-2-hydroxyethylpi-perazine-N'-2-ethanesulfonic

acid]

[pH 8],

1 mM

EDTA,

1%

Triton X-100, 0.02% sodium

azide,

30 mM sodium

PP1,

50 mM NaF, 1 mM sodium

orthovanadate,

and a mixture of

protease inhibitors

[20]).

The cells were stored at -70°C until used.

Purification and monitoring of EBNA1.

CV-lp

cells ex-pressing EBNA1 were

lysed by being

passed

twice

through

a French press at 800 lb/in2 in two

packed

cell volumes of

buffer D. Ammonium sulfatewas added to

30%

saturation,

and lysateswere left onice for 1 h and spunat

100,000

x g for 1 hat4°C. Supernatantswerediluted in bufferDto

give

an ammonium sulfate concentration of0.4 M.

Supernatant

from 109 cellswas

applied

to a10-ml

heparin-agarose

column and washed with 10 column volumes of0.4 M ammonium

sulfate, andtheEBNA1-containing fractionwaselutedwith 3 column volumes of0.7 M ammonium sulfate. The eluate was diluted to 0.3 M ammonium sulfate and applied to an

affinity column madeoftheBamHI FR

fragment

of

p304

as described by Kadanoga and

Tjian

(12). The column was

washedwith 10column volumesof buffer E (20 mM HEPES

[pH 8.0], 1 mM EDTA, 1 mM dithiothreitol)

plus

0.6 M

NaCl, and EBNA1 was eluted with 2 column volumes of buffer E plus 2 M NaCl. The eluate was concentrated

by

using carboxymethyl cellulose (Aquacide IV;

Calbiochem)

asrecommendedbythemanufactureranddialyzed in buffer

E with 0.2 MNaCl and 15% glycerol. The

purified

protein

was storedin this buffer at

-70°C.

Theelution characteristics of EBNA1 weremonitored

by

anenzyme-linked immunosorbentassay.Threefold dilutions

of 1 ,l of the protein-containing solution were bound to

nitrocellulose in 100 ,l of 10 mM sodium phosphate (pH

7.2)-S150

mMNaCI-20 ,ug of bovine serum albumin per ml. Twofold dilutions of 20 ng of a lambda

cro-EBNA1-,-galactosidase fusion protein (30) were similarly bound. The amounts of EBNA1 in purification fractions were

deter-mined by densitometric scanning of immunoblots probed

with antibodies made to the fusion protein (30).

The purity of the protein was determined by sodium

dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis,

followed by staining ofthe gel with Coomassie blue. The concentration of purified EBNA1 was determined by flu-oraldehyde assay as recommended by the manufacturer

(Pierce Chemical Co.).

DNA-binding assay. The DNA used was either p896 or the oligonucleotide used to construct p896. Twenty-five femto-moles of end-labelled (28) DNA was bound to protein in 50 ,l of 20 mM HEPES (pH

8.0)-i

mM EDTA-1 mM dithio-threitol-0.2 M NaCl-50 ,ug of bovine serum albumin per ml for 10 min at 20°C. The samples were filtered through

nitrocellulose by using a dot blotting apparatus from Schlei-cher & Schuell and washed with 400

Rl

of 20 mM HEPES (pH

8.0)-i

mM EDTA-1 mM dithiothreitol-0.2 M NaCl. Dots were cut out and counted with a scintillation counter. Introduction of purified EBNA into mammalian cells. EBNA1 was incubated with luciferase reporter plasmids underthe conditions used for the DNA-binding assay. The

DNA-EBNA1 mixture was introduced into 143 cells by electroporation by using a rise time of 8 ,us, a voltage peak of 760 V, and a time of 30 ms for decay of the voltage to one-third of its peak (15). Extraction of luciferase from the

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cells andmeasurementofluciferaseactivitywereperformed aspreviouslydescribed (5).

