An ATF/CRE Element Mediates both EBNA2Dependent and EBNA2Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Feb. 1998, p. 1365–1376 Vol. 72, No. 2

Copyright © 1998, American Society for Microbiology

An ATF/CRE Element Mediates both EBNA2-Dependent andEBNA2-Independent Activation of the Epstein-Barr

Virus LMP1 Gene PromoterANNA SJOBLOM,* WEIWEN YANG, LARS PALMQVIST, ANN JANSSON, AND LARS RYMO

Department of Clinical Chemistry and Transfusion Medicine, Goteborg University,Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden

Received 14 July 1997/Accepted 29 October 1997

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a viral oncogene whose expression isregulated by both viral and cellular factors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LMP1expression in human B cells, and several EBNA2 response elements have been identified in the promoterregulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMPresponse element (ATF/CRE) site in LRS is involved in EBNA2 responsiveness. We now establish the impor-tance of the ATF/CRE element by mutational analysis and show that both EBNA2-dependent activation andEBNA2-independent activation of the promoter occur via this site but are mediated by separate sets of factors.An electrophoretic mobility shift assay (EMSA) with specific antibodies showed that the ATF-1, CREB-1,ATF-2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun heterodimers whereas the Sp1 andSp3 factors bind to an adjacent Sp site. Overexpression of ATF-1 and CREB-1 in the cells by expression vectorsdemonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 pro-moter in an EBNA2-independent manner. The homodimers of ATF-2 and c-Jun did not significantly stimulatepromoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absenceof EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protein.Evidence for a direct interaction between the ATF-2/c-Jun heterodimeric complex and EBNA2 was obtained byEMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactiva-tion via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer.EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimericcomplex between the ATF-1 and CREB-1 factors.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvi-rus, consistently detected in several human malignancies, in-cluding endemic Burkitt’s lymphoma (BL), nasopharyngealcarcinoma (NPC), and posttransplantation lymphoma (50). Invitro infection of B lymphocytes by EBV, as well as explantculture of lymphocytes from seropositive adults, gives rise toimmortalized cell lines with the limited gene expression of sixnuclear proteins (EBNA1 to EBNA6) and three membraneproteins (LMP1, LMP2A, and LMP2B), as well as two smallnuclear RNAs (EBER1 and EBER2) (36). Mutagenesis of theviral genome has defined a subset of six genes required for(EBNA1 to EBNA3, EBNA6, and LMP1) or contributing to(EBNA5) B-cell immortalization (12, 30, 35, 45, 58, 70).

The EBNA2 protein transactivates the LMP1 gene but alsotransactivates other viral and cellular genes (1, 13, 19, 24, 38,57, 63–67, 71). However, since EBNA2 seems to lack se-quence-specific DNA-binding ability, the participation of cel-lular proteins is necessary for the recognition of specific pro-moters. Some of these proteins have been identified, includingC promoter binding factor 1 (CBF1), also designated Jk re-combination signal-binding protein (RBP-Jk) (26, 31, 43, 62,72), the Ets-related PU.1 factor (33), and a POU domainprotein (55). It has been suggested that EBNA2, when boundby cellular proteins, associates with specific regulatory sites inviral or cellular genomes and activates transcription by recruit-

ing basal transcription factors to nearby promoters. This iscorroborated by the recent demonstration of a direct interac-tion between EBNA2 and components of the RNA polymeraseII transcription initiation complex (59–61, 68).

Several lines of evidence indicate that the transforming ef-fect of LMP1 is explained to a large extent by its functionalsimilarity to an activated form of tumor necrosis factor familyreceptor (TNFR). This notion is based on the facts that LMP1has an intrinsic ability to aggregate in the plasma membraneand to associate with TNFR-associated factors (17, 42, 44, 48).The expression of the LMP1 gene in B cells is primarily due toactivation of a promoter designated EDL1 in the EBV genome(22, 23). In the present study, we have focused on the promot-er-proximal part of the LMP1 regulatory sequence (LRS)which contains a potential Sp site at position 233 and anactivating transcription factor/cyclic AMP (cAMP) responseelement (ATF/CRE) site at position 241 relative to the tran-scription initiation site (see Fig. 1). The Sp factor-bindingelement is one of the most widely distributed promoter ele-ments in cellular and viral genes. To date, four different Spproteins, designated Sp1, Sp2, Sp3, and Sp4, have been iden-tified. The members of this transcription factor family havestructural features in common, including zinc fingers and glu-tamine- and serine/threonine-rich amino acid stretches (27,37). A previous study showed that the ATF/CRE motif in LRSis likely to play a role as a mediator of the EBNA2 effect andpromoter activation by cAMP analogs (21). The ATF/CREsequence motif belongs to one of the major classes of regula-tory elements that participates in transcriptional regulationinduced by extracellular signals. Several proteins, including

* Corresponding author. Mailing address: Department of ClinicalChemistry and Transfusion Medicine, Sahlgrenska University Hospi-tal, S-413 45 Gothenburg, Sweden. Phone: 46-31-603054. Fax: 46-31-828458. E-mail: [email protected].

1365

on Novem

ber 27, 2014 by guesthttp://jvi.asm

.org/D

ownloaded from

http://jvi.asm.org/

ATF-1, ATF-2, ATF-3, ATF-a, CREB-1, CREB-2, and theCREM proteins, bind as homo- or heterodimers to this se-quence (15). This family of regulatory factors has been impli-cated in cAMP-, calcium-, and virus-induced modulation oftranscription (15, 25, 54). The AP-1 binding site (TRE), whichconfers responsiveness to tetradecanoyl phorbol acetate (2)differs by only 1 nucleotide from the ATF/CRE site. Thiselement is recognized by a group of proteins, including thoseencoded by the c-fos and c-jun gene families, that form homo-and heterodimers with each other (29). The members of theAP-1 and ATF/CREB factor families bind preferentially totheir respective sequence, but due to selective formation ofinterfamily heterodimers, new binding specificities arise. Forexample, c-Jun binds to an ATF/CRE site with considerablyhigher affinity as a heterodimer with ATF-2 than as a c-Junhomodimer (29).

The objective of the present study was to define the role ofthe Sp and ATF/CRE sites in EBNA2 responsiveness of theLMP1 promoter and to characterize the factors involved. Mu-tational analysis showed that both elements were required foran efficient response of the promoter. Electrophoretic mobilityshift assay (EMSA) and antibody supershift analysis demon-strated that Sp1 and Sp3 bound to the Sp site and that twodistinct heterodimeric complexes, ATF-1/CREB-1 and c-Jun/ATF-2, interacted with the ATF/CRE site. The results indicatethat the EBNA2 transactivation of the promoter is dependenton interaction with the c-Jun/ATF-2 heterodimer whereas thepreviously shown stimulatory effect of cAMP on LMP1 expres-sion probably is mediated by the ATF-1/CREB-1 heterodimer.

MATERIALS AND METHODS

Plasmids. All constructs made were verified by dideoxy sequencing utilizingthe Sequenase system (United States Biochemical Corp., Cleveland, Ohio). ThepSV2gpt, pEDA6, pIBI31(BYRF), pgCAT, pgLRS(254)CAT, pgLRS(2106)CAT, and pgLRS(2634)CAT constructs have been described previously (20, 52,55). The LRS is defined as nucleotides 169477 to 170151 of B95-8 EBV DNA,which corresponds to positions 2634 to 140 relative to the transcription initia-tion site.

To make a series of Sp and ATF/CRE mutated reporter plasmids, PCRamplifications were performed with the pgLRS(2152)CAT plasmid (55) as atemplate and primers that resulted in fragments with one end corresponding toposition 140 in LRS and the other end corresponding to position 258, withmutations in the Sp site (G to T in position 233 and 232) or the ATF/CRE site(C to A in position 240 and G to T in position 241). The PCR fragments werecloned into the TA cloning vector (Invitrogen, NV Leek, The Netherlands).Taking advantage of a synthetic HindIII site in one primer and a PstI site in theTA cloning vector, the PCR fragments were then cloned between the HindIIIand PstI sites in the pgCAT plasmid. To generate the pgLRS(2106)CAT plasmidwith the Sp or ATF/CRE site mutated, the pgLRS(258)CAT constructs weredigested with HindIII and MluI and the HindIII-MluI LRS fragments that con-tained the 254/140 part of LRS were isolated. The wild-type pgLRS(2106)CATwas cleaved with the same enzymes, and the MluI-HindIII fragment correspond-ing to positions 2106 to 255 was isolated and ligated with the mutated 254/140LRS fragments and HindIII-cleaved pgCAT, generating pgLRS(2106)(Spmut)CAT and pgLRS(2106)(ATF/CREmut)CAT. The mutated pgLRS(2634)CATconstructs were generated by ligating the mutation-containing HindIII-MluI frag-ments described above into the HindIII-MluI-digested pgLRS(2634)CAT vec-tor, resulting in pgLRS(2634)(Spmut)CAT and pgLRS(2634)(ATF/CREmut)CAT. The pgLRS(2106)DCAT vector used for in vitro transcription of the probein RNase protection experiments was made by cloning the PvuII-SalI LRSfragment of pgLRS(2106)CAT in the SmaI-SalI sites of the Gemini 3Zf(1)vector (Promega Corp., Madison, Wis.). The pCMV-Sp1 plasmid was a gift fromG. Suske (Klinikum der Philipps-Universitat Marburg, Marburg, Germany). ThecDNA for human ATF-2 was kindly provided by C. Svensson-Akusjarvi (UppsalaUniversity, Uppsala, Sweden). The ATF-2-encoding cDNA was amplified byPCR and cloned into the TA cloning vector. The ATF-2 gene-containing frag-ment was excised with BstXI and ligated into the pcDNAI/Amp plasmid (In-vitrogen), resulting in the pc(ATF-2) expression vector. An expression vector forhuman ATF-1, designated pc(ATF-1), was made by cloning the human cDNAfor ATF-1 obtained by XbaI cleavage of the pET-15b(ATF-1) plasmid, a giftfrom R. H. Goodman (Oregon Health Science University, Portland, Oreg.), inpc(ATF-1). An expression vector for rat CREB-1, designated pc(CREB-1), wasgenerated by cloning the CREB341 cDNA-containing BamHI-XbaI fragment ofpET-15b(CREB341), also provided by R. H. Goodman, in pcDNAI/Amp. To

generate an expression vector for human c-Jun, cDNA was isolated from thepCMV c-jun vector, kindly supplied by R. Tjian (University of California Berke-ley, Berkeley, Calif.) by cleavage with BamHI and PvuII and was cloned intoBamHI-EcoRV-digested pcDNAI/Amp. The resulting plasmid was designatedpc(c-jun).

