Department of Clinical Chemistry and
Transfusion Medicine, Sahlgrenska University Hospital,
Göteborg University, S-413 45 Göteborg, Sweden
The identification of the cellular factors that control the
transcription regulatory activity of the Epstein-Barr virus C promoter
(Cp) is fundamental to the understanding of the molecular mechanisms
that control virus latent gene expression. Using transient transfection
of reporter plasmids in group I phenotype B-lymphoid cells, we have
previously shown that the
248 to
55 region (
248/
55 region) of
Cp contains elements that are essential for
oriPI-EBNA1-dependent as well as
oriPI-EBNA1-independent activation of the promoter. We
now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together
with or without oriPI. The reporter plasmids were
transfected into group I phenotype Rael cells and group III phenotype
cbc-Rael cells, and the Cp activity measured was correlated with the
binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We
show that the NF-Y transcription factor interacts with the previously
identified CCAAT box in the
71/
63 Cp region (M. T. Puglielli,
M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758-5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the
119/
112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence
in the
99/
91 Cp region that is essential for
oriPI-EBNA1-independent as well as
oriPI-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our
results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to oriPI contains only the
111/+76 region of Cp still retains the ability to be activated by EBNA1.
 |
INTRODUCTION |
Epstein-Barr virus (EBV) is a
human lymphotropic herpesvirus that is the etiologic agent of
infectious mononucleosis, a self-limiting lymphoproliferative disorder
(35). In addition, EBV is associated with various
malignancies, including Burkitt's lymphoma (BL), Hodgkin's disease,
nasopharyngeal carcinoma (NPC), and lymphoma in immunocompromised
individuals (23). The virus can infect and establish
latency in B lymphocytes and is capable of adopting four programs of
latency (latency 0, I, II, and III). In healthy individuals, latent EBV
infection appears to be primarily confined to resting memory B cells
(3). The only EBV gene product that is consistently
detected in these cells is LMP2A, a pattern of gene expression at
present termed latency 0 (33, 34, 57). In BL biopsies,
only EBV nuclear antigen 1 (EBNA1) is expressed (latency I) (44,
57). In Hodgkin's disease, NPC, and T-cell lymphomas, EBNA1 and
variable combinations of the three members of the latent membrane
protein family (LMP1, LMP2A, and LMP2B) are expressed (latency II)
(44, 57). During acute infectious mononucleosis, in
lymphoproliferative syndromes in immunocompromised individuals, and in
lymphoblastoid cell lines (LCLs), all six nuclear antigens (EBNA1 to
EBNA6) are expressed (latency III) (5). In addition, all
three LMPs are expressed (20). Cell lines established from
BL cells can be divided into three groups (BL I to BL III) depending on
the expression of different B-lineage restricted surface antigens
(48, 49). Group I BL cell lines retain the phenotype of
the original biopsy cell, whereas group II and III BL cell lines
express the full spectrum of B-cell activation signals and adhesion
molecules. EBV-positive group I BL cell lines present a type I pattern
of latent viral gene expression. EBV-positive group II BL cell lines
express viral genes in a way that is intermediate between type II and
III latency. Viral gene expression in group III BL cell lines and group
III phenotype LCLs is an example of type III latency (44,
49).
After in vitro infection of B lymphocytes, EBNA5 and EBNA2 are the
first EBV genes expressed from a bicistronic transcription unit under
transcriptional control of the W promoter (Wp) in the BamHI
W repeat region of the EBV genome (1, 2, 47, 61). Within
36 h there is a switch in promoter usage from Wp to the upstream C
promoter (Cp) in the BamHI C region (62). EBNA2
appears to be required for the Wp-to-Cp switch (22, 56, 67,
68). One study supports a model in which EBNA1, expressed by an
unknown mechanism at early time points postinfection, also plays an
important role in the cascade of events that leads to successful
switching from Wp to Cp (53). However, from this study it
is unclear whether EBNA1 expression is obligate for the initial
upregulation of Cp. Transcription from Cp leads to a concomitant
expression of all EBNAs from a polycistronic transcription unit that is
spliced to yield the different EBNA molecules (5). The
restricted pattern of EBV gene expression in group I BL and NPC cells
is associated with a downregulation of Cp and Wp due to
hypermethylation (21, 31) and a parallel activation of the
Q promoter in the BamHI Q region for selective EBNA1 gene
transcription (52, 59, 70). The genome of EBV is
maintained in latently infected cells as an episome. EBNA1 activates
DNA replication once every cell cycle from the EBV origin of DNA
replication, oriP (64-66). The expression of
EBNA1 is thus essential to prevent loss of the EBV genome during multiple cell divisions, and EBNA1 is consistently detected in all
types of virus latency in growing cells (23).
