Identification of cellular factors that bind specifically to the Epstein-Barr virus origin of DNA replication.

0022-538X/91/010514-06$02.00/0

CopyrightC) 1991, AmericanSociety for Microbiology

Identification of Cellular Factors That Bind Specifically

to

the

Epstein-Barr Virus Origin of DNA Replication

SANG-JIN OH,THOMASCHITTENDEN, ANDARNOLD J. LEVINE* Department of Molecular Biology, Lewis ThomasLaboratory, Princeton University,

Princeton, New Jersey 08544-1014

Received 26 July 1990/Accepted 25 September 1990

Thespecific binding of HeLa cell factorstoDNAsequencesattheEpstein-Barrvirus(EBV) latent origin of

DNA replication was detected by gel shift experiments and DNase I footprinting analysis. These cellular

proteins protectedatleastfivediscreteregionsoftheDNAreplicationorigin. Theviralprotein requiredfor

EBV plasmid replication, EBV nuclearantigen 1 (EBNA-1), binds to specific sequences within the origin

region. The HeLa cellproteins competed withEBNA-1for bindingtoEBVoriginDNA invitro,leadingtothe

possibilitythat these cellularproteins regulateEBV DNAreplication bydisplacingEBNA-1attheoriginsites.

The herpesvirus Epstein-Barr virus (EBV) infects and

immortalizes human B lymphocytes in culture(reviewed in

references5 and 13).The EBV genomeis usuallymaintained as a multicopy plasmid in latently infected cells (7, 10).

Extrachromosomal maintenance of EBV plasmids requires onlytwoviralgenetic elements: 1,800bp oftheviralgenome thatfunctionsas anorigin ofreplication (oriP)(6, 11, 17,21) anda trans-actingfactor, EBV nuclearantigen 1 (EBNA-1)

(9, 11, 22). Maintenance ofEBV latent infection is depen-denton EBNA-1both forreplicationof EBV episomes and

fortransactivation oftranscription fromthe EBVBamHI-C latency promoter (18). Viral plasmid replication is

coordi-nately regulated with cellular DNA synthesis in that each

EBV genome duplicates only once pergeneration in the S

phase ofthe cellcycle(1,8).

The EBVorigin of replication,oriP,containstwoessential

components (11, 17, 21). Onecomponent is aregion

com-posed of20tandemly repeatedcopies ofa30-bp sequence. Each of these repeated sequences contains a 12-bp palin-dromiccorewhich constitutesanEBNA-1binding site.The

family of 30-bp repeats can function as a transcriptional

enhancer element in the presence of EBNA-1 (16, 18). The second essential component of oriP is a region of dyad

symmetry which constitutes the actual site of initiation of viral DNAreplication (6). Thisregion contains fourrelated

copies ofthe 30-bp repeats, of which two are embedded withina65-bp sequence of dyad symmetry and the

remain-ing two are located together at the base of the dyad se-quence. Previous studies using deletion mapping have

iden-tifiedacritical140-bp region encompassing the dyad element

(between the EcoRV and HpaI sites within oriP) as neces-saryincombination with the 30-bp repeats for EBV plasmid

maintenance(3, 11, 17).

With the exception of EBNA-1, EBV relies entirely upon the cellular DNA replication machinery. The properties of EBVplasmidreplication suggest that viral DNA synthesis is linkedtothecellularmechanisms thatgovern chromosomal DNA synthesis. Thus, it is possible that the virus exploits cellularfactors which bindto oriPand regulate the replica-tion of EBV. To identify cellular proteins possibly involved in EBV plasmid maintenance, protein-DNA gel retardation

experiments and DNase I footprinting were performed,

*Corresponding author.

using HeLa cell nuclear extracts and the 140-bp oriP dyad symmetry region. Recombinant oriP plasmids encoding EBNA-1 replicate autonomously in HeLa cells (11, 22), demonstrating that these cells express all trans-acting

fac-torsnecessaryfor EBVplasmidmaintenance. In thisstudy, weidentify thebinding sites of HeLa cellproteinswithin the EBVoriPdyadregion. These cellular factorscompetedfor

binding with a bacterially produced carboxy-terminal do-main of EBNA-1 (28,000-molecular-weight EBNA-1 [28K EBNA-1]) (14, 15).