The EBNA2 expression vector pc(BYRF) was constructed as follows. First,part of the BYRF open reading frame was subjected to PCR amplification witha sense oligonucleotide with two new restriction enzyme sites (XhoI-NdeI) andthe BYRF translation start sequence and an antisense primer corresponding tothe sequence around the BamHI Y/H cleavage site. Then the fragment wasexcised and subcloned between the XhoI and BamHI sites in pEDA8 (52),re-creating the complete BYRF sequence with the addition of a NdeI site closeto the translation initiation codon. The EBNA2-encoding NdeI-BglII fragment ofthis plasmid was cloned in the XbaI site of the pCI vector (Promega) with XbaIlinkers. Finally, the EBNA2-encoding EcoRI-SalI fragment of this plasmid wascloned between the EcoRI-XhoI sites in the pcDNAI/Amp plasmid, creating thepc(BYRF) vector. The pE300CY6 vector, which allows the expression of atruncated version of rat CD2, was most kindly provided by E. Lundgren (Uni-versity of Umeå, Umeå, Sweden), and the E1A 13S expression vector wasprovided by C. Svensson-Akusjarvi.

Cell culture, DNA transfections, and CAT assays. DG75 is an EBV genome-negative BL cell line (6). The lymphoid cells were maintained as suspensioncultures in RPMI 1640 medium (Life Technologies AB, Taby, Sweden) supple-mented with 10% fetal calf serum (Life Technologies AB), penicillin, and strep-tomycin. Transfections were generally performed with 5 3 106 DG75 cells, 6.0 to10 mg of DNA of the reporter construct to be tested, and 1.4 pmol of DNA of theEBNA2 expression vector pEDA6 or the pSV2gpt control plasmid by electro-poration at 260 V and 960 mF in 250 ml of cell culture medium with the Bio-RadGene Pulser and 4-mm-gap cuvettes (Bio-Rad, Hercules, Calif.). The cells wereharvested after 72 h, and aliquots of the cell lysates were assayed for chloram-phenicol acetyltransferase (CAT) activity (51). For Sp1 transfections (see Fig. 6),0.95 pmol of the pCMV-Sp1 vector or the pCMV control vector, 0.68 pmol of theEBNA2 expression vector pEDA6 or the pSV2gpt control, and 6.0 mg of thereporter plasmid were used. In the ATF-1 and CREB-1 transfections (see Fig.7A), 3.6 pmol of either pc(ATF-1) or pc(CREB-1) or, alternatively, 1.8 pmol ofeach of the vectors or 3.6 pmol of the pcDNAI/Amp (from now on designatedpc) control vector were used together with 0.68 pmol of EBNA2 expressionvector pEDA6 or 0.68 pmol of the pSV2gpt control vector and 5.0 mg of therespective reporter construct. In the ATF-2 and c-Jun transfections (see Fig. 7B),3.6 pmol of pc(ATF-2), or 0.20 pmol of pc(c-jun) and 3.4 pmol of pc, or 1.8 pmolof pc(ATF-2) and 0.10 pmol of pc(c-jun) and 1.7 pmol of pc, or 3.6 pmol of pcwas used together with 0.68 pmol of EBNA2 expression vector pc(BYRF) or 0.68pmol of the pc control vector and 5.0 mg of the respective reporter construct. Forthe study of the effect of EBNA2 on phosphorylation (see Fig. 8), 1.4 pmol ofpEDA6, pSV2gpt, and the E1A 13S expression vector, respectively, was cotrans-fected with 10 mg of the pE300CY6 plasmid into 107 DG75 cells. In the immu-noprecipitation experiments (see Fig. 10) 1.8 pmol of pc(ATF-2) and 0.1 pmol ofpc(c-jun) were cotransfected with 10 mg of the pE300CY6 plasmid into 107 DG75cells together with either 0.68 pmol of the EBNA2 expression vector pc(BYRF)or 0.68 pmol of the pc control vector.

RNase protection assay. Cytoplasmic RNA was prepared and analyzed by theRNase protection assay as described previously (51). 32P-labelled RNA wassynthesized by in vitro transcription of pgLRS(2106)DCAT with [a-32P]UTP(3000 Ci/mmol; Du Medical Scandinavia AB, Sollentuna, Sweden) and T7 RNApolymerase by following a standard procedure. Hybridization was performed at50°C.

EMSA. Nuclear extracts were prepared as described previously (18), exceptthat antipain (5.0 mg/ml), leupeptin (5.0 mg/ml), and aprotinin (2.0 mg/ml) wereadded to the buffer in the final homogenization and dialysis steps and phenyl-methylsulfonyl fluoride was substituted by Pefabloc (0.50 mM). Aliquots werefrozen in liquid nitrogen and stored at 270°C. The following double-strandedsynthetic oligonucleotides were used in the mobility shift assays: an oligonucle-otide corresponding to the 250 to 219 part of LRS; a similar oligonucleotide inwhich the Sp site was mutated by the introduction of G-to-T mutations betweennucleotides 233 and 232; and an oligonucleotide corresponding to an AP-1consensus sequence (59-CGCTTGATGACTCAGCCGGAA-39). The blunt-ended oligonucleotides were labelled with [g-32P]ATP (6,000 Ci/mmol, Du Med-ical Scandinavia AB) by using polynucleotide kinase (Boehringer MannheimScandinavia AB, Bromma, Sweden). The labelled probes were purified by elec-trophoresis in a 5% polyacrylamide gel (acrylamide/bisacrylamide, 30:1) in 0.53TBE (50 mM Tris, 50 mM boric acid, 1.0 mM EDTA [pH 8.3]). The wet gel wasautoradiographed, and the DNA fragments were excised, electroeluted by iso-tachophoresis (48a), and precipitated. Binding-reaction mixtures (in 25 ml) withcrude nuclear extract contained 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1.0 mMdithiothreitol, 1.0 mM EDTA, 5% glycerol, various amounts (0.30 to 7.0 mg) ofpoly(dA-dT), 6.0 fmol of [32P]DNA (approximately 70,000 cpm) and variousamounts (1.0 to 20 mg) of nuclear proteins (always added last). Binding-reactionmixtures (in 25 ml) with in vitro-translated proteins contained 10 mM HEPES(pH 7.9), 50 mM KCl, 6.0 mM MgCl2, 2.5 mM dithiothreitol, 100 mg of bovineserum albumin (BSA) per ml, 0.01% Nonidet P-40, 10% glycerol, 6.0 fmol of[32P]DNA (approximately 70,000 cpm), and 5.0 ml of programmed rabbit reticu-locyte lysate. In the competition experiment, a 200- or 300-fold excess of com-

1366 SJOBLOM ET AL. J. VIROL.

on Novem


.org/D

ownloaded from

http://jvi.asm.org/

peting oligonucleotide was added before the 32P-labelled probe. After incubationat room temperature for 25 min, the samples were electrophoresed on 5%polyacrylamide gels (acrylamide/bisacrylamide, 30:1) in 0.53 TBE. The unla-belled competitors used were as follows: probe oligonucleotide corresponding toLRS 250/219, 59-GAGGCTTATGTAGGGCGGCTACGTCAGAGTAA-39;nonspecific oligonucleotide, 59-ATGTTCGGTAACATCTCTCATTGCGCACAAAGAACCCTACATCCG-39; ATF/CRE consensus oligonucleotide, 59-AAGATTGCCTGACGTCAGAGAGCTAG-39; Sp consensus oligonucleotide, 59-ATTCGATCGGGGCGGGGCGAGC-39; The HindIII-MluI fragment of pgLRS(2106)(Spmut)CAT corresponding to LRS 254 to 140 with a mutated Sp site(two G nucleotides at positions 232 and 233 were replaced by two T nucle-otides); the corresponding HindIII-MluI fragment of pgLRS(2106)(ATF/CREmut)CAT with a mutated ATF/CRE site (CG at positions 240 and 241were replaced by AT); the corresponding HindIII-MluI fragment of pgLRS(2106)(Sp1ATF/CREmut)CAT with mutated Sp and ATF/CRE sites (C, T, G,and A at positions 237, 238, 241, 244, were changed to T, A, A, and T).

The antibodies against the transcription factors Sp1 (sc-59X), Sp3 (sc-644X),CREB-1 (sc-271X), CREB-2 (sc-200X), ATF-1 (sc-243X), ATF-2 (sc-187X),c-Jun (sc-822X), c-Jun/Jun B/Jun D (sc-44X), Jun B (sc-46X), and Jun D (sc-74X) (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used. The su-pershift analyses were performed as described above for the EMSA experimentsexcept that 3.0 to 8.0 ml of the respective antibody was added after the incubationat room temperature. The mixture was incubated at 4°C for 60 min and thensubjected to polyacrylamide gel electrophoresis (PAGE) (5.0% polyacrylamide)followed by autoradiography.

For in vitro expression, a fragment of pEDA6 containing the EBNA2-encodingopen reading frame, BYRF1, was subcloned into the pIBI 31 vector (IBI, NewHaven, Conn.). The cDNAs for ATF-2 and c-Jun were translated in vitro withthe pc(ATF-2) and pc(c-jun) constructs. The supercoiled DNA templates weresequentially transcribed and translated in the same reaction mixture containingrabbit reticulocyte lysate (Promega Corp.), amino acids, and the T7 RNA poly-merase, as recommended by the manufacturer. Translated proteins were ana-lyzed by sodium dodecyl sulfate (SDS)-PAGE.

CD2 selection. To introduce a marker for cell sorting, the CD2 expressionvector pE300CY6 was cotransfected with the EBNA2 expression vector(pEDA6), the E1A 13S expression vector, and the pSV2gpt control plasmid. Thetransfected cells were collected by centrifugation after 48 h and washed. Sortingfor CD2 expressing cells was performed with a mouse CD2-specific antibody(MCA154; Serotec, Oxford, United Kingdom) and magnetic beads linked to ratanti-mouse antibodies (Dynabeads M450; Dynal Ltd., Merseyside, United King-dom) as described by Pilon et al. (49). The cells were lysed in lysis buffer (20 mMimidazole-HCl [pH 6.8], 100 mM KCl, 1.0 mM MgCl2, 10 mM EGTA, 0.20%[vol/vol] Triton X-100, 10 mM NaF, 1.0 mM sodium vanadate, 1.0 mM sodiummolybdate, 5.0 mg of leupeptin per ml, 2.0 mg of aprotinin per ml), incubated for15 min at 4°C, and cleared by centrifugation. The protein concentration in thelysates was determined (Bradford protein assay; Bio-Rad), and aliquots weremixed with sample buffer containing 65 mM Tris-HCl (pH 6.8), 2.0% SDS, 10%glycerol, 5.0% b-mercaptoethanol, and 0.10% bromphenol blue (BPB). A 50-mgportion of protein from the respective sample was boiled and subjected toSDS-PAGE (10% polyacrylamide). Total c-Jun, c-Jun phosphorylated at Ser63or Ser73, total ATF-2, and ATF-2 phosphorylated at Thr71 were detected withthe PhosphoPlus antibody kits (New England Biolabs, Inc., Beverly, Mass.). Itshould be noted that the anti-c-Jun(Ser73) antibody also recognizes JunD phos-phorylated at Ser100. The negative control consisted of total-cell extract fromNIH 3T3 cells, and the positive controls were extracts of UV-treated (c-Jun) oranisomycin-treated (ATF-2) NIH 3T3 cells.