oriP consists of two subelements, the family of repeats and
the dyad symmetry, also termed oriPI and oriPII,
respectively (41, 43). oriPI comprises 20 copies of a 30-bp repeat that contains the EBNA1 binding motif and acts
as an EBNA1-dependent enhancer of transcription from heterologous
promoters (42). Notably, EBNA1 does not appear to contain
any transactivating domains, but it has been observed to participate in
homotypic and heterotypic protein interactions (15, 16)
and DNA linking (28).
The regulation of the Cp promoter has been the subject of several
investigations, and positive cis-acting transcription
regulatory elements have been identified in the regions upstream and
downstream of Cp (Fig. 1). oriPI in cis in
conjunction with EBNA1 in trans activates Cp
(55) and are essential for significant transcription from
both Cp and Wp in LCLs (36, 40). A
glucocorticoid-responsive element (GRE) has been identified in the Cp
upstream region (26). A third cis element
identified upstream of Cp is the EBNA2-responsive enhancer (E2RE)
(22, 56, 68). Cp also appears to require a properly
positioned CCAAT box for optimal activity in LCLs (40). A
weakly positive cis element has been identified within the
sequences from +2680 to +2880 relative to the Cp transcription
initiation site (39). A recent analysis of the Cp region
in the genomes of two primate lymphocryptoviruses and the alignment of
these sequences with that of EBV Cp has demonstrated preservation of the GRE, the E2RE, and the CCAAT box (17). It is
interesting, however, that the comparison revealed several additional
conserved stretches of nucleotides, suggesting that there might be
other candidate regulatory elements in this region.
The starting point of the present investigation was our previous
observation that the
248 to
55 region (
248/
55 region) of Cp
contains transcription regulatory elements that activate reporter
plasmids in group I phenotype cells. We also showed that the same
region seems to be involved in oriPI-EBNA1-induced promoter activation (36). Our finding that Cp exhibits a
significant oriPI-EBNA1-independent activity in group I
phenotype cells is compatible with the hypothesis that an
EBNA1-independent Wp-to-Cp switch might occur in newly infected cells
that have not yet adopted the characteristics of a group III phenotype.
In the present report we describe the results of a detailed mutational
analysis of the
248/
55 Cp region. We have identified
promoter-proximal transcriptional elements involved in the activation
of Cp both in an oriPI-EBNA1-dependent and an
oriPI-EBNA1-independent manner in group I and group III B
lymphoid cells.
 |
MATERIALS AND METHODS |
Plasmid constructions.
All constructs made were verified by
dideoxy sequencing (51) utilizing the Sequenase system
(United States Biochemical Corp., Cleveland, Ohio) or the ABI PRISM Big
Dye terminator cycle sequencing kit (PE Applied Biosystems). The series
of chloramphenicol acetyltransferase (CAT) reporter plasmids with
different 5' deletions of Cp has been described previously
(36). The Cp fragment together with the CAT gene was
transferred from these constructs as a SalI-BamHI fragment into the pGEM-3zf(+) plasmid (14). The names of
the resulting plasmids were changed as follows: p
C1CAT to
pgCp(
55)CAT, p
C2CAT to pgCp(
248)CAT, p
C3CAT
to pgCp(
1024)CAT, and p
C4CAT to pgCp(
3889)CAT.