ATPaseassay. ATPase activity was measured by absorp-tion of unhydrolyzed ATP by charcoal. Reacabsorp-tionscontained

200 mM KCl, 10 mM MgCl2, 50 ,ug of bovineserumalbumin

perml, 1 mM EDTA, 0.2% Triton X-100, 3% glycerol, 1 mM

dithiothreitol, 0.1 to 1 1xCi of [-y-32P]ATP, 50 mM HEPES (pH 8.0) (unless otherwiseindicated),1 pmolof EBNA1, and 0.2 pmol of DNA (unless otherwise indicated). A 0.2-pmol sample of the DNA containing EBNAl-binding sites

con-tained 10 pmol of binding sites. After incubationat37°C for 20 min, 200 ,ul of a 20% (wt/vol) suspension of activated charcoal (Norit; Sigma Chemical Co.) in 10 mM sodium

PP-2%trichloroacetic acidwasadded andmixed for10min.

Thecharcoalwaspelleted, and 50,ul of thesupernatantwas

removedforscintillation counting.

FR-DS associations. FR bound toagarose resin was pre-pared as previously described (12). EBNA1 was incubated with 10 fmol of end-labelled DNA containing

EBNA1-binding sites by using the reaction conditions described for thenitrocellulose filter bindingassay.This mixturewasthen incubated with 10

RI

of resin (500 fmol of DNA) for 60minat 4°C. The resin was pelleted and washed twice for 20 min

eachtime with 200 ,ul ofbinding buffer. The DS-containing DNAwaseluted by incubation for 10minat65°C in 20 ,ul of 10 mM Tris-HCl (pH 8.0)-i mM EDTA-0.1% SDS. The eluted material was electrophoresed on an agarose gel and visualized by autoradiography.

RESULTS

Construction ofanEBNA1 expression vector and purifica-tionof the protein. Expression of EBNA1 in EBV-positive lymphoblastoid cells is limited to about 5 x 104molecules percell(30). To facilitatepurification, apackageable

expres-sion vector containing the EBNA1 gene replacing the late

region of SV40 (provided by John Yates) was used. This

DNAwastransfected intoCV-lp cells along witha

comple-menting viral DNA containing a frameshift deletion in the

T-antigengene(provided by Janet Mertz)togeneratestocks ofinfectious virus. These viruses were then used to infect CV-lp cells for production of EBNA1. The infected cell populations typically contained approximately 106 EBNA1 moleculespercell, orabout 100 p,gof EBNA1 per 109 cells (datanot shown).

The scheme used to purify EBNA1 is shown in Fig. 2. Both protease and phosphatase inhibitors were required to maintainthe integrity of the protein through the first steps.

The purification required two chromatography steps, as

indicated inMaterials and Methods. Afterasinglepass over

theaffinitycolumn, EBNA1wastheonly band visibleonan SDS-polyacrylamide gel. A typical gel is shownin Fig. 3A. Immunoblotting of the purified material generallygave one

band that corresponded to the position of the Coomassie blue-stained band (Fig. 3B, lane 1). The exception to this

finding occurredinearly preparations inwhichphosphatase inhibitors were not used. In these cases, EBNA1 was

distributed among three to six bands spaced about 2 kDa apart, as shown in lane 2 ofFig. 3B. This observation is

consistentwith the interpretationthat the multiple forms of

EBNA1 detected resulted from its dephosphorylation after

thecells were harvested. The EBNA1 isolated in the

pres-enceofphosphatase inhibitors appearedtobehomogeneous, which indicates that its overexpression did not result in

heterogeneous phosphorylation of theprotein.

Binding of purifiedEBNA1 toDNA. EBNA1 binds

specif-SV40ori

Tantigen EBNAl

C

Cvi cells

SV40ori

1 ,VP1-3

f 48-96hours

Harvest cells

f

Frenchpress

f

[NH4]2504to30% saturation

100,000xg,2hours

Hepannagarosecolumn

[NH412S04

0.4 M 0.7M

FR-Sepharose column NaCI

0.3M 0.6M 2M

FIG. 2. Outline of the protocol used to express and purify EBNA1. Packageable SV40 vector398Y hasareplacement of its late region with the EBNA1 gene. 217M contains a frameshift mutation in theTantigen gene but supplies viral late gene products. Details of thepurificationaresupplied in Materials and Methods.

icallyto a 30-bpsequence thatis repeatedmultiple times in FRandDSoforiP. Retention of thisactivityin the purified

proteinwas tested by using a nitrocellulose binding assay.

