Proc. Nati. Acad. Sci. USAVol. 91, pp. 2843-2847, March 1994Medical Sciences

The cellular proteins that bind specifically to the Epstein-Barrvirus origin of plasmid DNA replication belong to a gene familySHENGJIA ZHANG AND MEIHAN NONOYAMA*Laboratory of Virology, Tampa Bay Research Institute, 10900 Roosevelt Boulevard, St. Petersburg, FL 33716

Communicated by Bernard Roizman, December 22, 1993 (received for review August 19, 1993)

ABSTRACT This laboratory has previously reported thata tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate ("12-0-tetradecanoylphorbol 13-acetate"), inducesBJAB cells, a Burkitt lymphoma cell line, to express cellularproteins that bind to the origin of plasmid DNA replication(oriP) of Epstein-Barr virus. These oriP-binding proteins in-terfere with the EBV-encoded nuclear antigen EBNA-1, whichbinds to oriP in Raji cells. To further characterize theseproteins, a A phage expression cDNA library made fromphorbol ester-induced BJAB cells was screened for fusionproteins which bind to oriP. Two recombinant phages contain-ing sequences encoding jt-galactosidase fusion proteins anddesignated A-OBP-1 and A-OBP-2 were identified. A-OBP-1and A-OBP-2 contained 0.43 kbp and 0.61 kbp of BJAB cellcDNA, respectively, of which 395 bp were shared. UsingA-OBP-1 as probe, two cDNAs of 1.4 kbp and 1.2 kbp,designated OBP-1 and OBP-2, respectively, were isolated.These cDNAs also shared the 395-bp sequence at the 3' end.With these cDNAs as probes, Northern blot analyses ofmRNAfrom BJAB cells gave 1.4, 2.4, and 3.4-kb bands, but aSouthern blot of human genomic DNA revealed one band. It islikely that the oriP-binding proteins were derived from splicedmRNA(s) of a gene family.

In latent infection of human B lymphocytes by Epstein-Barrvirus (EBV), the viral genome exists predominantly as plas-mids and is present in multiple copies (1-3). The origin ofplasmid replication (oriP) of EBV has been identified as theelement required for the replication and the extrachromo-somal maintenance of the viral genome in cells that expressthe EBV-encoded nuclear antigen EBNA-1 (4-6). oriP con-sists of two cis-acting DNA sequences (7), a family of 20copies of 30-bp direct imperfect repeats (FR), and a sequenceof four copies of the same repeat with dyad symmetry (DS).FR and DS are separated by 1 kbp, and each contains multipleEBNA-1 binding sites (7). Binding of EBNA-1 to FR and DScauses DNA conformational changes and brings FR and DSadjacent to each other (6). Exactly how this interactioncontributes to the replication and the extrachromosomalmaintenance of the EBV genome in cells is unclear.

Transcription of the EBNA-1 gene is regulated by one of itsthree promoters: the BWR1 promoter, Wp, located at theBamHI W repeat region of the EBV genome; the BCR2promoter, Cp, located in the BamHI C fragment; and the Fppromoter, located in the BamHI F/Q restriction fragment (8,9). Switching from one promoter to another that regulatesEBNA-1 gene transcription can occur in EBV-infectedB-lymphocytes (10). This promoter switching ensures theconstitutive expression of EBNA-1 in EBV-positive cells. Ithas been reported that the binding of EBNA-1 to FR, but notthe binding to DS, may enhance BCR2-promoted EBNA-1transcription (11, 12). The BCR2 promoter also regulates thetranscription of the other members of the EBNA gene family

(13, 14) during EBV latency. Thus, the EBNA-1-FR inter-action may also modulate the expression of the other mem-bers of the EBNA gene family. Apart from interaction withFR and DS of oriP, EBNA-1 also binds to a DNA sequenceadjacent to the Fp promoter (8, 9). However, the significanceof this EBNA-1-Fp promoter interaction is unknown.Two DNA-binding proteins of cellular origin have been

reported to compete with EBNA-1 and bind to FR and DS(15). The competition of these oriP-binding proteins (OBPs)with EBNA-1 may modulate the stability of the EBV genomeas plasmids and/or regulate the expression ofthe EBNA genefamily from the BCR2 promoter. This study reports thecloning of the cDNAs for these cellular OBPs.t