To detect cellularproteins that bind to the 140-bp oriP

dyad region, nuclearextractspreparedfrom HeLa cells (4) were used in a protein-DNA gel shiftassay (Fig. 1). Blue-script(Stratagene) plasmidscontaining subcloned oriP DNA

fragments (3) were used to generate end-labeled probes representing the 140-bp EcoRV-to-HpaI oriPdyad symme-tryregion (nucleotides 8897to9136;numberingasdescribed by Baer et al. [2]). Plasmids were linearized at polylinker

restriction sites adjacent toeither the EcoRV or the HpaI

end of the dyad region, end labeled with a-32P-labeled

deoxynucleoside triphosphates and Klenow fragment, and

digested with a second restriction enzyme to excise the

140-bp-labeledfragments. Labeled oriPfragmentswere

pu-rified after isolation fromagarosegels. Nuclearextracts(5to

10 ,ug) wereincubated for 30 minatroomtemperature with 0.5 to 1 ng of end-labeled 140-bp probe and 1

pug

of poly(dI-dC) in 12.5 ,ul of buffer containing 15 mM

N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.9),50 mMNaCl,0.5 mMEDTA,and1 mM

dithiothre-itol. Samples were electrophoresed at 200 V on 4% poly-acrylamide gels in 10 mM Tris hydrochloride (pH 8.0) containing0.1mM EDTAfor 2.5 h.

Three DNA bands ofreduced mobility wereobservedin thesenondenaturing gels (Fig. 1A),and thespecificityof the

majorshifted bands wasinvestigated bycompetition exper-iments. Two DNA fragments were employed as specific competitors: the homologous 140-bp EcoRV-to-HindIII dyadfragmentanda36-bp oligonucleotide correspondingto

the consensus 30-bp oriP repeat sequence that binds EBNA-1 (termed the EBNA-1 consensus oligomer; see

legend to Fig. 1B). As described above, four partial

ho-mologs ofthisconsensusrepeat sequencearepresentwithin the 140-bp dyad region. Addition of the homologous unla-beled 140-bp dyad fragment during the binding reactions caused the disappearance ofall bands(Fig. 1A). The36-bp

514

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Competitor 0 0 0 0 0

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-140bps

FIG. 1. Detection of cellular proteins that bindtc dyad region by protein-DNA gel shiftassays.A1-n,

end-labeled probe corresponding to the 140-bp

dyad region and 1,ug of poly(dI-dC) were added HeLa nuclear extract together with several diff double-strandDNAcompetitors,asindicated below binding reaction, the mixturewas electrophoresed

naturing polyacrylamide gel. Competition experin

formed by including in the binding reaction mixturt

gous(A)orheterologous (B and C)competitor DN excess over theprobeDNA indicated above each petitor DNAs were as follows: (A) the 140-bp oi

fragment; (B)a36-bpEBNA-1consensusoligomer(

TTAGGATAGCATATGCTACCCAGATATA-3 ATCTGGGTAGCATATGCTATCCTAATCTG-nonspecific oligonucleotide (5'-GGCACCAGCT'

ACAGTGTAAAAAAGGGCC-3':5'-GGCCCTTI TGACTGATTGAGCTGGTGCC-3'). Arrows in( of bands of altered mobilitywhich result fromspec

of HeLa nuclearproteins and the140-bp oriPdyad

EBNA-1 consensus oligomer also effectively

binding of cellular factors to the 140-bp dya( (Fig. 1B), whereas a 37-bp DNA fragment

sequence did not compete (Fig. 1C), indical complexes resulted from sequence-specific This result indicates that those cellular factoi specifically with the 140-bp dyad region als EBNA-1 consensus repeat oligomer. Furth findings suggest that the cellular factors bin recognition sites, sinceboththe 140-bp dyad EBNA-1 consensus oligomer sharethis sequt