Binding ofEBNA1 to 25fmol of eithera30-bp

oligonucleo-tidecontaining a consensusDNA-binding siteor a plasmid

containingonecopy of thisoligonucleotidewaslinear overa

10-fold range (Fig. 4). EBNA1 has been shown to bind to DNA as a dimer (2, 9), and it will be assumed here that a

dimer is the active form of EBNA1. These data show a

one-to-one correspondence between the moles of EBNA1

dimer added and the moles of probe DNA retained. This

binding was specificfor the EBNAl recognitionelement in that EBNA1 did not retain on nitrocellulose either the

parental plasmid lacking this 30-bp recognition site or an

oligonucleotide containing a recognition element for the estrogenreceptor(datanot shown).

Biological activity ofpurified EBNA1. FR acts as a

tran-scriptional enhancer when bound

by EBNA1,

both with heterologouspromoters andwithapromoterlocatedabout3

kbp away in the EBV genome (26,

32).

This element sup-ports DNA

replication

with thesame

flexibility

inits

position

and orientation as its

displays

in

supporting transcription

(33). Furthermore, mutations in EBNA1 that affect DNA replication affect RNA

transcription (23, 35).

Weused the transcriptional

activating

property ofEBNA1 and FRtotest

whether the

purified

EBNA1

protein

was

biologically

active.

EBNA1

protein

bound to a

plasmid

containing

FR,

the herpessimplexvirustype 1

thymidine

kinasepromoter,and

the firefly luciferase gene was

electroporated

into cells.

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A

Kd

97-68

-

43-

29-12

B

1

2

68-

43-FIG. 3. Gel electrophoresis ofpurified EBNA1. Samples were resolvedon an SDS-10%polyacrylamide gel. (A)Gel stained with Coomassie blue. Lanes: 1, supernatant from precipitation of an infected cellextractwith 30%(NH4)2SO4; 2, 1,ugof EBNA1 eluted fromthe DNAaffinitycolumn. Kd, kilodaltons. (B)Immunoblot of purified EBNA1. Lanes: 1, purification in the presence of phos-phataseinhibitors as indicated inMaterials andMethods;2, purifi-cation in the absence ofphosphatase inhibitors. Both lanes con-tainedapproximately 100ngofEBNA1.

These cellswere harvested 12 to72 hlaterandassayed for

luciferase activity.

We first tested whether EBNA1 remained bound to the DNAundertheconditionsusedforelectroporation. EBNA1

wasboundtotheDNAasdescribedearlier, diluted into the medium used for electroporation (but lacking serum), and

filtered through nitrocellulose. DNA retention was unaf-fectedby thistreatment, indicating that the DNA remained

boundby EBNA1 (datanotshown).

60

fmol EBNA1

FIG. 4. RetentionofDNA containingone EBNAl-bindingsite

on nitrocellulose after its incubation with purified EBNA1. A

25-fmol DNAprobesamplewasused foreachreaction.Theresults

shownarecombineddatafrom threeindependentexperiments using

either oftheprobesdescribed inMaterials andMethods.

The EBNA1-plasmid complex was then

electroporated

into 143 cells, an EBV-negative human osteosarcoma cell line. The effect ofthe ratio of EBNA1 to

plasmid

and the

time courseofaccumulation of luciferase

activity

areshown in Fig. 5. The amount of luciferase activity increased

lin-earily up to a fourfold molar excess ofEBNA1 dimer with respect tobindingsites,whereit reacheda

plateau (Fig.

5A).

Itis not known whetherthe molar excess ofEBNA1relative

tobinding siteswasneededtoachieve maximalsaturation of

binding sites or whether anincreased nuclearconcentration ofEBNA1 simply maintained

transcriptional

activation fora

longer time. Luciferase activity peaked

approximately

2 days after transfection, although a significant amount of activity remained at 3 days (Fig.

5B).