The Cp transcription initiation site at position 11336 of strain B95-8
EBV DNA (4) was numbered as +1. Two more 5' deletion
fragments were made, pgCp(
111)CAT and pgCp(
170)CAT, as AluI-SfiI (nucleotides 11225 to 11412) and
MvaI-SfiI (nucleotides 11166 to 11412)
restriction fragments, respectively, and subcloned into the unique
HindIII site immediately upstream of the CAT gene. The
3' end of the subcloned fragments all contained 76 bp downstream of the
Cp transcription initiation site (+76). The plasmids
pgCp(
112)CAT, pgCp(
122)CAT, pgCp(
132)CAT,
and pgCp(
144)CAT were made by synthesis of double-stranded
oligonucleotides that contained sequences from positions
112,
122,
132, and
144, respectively, to the Bsu36I site at
position
55 in Cp. The oligonucleotides contained a SalI site in the 5' end and a Bsu36I site in the 3' end. They
were cloned in pgCp(
111)CAT, replacing the
111/
55 region
between the unique SalI and Bsu36I sites.
A series of pgCp(
170)CAT derivatives with different internal
mutations, pgCp(
170/m1)CAT to pgCp(
170/m12)CAT, was
made by synthesizing four pairs of overlapping, complementary
oligonucleotides for each construct, with a SalI site at the
5' end (
170Cp) and a Bsu36I site at the 3' end (
55Cp).
These double-stranded oligonucleotides were ligated into a
SalI-Bsu36I opened pgCp(
248)CAT,
replacing the
248/
55 region. Mutations were introduced by using
oligonucleotides with purine-pyrimidine transversions in defined
regions. (The positions and sequences of the mutations are shown in
Fig. 3 and Table 1.) One spontaneous internal deletion mutant that
lacked the sequence between
140 and
74 was found and included in
the mutation series as Del-1. To make CAT reporter plasmids with
specific Sp and Egr mutations, PCR amplifications in two steps were
performed with the pgCp(
170)CAT as a template. Primers with
specific mutations in the Egr site (ACG to GAT in positions
94 to
92) and the three different Sp sites (CGG to ATT in positions
166
to
164, GCG to TAT in positions
140 to
138, and G to T in
position
98) were used. The first round of amplifications was
performed with the M13 reverse sequence primer in the pGEM-3zf(+)
vector 5' of the insert and a 3' primer including the desired mutation,
in parallel with a 5' primer including the mutation together with a 3'
primer in the CAT gene. These two overlapping amplicons were denatured,
annealed, and elongated in five cycles and subjected to a second round
of amplifications with the M13 reverse sequence primer and the primer
in the CAT gene. The resulting fragment was digested with
SalI and Bsu36I, isolated, and ligated into a
SalI-Bsu36I-opened pgCp(
248)CAT,
replacing the
248/
55 region and yielding
pgCp(
170/m13)CAT, pgCp(
170/m14)CAT,
pgCp(
170/m15)CAT, pgCp(
170/m16)CAT, and
pgCp(
170/m17)CAT (Table 1). To study sequence motifs
important for the oriPI-EBNA1-mediated activation of Cp, the
family of repeats of oriP (oriPI) was excised as
an EcoRI-SmaI fragment corresponding to the
4021/
3146 Cp region. PstI linkers were added, and the
fragment was ligated to the unique PstI site of the pgCAT
reporter constructs that contained the different deleted or mutated Cp
fragments, yielding the pgCp(oriPI/...)CAT series of
plasmids, where the dots in parentheses stand for the different Cp
fragments (see Fig. 7). The deletion of positions
247 to
57 and the
introduction of a 3-bp mutation in the Sp binding site in positions
308 to
306 (GCG to TAT) was made in a combined PCR with two pairs
of primers. The mutated region was excised as a 190-bp
AvrII-Bsu36I fragment and subcloned in two steps
into the pgCp(
3889)CAT reporter construct. The new plasmids were designated pgCp(
3889/Del-2)CAT for the construct
containing only the
247/
57 deletion and pgCp(
3889/Del-2/m18)
CAT for the one containing both the deletion and the 3-bp mutation in
the Sp site.