Immunoprecipitation and immunoblot analysis. DG75 cells were harvested48 h after transfection and subjected to CD2 selection as described above. B95-8cells and the selected DG75 cells were lysed as described above, sonicated, andcleared by centrifugation. Aliquots corresponding to 0.9 3 106 DG75 cells and1.8 3 106 B95-8 cells were immunoprecipitated with 30 mg of anti-ATF-2 (sc-187X; Santa Cruz Biotechnology, Inc.) per ml and 30 mg of anti-c-Jun (sc-822X;Santa Cruz Biotechnology, Inc.) per ml, respectively, in a total volume of 300 mland incubated at 4°C overnight. Samples without antibody were used as negativecontrols. Aliquots (50 ml) of 50% protein A/G agarose (Santa Cruz Biotechnol-ogy, Inc.) in lysis buffer containing 10 mg of BSA per ml were added, the samples

were rocked at 4°C for 2 h, and the protein A/G agarose beads were collected bycentrifugation. After the beads had been washed five times in lysis buffer plusBSA, the proteins were eluted by boiling in 40 ml of sample buffer, analyzed bySDS-PAGE (10% polyacrylamide), and blotted to Hybond C-extra nitrocellulosemembranes (Amersham Life Science, Little Chalfont, United Kingdom). Themembranes were incubated with rabbit anti-c-Jun antibodies (sc-822X), mouseanti-ATF-2 antibodies (sc-187X), or a human serum containing anti-EBNA2antibodies in phosphate-buffered saline (PBS; 180 mM NaCl, 3.6 mM KCl, 11mM Na2HPO4, 2.0 mM KH2PO4) containing 0.5% nonfat dry milk and, afterrepeated washings in PBS, incubated with horseradish peroxidase-conjugateddonkey anti-rabbit (Amersham Life Science), sheep anti-mouse (Amersham LifeScience), or goat anti-human (Bio-Rad) antibodies. The membrane was washedin PBS containing 0.3% Tween 20, and the proteins were visualized by enhancedchemiluminescence procedures as described by the manufacturer of the reagents(Amersham Life Science).

RESULTS

EBNA2-induced transactivation of the LMP1 promoter de-pends on intact Sp and ATF/CRE motifs in LRS. In previousstudies, we have demonstrated that the 2106 to 140 part ofLRS contains one or several elements that mediate EBNA2-induced upregulation of promoter activity (19, 21, 55). Twoshort subsequences in this region were defined with homologyto an Sp and an ATF/CRE site, respectively (Fig. 1) (21).Mutation analysis provided evidence for a role of the ATF/

FIG. 1. Schematic presentation of the LRS in the B95-8 EBV genome. Thescale refers to the position relative to the transcription initiation site from theEDL1 promoter. Transcription factor-binding sites previously identified as in-volved in regulation of the LMP1 promoter are indicated by open boxes. The Spand the ATF/CRE sites are defined in the present investigation.

FIG. 2. EBNA2-induced transactivation of LRS depends on intact Sp andATF/CRE motifs in LRS. Mutations were introduced in the pgLRS(2106)CATand pgLRS(2634)CAT plasmids, respectively, as indicated in Materials andMethods. The reporter plasmids were cotransfected with pEDA6 (1EBNA2) orwith an equivalent amount of pSV2gpt (2EBNA2) in the EBV-negative B-cellline DG75. The CAT activity is given as relative chloramphenicol acetylationexpressed as a percentage of the activity obtained with pgLRS(2634)CAT in thepresence of EBNA2. The 100% value corresponded to acetylation of 97% of thesubstrate in the assay. The values are the mean of three independent transfec-tions. The standard errors are indicated by error bars.

VOL. 72, 1998 REGULATION OF THE LMP1 PROMOTER VIA AN ATF/CRE SITE 1367

on Novem


.org/D

ownloaded from

http://jvi.asm.org/

CRE site in promoter transactivation (21). However, subse-quent binding studies revealed that this mutation preventedthe binding of factors to both the Sp site and the ATF/CREsite. To assess the relative contribution of the two sites topromoter activity and to further elucidate their role in theEBNA2-induced transactivation process, LRS-carrying re-porter plasmids were created with specific mutations of therespective sites. The pgLRS(2106)CAT plasmid contained the2106 to 140 part of LRS and was mutated as indicated inMaterials and Methods. To determine the effect of the muta-tions in the context of the complete LRS, mutated derivativesof the pgLRS(2634)CAT reporter plasmid were generated.The plasmids were cotransfected with an EBNA2 expressionvector or a control plasmid into the EBV-negative DG75 B-cellline. Mutation of the ATF/CRE site in pgLRS(2106)CATreduced the EBNA2-induced activity to 8.0% relative to thewild-type plasmid, i.e., close to the activity of the backgroundplasmid pgCAT (Fig. 2). Mutation of the Sp site left a lowresidual EBNA2-induced activity of 16% relative to the wild-type plasmid (Fig. 2). The corresponding mutations in the LRS(2634)CAT plasmids similarly resulted in a pronounced re-duction of activity (Fig. 2). Thus, the results clearly showedthat the two sites are important for the EBNA2-dependenttransactivation of the LMP1 promoter, especially in the con-text of the promoter-proximal part of LRS. The full-lengthLRS seemed to contain elements that to a certain extent couldcompensate for the loss of the ATF/CRE site at position 241.

To confirm that the observed transactivation of the reporterplasmids was due to correctly initiated transcripts from theEDL1 promoter, RNase protection analysis was performed.The result showed that transcription was initiated at the cor-rect LRS position in the constructs investigated (Fig. 3).

Identification of factors binding to the Sp and ATF/CREmotifs. We next characterized the regulatory factors in DG75cells that bound to the Sp and ATF/CRE elements by perform-

ing EMSAs with a double-stranded oligonucleotide corre-sponding to the 250 to 219 part of LRS. Competition exper-iments with unlabelled oligonucleotides containing intact Sp orATF/CRE consensus sequences or LRS fragments with muta-tions of the corresponding sites were carried out to correlatethe resulting EMSA bands with the binding sites. Five specificcomplexes were identified (Fig. 4, lanes 2 and 3). Bands thatwere not abolished by competition with unlabelled probe wereassumed to represent nonspecific complex formation. It mightbe noted that the same binding pattern was observed both withEBV-negative and EBV-positive B cells and with epithelialcells and T cells (data not shown). Competition with an LRSfragment that contained a mutated Sp site (lanes 6 and 7)removed three of the bands and left two bands. These com-plexes presumably represented binding to the Sp site. Compe-tition with an LRS fragment that contained a mutated ATF/CRE site (lanes 10 and 11) removed the two bands marked Spand left three bands. These were assumed to represent bindingto the ATF/CRE site. Furthermore, an LRS fragment mutated

FIG. 3. EBNA2-induced transcription initiates at the correct LMP1 pro-moter site in reporter plasmids. RNA was prepared from DG75 cells transfectedwith the EBNA2 expression vector pEDA6 or the pSV2gpt control vector and theindicated LRS CAT reporter plasmids and subjected to RNase protection anal-ysis with a 32P-labelled probe corresponding to positions 2106 to 140 of LRSand the first part of the CAT gene. Lanes: 1, probe only; 2, pgLRS(254)CATand pSV2gpt; 3, pgLRS(254)CAT and pEDA6; 4, pgLRS(2106)CAT andpSV2gpt; 5, pgLRS(2106)CAT and pEDA6; 6, pgLRS(2634)CAT andpSV2gpt; 7, pgLRS(2634)CAT and pEDA6; 8, pgCAT and pSV2gpt; 9, pgCATand pEDA6; 10, DNA size markers. The band corresponding to the commoninitiation site in the EDL1 promoter is indicated by the solid arrow. Bandscorresponding to nonspecific initiation upstream of LRS in the vector part of thereporter plasmids are indicated by dotted arrows. The lengths of these protectedfragments differ depending on the plasmid. The solid arrowhead indicates a bandpresent in all samples, probably due to incomplete RNase cleavage.

FIG. 4. Sp and ATF/CRE transcription factors in B-lymphoid cells bind toLRS. A 32P-labelled double-stranded synthetic oligonucleotide corresponding tothe 250 to 219 LRS region was incubated with nuclear extracts from DG75 cellsand subjected to EMSA. Lane 1 shows the binding pattern obtained with thenuclear extract. Competition reactions was carried out as indicated below theautoradiogram and described in Materials and Methods. In lanes 2 to 5, thebinding mixtures contain a 300-fold excess of unlabelled competitor over probe;in lanes 6, 8, and 10, they contain a 200-fold excess; and in lanes 7, 9, and 11, theycontain a 300-fold excess. Some of the competitors were mutated at the Spand/or the ATF/CRE sites as specified in Materials and Methods. Five com-plexes indicated by solid arrows are considered specific and designated Sp (bandsremaining after competition with an LRS fragment that contained a mutated Spsite) and ATF/CRE (bands remaining after competition with an LRS fragmentthat contained a mutated ATF/CRE site), respectively. Three nonspecific bandsthat were not abolished by competition with unlabelled probe are indicated bydotted arrows.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

in both the Sp and the ATF/CRE site did not compete with anyof the complexes (lanes 8 and 9). Competition with an oligo-nucleotide that contained an Sp consensus sequence removedthe two Sp bands (lane 5), and an oligonucleotide that con-tained an ATF/CRE consensus sequence removed the threeATF/CRE bands (lane 4). It was noted that although theEMSA band patterns were similar in qualitative terms, theintensity of the bands was weaker when DNA fragments (lanes6 to 11) were used as competitors compared with the bandsobtained with the corresponding synthetic oligonucleotides(lanes 3 to 5), even though similar molar amounts of compet-itor had been used. The reason is not clear, but we suggest thatthe effect was nonspecific and was related to the fact thatcompetitors of different lengths were used (the average lengthof the oligonucleotides was about 30 bp, and the length of theLRS fragments was about 90 bp).