EBNA1 lacks ATPase activity. An early step in DNA replicationis energy-dependentunwinding ofthe DNAatthe origin. We determined whether EBNA1 was an

oriP-depen-dent ATPase. First, [32P]phosphate release from ATP was

measured in the presence oforiPDNAfrom severalstepsin the purification ofEBNA1 (Fig. 6A). The supernatantfrom

ammonium sulfate precipitation contained ATPase

activity;

however, the fraction from the heparin-agarosecolumn that

contained EBNA1 did not. Thus, as assayed here, there were no detectable ATPase activities that copurified with EBNA1 through the last two purification steps. The pres-ence of ATPase activity was notaffectedby the absence of

DNA in the assay (data not shown).

We also examined the starting material and the 0.7 M eluate from the heparin-agarose column for ATPase

activi-ties over a pH range of 5 to 9 (Fig. 6B). With the starting

material, there was substantial ATPase activity over the entire pH range, with a maximum at pH 8. Again, in the fractions eluted from the column containing EBNA1 there was no detectable ATPase.

EBNAI-EBNAl interactions promote an association be-tween the two elements oforiP. The genetic requirement of

two groups of DNA-binding sites for EBNA1-dependent EBV oriPreplication activity (27) led us toexamine whether these two elements form a complex with each other via a bridge of EBNA1. In preliminary experiments, purified

EBNA1 was added to p972 which had been digested such that FR and DS resided on separate DNA fragments. Asso-ciation between these two fragments was analyzed by elec-trophoresis in 1% agarose gels. Increasing amounts of EBNA1 shifted the labelled DNA fragments to the wells of thegel, although the mobility of fragments lacking

EBNA1-binding sites was unaffected (data not shown). Thus, we could not determine whether complexes between the DS-andFR-containing fragments had formed.

To circumvent this problem, we immobilized an FR-containing fragment on agarose beads and then determined whether the DS element, when bound by EBNA1, was retained on this matrix. We titrated the amount ofEBNA1 needed toretain the DSfragment on the resin. A 10-fmol DS fragment sample (40 fmol of binding sites) was incubated with 10 to1,000 fmol of the EBNA1 dimer, and this mixture was added to 10 ,ulof resin containing 500 fmol of the FR fragment (10 pmol of EBNA1-binding sites). The binding reaction was allowed to proceed for 1 h at 4°C and was washed twice with 20 volumes (each time) of binding buffer toremove the unbound DS fragment. Binding was detectable with addition of 30pmol ofEBNA1 and increased through-out the entire range of EBNA1 concentrations (Fig. 7A). When 1,000 fmol of EBNA1 was added, 40% of the DS fragment was bound to the resin. This binding was absent when the samefragment lacking the DS element was used as

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2 4 6 8 EBNA1/binding sites for FR-tk-Luc

B 5

4

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Ic

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Hours after transfection

FIG. 5. Transcriptional activationin vivo by purifiedEBNA1. PurifiedEBNA1 wasboundto reporterplasmids and electroporated into 143 cells, andextractsofthecellswereassayedfor luciferase activity.(A)Titration of EBNA1. A3-,ugsample ofreporterplasmidswasbound bya0.5-to8-fold molarexcessofEBNA1relativetobinding sites in FR-tk-luciferase. After24 h,cellswereharvested and assayed. (B) Time

course of luciferase expression. One microgram ofFR-tk-luciferase was incubated with afourfold molar excess ofEBNA1 relative to DNA-binding sites for EBNA1, electroporated into 143 cells, harvested, and assayed for luciferase activity atthe indicated times after electroporation. RLU, relative lightunit.

theprobe, when EBNA1 wasomitted from the reaction, or

with addition of polyclonal antibodiesto EBNA1 (Fig. 7B).

Thus, the FR element was able to retain specifically the

DS-containing DNA inareactionthatrequiredboth the DS element and EBNA1.EBNA1-mediated binding of DStoFR wasnotafunction of thestructureoftheDS element. When aplasmidcontaining justoneEBNA1DNA-binding sitewas

substitutedforthe DS fragment, itwas also retained by the FRelement(Fig. 7B).

