Structure and function of the Epstein-Barr virus BZLF1 protein.

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JOURNAL OF VIROLOGY, May1990,p. 2110-2116 0022-538X/90/052110-07$02.00/0

Structure and Function of the Epstein-Barr

Virus BZLF1

Protein

GRAHAMPACKHAM, ANDROULLA ECONOMOU, CLIONAM. ROONEY,DAVID T. ROWE,

AND PAUL J. FARRELL*

Ludwig Institute for Cancer Research, St. Mary's HospitalMedicalSchool, Norfolk Place, London W2IPG, England Received 20 November 1989/Accepted 14 January 1990

FiveDNA-binding sites for the Epstein-Barr virus BZLF1 protein have been identified withinthreeof the early viral promoters, and four of thesebindingsites containaconsensusAP-1 site. Thepartof theBZLF1 protein required for sequence-specific DNA bindingtooneof these AP-1-like siteswas identifiedby deletion mapping. Site-directedmutagenesis of this DNA target suggests that BZLF1mayworkpartlybyovercoming

acellularrepressorof viral transcription.

Epstein-Barr virus (EBV) isahumanherpesvirusthatcan

follow either a latent (i.e., nonproductive) or aproductive life cycle. When EBV infectsan appropriate B lymphocyte invitro,thelymphocyte becomesimmortalizedandalatent infection is established (reviewed in reference 13). The switch tothe virus-productive infection can be induced in

vitro by treating the latently infected lymphocytes with a variety of chemical or biological inducers (e.g.,

12-O-tet-radecanoylphorbol-13-acetate, transforming growth factor

,B,

or

antiimmunoglobulin)

or

by superinfection

with certain

rearranged, defective EBV genomes (e.g., het virus). In

common with other herpesviruses, the productive-cycle

geneexpression of EBV is organizedas acascade ofgroups

ofgenes (reviewed in reference 6). Present evidence indi-catesthat expression of the BZLF1geneof EBV(also called

EB1 and ZEBRA) is the first step in the productive-cycle cascade (2, 5, 20, 21). Transfection of a transcriptionally active BZLF1constructissufficienttoinduce theproductive cycle (4, 5, 18). The BZLF1 and BRLF1 proteinsaregene regulatory molecules, both of whichcanactivate the BSLF2 +BMLF1 gene (also called EB2), and then various

combi-nations of these three gene products appear to activate further stages of the cascade (3, 8, 10-12, 15-18, 22). We showedpreviously that BZLF1 isasequence-specific

DNA-binding protein thatcanbindtoaDNAsequencewithinthe promoter region of the BSLF2+BMLF1 gene (7). This binding site contains a consensus recognition sequence for

the AP-1 transcription factor family (TGAGTCA; EBV position 84429). BZLF1 has localized protein sequence homologytoc-fos and c-jun, which are components of the AP-1factor, particularly inaregion called the "basic motif," which is thoughtto be thepartof the protein that interacts with DNA. We have shown elsewhere (7, 18) that approxi-mately theC-terminal half of BZLF1 (from the SmaI siteto the Cterminus) is sufficienttobind specificallytothetarget but will not activate transcription. N-terminal sequences within the BZLF1 protein aretherefore required forthefull activity of the protein butnotfor DNA bindingper se.Inthis

paper we have mapped five DNA-binding sites for the BZLF1 protein within the EBV genome; not all of these contain a consensus AP-1 site. We also delineate more precisely those parts ofBZLF1 thatare required for DNA

binding at one of the sites and for transactivation ofthat EBV promoter. Site-directed mutagenesis of this target

*Correspondingauthor.

indicates that BZLF1 may workpartly by counteracting a

cellular factor thatrepressesviraltranscription. MATERIALS ANDMETHODS

Construction of plasmids. The starting point for all the BZLF1 deletionmutantswastheplasmid SP64-BZLF1(18),

which isan870-base-pair BamHI-EcoRI cDNA for BZLF1

cloned between the BamHI and EcoRI sites of SP64.

SmaI-C.The N-terminalSmaIfragmentwasdeletedfrom

SP64-BZLF1 and replaced with adouble-stranded (ds) oli-gonucleotideofsequence CATCGATG, which suppliesthe

initiator methionine codon.

N-Hincd. SP64-BZLF1 was cut with HincIl, and a ds

oligonucleotide of sequence TAGAATTCTA was added. Afterdigestion with EcoRI, the DNA wascircularized and cloned.

N-PstI. SP64-BZLF1 was cutwithPstI, and the PstI ends wereligatedto adsadaptor ofsequence

GCGAATTCTTACTGCA CGCTTAAGAATG

After cleavage with EcoRI and BamHI, the resulting BamHI-EcoRI fragment containing the appropriate part of BZLF1 was cloned between theBamHI and EcoRI sitesof

SP64.

N-StyI. SP64-BZLF1wascutwithStyI, and the endswere

made blunt withmung bean nuclease. A dsoligonucleotide

ofsequence TAGAATTCTAwasligatedtothe ends. After digestion withEcoRI,theplasmid wascircularized.