To identify the members of the Sp and ATF/CREB tran-scription factor families that were involved in the formation ofa complex with the 250/219 LRS probe, we performed anti-body supershift analysis with different commercially availableantibodies (Fig. 5). To simplify the band pattern, the first seriesof EMSAs were carried out as competition experiments with a

300-fold molar excess of the unlabelled LRS probe containinga mutated Sp binding site, which allowed only Sp-related fac-tors to bind to the labelled probe. As illustrated in Fig. 5A, oneof the complexes supershifted with an anti-Sp1 antibody (lane2) and the other two supershifted with an anti-Sp3 antibody(lanes 3 and 4). One of the Sp3-containing complexes washidden behind the strong band corresponding to the Sp1 com-plex; therefore, the supershift became evident only when theanti-Sp1 and anti-Sp3 antibodies were added simultaneously.The anti-Sp3 antibody removed the two Sp3-containing EMSAcomplexes but did not give rise to supershifted bands in the gel,due to the inhibition of complex formation by the antibody.

The ATF/CREB antibody supershift analyses were carriedout with a labelled 250/219 LRS probe with a mutated Sp site,which allowed the formation of complexes only with the ATF/CRE site of the probe (Fig. 5B and C). Two of the threecomplexes were supershifted by both an anti-CREB-1 antibody(Fig. 5B, lane 2) and an anti-ATF-1 antibody (lane 4). Thethird ATF/CRE complex was removed by an anti-ATF-2 anti-body (Fig. 5C, lane 3) and an anti-c-Jun antibody (lane 5) andshifted by another anti-c-Jun antibody (lane 4).

FIG. 5. Identification of the transcription factors interacting with the Sp and ATF/CRE motifs in LRS. (A) Nuclear extract of DG75 cells was incubated underbinding conditions with a 32P-labelled double-stranded oligonucleotide corresponding to the 250 to 219 LRS region in the presence of a 300-fold molar excess of thecompetitor LRS 250/219 with a mutated Sp site. Antibody supershifts were carried out by incubation with a goat polyclonal antibody against Sp1, a rabbit polyclonalantibody against Sp3, and a mixture of the antibodies, as indicated below the autoradiogram. The reaction mixtures were analyzed by EMSA. Three specific complexesare indicated by solid arrows, one designated Sp1 and two designated Sp3. Two bands that were not abolished by competition are indicated by the dotted arrows. Theposition of the anti-Sp1 antibody-shifted complex is shown by the solid arrowhead. (B and C) EMSA and antibody supershift analysis were performed by incubatingnuclear extract of DG75 cells under binding conditions with a 32P-labelled double-stranded oligonucleotide corresponding to the LRS 250 to 219 region with a mutatedSp site and with antibodies as indicated below the autoradiogram. Two bands that were not abolished by competition are indicated by the dotted arrows. (B) Threespecific complexes are indicated by solid arrows, two of which are designated ATF-1, CREB-1 since they contain both factors. The positions of the antibody complexesare indicated by an open arrowhead for the anti-CREB-1 shift and solid arrowheads for the anti-ATF-1 shifts. (C) The third of the three specific complexes indicatedby solid arrows is identified as ATF-2, c-Jun. The positions of the immunologically shifted complexes are shown by the solid arrowheads for the anti-ATF-1 shifts andthe open arrowhead for the anti-c-Jun shift.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

Involvement of an Sp site in the regulation of the LMP1promoter. Our results showed that the Sp site at position 233in the promoter-proximal region of LRS had to be present toachieve EBNA2-induced transactivation of the LMP1 pro-moter and that both the Sp1 and Sp3 factors bound to this site.The ability of Sp1 to transactivate the promoter was assessedby transient transfections with Sp1 and EBNA2 expressionvectors and a pgLRS(2106)CAT reporter plasmid. In the ab-sence of EBNA2, the Sp1 vector induced a low level of trans-activation compared with the control plasmid (Fig. 6). Theactivation was, however, significantly higher than that obtainedwith a reporter plasmid in which the Sp site was mutated. TheSp1 expression vector did not add to the activity of the pgLRS(2106)CAT plasmid induced by EBNA2, although mutationof the Sp site largely abolished promoter activity. This suggeststhat Sp1 has an EBNA2-independent stimulatory effect onLMP1 promoter activity. Protein levels in the transfected cellswere checked by immunoblot analysis (data not shown). Theamount of Sp1 in the cells increased from a basal level aftertransfection with the expression vector.

ATF-1 and CREB-1 can activate the LMP1 promoter in anEBNA2-independent manner. We have previously reported onthe existence of a possible relationship between the EBNA2effect on LMP1 in B cells and the cAMP signal transductionpathway and provided evidence for the notion that the ATF/CRE site in the 2106/140 part of LRS is a possible target inthe EBNA2-induced activation of the promoter (21). TheEMSA results of the present study indicated that the CREB-1and ATF-1 factors are candidate mediators of the activating

effect. To investigate this question, ATF-1 and CREB-1 ex-pression vectors, separately or in combination, were cotrans-fected with the pgLRS(2106)CAT plasmid with or without anEBNA2 expression vector in DG75 cells. As illustrated in Fig.7A, overexpression of CREB-1 or ATF-1 activated the LRS(2106)-containing reporter plasmid independently of EBNA2.The activating effect was largely abolished when the ATF/CREsite was mutated. The residual ATF-1-mediated transactiva-tion of the mutated LRS reporter plasmid most probably didnot originate from the ATF/CRE site, since EMSA analysisshowed that the transcription factors no longer bound to themutated site (data not shown). Transfection with a mixture ofhalf the amount of each of the expression vectors resulted in alevel of activation intermediate between those obtained withthe ATF-1 and a CREB-1 expression vectors separately. Con-comitant expression of EBNA2 did not significantly increasethe activity of the LRS reporter plasmid. Protein levels in thetransfected cells were checked by immunoblot analysis (datanot shown). The ATF-1 and CREB-1 protein concentrationsincreased from a basal level, and the EBNA2 protein appearedwhen the respective expression vectors were introduced intothe cells. We suggest that the CREB-1 and ATF-1 factorsseparately and together are able to activate the LMP1 pro-moter via the ATF/CRE site at position 241 in LRS indepen-dently of each other and of EBNA2.

EBNA2 can transactivate the LMP1 promoter via the ATF/CRE site and a c-Jun/ATF-2 heterodimer. According to theEMSA results, the ATF-2 and c-Jun factors interact at theATF/CRE site at position 241 in the LRS. To investigate thepossible role of these factors in the EBNA2-dependent activa-tion of the EDL1 promoter, ATF-2 and c-Jun expression vec-tors were transfected together with an EBNA2 expression vec-tor and pgLRS(2106)CAT in DG75 cells. In the absence ofEBNA2, the homodimers of ATF-2 or c-Jun did not signifi-cantly increase the basal activity of the reporter plasmid (Fig.7B). The heterodimeric forms of the factors transactivated thepromoter about twofold. However, coexpression of ATF-2,c-Jun, and EBNA2 in the cells resulted in a strong and ATF/CRE site-dependent activation of the promoter. Immunoblot-ting control experiments showed that ATF-2 and c-Jun proteinlevels increased and that EBNA2 appeared in the cell extractsafter transfection (data not shown). It should be noted that acytomegalovirus promoter-driven EBNA2 expression vectorwas used in these experiments since the EBNA2 expression ofour standard pEDA6 plasmid was downregulated in the pres-ence of c-Jun. This new vector expressed EBNA2 less well,giving rise to seemingly lower EBNA2 inducibility of the re-porter constructs.

It has been established that phosphorylation of definedamino acid residues in c-Jun (Ser-63 and Ser-73) and ATF-2(Thr-69 and Thr-71) is required to generate efficient transac-tivational function of the factors. Therefore, the followingquestion arises: does EBNA2 modify the phosphorylation stateand/or the levels of these factors as a means of inducing acti-vation of the LMP1 promoter? This possibility was studied inEBNA2 cotransfection experiments with commercially avail-able antibodies with the ability to specifically identify phos-phorylated forms of the c-Jun and ATF-2 proteins (Fig. 8).Induction of phosphorylation and increased expression of c-Jun by the adenovirus E1A protein was used as an experimen-tal control. The results showed that EBNA2 did not signifi-cantly affect either the total level or the phosphorylation stateat the Ser-63/Ser-73 and Thr-71 residues, respectively, of c-Junor ATF-2. The DG75 cells apparently contained a significantendogenous level of the factors in phosphorylated form. Thus,the results support the notion that EBNA2 transactivation of

FIG. 6. Sp1 transactivates the LMP1 promoter independently of EBNA2.The pCMV-Sp1 expression vector or an equivalent amount of the empty pCMVcontrol plasmid was cotransfected with the EBNA2 expression vector pEDA6 orthe pSV2gpt plasmid with the pgLRS(2106)CAT reporter plasmid or the mu-tated derivative pgLRS(2106)(Spmut)CAT in DG75 cells. The CAT activity isexpressed as percent chloramphenicol acetylation, with the value obtained withthe pCMV plasmid together with pEDA6 and pgLRS(2106)CAT as 100%. Thestandard errors are indicated by error bars. The 100% value corresponded to22% conversion of substrate to product in the CAT assay. The values presentedare the mean of four independent transfections.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

the LMP1 promoter does not occur through the phosphoryla-tion of c-Jun or ATF-2.

To correlate the observed effects of overexpression of ATF-2and/or c-Jun on promoter activity with the binding of therespective factor at the ATF/CRE site and to decide whetherEBNA2 interacts with any of these factors, a series of EMSAswere performed with in vitro translation reaction mixturescontaining ATF-2, c-Jun, and EBNA2, respectively. Notably,the bands corresponding to the homomeric forms of in vitro-synthesized ATF-2 and c-Jun were not affected by the additionof EBNA2 (Fig. 9A, lane 5, Fig. 9B, lane 4). However, theheterodimeric complex formed by in vitro-translated ATF-2and c-Jun was removed by the addition of EBNA2 to theEMSA reaction mixture, showing that EBNA2 specifically in-teracts with the heterodimer but not with the homodimericforms of the two transcription factors (Fig. 9C, lane 6). Thereason why the in vitro-translated heterodimer of c-Jun/ATF-2migrated more slowly than the in vivo-synthesized heterodimerin the nuclear extracts is not clear. However, the results werealso corroborated by an EMSA experiment where addition ofin vitro-translated EBNA2 to the nuclear extract removed thec-Jun/ATF-2 complex from the EMSA pattern but left theATF-1/CREB-1 factors bound to the probe (data not shown).