In order to identify the nucleotide sequen

oriPdyadregion (nucleotides 8897to9136)th; factors, DNase I footprinting was performec

finalreaction mixture (50

RI)

contained 4ng(

labeled140-bporiPdyadprobe (seeabove), 1 dC), 8 mMHEPES(pH7.9),24 mMKCI, 1m

mM EDTA, 0.2 mM dithiothreitol, 8% glyce 200 pug of HeLa nuclear extract. A 46-pd re

that containedallcomponents exceptthelab incubatedonicefor15min. Theprobe DNA

c

4-pd

volume,

and the reactionmixturewasincubated at 25°C

for10min. DNase I was

added,

andincubationwas

contin-COg 0 ued at room

temperature

for 1 min. The concentration of DNase I required to obtain uniformdigestionateachextract

protein concentration was determined

empirically

and

rangedfrom 0.6 to 7.5

,ug/ml.

After theadditionof 300

RI

of 10 mM Tris hydrochloride (pH

7.4)-300

mM NaCl-20 mM

EDTA-1% sodiumdodecyl sulfate-50

p.g

of tRNA per

ml,

the DNA waspurifiedby extraction withphenoland chlo-roform andanalyzedon8%

polyacrylamide

sequencing gels.

_"*s1+iiiii

flok The sizes of the

digestion products

were compared with thoseof productsgenerated

by

specific

chemical

cleavage

of thelabeled DNA(12).

DNase Iprotectionanalysisdemonstrated that the

140-bp

oriPdyadregion containsmultiple

binding

sites for cellular factors. At least five DNase I-protected regions were de-tected within the 140-bp oriP dyad

region

on the

coding

strand, designated 1 to5(Fig. 2A), andatleast four

regions

were detected on the noncoding strand,

designated

1 to 4 (Fig. 2B). Anadditionalprotected

region

(broken

line in

Fig.

2A)was alsoobserved in the

coding

strand. The nucleotide sequenceofeachofthese

protected

regions

is summarized in Fig. 2C.Mostoftheprotected

regions

areflanked

by

DNase 1 a 3 4 I-hypersensitivesites. One of theseregions

corresponds

toa

Z

the 140-bp oriP sequence

previously

identified

by

deletion

analysis

as

essen-thamount

of

32p_ tial for

plasmid

maintenance

(region

1 in

Fig. 2C) (3).

Two

EcoRV-to-HpaI protected

regions,

2 and

5,

are located onthe stem of the

to 5 to 10 ,ug of 65-bp dyad. Footprints 1, 3, 4, and the broken-line region

rerent unlabeled overlapwith the EBNA-1recognitionsequences and sharea e. After 30minof core ATATsequenceand similar adjacent sequences. These on a 4%nonde- findings and the results of gel shift assays (Fig. 1) suggest nents were per- that one or more proteins in HeLa cell extracts bind to

eeitherhomolo- sequences within oriP thatcoincide with the EBNA-1 rec-[As inthemolar ognition

sites.

.riP

dyad region Consistent with thispossibility, specific HeLa cell-protein 'S'-GATCCAGA interactions with the EBNA-1 consensus oligomer were

':5'-GATCTAT detected by gel shift assays (Fig. 3). A nuclear extract from -3'); (C) a 37-bp HeLa cells was

subjected

to

chromatography

on a

cation-CAATCAGTC exchange matrix

(SP-5PW),

and fractions were tested

by

a

rTTTACACTG band shift assay using a

32P-5'-end-labeled

EBNA-1

consen-dicate positions sus oligomer (Fig. 3). This analysis revealed three

protein-cific

interactions DNA complexes of reduced

mobility (arrows

a,

b,

and c,

Iprobe. Fig. 3). The activities giving rise to the a and bcomplexes (fraction 9) were separated from those

producing

the c

complex (fraction 13,

Fig. 3A).