DISCUSSION

While the replication origins of several mammalian lytic viruses have been studied intensively, our knowledge of origins thatarereplicatedoncepercellcycle is much more limited. The structural organization of oriP is similartothat ofanumberof prokaryoticorigins, in particular, origins that are understrict copy number control (21). It is not known whether this organization extends to mammalian chromo-somalorigins, althoughautonomously replicatingsequences derived fromyeastchromosomes do share this organization (22).

In common with these prokaryotic origins, oriP binds a

replicon-encoded protein that recognizes the repeat ele-ments that make up the origin. We purified oriP-binding

protein EBNA1 from mammalian cells. Thepurified protein retained biological activity, as Tmeasured by its capacity to stimulate a reporter gene when EBNA1 and a reporter

plasmid containing the FR enhancer and the firefly luciferase

gene wereintroduced intocells. Purified EBNA1stimulated

luciferaseactivity 200-foldoverthatseenin itsabsence. One uncertainty regarding this assay is the extent to which the stimulationof luciferaseactivity results fromanincreasein

transcriptional activation by EBNA1.AdditionofEBNA1to this reporter plasmid provides for attachment of up to 21

EBNA1 dimers possessing up to 42 nuclear localization signals (2, 27).These nuclearlocalization motifs maytarget

the electroporated DNA to a compartment of the cell in which it is transcribed efficiently. However, ifthistargeting

were the only mechanism by which EBNA1 stimulates

transcription of the reporter plasmid, then-introduction of

the reporter plasmid bound by EBNA1 into cells already expressingEBNA1 would result inanincrease in luciferase

activity relative to that caused by introduction of naked

DNA. We didnotdetect suchanincrease (datanotshown). This finding would be expected if the increase in luciferase activity observed (Fig. 5) resulted largely fromactivation of transcription and notsimply from targeting of the reporter DNAtothenucleus. Theamountof EBNA1 thatenterseach celluponelectroporation is likelytobe small relativetothe approximately 40,000 molecules already in these EBNA1-positive cells and thereforecannotcontributesignificantlyto thetranscriptional activation observed.

Purified EBNA1 lacksdetectable DNA-dependent ATPase activity (Fig. 5), which makes it unlikelytohaveanintrinsic helicase activity. EBNA1 purified from insect cells also lacks detectable helicase activity (9). These observations indicate that EBNA1 probably contributes functions tothe EBVplasmid replicon other than ATP-dependent unwinding of the DNA. Because localizedunwinding of the DNA bya helicase is likelyto be an early enzymatic activity required for DNAreplication,oneof theactions of EBNA1 maybeto bind a cellular helicase to the origin. The availability of purified EBNA1 allows this hypothesistobe tested.

Purified EBNAl can link the two cis-acting elements of oriP, FR and DS(Fig. 6). We imagine that this association

occursviainteractions ofEBNA1dimers boundtoFR with

those bound toDS, although wecannot ruleout the possi-bility thatasingle dimer is capableofbindingtositesonboth

molecules. The interaction measured herewas

intermolecu-lar. Intramolecular association betweenDS andFRhas also

beenvisualizedby electron microscoy (9a, 31a).

Anotable feature of the observed intermolecular

associa-tion was the relatively small number of EBNA1 molecules needed to mediate it. Forthe binding assay, EBNA1 was incubated with the DS element and then this mixture was incubatedwiththe immobilizedFR element. BindingofDS to FR was readily detectable when there was a 300-fold excessofFR-binding siteswithrespecttoEBNA1,and 40%

of theDSfragmentwasbound witha1:10 ratio ofEBNA1to binding sites. This indicates that a singledimer ofEBNA1

bound to FR is probably sufficient to mediate association

with DS boundby EBNA1 (Fig. 6A).A relatedphenomenon was first noted by Milman and Hwang (19), who used a fragmentof EBNA1 that includedthe carboxy-terminal 191 amino acids. This portion of EBNA1 was sufficient to associatetwoDNAfragments containing single high-affinity

consensus EBNA1-binding sites. Figure 6B confirms this

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FIG. 6. ATPase activities detected during purification of EBNA1. ATPase activity was measured by incubation of protein with ATP andseparation of unhydrolyzed ATP from phosphate by absorption by charcoalasdescribed in Materialsand Methods.(A) ATPaseactivity associated with severalstagesofpurification. The fractionsassayedareindicated below thebargraph. Theamountof each fraction usedcorrespondedtotheextractfromapproximately 105, 105, and 4 x 105 cells for the 100,000 x gsupernatant (sup), heparin-agarose, and purified EBNA1 fractions, respectively, after correction forrecoveryof EBNA1ateachstep.(B) Effect of pHon