HindIll-delta. SP64-BZLF1 was cut with HindIII, giving three pieces. The N-terminal HindIII fragmentwas cloned into the large vector/C-terminal piece, omitting the small centralHindIIIfragment.

BsmI-C. SP64-Sma-Cwasdigested with EcoRI and BsmI. An adaptorconsisting of the ds oligonucleotide

GATCCAAGATGTGCG GTTCTACAC

wasusedtojoin theBsmIendtothe BamHI site in thelarge BamHI-EcoRI fragment of SP64.

BsmI-HincII. SP64-BsmI-C was digested with PstI. The

smallerfragmentwascloned into thelarge PstI fragment of SP64-N-HincII.

The aboveconstructsin SP64werefor in vitro transcrip-tion and translation. To assay the activity of the BZLF1

deletion mutants by transfection, the sequences derived from BZLF1 in the SP64 constructs were subcloned as 2110

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STRUCTURE AND FUNCTION OF EBV BZLF1 PROTEIN 2111

XbaI-EcoRI fragments into the 3.1W plasmid, as for 3.1W-BZLF1 in (18). This results in the EBV BamHI W latent

cycle promoterdriving expression ofthe BZLF1 genes. The S-CAT plasmid, the BglII derivative of pSVOCAT, and CMV-BZLF1 have been described elsewhere (18).

CMVLTR-BRLF1 contained EBV sequences 105415 to 103081 (BglII toHindIII) cloned between a simian virus 40

polyA site andastrongpromotercomprisingahuman T-cell leukemia/lymphoma virus 1 long terminal repeat and the

cytomegalovirus immediate early enhancer.

In vitro transcription and translation of BZLF1 mutants. TheSP64-BZLF1 constructs werelinearizedwithEcoRI and

transcribed with SP6 polymerase at 40°C for 45 min.

Tran-scriptionreactionscontained2.5 ,ugofDNA;0.5 mM eachof

ATP, GTP, CTP, and UTP; 100 ILM 7MeGpppG; 10 mM

dithiothreitol; 40 mM Tris hydrochloride (pH 7.5); 6 mM

MgCl2; 2 mM spermidine hydrochloride; 62.5 U ofRNasin (Promega);and 25 Uof SP6polymerase inavolumeof50

RI.

Afterthe transcription, the templateDNA wasremovedby digestionwith DNase I(Pharmacia; fastprotein liquid

chro-matographygrade, 2.5 U)at37°C for15 min. The RNA was

phenol-chloroform extracted, ethanol precipitated, and

dis-solved in 20

RI

of water. Translation was in the

mRNA-dependent reticulocyte lysate (Amersham) with 1.5 ,ul of

RNA in a totalvolume of15 ,ulat 30°C for 35 minorin the wheat germ system(Amersham)with 1 ,ulofRNA inatotal

volume of13

pul

at25°Cfor60min. Asamplewasdiluted in sodiumdodecyl sulfategelsample bufferandanalyzedon a

20%polyacrylamide gel. The remaining translation product

wasfrozenat -70°C forDNA-bindingassays.

Site-directed mutagenesis of the promoter for BSLF2+ BMLF1. The 257-CAT, AE4-CAT, and AE4K-CAT plas-mids were prepared, starting with an M13 clone (257.RIF) from the DNA-sequencing program. This contained EBV sequences 84325 to 84767 (1) in M13mp8 at the SmaI

site,

oriented with the 84325 end close totheBamHI siteof the M13

polylinker.

Position84325 is the

transcription

initiation nucleotide ofthe spliced RNA encoding BSLF2+BMLF1,

and 257.RIFincludesthe promoter

region

forthisRNAfrom +1 to -442 relative to the transcription start. A

synthetic

oligonucleotide of sequence GCGAAGCACTCTCGAGTG AAGGTGAC wasusedfor site-directed

mutagenesis

(14) of

257.RIF, resulting in the replacement of the GACTCA at EBV 84430 with CTCGAG (an XhoI site). This gave M13AE4. ds DNA from 257.RIF and M13AE4 was

pre-pared, and theEBV sequencescouldbeexcised

by using

the

flanking BamHI and EcoRI sites in the

polylinker.

The EcoRI site was filled in with Klenow DNA

polymerase;

a BamHIlinkerwasadded,and theresulting BamHI

fragment

wasclonedinto theBglIIsite ofpSVOCAT,giving257-CAT and AE4-CAT, resulting in either the

wild-type

or mutant promoterdrivingthe CATgene. AE4-CATwasthen cutat theunique XhoIsite,filled in with Klenowpolymerase,and recircularized with DNA ligase, giving AE4K-CAT. The sequencesof257.RIF, AE4, and AE4K around the

point

of

mutagenesis werecheckedby DNA

sequencing.

Footprinting. DNase I footprinting used the same prepa-ration ofBZLF1 fusion protein

(pREX-SmaA),

which is a fusion ofproteinAtotheSmaI-CBZLF1 sequences,as was described elsewhere (7, 18). Reaction conditions for

foot-printingwere asdescribedpreviously (7).