Competition

experiments

competed for wereperformedwith the EBNA-1 consensus

oligomer

probe

d region probe and either fraction 9

(Fig. 3B)

or fraction 13

(Fig.

3C).

t of unrelated Complexes a, b, and c were inhibited

by

the addition of

ting that these unlabeled

homologous

DNA

during

the

binding

reaction but interactions. were notinhibited

by

a

nonspecific

oligonucleotide

(Fig.

3B rs thatinteract and C). Nonspecific

oligonucleotides

containing

single-io bind to the stranded DNA end structures similar to the EBNA-1

oligo-termore, these mer also did not compete for

binding

to these

complexes

id to EBNA-1 (data not shown). These results indicate that allthree

activ-region and the ities resulted from

sequence-specific

interactions.

ence. Rawlins et al.

(15)

demonstrated that a 28-kDa fusion

Ices within the protein

containing

the

carboxyl-terminal

one-third of atbind cellular EBNA-1 (28K

EBNA-1)

is

capable

of

binding

to

recognition

I (Fig. 2). The sites within the tandem

30-bp

repeat

region

and the

dyad

of the

32P-end-

symmetry

region

of oriP. To examine the

relationship

be-.

jig

ofpoly(dI- tween EBNA-1

binding

and the HeLacellfactors

binding

to

M

MgCl2,

0.08 oriP, the in vitro interactions between the HeLa cellular -rol, and 25 to proteins and 28K EBNA-1 with the EBNA-1 consensus

action volume oligomer were studied. 28K EBNA-1 was

expressed

in eled DNA was Escherichia coli

harboring

pNAK28

and

partially purified

was added in a according to the method of Milman et al.

(14).

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FIG. 2. DNase Ifootprinting of theoriPdyadsymmetryregion (nucleotides8897 to9136)of EBV. The140-bp EcoRV-to-HpaIoriPdyad regionwas32Pendlabeledasdescribedinthetext.(A) Coding strand.Theprobewasincubated withnoprotein (lane 1)orwith HeLa extract (lane 2, 25 ,ug; lane 3, 100 ,ug; lane 4,200 jLg) anddigested with DNaseI. DNAwasfractionated by electrophoresis on8% denaturing polyacrylamide gels, and the cleavage products were visualized by autoradiography. Lanes G+A correspond to apurine-specific DNA

sequencemarkerasdescribed by Maxam and Gilbert (12). Arrows indicate the 65-bp dyadsymmetrystructure.Proteinprotected regionsare

numbered 1to5.Thebroken line indicates weaker levels of protein protection. (B) Noncodingstrand. Lane1,Noprotein;lanes 2and3,100 and200 jigofHeLa nuclearextract,respectively. (C) Summary of protected regions within the140-bporiPdyadsymmetryregion.Thick

horizontallinesrepresentfootprints 1 through 5 shown in panel A. Four partial homologs of the30-bp repeatedmotif(EBNA-1binding sites)

aremarked Athrough D. Arrows indicate the 65-bp dyad symmetry structure. NucleotidenumberingisasdescribedbyBaeretal.(2).

ally synthesized 28K EBNA-1 bound to the consensus

oligomerDNA inagelshiftassay(Fig. 4A).Twospeciesof reduced mobility (Fig. 4A, arrows) were shown to be the

resultsofspecific interactions between 28K EBNA-1 protein

and DNA by competition experiments, while the third

ap-pearedtobenonspecific (lower band, Fig. 4A). In order to determine whether 28K EBNA-1 could compete for the

same sites on the oligomer which are recognized by the HeLa cell factors, increasing concentrations of 28K EBNA-1 were added toa fixed level of HeLa cell extract