The results of the EMSA analysis suggested that EBNA2 hasthe ability to interact with the c-Jun/ATF-2 heterodimer. Tocorroborate this observation, experiments aimed at identifyinga complex between EBNA2 and c-Jun and ATF-2 in cell ex-tracts by immunoprecipitation were performed (Fig. 10). Anti-

ATF-2 and Anti-c-Jun antibodies were used to precipitate theputative complex, the precipitates were analyzed by SDS-PAGE and immunoblotting, and the proteins were identifiedwith anti-ATF-2 (Fig. 10A), anti-c-Jun (Fig. 10B), and anti-EBNA2 (Fig. 10C) antibodies. The results showed that ATF-2and c-Jun were precipitated by the respective antibody andthat EBNA2 coprecipitated with both ATF-2 and c-Jun (Fig.10C, lanes 4, 6, 7, and 9). In the controls without the primaryantibody (lanes 10 to 12), none of the proteins were detected.The recombinant c-Jun protein had a somewhat lower molec-ular weight than endogenously expressed c-Jun, but the reasonfor this is not clear. It should be noted that the EBNA2/c-Jun/ATF-2 complex was identified not only in a situation of over-expression of the respective factors but also in nontransfected,EBV-infected cells (B95-8 cells). Together, the results of theEMSA and the immunoprecipitation experiments strongly sug-gest that EBNA2 can transactivate the LMP1 promoter via theATF/CRE motif and that a direct interaction between EBNA2and a heterodimeric complex of c-Jun and ATF-2 constitutesan essential step in the activation process.

DISCUSSION

We have shown in previous studies that EBNA2-inducedtransactivation of the LMP1 promoter in lymphoid cells de-pends to a significant extent on transcriptional cis-elements inthe 2106/140 part of LRS and that an ATF/CRE site in thisregion most probably plays a role as a mediator of the EBNA2

FIG. 7. The LMP1 promoter can be transactivated by CREB-1 and ATF-1 homo- and heterodimers independently of EBNA2 and by a c-Jun/ATF-2 heterodimerin an EBNA2-dependent manner. (A) The pc(ATF-1), and pc(CREB-1) expression vectors, separately or mixed, or the pc control plasmid was cotransfected with theEBNA2 expression vector pEDA6 or an equivalent amount of pSV2gpt and the reporter plasmids pgLRS(2106)CAT or pgLRS(2106)(ATF/CREmut)CAT into DG75cells, as detailed in Materials and Methods. The CAT activity is expressed as the percent chloramphenicol acetylation relative to the value obtained in transfectionswith the pc plasmid together with pEDA6 and pgLRS(2106)CAT. The 100% value corresponded to 21% conversion of substrate to product in the CAT assay. Thestandard errors are indicated with error bars. The values shown are the mean of three independent transfections. (B) The pc(ATF-2) and pc(c-Jun) expression vectors,separately or in combination, or the pc control plasmid was cotransfected with the EBNA2 expression vector pc(BYRF) or an equivalent amount of the pc plasmidand the reporter plasmids pgLRS(2106)CAT or pgLRS(2106)(ATF/CREmut)CAT into DG75 cells, as detailed in Materials and Methods. The CAT activity isexpressed as the percent chloramphenicol acetylation relative to the value obtained in transfections with the pc plasmid together with pc(BYRF) and pLRS(2106)CAT.The 100% value corresponded to 12% conversion of substrate to product in the CAT assay. The standard errors are indicated by error bars. The values shown are themean of three independent transfections.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

effect and in promoter activation by cAMP analogs (21, 55). Inthis study, using the EBV-negative DG75 cell line as a modelsystem for B cells, we demonstrated that the LMP1 promotercan be activated in an EBNA2-independent manner via a pro-cess that includes the binding of ATF-1 and CREB-1 homo- orheterodimers to the ATF/CRE site whereas EBNA2-depen-dent activation of the promoter, on the other hand, occursthrough a pathway that involves a direct interaction betweenEBNA2 and ATF-2–c-Jun heterodimers at the same site. Inaddition, the activity of the promoter is modulated in anEBNA2-independent way by the interaction of Sp1 and possi-bly Sp3 with an adjacent Sp element. We have previouslycompared the activity of the LRS in a variety of cell lines ofepithelial and B-cell origin, including DG75 (20). That studywas not as detailed as the present one, but an important con-clusion was that all group I BL cell lines investigated, bothEBV negative and EBV positive, followed the same patternwith regard to EBNA2-induced transactivation of LRS. Wetherefore consider the DG75 cell line to be representative ofits category of B cells and useful for investigations of theregulation of the LMP1 promoter. The reason why severalother investigations have failed to detect EBNA2 responsive-

ness in the proximal LMP1 promoter region is not clear to us.It has been suggested that a cryptic RBP-Jk site in our reporterconstructs is involved. A search for structural motifs in theproximal part of the LRS with reasonable identity to a RBP-Jksite revealed the presence of a TTGGGAT sequence at posi-tions 267 to 261. However, the results of EMSA competitionanalysis and site-directed mutagenesis strongly argued againstthe possibility that RBP-Jk or any other factor binds to thismotif (unpublished results). Furthermore, we have consistentlyobtained the same EBNA2-induced promoter response withthe 2106/140 LRS fragment inserted in an unrelated plasmidcarrying the luciferase reporter gene. The EBNA2 expressionvector used in our previous studies is a genomic construct thatwould hypothetically allow the synthesis of a mini-version ofEBNA5 or some other unidentified product and might in thisway be responsible for discrepancies between our observationsand those of others. To eliminate this possibility, we repeatedthe experiments with another expression vector in which thecytomegalovirus early promoter was placed immediately up-stream of a DNA fragment containing only the EBNA2-en-coding BYRF1 open reading frame from the B95-8 EBVstrain. This construct was considerably less efficient in EBNA2expression than was our standard EBNA2 expression vectorpEDA6 but produced the same result in qualitative terms withregard to EBNA2 responsiveness of the 2106/140 LRS region(data not shown). Furthermore, we have never detected pep-tide material reacting with anti-EBNA5 antibodies in EBV-negative cells (DG75 and COS-1 cells) transfected with thepEDA6 plasmid by immunoblot or immunofluorescence anal-ysis (data not shown).

EBNA2-independent activation of LRS via the Sp and theATF/CRE sites. Mutational analysis and EMSA binding stud-ies showed that an Sp element at position 233 in LRS isrequired for efficient EBNA2-dependent and EBNA2-inde-pendent transactivation of the LMP1 promoter and that theSp1 and Sp3 transcription factors bind to this site. Sp1 is a well-known transcriptional activator. The limited effect of overex-pression of Sp1 on promoter activity in the absence of EBNA2obtained in our transfection experiments might be explained bythe fact that DG75 cells contain a high endogenous level of theSp1 factor that diminishes the relative contribution of theexogenously added protein. The Sp3 transcription factor hasbeen shown to function as a repressor of Sp1-mediated tran-scriptional activation (28). Multiple Sp3-containing complexessimilar to those observed in our EMSA analyses have previ-ously been found in another system (16). We suggest that thestimulatory effect of Sp1 on the LMP1 promoter is indepen-dent of EBNA2 but is a prerequisite for EBNA2 inducedtransactivation. The interaction of the Sp1 and Sp3 factors withtheir binding site in LRS might constitute an EBNA2-indepen-dent regulatory system in which the balance between the pos-itively acting Sp1 and the negatively acting Sp3 factors is one ofthe factors that determines the final level of activity of theLMP1 promoter.

It is now generally believed that bZIP proteins like ATF-1and CREB-1 bind to DNA only as dimers and not as mono-mers. The results of the EMSA and antibody supershift exper-iments in this study suggested that the ATF-1 and CREB-1proteins in DG75 cells bind to the ATF/CRE site at position241 in LRS as a heterodimer, since the homomeric forms ofthe factors were not detected. The presence of two ATF-1/CREB-1 complexes with different mobilities in the electro-phoretograms is explained by the previous observation thatphosphorylation drastically changes the conformation of ATF-1 and, as a consequence, the electrophoretic mobility of thecorresponding EMSA complex (46). It should be noted, how-

FIG. 8. EBNA2 does not affect the level or phosphorylation state of c-Jun orATF-2 in DG75 cells. The EBNA2 expression vector pEDA6 or equivalentamounts of the pSV2gpt control plasmid or the E1A 13S expression vector weretransfected together with the CD2 expression vector pE300CY6 in DG75 cells.The transfected cells were selected for their CD2 expression with magneticbeads. The cells were lysed and equal amounts of protein extract were analyzedby SDS-PAGE and immunoblotting. The antibodies used in panel A were anti-c-Jun, anti-phospho-c-Jun(Ser63), and anti-phospho-c-Jun(Ser73), and thoseused in panel B were anti-ATF-2 and anti-phospho-ATF-2(Thr71). NIH 3T3 cellextracts containing nonphosphorylated or phosphorylated forms of c-Jun (panelA, lanes 1 and 2) and ATF-2 (panel B, lanes 1 and 2), respectively, were used ascontrols of antibody activity. The anti-c-Jun(Ser73) antibody also detected JunDphosphorylated at the Ser100 residue.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

ever, that overexpression of the factors in the cells by trans-fection with expression vectors under conditions that favoredthe formation of the homodimeric forms showed that thesewere as efficient in inducing promoter activity as was the het-erodimeric form (Fig. 7A).

The ATF-1 and CREB-1 factors are phosphorylated by pro-tein kinase A (PKA), which in most cases appears indispens-able for activation (for a review, see reference 47). The majoreffect of phosphorylation seems to occur at the level of thetransactivating function, while the effects on dimerization andDNA binding are less certain. Protein phosphatase 1 (PP-1)dephosphorylates ATF-1 and CREB-1 and correspondinglyattenuates the transactivational activity of the factors. Thephosphatase inhibitor protein-1 (IP-1) is a specific inhibitor ofPP-1, and its activity is dependent on phosphorylation by PKA.Thus, activation of PKA by cAMP would result in the phos-phorylation and activation of ATF-1/CREB-1 and IP-1, withthe latter leading to the inhibition of PP-1. Studies of purifiedATF/CREB proteins have demonstrated that the negatively

charged and phosphorylated kinase-inducible domain of thefactors is responsible for the interaction with components ofthe basal transcription apparatus. We have previously sug-gested a model for EBNA2-induced transactivation of theLMP1 promoter that involved the ATF/CRE site and a directinhibition of PP-1-catalyzed dephosphorylation of CREB-1 byEBNA2 in an IP-1-analogous manner (21). However, ourpresent investigation does not support the hypothesis that ac-tivation of the ATF/CRE site would occur through an EBNA2-induced increase of the phosphorylated form of CREB-1 orATF-1 at the binding site. EBNA2 did not significantly in-crease the stimulatory effect of ATF-1 and CREB-1 on LRS-CAT reporter plasmids in DG75 cells (Fig. 7A) or change thephosphorylation status of the transcription factors (data notshown).