The expression vector for Egr-1 was made by using a random hexamer
primer to synthesize cDNA from total RNA prepared from cbc-Rael cells
as described by Chirgwin et al. (7). This cDNA was
amplified in two steps with Egr-1-specific primers. In the second round
of amplifications, the 5' primer contained an EcoRI site and
the 3' primer contained a SalI site. The PCR products were
digested with EcoRI and SalI and separated on an
agarose gel, and the band corresponding to the expected full length was excised and isolated by isotachophoresis (37). The
purified fragment was cloned into the pCI expression vector (Promega,
Madison, Wis.) directly in the right orientation under the control of
the cytomegalovirus immediate-early enhancer/promoter. Protein
expression was verified by transfection of Rael cells, and analysis of
cell extracts was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (data not shown). The pPacSp1, pPacSp3, and parent pPac expression vectors for transfection of SL2
insect cells were kindly provided by G. Suske (Klinikum der Philipps-Universität Marburg, Marburg, Germany). The EBNA1
expression vector was constructed by utilization of the pPacSp1 that
contains the Drosophila ubx leader for efficient
gene expression. The EBNA1-encoding sequence was prepared as two
fragments divided by the unique NcoI site at position 108064 in the B95-8 genome (4). The first 160 bp of the EBNA1
exon was PCR amplified, introducing an XhoI site 5' of codon
11 of the EBNA1 gene (position 107980 in the EBV genome). A cloned
BamHI K fragment of the EBV genome was used as a template,
and the sequence of the 5' PCR primer was
5'-CGGGATCCCTCGAGGGAAATGGCCTAGGAGAGA-3' (the EBV
sequence is underlined). The amplified material was cleaved with
XhoI and NcoI, generating a 90-bp fragment. An
NcoI-AccI fragment (nucleotides 108064 to 109951)
was excised from the BamHI K fragment, and XhoI
linkers were added to the AccI end. The two fragments were
ligated at the NcoI site and cloned into the
XhoI-digested pPacSp1 plasmid, replacing the Sp1 gene.
The plasmid was designated pPacEBNA1. EBNA1 expression was
verified by transfection of SL2 cells and analysis of cell extracts by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
immunoblotting (data not shown). Intranuclear expression of EBNA1 in
transfected SL2 cells was confirmed by immunostaining using polyclonal
human serum CW with high titers of antibody to EBNA1, diluted 1:10,
1:50, and 1:200, and fluorescein isothiocyanate-conjugated secondary
(F0202; DAKO, Glostrup, Denmark) and tertiary (F0205; DAKO) antibodies
diluted 1:50 (data not shown).
Cell culture, transient transfections, and CAT assay.
Rael
is an EBV-positive BL cell line with a group I phenotype
(24). The cbc-Rael line was obtained by in vitro infection of cord blood cells with the Rael virus strain and has a group III
phenotype (13). Cells were grown as suspension cultures in
RPMI 1640 (Gibco, Life Technologies Inc., Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco, Life Technologies Inc.), streptomycin, and penicillin. Schneider's Drosophila
line 2 (SL2) is a Drosophila cell line that lacks endogenous
Sp factors (9). The SL2 cells were cultured in room
temperature in Schneider's Drosophila medium (Gibco, Life
Technologies Inc.) supplemented with 10% fetal calf serum (Gibco, Life
Technologies Inc.), streptomycin, and penicillin. The Rael cell line
was transfected by the DEAE-dextran method (30) or by
electroporation (38) with a Gene Pulser system (Bio-Rad
laboratories, Hercules, Calif.). Transfections were performed with
5 × 106 cells and 10 µg of the reporter
plasmids. The cbc-Rael cell line was transfected by electroporation
(38) with a Gene Pulser system using 7 × 106 cells and 14 or 20 µg of the reporter
plasmids. Three days after transfection, the cells were harvested. Cell
extracts were prepared by three rounds of freezing and thawing and
analyzed for CAT activity as described by Ricksten et al.
(45). Storage phosphor screens were exposed, scanned in a
PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.), and quantified
using ImageQuant software (Molecular Dynamics). The SL2 cells were
cotransfected with 10 µg of different CAT reporter constructs and
various amounts of either pPacSp1, pPacSp3, pPacEBNA1 or the parent
pPac in 60-mm dishes using the calcium phosphate-DNA precipitation
method (11). Cells were harvested 2 days after
transfection. Cell extracts were prepared and the CAT activity was
analyzed as described above.
EMSAs.
Nuclear extracts were prepared essentially as
described by Dignam et al. (10) except that antipain (5 µg/ml), leupeptin (5 µg/ml), and aprotinin (2 µg/ml) were added
to the buffer in the final homogenization and dialysis steps and
phenylmethylsulfonyl fluoride was replaced by Pefabloc (0.5 mM).