MATERIALS AND METHODSCells and Activation by Phorbol Ester. BJAB (EBV-

negative) and Raji (EBV-positive) are African Burkitt lym-phoma cell lines. They were maintained in RPMI 1640medium (GIBCO/BRL) supplemented with 2 mM L-gluta-mine, penicillin (50 units/ml), streptomycin sulfate (50 ug/ml), and 10% (vol/vol) heat-inactivated newborn calf serum(Intergen, Purchase, NY) at 370C in an atmosphere of 5%CO2. Phorbol 12-myristate 13-acetate (" 12-O-tetrade-canoylphorbol 13-acetate," TPA) at 20 ng/ml was used toactivate Raji or BJAB cells (5 x 105 cells per ml) for 3 daysat 370C.

Plasmids. The 140-bp EcoRV-HincII subfragment whichcontains the entire DS of oriP, located in the BamHI Cfragment of the EBV genome (16), was blunt-end ligated intoplasmid pUC18 at the Sma I site to give the pEC140. PlasmidpUCoriP2 (17), containing 11 of the 20 copies of the 30-bprepeats in FR, and A-EB phage (17), encoding a f3-galacto-sidase fusion protein that contains the oriP-binding domain ofEBNA-1 (7), were generous gifts from H. Singh (Universityof Chicago).

Preparation and Screening of cDNA Library. Total cellularRNA was prepared from TPA-induced BJAB cells by theguanidine thiocyanate method (18). Poly(A)+ RNA isolatedby use of the streptavidin/biotin/magnetic bead techniqueprovided in the PolyAtract mRNA isolation system I(Promega) was used to synthesize oligo(dT)-primed cDNA.Double-stranded cDNAs were ligated to Agtll Sfi I-Not Ivector (Promega), and packaged with Gigapack II Goldpackaging extract (Stratagene). The cDNA expression librar-ies were screened with labeled FR and DS probes by themethod described by Singh et al. (17), except poly(d1-dC)-(dl-dC) was replaced with sheared and denatured salmon sperm

Abbreviations: EBV, Epstein-Barr virus; oriP, origin of latent viralDNA replication; OBP, oriP-binding protein; DS, a sequence withdyad symmetry in the EBV oriP; FR, a family of repeats with 20tandem copies of 30-bp direct imperfect repeats in the EBV oriP;TPA, "12-O-tetradecanoylphorbol 13-acetate" (phorbol 12-myristate 13-acetate).*To whom reprint requests should be addressed.tThe sequences reported in this paper have been deposited in theGenBank data base (accession nos. L29095 and L29096).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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DNA (10 yg/ml). Positive phage clones expressing f-galac-tosidase fusion proteins that bound to FR and DS werepurified by repeatedly screening the plaques with the labeledFR and DS probes until pure single clones were obtained.Gel Retardation Assay. DNA-protein binding reactions

were performed at room temperature. Each incubation mix-ture contained 4 pul of 5x binding buffer [5 x is 100 mM TrisCl, pH 7.5/250 mM NaCl/15 mM MgCl2/5 mM dithiothrei-tol/0.03% Nonidet P-40/100 mM EDTA/25% (vol/vol) glyc-erol with bovine serum albumin at 250 pg/ml], 2.5 jil of 0.1%poly(dl-dC)-(dl-dC), radioactive probe, water, and 10 pg ofprotein extract to give a final volume of 20 p.l. Protein extractwas always added last and the mixtures were then incubatedfor 15 min before being loaded directly onto a 5% polyacryl-amide gel in 0.25x TBE (lx TBE is 90 mM Tris/64.6 mMboric acid/2.5 mM EDTA, pH 8.3). After electrophoresis,gels were fixed with 10%6 acetic acid/10% methanol for 20 minand then dried onto Whatman 3MM paper and visualized byautoradiography on Kodak XAR-5 film.