Gel retardation.

32P-labeled

DNA substrates were either the 261-base-pair 374RIF fragment from the promoter for BSLF2+BMLF1 (7) ords

oligonucleotides

overlapping

its consensus AP-1site. The wild-type

oligonucleotide

292/293 had ds sequence

GAAGCACTGACTCATGAAG,

and the

AE4

oligonucleotide

was ds GCGAAGCACTCTCGAGTG AAGGTGAC.

Binding

was

performed

at roomtemperature as described

previously (7).

The fusion

protein

orin vitro translation

product

(usually

1to3

pI)

wasdiluted in 18

pul

of

100 mMKCl-20mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid)

(pH 7.9)-10%

glycerol-0.2

mM

EDTA-4 mM

dithiothreitol,

and to

this,

4 ,ulof

premixed

DNA

[i.e.,

poly(dIC),

32P-labeled

target

DNA,

and any

competitor

oligonucleotide]

was added. This resulted in a

final concentration of 100

pug

of

poly(dIC)

per ml. Concen-trations of

32P-labeled

DNAsand

competitors

are

given

in the

figure

legends.

Afterincubationatroomtemperaturefor

20to30min,3

pu1

of

dye

(xylene

cyanol

FFand

bromophenol

blue in thesame

buffer)

wasadded and the whole

sample

was

loadedon toeithera1%agarose

gel

or a4%

polyacrylamide

gel.

Gelswere runeither in 0.5x TBEorbuffer G

(380

mM

glycine,

50 mM

Tris,

5 mM EDTA

[pH

8.5]).

The shifted

complexes

were detected

by

autoradiography

of the dried

agarose

gel

orthewet

polyacrylamide gel.

Transfection

experiments. (i) Assay

of BZLF1 deletion mutants. 3.1W-BZLF1 orthe3.1W-BZLF1 deletion mutant DNAs were

electroporated

with S-CATin BL41/CL16 cells as

previously

described

(18).

Cells were harvested after 1

day,

andCAT

activity

wasdetermined

(18).

(ii) Assay

of site-directedmutantsin AP-1 siteupstreamof BSLF2+BMLF1.

S-CAT, 257-CAT,

AE4-CAT,

and

AE4K-CATwere

electroporated

withorwithout

CMV-BZLF1,

or

CMVLTR-BRLF1,

into BL41cellsas

previously

described

(18).

Cells were harvested after 2

days,

and CAT

activity

was

assayed (18).

RESULTS

Deletion

mapping

the part of BZLF1

required

for DNA

binding.

A cDNA

encoding

the BZLF1

protein

was cloned

previously

between theBamHIand EcoRIsitesof

SP64,

and this clone could be transcribed and translated in vitro to

produce

authentic BZLF1

protein

(18).

Various deletion mutants were

prepared by

restriction

digestion,

using

syn-thetic

oligonucleotides

to

repair

the resected ends

(Fig.

1).

By

this means, aninitiatormethionineresiduewas

supplied

totheN-terminal deletionsandatranslationterminatorwas

restored tothe C-terminal deletions. All thedeletionswere

precise

truncations ofthe BZLF1 sequence, and no extra-neous aminoacids were introduced

(except

forthe initiator methionine residues in the N-terminal

deletions).

All ofthe

deletions ofBZLF1 weretranscribedwith SP6

polymerase,

and the

resulting

RNAwastranslatedineitherthe

reticulo-cyte

lysate

or wheat germ translation

system,

using

[35S]cysteine

astheradioactive label.

Analysis

of the

trans-lation

products

on sodium

dodecyl

sulfate

gel

electrophore-sisshowed that allofthemutantsgave

proteins

of

appropri-ate relativesizes

(Fig.

2; BsmI-HincII

isnot

shown).

Itwas

necessary to use the wheat germ system to demonstrate translation of the smaller

proteins

because the

high

concen-tration of

globin

in the

reticulocyte

lysate

distorted the sodium

dodecyl

sulfate

gel

in that size range. The in

vitro-translated

proteins

were thenused in

gel

retardationassays to test their

ability

to bind

specifically

to a

261-base-pair

32P-labeled

fragment

of EBV DNA

spanning

the

promoter

region

of BSLF2+BMLF1 and

containing

the consensus

AP-1 site. This is the same DNA

fragment

to which we

previously

showed

binding

of BZLF1 fusion

proteins

and in

vitro-translatedBZLF1

(7).

The

full-length

BZLF1,

SmaI-C,

BsmI-C,

and N-HincIImutantsallshifted the

target,

butthe N-PstI mutant did not

(Fig.

3).

Because ofits small

size,

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2112 PACKHAM ET AL.

0 200 400 600 800bp

I I I

BamHI Hindlil Hindill Smal Styl Bsmi

BZLF1

BZLF1 N-Hincil

BZLF1 Hindlil defta

BZLF1 N-Psti

BZLF1 Smal-C

BZLF1 N-Styl

BZLF1 BsmI-C

BZLF1 Bsml-Hincil

I I

PstI Hincil EcoRt

boundaries

MMD... LNF

- MMD ... LDV

..FVQAYA..