The importance of this ATF/CRE site in LRS is also em-phasised by the results of Chen et al. (7). They showed that asequence variant found in the corresponding ATF/CRE motifof an NPC EBV isolate, when transferred into a B95-8 LRS

FIG. 9. In vitro-translated EBNA2 abrogates the binding of the in vitro-translated heterodimer c-Jun/ATF-2, but not the respective homodimeric forms, to theATF/CRE site. The 32P-labelled oligonucleotide probes indicated in the figure were incubated with DG75 nuclear extract and/or in vitro-translated proteins andanalyzed by EMSA. The three specific complexes obtained with DG75 nuclear extract and identified above are indicated by solid arrows and designated ATF-1, CREB-1and ATF-2, c-Jun. In vitro-translated proteins are denoted by the prefix IVT, and the positions of the corresponding complexes are shown by solid arrows. Nonspecificbands that were not abolished by competition are indicated by the dotted arrows. (A) The binding-reaction mixtures contained a 32P-labelled 250 to 219 LRSoligonucleotide probe with a mutated Sp site and DG75 nuclear extract (lane 1) or the same probe with in vitro-translated ATF-2 (lanes 2 to 6). Antibody supershiftswere performed by incubation with a rabbit polyclonal antibody against ATF-2 (lane 3) or a rabbit polyclonal antibody against CREB-2 (lane 4). A supershifted bandis indicated by a solid arrowhead. In lanes 5 and 6, aliquots of reticulocyte in vitro translation reactions with EBNA2 DNA or control DNA were added to thebinding-reaction mixtures. (B) The binding-reaction mixtures contained a 32P-labelled AP-1 consensus oligonucleotide probe and in vitro-translated c-Jun protein (lane1). Antibody supershifts were performed by incubation with a mouse monoclonal antibody against c-Jun (lane 2) or a goat polyclonal antibody against Jun B (lane 3).A supershifted band is indicated by a solid arrowhead. In lanes 4 and 5, aliquots of reticulocyte in vitro translation reaction mixtures with EBNA2 DNA or control DNAwere added to the binding-reaction mixtures. (C) The binding-reaction mixtures contained 32P-labelled 250 to 219 LRS oligonucleotide probe with a mutated Sp siteand DG75 nuclear extract (lane 1) or in vitro-translated ATF-2 and c-Jun protein (lanes 2 to 7). Antibody supershifts were performed by incubation with a rabbitpolyclonal antibody against ATF-2 (lane 3), a mouse monoclonal antibody against c-Jun (lane 4), or a goat polyclonal antibody against Jun B (lane 5). Supershiftedbands are indicated by solid and open arrowheads. In lanes 6 and 7, aliquots of reticulocyte in vitro translation reaction mixtures with EBNA2 DNA or control DNAwere added to the binding-reaction mixtures.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

sequence, conferred a threefold reduction of the activity of theLMP1 promoter both in B cells and in epithelial cells. Inter-estingly, the NPC sequence variant diminished the absolutelevels of activity of a reporter plasmid that carried the 2495/120 part of LRS in both the absence and presence of EBNA2but did not change the relative level of EBNA2 responsivenessin B cells. This indicates that the EBNA2-independent pro-moter activation pathway is disrupted by this sequence variant.It is consistent with our conclusion that the ATF/CRE site isone of the limiting factors that determine the final level ofactivity of the LMP1 promoter under different induction con-ditions and cellular environments.

Treatment of BL lines in the EBNA1-positive form of la-tency (latency I) with anti-immunoglobulin or with tetrade-canoyl phorbol acetate induces the lytic cycle via the proteinkinase C (PKC) signal transduction pathway. The switch to thelytic cycle involves the rapid upregulation of LMP1 in the cellsand occurs independently of EBNA2 expression (53). It hasbeen shown that CREB-1 and possibly ATF-1 can be activated

by phosphorylation via the PKC pathway in B lymphocytes(69). It is thus conceivable that the LMP1 promoter can beactivated through the PKC pathway as well as the PKA path-way and that the signal to the general transcriptional machin-ery is mediated in both cases by ATF-1/CREB-1 dimers boundto the ATF/CRE site.

EBNA2-dependent activation of LRS via the ATF/CRE site.The results of this study lend strong support to the notion thatEBNA2 can activate the LMP1 promoter via a mechanism thatis different from the ATF-1/CREB-1 pathway discussed aboveand that involves the binding of the ATF-2 and c-Jun factors asa heterodimer to the ATF/CRE site. EBNA2 is required forthe activation (Fig. 7B) and, judging from the EMSA (Fig. 9C)and coimmunoprecipitation (Fig. 10) experiments, seems tomake a direct contact with the c-Jun/ATF-2 dimer complex.Thus, the question arises of how this interaction may lead topromoter activation. Does EBNA2 induce a modification ofthe ATF-2/c-Jun dimer and/or its binding site or change theconcentration of the factors in the cell nucleus in a way thatfavors promoter activation through the activating domains ofATF-2 and c-Jun? Or is EBNA2 recruited to the LMP1 pro-moter by protein-protein interactions with the ATF-2/c-Jundimer to bring the EBNA2 transactivational domain in thecorrect position for a productive contact with one or severaldistinct general transcription factors? The possibility also existsthat the interaction between EBNA2 and the c-Jun/ATF-2dimer decreases the affinity of this complex for the ATF/CREsite, leading to an increased binding of the ATF-1 and CREB-1factors and activation of the LMP1 promoter through thispathway. The fact that overexpression of ATF-2 and c-Jun inthe presence of EBNA2 has a pronounced activating effect onthe LMP1 promoter (Fig. 7B) strongly argues against such ahypothesis. With regard to the first alternative, we have notbeen able to detect any change in the phosphorylation status orthe levels of ATF-2 and c-Jun in parallel with the EBNA2-induced activation of the LMP1 promoter (Fig. 8). In addition,it has been demonstrated in several studies that the C-terminalacidic domain of EBNA2 is required for transcriptional trans-activation by EBNA2 (10, 11, 56), and the activating domain ofEBNA2 has been found to make physical contact with severalgeneral transcription factors, including TFIIB, TAF40, andTFIIH (59, 61). Thus, it seems quite likely that EBNA2, atleast in the context of the 2106/140 part of LRS, functions ina manner analogous, in several respects, to the transcriptionalcoactivators CBP (CREB-binding protein) and the adenovirusE1A-associated cellular protein p300 with regard to the ATF/CRE. Neither CBP nor p300 by itself binds to DNA, but theycan be recruited to promoter elements by interaction with amultitude of sequence-specific activators. These interactionsinclude CBP-CREB (9, 39), p300-CREB (3), CBP–c-Jun (4),p300-YY1 (41), CBP-Fos (5), CBP–c-Myb (14), and CBP-nu-clear receptors (34). CBP can activate transcription through aglutamine-rich region in the C-terminal part of the protein,and the activation domain has been shown to interact withcomponents of the basal transcription machinery (39). Thus,CBP and p300 are transcriptional coactivators that provide acrucial link between transcriptional activators stimulated bysignalling cascades and initiation of transcription. EBNA2seems to function through a similar mechanism.

EBNA2 interacts with several other transcriptional regula-tory elements in the LMP1 promoter. These factors include theRBP-Jk (26, 31, 43, 62, 72), the Ets-related PU.1 factor (33),and an unidentified member of the POU domain-containingprotein family (55). RBP-Jk is a transcriptional repressorwhich binds to DNA sequences (GTGGGAA) in LRS. It hasbeen shown that EBNA2 can act by targeting DNA-bound

FIG. 10. EBNA2 coimmunoprecipitates with the c-Jun/ATF-2 heterodimer.The EBNA2 expression vector pc(BYRF) or equivalent amounts of the pccontrol plasmid were transfected together with pc(c-Jun) and pc(ATF-2) and theCD2 expression vector pE300CY6 in DG75 cells. The transfected cells wereselected for CD2 expression with magnetic beads. After lysis of the cells, theproteins were immunoprecipitated with specific antibodies and adsorption toprotein A/G agarose, eluted, and analyzed by SDS-PAGE and immunoblotting.Cell extracts that had not been subjected to immunoprecipitation were analyzedin lanes 1 to 3, and control immunoprecipitations without the specific antibodybut including the adsorption step with protein A/G were analyzed in lanes 10 to12. Lanes: 1, DG75 cells transfected with pc(BYRF); 2, DG75 cells transfectedwith the pc plasmid; 3, B95-8 cells; 4, anti-ATF-2 precipitate from DG75 cellstransfected with pc(BYRF); 5, anti-ATF-2 precipitate from DG75 cells trans-fected with the pc plasmid; 6, anti-ATF-2 precipitate from B95-8 cells; 7, anti-c-Jun precipitate from DG75 cells transfected with pc(BYRF); 8, anti-c-Junprecipitate from DG75 cells transfected with the pc plasmid; 9, anti-c-Jun pre-cipitate from B95-8 cells; 10, protein A/G agarose eluate from DG75 cellstransfected with pc(BYRF); 11, protein A/G agarose eluate from DG75 cellstransfected with the pc plasmid; 12, protein A/G agarose eluate from B95-8 cells.Antibodies used for visualizing the proteins on the immunoblots were anti-ATF-2 (A), anti-c-Jun (B), and a human serum containing anti-EBNA2 anti-bodies (C). The positions of ATF-2, c-Jun, EBNA2, and immunoglobulin heavychains (Ig H) are indicated by the solid arrowheads.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