ATPase activity. Symbols: E3, material applied to heparin-agar-ose;*, material eluted from heparin-agarose.

finding and shows further that the lower-affinity sites which

composeDScanalsoformacomplex undertheconditions in

which the single higher-affinity sitewas bound.

This result indicates that not all of the multiple binding sites found in FRareneeded for EBNA1-mediated

associa-tion with DS. Wesuggest twoadditional propertiesthat FR

mayprovide this origin. One is that the multiple binding sites of FRbound by EBNA1 are needed tostabilizean

alterna-tive structure of the DS element bound by EBNA1 that is

obligatory for initiation of replication. Forexample, the DS element is theoretically capable of forming a cruciform structurethat extendsoverapproximately65bp. Chittenden etal. (7) have demonstrated that deletions that disrupt this potential cruciform but do not affect the EBNA1-binding sites render the DS element nonfunctional. Interactions of

FIG. 7. Associationof FR and DS elements via bound EBNA1. EBNA1wasincubatedwitha32P-labelled DNAfragment (3,152 bp) which contains the DS element, and this mixture was added to

immobilizedFR. Theretained DSfragmentwas eluted withSDS, electrophoresed,andexposedtofilm. Theresultingautoradiographs

areshown.(A) Titrationof the amounts ofEBNA1 needed to retain DS. The amounts ofEBNA1 added are indicated at the top. (B) RequirementforEBNA1- and DNA-binding sites for this associa-tion. Eachanalysiswasperformedinduplicatetoyieldtwo laneson

theautoradiogram. Probe DNAs: DS, plasmid containing the DS element;1 site, plasmid896 containingonecopyoftheconsensus

EBNA1-binding site; no sites, pBluescript KS' containing no binding sites. For EBNA1, treatments with added EBNA1

con-tained 100 fmol of the EBNA1 dimer. Foranti-EBNA1, DS and EBNA1 were incubated for 10 min, 1.5 ,ug of rabbit polyclonal anti-EBNA1 antibodieswasadded,andthe incubationwas contin-ued for an additional 20 min before addition of the mixture to

immobilized FR.

EBNA1molecules boundtoDSwiththoseboundtoFRmay

help stabilize suchastructure. Asimilar roleindistortingthe structureof lambda DNAnearitsorigin byits0proteinhas

been proposed (29).

Asecond candidatepropertyispromotion ofthe accumu-lation of cellular proteins needed to form a replication complexattheorigin by concentrating EBNA1 atthis site.

This effect could occur either by direct interaction of EBNA1withspecific replicationproteinsor,moregenerally, by bringingtheorigintoanuclear sitewhere theseproteins arelocalized.Thissuggestedpropertyof the FR element and EBNA1 would explain the apparently similar roles of EBNA1 inactivating transcription and replication. Multiple copies of EBNA1 localizedatFRwould activate transcrip-tion by forming heterologous complexes with other tran-scriptional activators, of viral or cellular origin. Similar predicted interactions form the basis for the looping model of

enhancer function (24). Given the similarity in the

charac-teristics of EBNA1 as anactivatoroftranscription andas a

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componentof the replicationapparatus, anunderstanding of

howEBNA1 facilitates replication may

also

aid our

under-standing ofhow transcriptional activators work. ACKNOWLEDGMENTS

We thank Julie Breister, David Anderson, and StephenHussey for technical assistance and Toni Gahn and Paul Lambert for comments on the manuscript. We thank Janet Mertz for providing plasmid 217Mand'JohnYates for providing plasmid 398Y.

This work was supported by Public Health Service grants CA-22443 and CA-07175 from the National Cancer Institute (B.S.), American Cancer Society grant IN-35-31-19, and a fellowship from the Cancer Research Institute (T.M.).

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