MMD ...LLQ

MGA ...LNF

MMD ... GAN

MCD... LNF

- MCD... LDV

RegionofhomologywithfosrjunDNAbindingdomain

Non-codingsequences

Codingsequences

Bsml Pstl Hincll

v

+

~~~+

v

+

BZLF1 SLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVD

v <--baSiC Otif --> L v L L L L

C-FOS LSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETDQLEDEKSALQTEIANLLKEKEKL

FIG. 1. BZLF1 deletion mutants. The structures of the BZLF1 deletion mutants are shown beneath a restriction map of the EBV sequences inthe BZLF1 cDNA(SP64-BZLF1). The amino acidsatthe relevant boundaries of the deletionsareindicated,andthe amino acid sequenceof theBsmI-HincIIportion that isrequired forDNAbinding is shown below. This sequence iscomparedwith part ofc-fosto illustrate the basic motif, the leucine zipper region (L) of c-fos, and the boundaries(V)ofexon2ofBZLF1andexon3ofc-fos.Conserved residues within the basic motif are marked with dots (R-K substitutions are consideredconserved).

BsmI-HincII was tested by using an oligonucleotide as the target(seebelow).

The shifts represent site-specific binding because assays were performed in the presence of excess poly(dIC) and

because the binding was abolished by an excess ofa ds

oligonucleotide containing the AP-1 site but not by an unrelated dsoligonucleotide. The results indicate that pro-tein sequences essential for binding lie between the BsmI and HincII sites. This region of 58 amino acids includes the

basic motifand, in c-jun and c-fos, the leucine zipper (the

leucinezipper is not apparent in BZLF1). Deletion from the

C terminustothe PstIsite removes the region equivalent to the leucine zipper and abolishes binding. Although the

BsmI-C mutant retains DNA binding, the protein-DNA

complex is less stable to salt than, for example, SmaI-C. This isillustrated in Fig.4, wheregelretardation assays of a

ds oligonucleotide covering the AP-1 site in the BSLF2+ BMLF1 promoter were performed in either 0.5x TBE or

bufferG(highersalt). Although the complex is seen in both

buffer systems with SmaI-C (lane 9), the BsmI-C complex

(lane 10) is only stable in the lower-salt buffer. A similar

phenomenonwasobservedby comparing full-lengthBZLF1 with N-HincII (data not shown). The BsmI-HincII mutant (lane 11) did notgive a significant retardation complex in either buffer system(an extremely weaksignalwas seenon theoriginal autoradiograph).

BZLF1 mutants that retain transactivation retain DNA binding. The BZLF1 cDNA and the deletion mutants HindIIIdelta, N-HincII, N-StyI, SmaI-C, andN-PstI were recloned downstreamofastrongEBV promoter(thelatent promoter in BamHI W) so as topermit expression of the BZLF1 protein. These constructs were

electroporated

into

BL41/CL16cells (whichcontain EBV) withaplasmid

con-tainingthe promoter for BSLF2+BMLF1 fusedtothe CAT gene. This promoter is dependent on transactivation from BZLF1 or BRLF1. The BZLF1 deletion mutant N-HincII was aseffective asfull-lengthBZLF1intransactivatingthis promoter, but N-PstI, N-StyI, and SmaI-C were inactive J. VIROL.

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FIG. 2. In vitro translation of BZLF1 deletionmutants.DNAfor the various BZLF1 deletion clones in SP64 was linearized with EcoRIandtranscribed with the SP6 polymerase, and the resulting RNA was translated in the mRNA-dependent reticulocyte lysate (lanes BZLF1, N-HincII, HindIII delta, and N-PstI) orthe wheat

germ system (no RNA, SmaI-C, BsmI-C, and N-StyI), using

[35S]cysteineasthe radioactivelabel. The translation productswere

detectedbysodium dodecyl sulfate gel electrophoresis and fluorog-raphy. The positions to which radioactive protein size markers migratedareshownonthe right in kilodaltons.

(Fig. 5). These results are consistent with a model of the BZLF1protein in which sequence-specific DNA binding is mediatedby amino acids between the BsmI andHincII sites butsequences N terminal of the BsmI sitearealso required

fortransactivation. The deletionof amino acids between the HindIIIsitesresults only inapartial loss of the transactiva-tionfunctionofthe N-terminaldomain (Fig. 5, lane 2).

BZLF1binds specificallytoDNAsequencesupstreamof the promotersforBZLF1 andBRLF1.Since BZLF1 is thoughtto stimulate expression of BZLF1, BRLF1, and BSLF2+ BMLF1,wetested theability ofa BZLF1fusion proteinto footprintsequencesaround theBZLF1and BRLF1

promot-ers. Inthese experiments, the SmaI-C fusionto protein A

wasused, sincewehadpreviously shown this footprintedto the consensus AP-1 site in the promoter for BSLF2+ BMLF1 (7). The fusion protein was titrated into the foot-printing reactions, and this gave some measure of the

relativeaffinity ofbindingatdifferent sites. Two clear sites werereproducibly found upstream of the BRLF1promoter (probes RBR.511 and 7.FAL, Fig. 6). Some other sites of lowaffinity werealso observed;thesegave variable results

in footprinting. Upstream of the BZLF1 promoter (probe 48.BAZ), one region was found to footprint but this was unusuallywidecompared withthe other footprints, and the titration (lanes 2 through 4) indicates two binding sites of different affinitiesadjacent toeach other. The sequences of

allthe sitesaresummarizedinFig.7andarecomparedwith the site (M) previously mapped upstream of the BSLF2+ BSLF1promoter. All the sitesexceptfor thehigher-affinity

FIG. 3. Gel retardationassayforBZLF1 deletion mutants bind-ingto the promoter for BSLF2+BMLF1. The 32P-labeled probe from374.RIF (1 ngper assay,final concentration3 nM) wasmixed with variousBZLF1deletion mutants made by in vitro translation in the reticulocytelysate or wheat germ system (see Materials and Methods). Allreactions contained100

jig

ofpoly(dIC)per ml. Lanes 0, No oligonucleotide competitor; lanes 1, cognate 292/293 ds oligonucleotide GAAGCACTGACTCATGAAG (0.5 ,uM); lanes 2, anunrelated ds oligonucleotide sequence GGGTACCC (0.5

JIM).

Electrophoresiswason a1%agarosegelin0.5x TBE.

site upstreamofBZLF1 arecentered on a close match to the consensusAP-1site. In thepossible alignment shown in Fig. 7, it can beseen that someother nucleotides also match in theflankingsequences thatarecoveredby the footprint.The

high-affinity site upstream of BZLF1 does not match the AP-1 consensus. A possible alternative consensus which matches all of the sites is indicated.

Site-directed mutagenesis of theconsensus AP-1 site in the promoterfor BSLF2+BMLF1. To assess thesignificanceof theconsensus AP-1 siteat 84429 in the promoterregionof

BSLF2+BMLF1, six nucleotides of the AP-1 site were

replaced by unrelated nucleotides without changing the

surrounding sequence. The site was changed from TGAC TCA to TCTCGAG. Analogous CAT fusions ofthe wild-type promoterand themutant(called AE4)wereprepared.A further mutant (AE4K) was made by cutting the XhoI site

(CTCGAG) introduced to AE4, repairing the ends with Klenow polymerase, and religating. This resulted in the inclusion of fourextranucleotides; therefore,in this mutant, the sequencewaschanged from TGAGTCAtoTCTCGATC GAG. These constructs were transfected into BL41 cells with or without CMV-BZLF1 to assay their sensitivity to

transactivation. Our principal objective here was to study

the effectofthesemutationson BZLF1transactivation,but

for illustrationandcomparison, the greatereffect of BRLF1 onthis promoter is shown inoneexperiment(Fig. 8).BZLF1 activated CATexpressionfrom thewild-typepromoter,but no effectwas seen on the AE4 construct because the AE4 promoterwas alreadyactive evenwithout BZLF1

(Fig.

8).

The AE4K promoter was inactive with or without BZLF1

(but could still be activated by BRLF1). These results suggest that BZLF1isactingonthis target in BL41cells

by

counteractingacellular repressorthatrecognizessequences

involving the consensus AP-1 site. When the consensus AP-1site isremoved, theBZLF1 nolongerhasaneffect in

VOL.64, 1990

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2114 PACKHAM ET AL.

1 2 3 4 5 6 7 8 9 10 11 12

S

A 7FAL 48BAZ 1234512345

tiEIf lA

{B..

U ..

_-rn

.-*abI a

aW

1 2 3 4 5 6 7 8 9 10 11 12

S V.

B RBR511 Ml 2345M

be4 .-529

_-406 m -406

1111