RBP-Jk within the nucleus and abolishing RBP-Jk-mediatedrepression through masking of the repression domain (26, 31,32, 43, 62, 72). Furthermore, EBNA2 interactions with PU.1and the POU domain protein seem to be essential for theefficient upregulation of the LMP1 promoter, and the elementsmight act in a cooperative manner (33, 55). The number ofEBNA2 molecules bound to the LMP1 promoter in its acti-vated configuration is not known. However, in a simplisticmodel, Johannsen et al. (33) have suggested that one EBNA2molecule (presumably as a dimer) binds to the regulatory re-gion via multiple protein-protein interactions with a number oftranscriptional activators including PU.1, LBF3, LBF5, LBF6,LBF7, and RBP-Jk. In the light of our investigations, we wouldsuggest that the c-Jun/ATF-2 heterodimer and the POU do-main protein should also be included. The biochemical func-tion of these multiple contact points would then be to increasethe stability of the promoter-EBNA2 complex and hence thespecificity and efficiency of the induction. Functional studiesare consistent with the notion that some of the activatorssurrounding the PU.1-binding site cooperate with PU.1 in thebinding of the same EBNA2 dimer (33, 40, 55). Independentbinding of separate EBNA2 molecules to multiple sites in vivothrough different factors including RBP-Jk, LBF3, LBF5,LBF6, LBF7, PU.1, the POU domain protein, and the c-Jun/ATF-2 heterodimer is, however, also possible, although wehave no data to support such an assumption. It has recentlybeen demonstrated in a model system that multiple EBV ZE-BRA molecules bound upstream of the TATA box and initi-ation site synergistically interact with TFIID and TFIIA, re-sulting in the assembly of a preinitiation subcomplex (theDA complex) and a concomitant isomerization (8). Onceisomerized, the complex binds TFIIB and the remaining gen-eral factors. Interestingly, the recruitment of the DA complexrequired multiple contacts and is therefore the basis for tran-scriptional synergy in the system. Recruitment and isomeriza-tion of the DA complex may be a general effect of manyactivators and coactivators, including EBNA2.

ACKNOWLEDGMENTS

We gratefully acknowledge Carina Strom and Jane Lofvenmark forskillful technical assistance. We thank G. Suske for the generous gift ofthe pCMV-Sp1 plasmid, C. Svensson-Akusjarvi for the ATF-2 cDNAand the E1A 13S expression vector, R. Tjian for the pCMV c-junplasmid, R. H. Goodman for the pET-15b(ATF-1) and pET-15b(CREB 341) plasmids, and E. Lundgren for the pE300CY6 plasmid.

This study was supported by grants from the Swedish Medical Re-search Council, the Swedish Cancer Society, and the Sahlgrenska Uni-versity Hospital.

REFERENCES

1. Abbot, S. D., M. Rowe, K. Cadwallader, A. Ricksten, J. Gordon, F. Wang, L.Rymo, and A. B. Rickinson. 1990. Epstein-Barr virus nuclear antigen 2expression of the virus-encoded latent membrane protein. J. Virol. 64:2126–2134.

2. Angel, P., and M. Karin. 1991. The role of Jun, Fos and the AP-1 complexin cell-proliferation and transformation. Biochim. Biophys. Acta 1027:129–157.

3. Arany, Z., D. Newsome, E. Oldread, D. M. Livingston, and R. Eckner. 1995.A family of transcriptional adaptor proteins targeted by the E1A oncopro-tein. Nature 374:81–84.

4. Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smeal, M. Karin, J.Feramisco, and M. Montminy. 1994. Activation of cAMP and mitogen re-sponsive genes relies on a common nuclear factor. Nature 370:226–229.

5. Bannister, A. J., and T. Kouzarides. 1995. CBP-induced stimulation of c-Fosactivity is abrogated by E1A. EMBO J. 14:4758–4762.

6. Ben-Bassat, H., N. Goldblum, S. Mitrani, T. Goldblum, J. M. Yoffey, M. M.Cohen, Z. Bentwich, B. Ramot, E. Klein, and G. Klein. 1977. Establishmentin continuous culture of a new type of lymphocyte from a “Burkitt-like”malignant lymphoma (line D.G-75). Int. J. Cancer 19:27–33.

7. Chen, M.-L., R.-C. Wu, S.-T. Liu, and Y.-S. Chang. 1995. Characterization of

59-upstream sequence of the latent membrane protein 1 (LMP-1) gene of anEpstein-Barr virus identified in nasopharyngeal carcinoma tissues. VirusRes. 37:75–84.

8. Chi, T., and M. Carey. 1996. Assembly of the isomerized TFIIA-TFIID-TATA ternary complex is necessary and sufficient for gene activation. GenesDev. 10:2540–2550.

9. Chrivia, J. C., R. P. S. Kwok, N. Lamb, M. Hagiwara, M. R. Montminy, andR. H. Goodman. 1993. Phosphorylated CREB binds specifically to the nu-clear protein CBP. Nature 365:855–859.

10. Cohen, J. I., and E. Kieff. 1991. An Epstein-Barr virus nuclear protein 2domain essential for transformation is a direct transcriptional activator.J. Virol. 65:5880–5885.

11. Cohen, J. I., F. Wang, and E. Kieff. 1991. Epstein-Barr virus nuclear protein2 mutations define essential domains for transformation and transactivation.J. Virol. 65:2545–2554.

12. Cohen, J. I., F. Wang, J. Mannick, and E. Kieff. 1989. Epstein-Barr virusnuclear protein 2 is a key determinant of lymphocyte transformation. Proc.Natl. Acad. Sci. USA 86:9558–9562.

13. Cordier, M., A. Calender, M. Billaud, U. Zimber, G. Rousselet, O. Pavlish,J. Banchereau, T. Tursz, G. Bornkamm, and G. M. Lenoir. 1990. Stabletransfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphomacells containing the EBV P3HR1 genome induces expression of B-cell acti-vation molecules CD21 and CD23. J. Virol. 64:1002–1013.

14. Dai, P., H. Akimaru, Y. Tanaka, D.-X. Hou, T. Yasukawa, C. Kanei-Ishii, T.Takahashi, and S. Ishii. 1996. CBP as a transcriptional coactivator of c-Myb.Genes Dev. 10:528–540.

15. Delmas, V., C. A. Molina, E. Lalli, R. De Grot, N. S. Foulkes, D. Masquilier,and P. Sassone-Corsi. 1994. Complexity and versatility of the transcriptionalresponse to cAMP. Rev. Phys. Biochem. Pharmacol. 124:1–28.

16. Denning, J., G. Hagen, M. Beato, and G. Suske. 1995. Members of the Sptranscription factor family control transcription from the uteroglobin pro-moter. J. Biol. Chem. 270:12737–12744.

17. Devergne, O., E. Hatzivassiliou, K. M. Izumi, K. M. Kaye, M. F. Kleijnen, E.Kieff, and G. Mosialos. 1996. Association of TRAF1, TRAF2, and TRAF3with an Epstein-Barr virus LMP1 domain important for B-lymphocyte trans-formation: role in NF-kB activation. Mol. Cell. Biol. 16:7098–7108.

18. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcrip-tion initiation by RNA polymerase II in a soluble extract from isolatedmammalian nuclei. Nucleic Acids Res. 11:1475–1489.

19. Fåhraeus, R., A. Jansson, A. Ricksten, A. Sjoblom, and L. Rymo. 1990.Epstein-Barr virus-encoded nuclear antigen 2 activates the viral latent mem-brane protein promoter by modulating the activity of a negative regulatoryelement. Proc. Natl. Acad. Sci. USA 87:7390–7394.

20. Fåhraeus, R., A. Jansson, A. Sjoblom, T. Nilsson, G. Klein, and L. Rymo.1993. Cell phenotype dependent control of Epstein-Barr virus latent mem-brane protein 1 (LMP1) gene regulatory sequences. Virology 195:71–80.

21. Fåhraeus, R., L. Palmqvist, A. Nerstedt, S. Farzad, L. Rymo, and S. Laın.1994. Response to cAMP levels of the Epstein-Barr virus EBNA2-inducibleLMP1 oncogene and EBNA2 inhibition of a PP1-like activity. EMBO J.13:6041–6051.

22. Farrell, P. J., A. Bankier, C. Seguin, P. Deininger, and B. G. Barrell. 1983.Latent and lytic cycle promoters of Epstein-Barr virus. EMBO J. 2:1331–1338.

23. Fennewald, S., V. van Santen, and E. Kieff. 1984. Nucleotide sequence of anmRNA transcribed in latent growth-transforming virus infection indicatesthat it may encode a membrane protein. J. Virol. 51:411–419.

24. Finke, J., R. Fritzen, P. Ternes, P. Trivedi, K. J. Bross, W. Lange, R.Mertelsmann, and G. Dolken. 1992. Expression of bcl-2 in Burkitt’s lym-phoma cell lines: induction by latent Epstein-Barr virus genes. Blood 80:459–469.

25. Flint, K. J., and N. C. Jones. 1991. Differential regulation of three membersof the ATF/CREB family of DNA-binding proteins. Oncogene 6:2019–2026.

26. Grossman, S. R., E. Johannsen, X. Tong, R. Yalamanchili, and E. Kieff.1994. The Epstein-Barr virus nuclear antigen 2 transactivator is directed toresponse elements by the Jk recombination signal binding protein. Proc.Natl. Acad. Sci. USA 91:7568–7572.

27. Hagen, G., S. Muller, M. Beato, and G. Suske. 1992. Cloning by recognitionsite screening of two novel GT box binding proteins: a family of Sp1 relatedgenes. Nucleic Acids Res. 20:5519–5525.

28. Hagen, G., S. Muller, M. Beato, and G. Suske. 1994. Sp1-mediated tran-scriptional activation is repressed by Sp3. EMBO J. 13:3843–3851.

29. Hai, T., and T. Curran. 1991. Cross-family dimerization of transcriptionfactors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc. Natl.Acad. Sci. USA 88:3720–3724.

30. Hammerschmidt, W., and B. Sugden. 1989. Genetic analysis of immortaliz-ing functions of Epstein-Barr virus in human B lymphocytes. Nature 340:393–397.

31. Henkel, T., P. D. Ling, S. D. Hayward, and M. G. Peterson. 1994. Mediationof Epstein-Barr virus EBNA2 transactivation by recombination signal-bind-ing protein Jk. Science 265:92–95.

32. Hsieh, J. J.-D., and S. D. Hayward. 1995. Masking of the CBF1/RBPJk


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

transcriptional repression domain by Epstein-Barr virus EBNA2. Science268:560–563.

33. Johannsen, E., E. Koh, G. Mosialos, X. Tong, E. Kieff, and S. R. Grossman.1995. Epstein-Barr virus nuclear protein 2 transactivation of the latent mem-brane protein 1 promoter is mediated by Jk and PU.1. J. Virol. 69:253–262.