G

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0

5

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a CM a v- 0*a 0la

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FIG. 4. Interaction of HeLa cell proteins and 28K EBNA-1 with the EBNA-1 consensus oligomer. (A) Gel mobility shift assay. Competition experiments were performed with the homologous specificEBNA-1 oligomer andaheterologous nonspecific oligomer (see

legendtoFig. 1B and C). Arrows indicate positions of bands of altered mobility which result from specific interactionsof the bacterial28K EBNA-1protein (0.2 ,ugperreactionmixture) and the EBNA-1consensusoligomer. (B)Gel retardationassaywith variousconcentrations of bacterial28K EBNA-1 protein. Binding reactionswerecarriedoutwith 4,ug ofpoly(dI-dC) ina10-pl reaction volume containingaconstant

amountof HeLa cellextract(12 ,ug) and increasing concentrations ofanextractcontaining the 28K EBNA-1protein. Finally,the32P-labeled EBNA-1consensusoligomerwasadded, and the reaction mixtureswereincubated for 30minatroomtemperature.DNA-protein complexes

wereseparated byelectrophoresison a4%polyacrylamide gel. Lane 1, Noextract;lane 2, HeLaextract(12 ,ug); lanes 3to7, binding reaction mixturescontainingconstantHeLa nuclearextract(12

jig)

and increasingamountsof 28K EBNA-1fusionproteinextract(lane3through 7, 4, 8, 12, 16, and 20 ,ug, respectively). Arrows indicate positions of DNA-protein complexes whichwereinhibitedby competition.Extracts

containing the cellular DNA-binding proteins and EBNA-1 28K proteinwerenotpurifiedorenriched.

togetherwiththeEBNA-1consensusoligonucleotide probe. Competition experiments were carried out with concentra-tions of DNAoligomer probesandproteinatwhichallofthe

oligomer is bound (and is limiting in the reaction). As the amountof 28K EBNA-1wasincreased,thebands represent-ing HeLa cellprotein-DNA complexeswereeliminated (Fig.

4B). Under the conditions used in these experiments, the nonspecific third bandfrom 28K EBNA-1(Fig. 4A)wasnot apparent. The reciprocal experimentwasperformed with a fixedlevel of 28KEBNA-1and increasingconcentrations of HeLaextract. Inthis experiment, atleastoneband derived

from the 28K EBNA-1-oligomer complex was inhibited by the addition of HeLa extract (data not shown). These findingsindicate thatHeLacell factors competed with 28K EBNA-1 in vitro for binding to the EBNA-1 consensus

oligomer.

It ispossiblethatcompetition between cellular factors and

EBNA-1 could regulate viral DNA replication in vivo by excluding EBNA-1fromthe binding sites withinoriP. Con-ceivably, theinteraction of such cellular proteins withoriP could be involved in the control of EBV replication within

the cell cycle or could govern the copy number ofEBV

plasmids. It has been reported that two cellular factors

produced bya12-O-tetradecanoylphorbol-13-acetate

(TPA)-treated, EBV-negative Burkitt's lymphomacellline(BJAB)

are able to compete for and uncouple EBNA-1 binding to oriP(19). It remains tobe determined whether these TPA-induced cellularfactors are the same as, or relatedto, the

HeLacellfactorsdetected in this study. Binding ofEBNA-1

tooriPappearstoberequiredbothfor EBVreplicationand for activation of the30-bprepeatenhancerelement(16, 18, 20). Recent experiments by Sugden and Warren (18) dem-onstrated that EBNA-1 can trans activate the BamHI-C

latency promoter via the oriP 30-bp repeat enhancer ele-ment. Thus, cellular factor(s) that compete for EBNA-1

binding sites could also playan importantrole in the regu-lation of viralgene expression inlatently infected cells.

WethankUlrich Muller forhelpful discussions,Matthew Marton for providing SP-5PW fractions of HeLa cell extract, Gregory Milman forsupplying the plasmidpNAK28, and EduardoMontalvo

forreviewing the manuscript.

S.-J.OhwassupportedinpartbyaKorea Science and Engineer-ing Foundation scholarship. This work was funded by grant CA49271from the National CancerInstitute.

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