*

~~~~~~~-

311 |**-1

.g- 240 .11:1 w-240

-219

i-182 * -0

*-162 . -182

*-i49 e *-162

R t , !r-~149

-125

*-112 -1251

92~ _7.. £ -92 * > 9

78 4-92

-, - 69

.. -6

- -_ _ . -69 1*Zi*ft

FIG. 4. Salt sensitivity ofgeJretardation complexes. Gel retar-dation assays were analyzed on polyacrylamide gels in0.5x TBE (upper panel) or buffer G (lowerpanel). Lanes 1 through 6, AE4 ds oligonucleotide (0.4 ,ug/ml)as acontrol; lanes 7 through 12,292/293 dsoligonucleotide (0.4 ,ug/ml). Lanes 1 and 7, No added protein; lanes 2 through 5 and 8 through 12, 2 ,u1 ofreticulocyte lysate translations of: lanes 2 and 8, no RNA; lanes 3 and 9,SmaI-C;lanes 4and 10,BsmI-C; lanes 5 and 11,BsmI-HincII.Lanes 6and 12, 1pI (0.5 pug)ofpREX-SmaA&fusion protein. Allreactions contained 100

pig

of poly(dIC) per ml.

this assay system. An important promoter element must, however, surround or overlap this region because

introduc-ing anextrafour nucleotides in AE4K destroys this

basal-level promoter activity. The pREX-SmaA BZLF1 fusion

9

4lb

...