34. Kamei, Y., L. Xu, T. Heinzel, J. Torchia, R. Kurokawa, B. Gloss, S.-C. Lin,R. A. Heyman, D. W. Rose, C. K. Glass, and M. G. Rosenfeld. 1996. A CBPintegrator complex mediates transcriptional activation and AP-1 inhibitionby nuclear receptors. Cell 85:403–414.

35. Kaye, K. M., K. M. Izumi, and E. Kieff. 1993. Epstein-Barr virus latentprotein 1 is essential for B-lymphocyte growth transformation. Proc. Natl.Acad. Sci. USA 90:9150–9154.

36. Kieff, E. 1996. Epstein-Barr virus and its replication, p. 2343–2396. In B. N.Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lip-pincott-Raven Publishers, Philadelphia, Pa.

37. Kingsley, C., and A. Winoto. 1992. Cloning of GT box-binding proteins: anovel Sp1 multigene family regulating T-cell receptor gene expression. Mol.Cell. Biol. 12:4251–4261.

38. Knutson, J. 1990. The level of c-fgr RNA is increased by EBNA-2, anEpstein-Barr virus gene required for B-cell immortalization. J. Virol. 64:2530–2536.

39. Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bach-inger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman.1994. Nuclear protein CBP is a coactivator for the transcription factorCREB. Nature 370:223–226.

40. Laux, G., B. Adam, L. J. Strobl, and F. Moreau-Gachelin. 1994. The Spi-1/PU.1 and Spi-B ets family transcription factors and the recombination signalbinding protein RBP-Jk interacts with an Epstein-Barr virus nuclear antigen2 responsive cis-element. EMBO J. 13:5624–5632.

41. Lee, J.-S., K. M. Galvin, R. H. See, R. Eckner, D. Livingstone, E. Moran, andY. Shi. 1995. Relief of YY1 transcriptional repression by adenovirus E1A ismediated by E1A-associated protein p300. Genes Dev. 9:1188–1198.

42. Liebowitz, D., D. Wang, and E. Kieff. 1986. Orientation and patching of thelatent infection membrane protein encoded by Epstein-Barr virus. J. Virol.58:233–237.

43. Ling, P. D., D. R. Rawlins, and S. D. Hayward. 1993. The Epstein-Barr virusimmortalizing protein EBNA-2 is targeted to DNA by a cellular enhancer-binding protein. Proc. Natl. Acad. Sci. USA 90:9237–9241.

44. Mann, K. P., D. Staunton, and D. A. Thorley-Lawson. 1985. Epstein-Barrvirus-encoded protein found in plasma membranes of transformed cells.J. Virol. 55:710–720.

45. Mannick, J. B., J. I. Cohen, M. Birkenbach, A. Marchini, and E. Kieff. 1991.The Epstein-Barr virus nuclear protein encoded by the leader of the EBNARNAs is important in B-lymphocyte transformation. J. Virol. 65:6826–6837.

46. Masson, N., J. John, and K. A. W. Lee. 1993. In vitro phosphorylation studiesof a conserved region of the transcription factor ATF1. Nucleic Acids Res.21:4166–4173.

47. Meyer, T. E., and J. F. Habener. 1993. Cyclic adenosine 39,59-monophos-phate response element binding protein (CREB) and related transcription-activating deoxyribonucleic acid-binding proteins. Endocr. Rev. 14:269–290.

48. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. VanArsdale, C. Ware, andE. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engagessignaling proteins for the tumor necrosis factor receptor family. Cell 80:389–399.

48a.Ofverstedt, L. G., K. Hammarstrom, N. Balgobin, S. Hjerten, U. Pettersson,and J. Chattopadhyaya. 1984. Rapid and quantitative recovery of DNAfragments from gels by displacement electrophoresis. Biochim. Biophys.Acta. 782:120–126.

49. Pilon, M., M. Gullberg, and E. Lundgren. 1991. Transient expression of theCD2 cell surface antigen as a sortable marker to monitor high frequencytransfection of human primary B cells. J. Immunol. 146:1047–1051.

50. Rickinson, A. B., and E. Kieff. 1996. Epstein-Barr virus, p. 2397–2446. InB. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed.Lippincott-Raven Publishers, Philadelphia, Pa.

51. Ricksten, A., A. Olsson, T. Andersson, and L. Rymo. 1988. The 59 flankingregion of the gene for the Epstein-Barr virus-encoded nuclear antigen 2contains a cell type specific cis-acting regulatory element that activates tran-scription in transfected B-cells. Nucleic Acids Res. 16:8391–8410.

52. Ricksten, A., C. Svensson, C. Welinder, and L. Rymo. 1987. Identification ofsequences in Epstein-Barr virus DNA required for the expression of thesecond Epstein-Barr virus-determined nuclear antigen in COS-1 cells.J. Gen. Virol. 68:2407–2418.

53. Rowe, M., A. L. Lear, D. Croom-Carter, A. H. Davies, and A. B. Rickinson.1992. Three pathways of Epstein-Barr virus gene activation from EBNA1-positive latency in B lymphocytes. J. Virol. 66:122–131.

54. Sheng, M., M. A. Thompson, and M. E. Greenberg. 1991. CREB: a Ca(21)-regulated transcription factor phosphorylated by calmodulin-dependent ki-nases. Science 252:1427–1430.

55. Sjoblom, A., A. Jansson, W. Yang, S. Laın, T. Nilsson, and L. Rymo. 1995.PU box-binding transcription factors and a POU domain protein cooperatein the Epstein-Barr virus (EBV) nuclear antigen 2-induced transactivation ofthe EBV latent membrane protein 1 promoter. J. Gen. Virol. 76:2679–2692.

56. Sjoblom, A., A. Nerstedt, A. Jansson, and L. Rymo. 1995. Domains of theEpstein-Barr virus nuclear antigen 2 (EBNA2) involved in the transactiva-tion of the latent membrane protein 1 and the EBNA Cp promoters. J. Gen.Virol. 76:2669–2678.

57. Sung, N. S., S. Kenney, D. Gutsch, and J. S. Pagano. 1991. EBNA-2 trans-activates a lymphoid-specific enhancer in the BamHI C promoter of Epstein-Barr virus. J. Virol. 65:2164–2169.

58. Tomkinson, B., E. Robertson, and E. Kieff. 1993. Epstein-Barr virus nuclearproteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growthtransformation. J. Virol. 67:2014–2025.

59. Tong, X., R. Drapkin, D. Reinberg, and E. Kieff. 1995. The 62- and 80-kDasubunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2. Proc. Natl. Acad. Sci. USA 92:3259–3263.

60. Tong, X., R. Drapkin, R. Yalamanchili, G. Mosialos, and E. Kieff. 1995. TheEpstein-Barr virus nuclear protein 2 acid domain forms a complex with anovel cellular coactivator that can interact with TFIIE. Mol. Cell. Biol.15:4735–4744.

61. Tong, X., F. Wang, C. J. Thut, and E. Kieff. 1995. The Epstein-Barr virusnuclear protein 2 acidic domain can interact with TFIIB, TAF40, and RPA70but not with TATA-binding protein. J. Virol. 69:585–588.

62. Waltzer, L., F. Logeat, C. Brou, A. Israel, A. Sergeant, and E. Manet. 1994.The human Jk recombination signal sequence binding protein (RBP-Jk)targets the Epstein-Barr virus EBNA2 protein to its DNA responsive ele-ments. EMBO J. 13:5633–5638.

63. Wang, F., C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A.Rickinson, and E. Kieff. 1990. Epstein-Barr virus latent membrane protein(LMP1) and nuclear proteins 2 and 3C are effectors of phenotypic changesin B lymphocytes: EBNA-2 and LMP1 cooperatively induce CD23. J. Virol.64:2309–2318.

64. Wang, F., C. D. Gregory, M. Rowe, A. B. Rickinson, D. Wang, M. Birken-bach, H. Kikutani, T. Kishimoto, and E. Kieff. 1987. Epstein-Barr virusnuclear antigen 2 specifically induces expression of the B-cell activationantigen CD23. Proc. Natl. Acad. Sci. USA 84:3452–3456.

65. Wang, F., H. Kikutani, S.-F. Tsang, T. Kishimoto, and E. Kieff. 1991. Ep-stein-Barr virus nuclear protein 2 transactivates a cis-acting CD23 DNAelement. J. Virol. 65:4101–4106.

66. Wang, F., S.-F. Tsang, M. G. Kurilla, J. I. Cohen, and E. Kieff. 1990.Epstein-Barr virus nuclear antigen 2 transactivates latent membrane proteinLMP1. J. Virol. 64:3407–3416.

67. Woisetschlaeger, M., X. W. Jin, C. N. Yandava, L. A. Furmanski, J. L.Strominger, and S. H. Speck. 1991. Role for the Epstein-Barr virus nuclearantigen 2 in viral promoter switching during initial stages of infection. Proc.Natl. Acad. Sci. USA 88:3942–3946.

68. Wu, D. Y., G. V. Kalpana, S. P. Goff, and W. H. Schubach. 1996. Epstein-Barr virus nuclear protein 2 (EBNA2) binds to a component of the humanSNF-SWI complex, hSNF5/Ini1. J. Virol. 70:6020–6028.

69. Xie, H., Z. Wang, and T. L. Rothstein. 1996. Signaling pathways for antigenreceptor-mediated induction of transcription factor CREB in B lymphocytes.Cell. Immunol. 169:264–270.

70. Yates, J. L., N. Warren, and B. Sugden. 1985. Stable replication of plasmidsderived from Epstein-Barr virus in a variety of mammalian cells. Nature313:812–815.

71. Zimber-Strobl, U., E. Kremmer, F. Grasser, G. Marschall, G. Laux, andG. W. Bornkamm. 1993. The Epstein-Barr virus nuclear antigen 2 interactswith an EBNA2 responsive cis-element of the terminal protein 1 gene pro-moter. EMBO J. 12:167–175.

72. Zimber-Strobl, U., L. J. Strobl, C. Meitinger, R. Hinrichs, T. Sakai, T.Furukawa, T. Honjo, and G. W. Bornkamm. 1994. Epstein-Barr virus nuclearantigen 2 exerts its transactivating function through interaction with recom-bination signal binding protein RBP-Jk, the homologue of Drosophila Sup-pressor of Hairless. EMBO J. 13:4973–4982.


on Novem


.org/D

ownloaded from

http://jvi.asm.org/

An ATF/CRE Element Mediates both EBNA2Dependent and EBNA2Independent Activation of the Epstein-Barr Virus LMP1 Gene Promoter

Documents