Aliquots were frozen in liquid nitrogen and stored at
80°C. Three
different double-stranded, blunt-ended, synthetic oligonucleotides were
used as probes in electrophoretic mobility shift assay (EMSAs):
(
80/
55)Cp (nucleotides 11256 to 11281), (
107/
84)Cp (nucleotides
11229 to 11252), and (
150/
105)Cp (nucleotides 11186 to 11231). In
competition experiments, the following double-stranded consensus
oligonucleotides were used: AP-2/Sp consensus,
5'-ACGGGCCGCGGGCGGTCAGTTCGATC-3'; AP-1 consensus,
5'-CGCTTGATGACTCAGCCGGAA-3'; C/EBP consensus,
5'-TGCAGATTGCGCAATCTGCA-3'; PEA3 consensus,
5'-GTATCTAAGGAAGTAGATAC-3'; Myb consensus,
5'-ATCACGTCAGTTATCTGCAT-3'; and Egr consensus,
5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3'. The nonspecific competitor was either an unrelated DNA sequence or, in the case of the
(
150/
105)Cp probe, the
150/
105 region of Cp transversely mutated. One strand of the oligonucleotide probe was labeled with [
-32P]ATP (6,000 Ci/mmol; NEN Life Science
Products, Brussels, Belgium) using polynucleotide kinase (Boehringer
Mannheim GmbH, Mannheim, Germany) and annealed to the complementary
strand. The labeled probe was purified by electrophoresis in an 8%
polyacrylamide gel in TBE (0.1 M Tris, 0.1 M boric acid, 2 mM EDTA; pH
8.3). The wet gel was autoradiographed, and the DNA fragment was
excised, electroeluted by isotachophoresis (37), and
precipitated. Binding reactions were carried out in a volume of 30 µl
containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, various amounts of poly(dA-dT) or
poly(dI-dC) (2 to 4 µg), 5 fmol of labeled probe (approximately
70,000 cpm), and various amounts of crude nuclear extracts from Rael or
cbc-Rael cells (5 to 10 µg). In competition experiments, 1 to 10 pmol
of unlabeled oligonucleotides was added to the reaction mixture. After
incubation at room temperature for 30 min, the samples were loaded on a
6% polyacrylamide gel (acrylamide-bisacrylamide, 29:1) in TGE (25 mM
Tris-HCl, 0.19 M glycin, 1 mM EDTA; pH 8.3). After electrophoresis,
gels were dried and autoradiographed. The supershift experiments were
performed as described above for the EMSAs except that 2 to 10 µl of
the respective antibody was added after the incubation at room
temperature. A second incubation was carried out at 4°C for 2 h
before the samples were loaded onto the gel. Antibodies used for
supershift experiments were NF-Y (PharMingen, San Diego, Calif.), NF-1
(sc-870X), YY1 (sc-1703X), Sp1 (sc-59X), Sp3 (sc-644X), Egr-1
(sc-110X), Egr-2 (sc-190X), C/EBP
(sc-61X), C/EBP
(sc-150X),
C/EBP
(sc-7659X), C/EBP
(sc-636X), and C/EBP
(sc-158X) (Santa
Cruz Biotechnology Inc., Santa Cruz, Calif.).
 |
RESULTS |
Activity of proximal Cp sequence elements in group I and III B
cells.
In a previous report it was shown that the
248/
55
region of the Cp (BCR2) promoter contains transcriptional elements that activate reporter plasmids in cells expressing BL group I phenotype and
are involved in oriPI-EBNA1-induced promoter activation
(36). This region was subjected to further analysis by
creating a series of reporter plasmids, pgCp(
3889)CAT,
pgCp(
1024)CAT, pgCp(
248)CAT, pgCp(
170)CAT, pgCp(
144)CAT,
pgCp(
132)CAT, pgCp(
122)CAT,
pgCp(
111)CAT, and pgCp(
55)CAT, containing
5'-deletion-containing fragments of the Cp region. The reporter
plasmids were transfected into the group I phenotype Rael cell line and
the group III phenotype cbc-Rael cell line. The plasmid with the
shortest fragment that could still induce expression of the reporter
gene in Rael cells contained the
144/+76 part of the Cp region (Fig.