4-36

I.,afff *

MD .i ifs

_^ ^N

4b:JI.

_I&a

_&.

-36

FIG. 6. Footprinting of BZLF1 SmaI-C (pREX-SmaA) fusion proteintopromoterregionsofBZLF1and BRLF1. 32P-end-labeled DNAswereprepared forfootprinting as describedpreviously (7) by usinga32P-end-labeledprimertocopyasecond strandonM13 ss DNA. Theprimerwasthe 17-base sequencing primer; the clones usedwere (A) 48BAZ(BZLF1 promoter) and (B)7 FAL(BRLF1 promoter)andRBR511(BRLF1 promoter), and allwere cutwith EcoRI.TheEBV contentof these clones is 48BAZ, 103188toabout 103650;RBR511, 106107 toabout106750; 7FAL, 106192toabout 106670.ProteinDNAcomplexeswereformedasforgelretardation, and then 1 pu1of25 mM MgCl2and1 pulofDNase (4 U/,u) were added. Afterdigestion for60 s at roomtemperature,phenol extrac-tion, and ethanol precipitaextrac-tion, samplesweredissolved in formam-ide andelectrophoresedon6% polyacrylamide gels in 8M urea.Size markers areanMspI digest of pBR322, endrepaired with Klenow DNA polymerase, and

[a-32P]dCTP.

Lanes 1 and 5, No added protein;lane2,1pul(0.5pug)of protein;lane3,2pA of protein; lane 4,4,ulof protein.

protein bound an oligonucleotide spanning the consensus AP-1siteofthe standard promoter in agelretardation assay butdidnotbindto an oligonucleotide correspondingtothe AE4 mutant sequence (Fig. 4, lanes 6 and 12).

DISCUSSION

* * * *v Deletionanalysisof the BZLF1proteinhas shown thatthe

part ofthe protein between the BsmI and Hincll sites is required for sequence-specific DNA binding by BZLF1 at

C

i 2 3 4 5 6 the site

upstream

of the

promoter

forBSLF2+BMLF1. This

isarefinement ofourearlier

mapping

of thisfunction. The

FIG. 5. CATassay of induction of S-CAT construct by BZLF1

deletion

mutant that lostDNA binding(N-PstI) also lostthe deletion mutants. p3.1W-BZLF1 fusions of the BZLF1 deletion

ability

to activate the promoter for

BSLF1+BMLF1,

con-mutants(20 ,ug) were electroporated with the S-CATplasmid (20

p.g)

sistent with the idea thatsequence-specific DNA binding is intoBL41/CL16cells. CAT activity was assayed 1 daylater. Lane part of the mechanismbywhich BZLF1 induces

productive-C, No BZLF1; lane 1, BZLF1; lane 2, HindIII delta; lane 3, cycle transcription.

N-HincII; lane 4,N-StyI;lane 5,SmaI-C; lane 6,N-PstI. Although the BZLF1 sequences involved in the protein-J. VIROL.

6 .,

.--.'.- M'41-. 4b

M.-Ai..

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Promoter for BZLF1, probe 48.BAZ

170 180 190

103310 103320 103330

TACATTAGCAATGCCTGTGGCTCATGCATAG

<---ACCGAGT-->

ATGTAATCGTTACGGACACCGAGTACGTATC

Promoter for BRIE1, probe RBR.511

150 160 170

106210 106220 106230 CTGGTCTTTTATGAGCCATTGGCATGGGCG

<---TGAGCCA--->

8 -7 -6 -5

-4

-3 -2

-310 320 330

106370 106380 106390 AACTAAGCTTATGAGCGATTTTATCACAGG

<---TGAGCGA--->

TTGATTCGAATACTCGCTAAATAGTGTCC

Promoter for BRLF1, probe 7.FAL

60 70 80

106210 106220 106230 CTGGTCTTTTATGAGCCATTGGCATGGGCG

<----TGAGCCA--- -> GACCAGAAAATACTCGGTAACCGTACCCGC

220 230 240

106370 106380 106390 AACTAAGCTTATGAGCGATTTTATCACAGG

<---TGAGCGA--->

TTGATTCGAATACTCGCTAAAATAGTGTCC

1

F

r-

,n

- + - + - + - + BZLF1

S 257 AE4 AE4K

CAT CAT CAT CAT

Normalised data S CAT

S CAT+BZLF1 S CAT+BRLF1

%conversion Expt 1 Expt2 Expt 3

1.11 0.49 1.0 3.62 0.86 6.5 27.4

average 0.87 3.66

Alignment

AE4mutant ttcATctcgaggt

R R R

z

?consensus? AP1

ttcATGAGTCAgt cttATGAGCGAtt tttATGAGCCAtt tgcATGAGCCAca tacATtAGCaAtg Y-YAT-AGY-A- -TGAGTCA

FIG. 7. Relationship of footprints seen in Fig. 6 to DNA se-quence ofEBV. Theextent of each high-affinity footprint is indi-cated by the arrows and dotted lines. The numbers above each segmentrelate the EBVgenomenumbers(1) to thegel size markers inFig.6.Below this is shownanalignment of thefootprintswiththe previously established footprint(M;seereference7)in the promoter region of BSLF2+BMLF1. Thesequenceofthe AE4 mutantof the M sequence, which does not bind BZLF1, is also shown. The alignment of four ofthefootprint targets to a consensus AP-1 site is indicated together with a possible alternative consensus that matchesallfive bindingsites.

DNA interaction apparently lie between the BsmI and HincII sites, the SmaI-BsmI and HincII-C parts seem to

contribute significantly to the stability of this protein

do-main. We presumethatthe virtual absence ofaretardation complexwith BsmI-HincII isaconsequenceofthe instabil-ity oftheproperly folded structure (the DNA-protein

inter-action is

evidently

not very salt sensitive, since SmaI-C

binds well in both high and low salt). The salt sensitivity

impliesthationic interactions areimportantinstabilizingthe

protein structure of the DNA-binding domain. There are several acidic residues in the SmaI-BsmI and HincII-C sections which may be involved in interactions with basic

amino acids in the basic-motif region stabilizingthe struc-ture. Inourassays, theSmaI-C segmentseemstobindjust

aswellasthe wholeproteinand,therefore, we conclude that the wholeDNA-binding domain liesC terminal of theSmaI site.

257CAT 257CAT+BZLFI 257 CAT+BRLF1 AE4CAT AE4CAT+BZLF1 AE4 CAT+BRLF1 AE4KCAT AE4KCAT + BZLF1 AE4KCAT+BRLF1

1.55 5.90 41.5

7.48 4.26 89.9

0.52 0.85 23.4

1.10 1.1 1.25 5.90 5.90 5.90

8.60 4.2 6.76 11.43 7.2 7.63

0.24 0.5 0.42 0.98 0.8 0.88

FIG. 8. Site-directed mutagenesis of the AP-1 site in the pro-moterforBSLF2+BMLF1. ReporterconstructsS-CAT, 257-CAT, AE4-CAT, and AE4K-CAT were electroporated alone or with CMV-BZLF1orCMVLTR-BRLF1 intoBL41 cells. After 2 days, extractsof the cellswereassayed for CATactivity.A totalof three experiments were performedwithBZLF1, butonly one was per-formed withBRLF1. The percentconversion ofchloramphenicolto acetyl chloramphenicol was determined, and to make the three experimentscomparableandgiveequal weighttoeachexperiment, the resultswerenormalizedonthe 257CATplusBZLF1figure. The true percentconversion figures for 257CAT plus BZLF1 were as follows: experiment 1, 2.02; experiment 2, 4.8; and experiment 3, 5.90. The normalizedfigureswereaveraged,and these averagesare plotted inthehistogramatthe top.

We identified binding sites for BZLF1 upstream ofthe promotersfor BRLF1, BZLF1, and BSLF2+BMLF1. Four of the five sitescontain close matches tothe AP-1 consen-sus, but the fifth site(inthe BZLF1promoter) doesnot. This site has also beenmappedaccurately by E.Flemingtonand S. H. Speck (submitted for publication). The variation in sites suggestsoneof threepossibilities:

(i)

twoDNA-binding

domains are present in BZLF1, (ii) the same part of the BZLF1proteincanrecognizetwoapparentlyquitedifferent DNA sequences, or (iii) the truebinding site for BZLF1 is common to all the targets and is overlapping a consensus AP-1site infour of the five targetsmapped. Alignmentofall five sites shows a possible consensus that matches all the VOL.64, 1990

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2116 PACKHAM ET AL.

targets, but ourfootprintingisnotyetsufficiently precise to determine the important nucleotides for binding. This will

require mutagenesis of individual nucleotides in the target

regions. We cannot yetexclude the possibilitythat BZLF1

contains two differentDNA-binding domains, but this can nowbe tested byusing our deletionmutantsand synthetic

DNAscontainingan AP-1-likeBZLF1 target orthe BZLF1 promoter target thatlacks the consensus AP-1 site. So far

our experiments on mapping the part of the protein that

binds DNA have all used one ofthe AP-1-like targets (the

oneupstreamof BSLF2+BMLF1).

In the EBV-negative Burkitt's lymphomacell line BL41, bothBZLF1andBRLF1areabletotransactivate the BSLF2

+BMLF1 promoter but it is clear that the activation by

BRLF1 isquantitively muchgreater. In Blymphocytelines

carrying EBV, the major routeby which BZLF1 activates

BSLF2+BMLF1 is, therefore, presumed to be through

BZLF1first activating BRLF1,whichthenactivates BSLF2

+BMLF1. Nevertheless, thereisamodest effect ofBZLF1 on thepromoterfor BSLF2+BMLF1 andwe have

investi-gatedthis. Oneinteresting explanation forourresults is that

atleastpartof the effect ofBZLF1onthispromoterinvolves counteracting acellularrepressorof thepromoterfunction.