RESULTSCloning and Sequencing of cDNA Encoding OBPs. TPA

induces BJAB cells to express OBPs that bind specifically toFR and DS (15). To clone the cDNAs of these OBPs, a Aphage expression cDNA library containing 1.5 x 106 cloneswas made from TPA-induced BJAB cells. By use of FR andDS as sequence-specific binding probes, 4 x 105 Agtll phagesfrom this library were screened for fusion proteins whichbound to FR and/or DS. Two recombinant phages containingsequences encoding P-galactosidase fusion proteins and des-ignated as A-OBP-1 and A-OBP-2 were identified. As shownin Fig. 1, both clones, like A-EB, which encodes a fusionprotein containing the DNA-binding domain of EBNA-1,reacted with the FR and the DS probe, but not with thenonspecific probe carrying a 102-bp Kpn I-Sac I multiplecloning site from pBluescript SK(+) vector (data not shown).Sequence analysis showed that the two cDNA clones shared395 nucleotides, excluding the polyadenylylation signal, atthe 3' end (Fig. 2). The available nucleotide sequencespredicted that both proteins had helix-loop-helix (HLH)structures and possessed protein kinase C as well as caseinkinase phosphorylation sites. The absence ofthe ATG codon,however, suggested that neither cDNA clone was full-length.There was no homology with other proteins or cDNA clonesin the amino acid (Swiss-Prot 27) and nucleic acid (GenBank80) data banks.

In an attempt to isolate the full-length cDNAs ofOBPs, theoriginal cDNA library was ligated into Agtll phage andscreened by use of A-OBP-1 as probe. A 1.4-kb OBP-1 and a1.2-kb OBP-2 cDNA clone were isolated. Sequence analysesagain revealed shared 3' ends, but the absence of ATGindicated that neither clone was full-length (data not shown).However, the isolation of four partial cDNA clones havingshared 3' sequence, but with unique 5' nucleotide sequence,suggests that these cDNAs may have derived from relatedtranscripts.

Characterization of Fusion Proteins from A-OBP-1 andA-OBP-2. Lysogens from A-OBP-1 and A-OBP-2 were pre-pared by use of Escherichia coli strain Y1089(r-) (20).Extracts prepared from A-OBP-1 and A-OBP-2 lysogens gavebands of approximately 129 and 136 kDa, respectively, ondenaturing polyacrylamide gel electrophoresis (SDS/PAGE)as compared with a 116-kDa band corresponding to ,B-galac-tosidase for the control Agtll lysogen. Thus, A-OBP-1 en-coded a 13-kDa polypeptide fused to f-galactosidase,whereas A-OBP-2 encoded a 20-kDa polypeptide fused tof-galactosidase. Gel retardation assay with 32P-labeled DS(Fig. 3A) and FR (Fig. 3B), using lysates from lysogens ofA-OBP-1 (lanes 6-8) and A-OBP-2 (lanes 9-11), demonstrated

IT --I r-.-.-.IT. 5r?-tr 1 1 1 H I Tr II--4I-

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;~1* T.* .::' iJ*' h.a .IA*fly00 :0,4lb 0rwt, 0At41 .1,FIG. 1. Isolation of two recombinant phages, A-OBP-1 and

A-OBP-2, encoding fusion proteins.that bound specifically to the oriPsequences of the EBV genome. An EcoRI-Hpa II DNA fiagment,containing 2 of the 11 copies of the 30-bp repeats of FR in plasmidpUCoriP2, was ligated into the Sma I site of pBluescript SK(+) togive pBSFR2. For screening of 4 x 105 recombinant phages of acDNA library, FR and DS DNA fragments were excised fromplasmids pBSFR2 and pEC140, respectively, and used as protein-binding labeled probes. Multiple screening of the positive phageclones expressing ,B-galactosidase fusion proteins (17) that bound toFR and DS was performed as described in Materials and Methods.TR, terminal repeat, IR, internal repeat; U, unique sequence.