2). The activity of the reporter plasmids
increased with increasing length of the fragments to reach a maximum
level with the pgCp(
3889)CAT construct, which encompassed
oriPI in its natural configuration. Moreover, pgCp(
3889)CAT was the only plasmid in the deletion series
that displayed significant reporter gene expression in cbc-Rael cells (Fig. 2), corroborating our earlier observation that oriPI
is essential for detectable Cp activity in cells expressing a group III
phenotype (36).
To identify the individual transcription factors and the corresponding
binding sites important for the activity of the
144/+76 Cp construct
in the Rael cell line, a series of derivatives of the
pgCp(
170)CAT plasmid with point
mutations at selected positions (Table 1
and Fig. 3A) was created. As a generic
term we used the designations pgCp(
170/m1)CAT to
pgCp(
170/m12)CAT, where m1 to m12 are mutant designations.
The activity of the resulting plasmids in Rael cells was reduced or
abolished by several of the mutations (Fig. 3B). The point mutations in
the m3-, m4-, m5-, m7-, m10-, and m11-containing plasmids and the
deletion mutation in the Del-1 plasmid reduced the CAT activity to
background levels. Mutations in the m1 and m6 constructs reduced the
activity to approximately 60 and 35%, respectively, of that of the
wild type. The results are consistent with the notion that the
142/
92 and
71/
63 regions of Cp contain elements that are
essential for promoter activity in BL group I cells. A database search
revealed the presence of possible binding sites for several
transcription factors in the two regions (Fig. 3A).

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FIG. 1.
Sequences involved in the regulation of the EBV
Cp. (A) Schematic illustration of the region upstream of Cp. The
sequence coordinates are from the DNA sequence of the B95-8 EBV genome
(4). The bent arrow indicates the Cp transcription
initiation site at position 11336. Boxes indicate the positions of
previously identified cis-acting elements, including
oriP, which is composed of oriPI (family
of repeats [FR]) and oriPII (dyad symmetry [DS]), a
GRE, and the E2RE. (B) Fragments of the region upstream of Cp present
in different CAT reporter plasmids. (C) Detailed map of upstream
Cp-proximal transcriptional elements described in this paper and their
relation to fragments present in the different CAT reporter plasmids.
Open boxes indicate putative transcriptional elements. Numbers in
panels B and C are positions in relation to the Cp transcription
initiation site (+1).
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FIG. 2.
Activity of pgCp(...)CAT reporter plasmids in
the Rael and cbc-Rael cell lines. CAT activities are percentages of the
activity obtained with pgCp( 3889)CAT. The 100% values
correspond to acetylation of 16.2 and 1.4%, respectively, of the
substrate; the background level was 0.2%. The values are means of at
least two independent transfection series with double samples. Error
bars indicate standard errors of the means.
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FIG. 3.
Transcriptional elements involved in the regulation of
Cp. (A) Sequence of the promoter-proximal region between positions
171 and 55 relative to the Cp transcription initiation site (+1).
Boxes indicate putative transcriptional elements. The corresponding
transcription factors shown to bind to the regions are indicated at the
top of the panel. Del-1 was the result of a spontaneous internal
deletion between positions 140 and 74. The unmutated sequence of
Del-1 is represented by a solid line and the deleted sequence is
represented by a dotted line. (B) Activity of
pgCp( 170/...)CAT reporter plasmids (Table 1 and Fig. 3A)
in the Rael cell line. CAT activities are percentages of the activity
obtained with the wild-type pgCp( 170)CAT. The 100% value
corresponds to acetylation of 3.6% of the substrate; the background
level was 0.2%. The values are means of five independent transfection
series. Error bars indicate standard errors of the means.
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|
Sp1 binding site in the
141/
136 region of Cp.
A putative
Sp factor binding site was found at positions
141/
136 of the Cp
region. EMSA analysis using Rael nuclear extracts and a probe
corresponding to the
150/
105 Cp region revealed six specific
complexes (Fig. 4A). A minor band was
assumed to correspond to a nonspecific complex since it did not
disappear after the addition of excess amounts of unlabeled probe in
competition experiments (Fig. 4A, lane 2). Competition experiments with
an excess of an oligonucleotide that contained a consensus Sp binding site suggested that at least three of the bands contained factors that
belong to the Sp family. The same bands were abolished by competition
with a (
150/
125)Cp oligonucleotide. The two fastest-moving bands
were not affected by competition with the (
150/
125)Cp oligonucleotide but were removed by a (
130/
105)Cp oligonucleotide as well as a C/EBP consensus oligonucleotide. The slowest-moving minor
band displayed the same behavior. To establish the identity of the
factors in the complexes, antibody supershift analysis was performed.
Both anti-Sp1 and anti-Sp3 antibodies gave rise to supershifted bands
(Fig. 4B, lanes 2 and 3). cbc-Rael nuclear extract gave rise to the
same binding pattern regarding the Sp factor complexes (data not
shown). It should be noted that a putative Sp site was also found at
positions
168 to
163. The site was investigated with EMSA using a
probe corresponding to the region from position
171 to
143.
Antibody supershift analysis showed one specific Sp1-containing complex
and two specific Sp3-containing complexes with nuclear extracts from
Rael and cbc-Rael (data not shown). However, judging from the results
of the deletion and site-directed mutational analyses described above,
this Sp site was not essential for Cp activity.

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FIG. 4.
Identification of transcription factors that interact
with the Sp and C/EBP motifs in the 150/ 105 region in the group I
BL cell line Rael. A 32P-labeled double-stranded synthetic
oligonucleotide corresponding to the 150/ 105 region was incubated
with nuclear extracts from Rael cells. The reaction mixtures were
analyzed by EMSA. (A) Competition experiments. Lane 1, binding pattern
obtained with the nuclear extract. Competition reactions were carried
out with an excess of unlabeled competitors as indicated below the
autoradiogram and as described in Materials and Methods. Six complexes
(solid arrows) are considered specific and designated Sp and C/EBP,
since they were abolished by Sp and C/EBP consensus sequences. One
noncompetable, nonspecific band is indicated by a dashed arrow. (B)
Supershift analysis. Seven complexes (solid arrows) are considered
specific and designated Sp1, Sp3, and C/EBP. Identification of the
C/EBP isoform that bind to the 119/ 112 sequence was determined by
addition of specific antibodies to C/EBP , - , - , - , and
- , respectively. The positions of the antibody-shifted complexes are
shown by arrowheads. The dashed arrow indicates a nonspecific band. As
a control, the anti-Sp1, anti-Sp3, anti-C/EBP , and anti-C/EBP
antibodies were incubated alone with the labeled oligonucleotide. There
was no nonspecific binding from the antibody preparations to the
oligonucleotide (data not shown). The Sp1 and Sp3 supershifts were
confirmed by incubation of the labeled oligonucleotide and nuclear
extracts with a mixture of anti-Sp1 and anti-Sp3 antibodies, which
shifted all Sp-specific bands (data not shown).
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C/EBP binding site in the
119/
112 region of Cp.
A putative
C/EBP binding site was found in the
119/
112 Cp region. EMSA
analysis using Rael nuclear extracts and a probe corresponding to the
150/
105 Cp region revealed six specific complexes (Fig. 4A). The
two fastest-moving bands were not affected by competition with an
oligonucleotide corresponding to the
150/
125 Cp region but were
removed by an oligonucleotide corresponding to the
130/
105 region
and by a C/EBP consensus motif-containing oligonucleotide. The
slowest-moving minor band displayed the same behavior. To establish the
identity of the factors in the complexes, antibody supershift analysis
was performed (Fig. 4B). Antibodies against different C/EBP isoforms
were used and C/EBP
shifted the two fastest-moving complexes as well
as the slowest-moving band. C/EBP
also shifted a part of the second
fastest-moving band. Nuclear extract from cbc-Rael contained less C/EBP
and gave rise to weaker C/EBP complexes. In cbc-Rael nuclear extracts, only C/EBP
could be detected by antibody supershift analysis (data
not shown). We conclude that the C/EBP
and C/EBP
transcription factors are both present in Rael cells, in contrast to cbc-Rael cells,
which contain only C/EBP
. Furthermore, the factors interact with the
C/EBP binding site in the
119/
112 Cp region, and judging from the
mutational analysis (m5) (Fig. 3), this interaction is essential for
the Cp activity.
Sp1 binding site in the
99/
95 Cp region.
The binding of
transcription factors to the putative Sp site in the
99/
95 Cp
region was investigated with EMSA and antibody supershift analysis
using a probe corresponding to the
107/
84 Cp region and nuclear
extracts of Rael and cbc-Rael cells. Two major and one minor EMSA bands
were evident in the Rael extracts (Fig.
5A). A fourth band was assumed to
represent nonspecific complex formation, since it was not removed by
the addition of excess amounts of unlabeled probe. The major band with
the lowest mobility was composed of two poorly separated bands, both of
which could be removed by AP-2/Sp consensus and Egr consensus
oligonucleotides (Fig. 5, lanes 4 and 5). Antibody supershift analysis
showed that anti-Sp1 antibodies removed one of the bands in the double
band as well as the minor band and that anti-Sp3 antibodies reacted with the other band in the double complex (Fig. 5, lanes 8 and 9).
Addition of antibodies against two other transcription factors, Egr-1
and Egr-2, did not affect any of the complexes obtained with the Rael
cells (Fig. 5A, lanes 7 to 9). cbc-Rael extracts gave rise to the same
binding pattern regarding the Sp complexes (Fig. 5B). In summary, our
results demonstrate that the Sp1 and Sp3 transcription factors are
present in Rael and cbc-Rael and interact with the Sp binding sites in
the
168/
163,
141/
136, and
99/
95 Cp regions.

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FIG. 5.
Identification of transcription factors that bind to the
107/ 84 region. A 32P-labeled double-stranded synthetic
oligonucleotide corresponding to the 107/ 84 region was incubated
with nuclear extracts from Rael (A) and cbc-Rael (B) cells. The
reaction mixtures were analyzed by EMSA. Lanes 1, binding pattern
obtained with the nuclear extract; lanes 2 to 5, patterns obtained with
a 400-fold excess of unlabeled competitors indicated below the
autoradiogram; lanes 6 to 9, patterns obtained after incubation with
the antibodies indicated below the autoradiogram. The specific
complexes are indicated by solid arrows. One band that was not
abolished by competition or antibody reactions is indicated by a dashed
arrow.
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Egr-1 binding site in the
99/
91 region of Cp.
The EMSA
pattern obtained with nuclear extracts of cbc-Rael cells and the
(
107/
84)Cp probe (Fig. 5B) contained an extra band in addition to
those identified as being the result of the binding of the Sp1 and Sp3
factors in the Rael extracts. This complex was shown to contain the
Egr-1 transcription factor by competition experiments and antibody
supershift analysis. We conclude that cbc-Rael contains the Egr-1
transcription factor and that Egr-1 interacts with the overlapping
binding sites for Sp1/Sp3 and Egr-1 in the
99/
91 Cp region.
CCAAT box in the
71/
63 region of Cp.
The
71/
63 Cp
region contains a CCAAT box consensus sequence. A detailed mutation
analysis defined the sequence necessary for the activating capability
of the element as that between positions
71 and
63
(5'-AACCAATTG-3') (Fig. 3 and
6). Transcription factors binding to this
sequence were identified with EMSA using a probe corresponding to the
80/
55 Cp region and nuclear extracts from Rael and cbc-Rael cells
(Fig. 6). Two complexes, one major and one minor, were detected and
considered to be specific. Competition experiments with
oligonucleotides containing mutations m10, m11, and m12 (Table 1)
showed that m10 and m11 could not remove any of the bands while m12
competed for both specific bands. The difference in the positions of
the mutations is only 1 bp from the CCAAT box in the 3' direction. This
corresponds well with the activity data shown in Fig. 3B, where m10 and
m11 abolished activity of the reporter plasmids. m12, on the other
hand, did not interfere with the CCAAT box, and the reporter plasmid
with this mutation was fully active. Notably, a standard CCAAT
consensus sequence oligonucleotide could not remove any of the bands,
which confirms that the whole sequence between positions
71 and
63
is necessary for complex formation. To identify the binding
transcription factors, antibody supershift analysis was performed with
commercially available antibodies. The complexes were supershifted by
an anti-NF-Y antibody (Fig. 6A and B, lanes 9). Antibodies against
three other CCAAT box binding factors (C/EBP, NF-1, and YY1) did not
interact with the complexes. