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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).
2843
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-
CL_ ~ ~~~~~~~~~~,.;@La
~~~~~~~.
CM ;.CL * .*"
;~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
X-OBP-2
X-OBP-1
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
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2846 Medical Sciences: Zhang and Nonoyama
BJAB-CL2-6
m---i_ + TPA
(Kb)-9.49-
-7.46-
-4.40-
...
-2.37-
-1.5-._
- 1.35-
A
ARaji(P)m cO E E
Lu m
(Kbp)
B
lane A(Kbp):9.49
7,46 --23.1-
9.4- _
........
6.6_4.40..I
.pb
2.37 -
4.4-
1 35-
2.3-
2.0 -
I.
B
B 1C "D--ti
.
_100 _wo 3-Actin
C D
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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