That repressor seems to require sequences overlappingthe consensus AP-1 site in the promoter

region. Although

we have not studied this repression function directly, obvious candidatestomediate suchrepression wouldbe membersof

the cellular AP-1 family of transcription factors. There are well-established precedents for these factors acting as re-pressors, aswell as in their more usualactivating function, for example, in the promoters ofc-fos (19) and c-myc (9).

The effectiveness of such repression would presumably be

influencedby endogenous levels ofthe appropriatecellular AP-1factors and the mode of presentation andquantity of

DNA in transfection assays. These variables may account

for differences between cell lines and another group not

observing

thiseffect ofBZLF1onthe promoterforBMLF2 +MLF1 (10).

At present, we do nothave the methods to testwhether

this apparent ability of BZLF1 to counteract a cellular

repressoris importantin the EBV genomeduring anormal infection. The EBVproductivecycleispresumablyinduced

by cellular changeswhicharereflectedin alteredpatternsof cellulartranscription factors.Theasyetunidentified factors

that switch on BZLF1 transcription are presumably the

primary trigger, butautoactivation by BZLF1 andaloss of repressionby cellular factors that recognize sequences

con-tainingtheconsensusAP-1sitesmayassist theswitchof the

virus from being latent to fully productive. Perhaps the

high-affinitynon-AP-1-likesite upstreamof BZLF1, which is

involved in autoactivation of BZLF1 in B lymphocytes

(Fleming and Speck, submitted) permits accumulation of

sufficient BZLF1 to overcome repressing cellular factors fromother AP-1-like targets and thenswitchonthe produc-tivecycle.

LITERATURE CITED

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ge-nome.Nature(London)310:207-211.

2. Biggin,M., M. Bodescot, M. Perricaudet, and P.Farrell. 1987. Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.J.Virol.61:3120-3132.

3. Chevallier-Greco, A., E. Manet, P. Chavrier, C. Mosnier, J.

Daillie,and A.Sergeant.1986. BothEpstein-Barrvirus

(EBV)-encoded transacting factors, EB1 and EB2, are required to activatetranscriptionfromanEBVearlypromoter. EMBO J. 5:3243-3250.

4. Countryman, J., H.Jenson, R. Seibi,H.Wolf,and G. Miller. 1987.Polymorphicproteinsencoded within BZLF1 of defective and standard Epstein-Barr viruses disrupt latency. J. Virol. 61:3672-3679.

5. Countryman,J., and G. Miller. 1985. Activation ofexpression

of latent Epstein-Barr herpes virus aftergene transfer with a smallclones subfragment ofheterogeneous viral DNA. Proc. Natl.Acad. Sci. USA 82:4085-4089.

6. Farrell,P.J. 1989. TheEpstein-Barrvirusgenome.Adv. Viral Oncol. 8:103-132.

7. FarreUl,P. J., D. T.Rowe, C.M. Rooney, and T.Kouzarides. 1989. Epstein-Barr virus BZLF1 trans-activator specifically

bindsto a consensusAP-1site and is relatedtoc-fos. EMBO J. 8:127-133.

8. Hardwick, J. M.,P. M.Lieberman,and S. D.Hayward.1988. A newEpstein-Barrvirustransactivator, R,inducesexpressionof acytoplasmic early antigen. J. Virol. 62:2274-2284.

9. Hay, N.,M.Takimoto,andJ.M.Bishop.1989.AFOSprotein

is present inacomplexthat bindsanegative regulatorof MYC. GenesDev. 3:293-303.

10. Kenney,S.,E.Holley-Guthrie,E.-C.Mar,and M. Smith.1989. TheEpstein-BarrvirusBMLF1promotercontainsanenhancer elementthat isresponsivetothe BZLF1 and BRLF1

transacti-vators.J. Virol. 63:3878-3883.

11. Kenney, S., J. Kamine, E. Holley-Gutherie, J.-G. Lin, E.-C. Mar, and J. Pagano. 1989. The Epstein-Barr virus (EBV)

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12. Kenney,S.,J.Kamine,E.HoUley-Guthrie,E. C.Mar,J.C.Lin, D. Markovitz, and J. Pagano. 1989. The Epstein-Barr virus immediate-early gene product, BMLF1, acts in trans by a

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13. Knutson, J. C., and B. Sugden. 1989. Immortalization of B lymphocytes by Epstein-Barr virus: what does the virus con-tributetothecell? Adv. Viral Oncol. 8:151-172.

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without phenotypic selection. Proc. Natl. Acad. Sci. USA 82:488-492.

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17. Oguro,M.O.,N.Shimizu,Y.Ono,and K. Takada.1987.Both therightwardand theleftwardopenreadingframeswithin the BamHI M DNAfragmentofEpstein-Barr virus act as trans-activators ofgeneexpression. J. Virol.61:3310-3313.

18. Rooney,C.M.,D. T.Rowe,T. Ragot,and P.J. Farrell.1989. ThesplicedBZLF1 geneofEpstein-Barrvirus(EBV) transac-tivatesanearlyEBVpromoterand inducesthe virusproductive cycle.J.Virol.63:3109-3116.

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