the formation of DNA-protein complexes. Competition withunlabeled homologous DNA fragments (lanes 7, 8, 10, and 11)in the gel retardation interfered with the binding of theA-OBP-1 and A-OBP-2 fusion proteins to the radiolabeled DSand FR DNA fragments, whereas nonspecific DNA such asA or pBR322 did not (data not shown). These results verifiedthe specific binding of the A-OBP-1 and A-OBP-2 fusionproteins to the EBV oriP. Likewise, A-EB (lanes 3-5) boundspecifically to FR and DS, but lysate protein from Agtlllysogen (lane 2) did not. Interestingly, A-OBP-1 and A-OBP-2fusion proteins also bound to the EBV BamHI Q fragment(Fig. 3C), which contains two distinct but low-affinity bind-ing sites for EBNA-1 (7, 23, 24).

Analyses of OBP RNA. To examine whether OBP mRNAwas more abundant in Burkitt lymphoma cells treated withTPA, poly(A)+ RNA was isolated from BJAB (Fig. 4 A andC) and Raji cells (Fig. 4 B and D) that were induced or notinduced with TPA. The poly(A)+ RNA samples were ana-lyzed by Northern blot hybridization with the labeledA-OBP-1 probe (Fig. 4 A and B). Simultaneous detection of3-actin mRNA by a labeled 3-actin probe served to verifythat equal amounts ofRNA had been loaded in each lane (Fig.4 C and D). A-OBP-1 was selected as labeled probe becauseit was the smallest cDNA clone isolated and 395 of its 426 bp(92.7%) were shared by the other OBP cDNAs. Hybridiza-tion with A-OBP-1 should detect all members in this family oftranscripts sharing the same common 3' sequence. Theintensity of the hybridization signals also should reflect thetrue ratio of the members in this family of transcripts. Three

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X-OBP-2 gaattccgttgccgtcgAGATGTTGGCCCAGAAGGCTGAGGAAAAGGAGE M L A Q K A E E K E

X-OBP-2 AACCATTGTCCCACAATGClTCCGGCCCCTTTCACATCGCACAGTCACAN H C P T M L R P L S* H R T V T

GGGGCAAAGCCCCTGAAAAAGGCTGTGGTGATGCCCCTACAGCTAATTG A K P L K K A V V M P L Q L I

CAGGAGCAGGCAGCATCCCCAAATGCCGAGATCCACATCCTGAAGAATQ E Q A A S P N A E I H I L K N

gaattccgttgct

K G R K R K L E S L D A L E P EgtcgCCTCTGCCTCCCAGCTGGAGTCCCTGGATGCCCTAGAGCCTGAG

A S A S* O L E S L D A L E P E

GAGAAGGCTGAGGACTGCTGGGAGCTACAGATCAGCCCGGAGCTACTGE K A E D C W E L Q I S P E L L

32 om etitor - I- - + + - I + - + +Lane 1 21 3 4 5_ 7 5 91011A DS

80

128

176

224

44

27292

X-OBP-2 GCTCATGGGCGCCAAAAAATACTGGATCTGCTGAACGAAGGCTCAGCC 320X-OBP-1 GCTCATGGGCGCCAAAAAATACTGGATCTGCTGAACGAAGGCTCAGCC 140

A H G R Q K I L D L L N E G S* A

R D L R S L O R I G P K K A Q L

ATCGTGGGCTGGCGGGAGCTCCACGGCCCCTTCAGCCAGGTGGAGGACI V G W R E L H G P F S* Q V E D

CTGGAACGCGTGGAGGGCATAACGGGGAAACAGATGGAGTCCTTCCTGCTGGAACGCGTGGAGGGCATAACGGGGAAkCAGATGGAGTCCTTCCTGL E R V E G I T* G K Q M E S F L

AAGGCAAACATCCTGGGTCTCGCCGCCGGCCAGCGCTGTGGCGCCTCCAAGGCAAACATCCTGGGTCTCGCCGCCGGCCAGCGCTGTGGCGCCTCCK A N I L G L A A G Q R C G A S

TGACCGTCGTCTCCTCACTCCGCCTTTTCAAATTTTTGTATAACCCCG

B FR

368188

416236

464284

AA_a L512332

C 0560380

X-OBP-2 TGTTGTGTAAATACAGTTTTTGCTCCGGTAAAAAAAAAAAAAAAAAX-OBP-1 TGTTGTGTAAATACAGTTTTTGCTCCGGTGAAAAAAAAAAAAAA

FIG. 2. Nucleotide sequences of A-OBP-1 and A-OBP-2 andamino acid sequences deduced from them. The cDNA inserts inA-OBP-1 and A-OBP-2 phage clones were purified and subcloned intothe pBluescript SK(+/-) vector (Stratagene). Sequencing in bothdirections was performed by the dideoxy chain-termination proce-dure (19) using a Sequenase kit (United States Biochemical), initi-ating SK and KS primers for chain elongation, and specific syntheticoligonucleotides prepared with an Applied Biosystems 380B DNAsynthesizer as the continuing primers. Ambiguous sequences wereconfirmed with a thermostable (65-70°C) DNA polymerase fromBacillus caldotenax by use of a Ladderman DNA sequencing kit(Takara Biochemical, Berkeley, CA). The EcoRI adaptor is given inlowercase letters. The shared sequence is given in bold type. Theamino acid sequence (one-letter symbols) was predicted from theavailable nucleotide sequence by use of the TRANSL program (Intel-liGenetics). Potential phosphorylation sites are denoted by stars.Regions with helical conformation are underlined. Stop codon isshown by three dashes.

transcripts corresponding to 1.4, 2.4, and 3.4 kb were de-tected by the A-OBP-1 probe, of which the 2.4-kb transcriptwas most abundant. All the transcripts were more abundantin the TPA-induced BJAB cells than in the untreated BJABcells (Fig. 4A). Likewise, all three transcripts detected by theA-OBP-1 probe were more abundant in TPA-activated Rajicells than in the Raji controls (Fig. 4B).To study the relationship between the OBP mRNA species

and the four partial cDNA clones, oligonucleotide sequencesunique to the four cDNA clones located at the 5' endupstream ofcDNAs were used as labeled probes in Northernblot analyses of poly(A)+ RNA from TPA-activated BJABcells. The unique sequence for A-OBP-1 detected a 1.4-kbmRNA (Fig. SB, lane A), whereas unique sequence forOBP-2 detected a 3.4-kb mRNA (lane D). In addition, a2.4-kb mRNA was detected by both the OBP-1 (lane B) andthe A-OBP-2 (lane C) probe.To confirm the genomic location ofOBP transcripts, BJAB

genomic DNA was digested with BamHI, EcoRI, or HindIIIrestriction enzyme and hybridized with the A-OBP-1 probe.Southern blot analyses of the BJAB genomic DNA gave a

FIG. 3. Analyses of the polypeptides encoded by A-gtll, A-EB,A-OBP-1, and A-OBP-2 by gel mobility-shift assay. Extracts fromAgtll (lane 2), A-EB (lanes 3-5), A-OBP-1 (lanes 6-8), and A-OBP-2(lanes 9-11) lysogens were prepared as described (20). Each lysogenextract was incubated with a 32P-labeled DNA probe containing the140-bp DS from the pEC140 plasmid (A), the EcoRI-BamHI frag-ment from pBSFR2 containing two copies of the 30-bp repeats ofFR(B), and a 260-bp PCR product containing the enhancer region of theEBV Fp promoter from the BamHI Q restriction fragment of EBV(C). Synthetic oligonucleotide primers 1 (5'-GGATCCGGAGGG-GACCACTAG-3') and 2 (5'-GCATGCGGGTCCCCAAACATA-3')specific to the BamHI Q fragment (8) of the B95-8 strain of EBV andtotal genomic DNA (template) from Raji cells were used to generatethe 260-bp DNA fragment by 30 cycles of PCR; each cycle consistedof 1 min at 95°C, 2 min at 55°C, and 3 min at 72°C in a Perkin-Elmer/Cetus DNA Thermal Cycler. After the 30th cycle, the mixturewas held at 72°C for 15 min to ensure complete polymerization. Theprotein-DNA complexes formed were resolved as described (21, 22).+ and + + indicate 25-fold and 100-fold molar excess, respectively,of unlabeled homologous competitors.

single hybridization band (Fig. 5A). These results, togetherwith sequence analysis of cDNA and the Northern blothybridization, indicate that the three mRNAs and the fourpartial cDNA clones are splicing products from the samegene family.

DISCUSSIONHuman B lymphocytes immortalized by EBV and EBV-positive Burkitt lymphoma cells usually express EBNA-1(27, 28). During EBV latency, EBNA-1 binding to oriPmaintains the viral genome in these lymphoblastoid cells asstable plasmids (1-6). The EBNA-1-oriP interaction also

X-OBP-2

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X-OBP-2

X-OBP-1

X-OBP-2X-OBP-1

X-OBP-2X-OBP-1

X-OBP-2X-OBP -1

X-OBP-2X-OBP-1

X-OBP-2X-OBP-1

X-OBP-2X-OBP-1

606426

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BJAB-CL2-6

m---i_ + TPA

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FIG. 4. Northern blot detection of OBP transcripts in AfricanBurkitt lymphoma cell lines. Poly(A)+ RNA was isolated fromEBV-negative BJAB cells (A) or EBV-positive Raji cells (B) treatedor not treated with TPA as previously described (25). Poly(A)+ RNA(3 Mg) was electrophoresed through a 1% agarose gel after denatur-ation with 1 M glyoxal in aqueous 50%o dimethyl sulfoxide (26) andtransferred to nylon membranes (Nytron, Schleicher & Schuell). Theblots were probed with an [a-32P]dCTP-labeled DNA randomlyprimed from the Sfi I-Not I subfragment (426 nucleotides andcontaining the shared sequence as shown in Fig. 2) from A-OBP-1(100 ng; specific activity, 105 cpm/ng) and exposed to x-ray film for16 hr at -800C. The membrane was then stripped by treatment with0.1% SDS and reprobed with a 32P-labeled P-actin cDNA (100 ng;specific activity, 8.5 x 10W cpm/ng) as internal control (C and D), andexposed to x-ray film for 1 hr at room temperature. Transcripts of 1.4,2.4, and 3.4 kb were detected by A-OBP-1. RNA size standards (inkilobases) are indicated.

modulates the expression of EBNA-1 and other members ofthe EBNA family from the BCR2 promoter (13, 14). Treat-ment of these latently infected cells with TPA can interruptlatency and activate the viral genome, presumably throughthe activation of the EBV BZLF-1 gene (29, 30). Thislaboratory has previously reported that treatment of Burkittlymphoma cells with TPA induces expression of cellularOBPs (15, 31). These OBPs interfere with the binding ofEBNA-1 to oriP (15, 31). We have exploited this specific FR-and DS-binding characteristic of the OBPs to isolate cDNAclones expressing fusion proteins that bind to oriP. A-OBP-1,A-OBP-2, OBP-1, and OBP-2 are partial cDNA clones whichshare the 395 nucleotides at the 3' end. Analysis of theavailable nucleotide sequences revealed no homology withany known cDNA or gene. Northern blot analyses ofmRNAfrom BJAB cells, using unique sequences from each clone aslabeled probes, showed that the A-OBP-1 clone was derivedfrom a 1.4-kb mRNA, and OBP-2 from a 3.4-kb message,whereas A-OBP-2 and OBP-1 most likely were derived fromthe same 2.4-kb transcript. Southern blot analysis of humangenomic DNA, however, detected a single band. Takentogether, these observations suggest that the OBPs are en-coded by differentially spliced transcripts derived from asingle gene. The sharing of 395 bp of sequence encoding theC-termini gave rise to their shared oriP DNA-binding do-main. Of note is the ability of each of these proteins to bindto the FR and DS region of the EBV oriP and to the enhancer

FIG. 5. Hybridization ofDNA and mRNA from BJAB cells withOBP cDNAs as probes. (A) Southern blot analysis. Genomic DNAwas isolated from EBV-negative BJAB cells. Restriction digests ofgenomic DNA were resolved in a 0.7% agarose gel and transferredto nylon membranes (Nytran; Schleicher & Schuell). Hybridizationwas performed with the same A-OBP-1 cDNA probe (containing theshared DNA sequence) described in the legend of Fig. 4. DNA sizemarkers (in kilobase pairs) are indicated at left. (B) Northern blotanalysis by use of oligonucleotide probes. Synthetic oligonucleotideprobes (21-mers) unique to A-OBP-1 (lane A), A-OBP-2 (lane B),OBP-1 (lane C), and OBP-2 (lane D), because they did not contain theshared DNA sequence, were end-labeled with [y-32PIATP. Poly(A)+RNA from BJAB cells was prepared and electrophoresed as de-scribed in the legend of Fig. 4. After transfer, membranes werehybridized with probe in the presence of 6x standard saline citrate(SSC)/5x Denhardt's solution, 0.15% sodium pyrophosphate/0.1%SDS containing denatured and sheared salmon sperm DNA (100pg/ml). Filters were washed in 0.5x SSC/0.1% SDS at 6(YC beforeexposure to x-ray film at -80oC for various times: lane A, 72 hr; lanesB and C, 16 hr; lane D, 36 hr.

sequence in the EBV BamHI Q fragment as well. Preliminarystudies of DNase I footprinting suggested that the binding ofA-OBP-1 to FR was comparable to what has been previouslyreported for EBNA-1 (7). It will be interesting to see whetherthese OBPs also protect the same DS and BamHI Q fragmentfrom DNase I digestion as does EBNA-1. The function(s) ofthese OBPs is unclear. Detection of the constitutive expres-sion of OBP mRNA in Raji cells also suggests that the OBPsmay interact with EBNA-1 and play a role in maintaining theEBV genome as stable plasmids. Treatment with TPA en-hances the expression of OBPs. OBPs in excess may disruptthis OBP-EBNA-1 interaction and lead to destabilization ofthe EBV genome as plasmids (15, 31) and/or the dysregula-tion of the EBNA-1 modulation of the expression of theEBNA gene family.The ability ofa virus to survive evolutionary pressure often

depends on its ability to insinuate its life cycle into theregulatory pathway ofcellular proliferation and function. Ourfinding that the OBPs are derived from spliced cDNAs of agene family and that they bind to the same regions of EBVDNA as EBNA-1 suggests that like many latent viruses, EBVhas the uncanny ability.to adapt its life cycle to the regulationof cellular factors. Likewise, the identification ofthese OBPsbelonging to a cellular gene family raises the possibility thatoriP-like cis-acting sites may prevail in cellular DNA. Theinteraction of OBPs with these sites may regulate the repli-cation or transcription of these cellular DNA. It will beimportant to isolate the full-length cDNAs of these OBPs,

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determine precisely the number of members in the OBPfamily of proteins, and study their role in the EBV life cycleas well as their normal physiological functions.

We thank J. Donovan for excellent technical assistance. We thankDr. H. Singh for the plasmid pUCoriP2 and for the Agtll-EBNA-1recombinant A-EB. We are grateful to Drs. A. Tanaka and G. Bradleyfor their generous advice and many helpful discussions throughoutthe course of this work. We thank Dr. Patrick K. Lai for hisassistance in preparing the manuscript and for his critical review ofit.

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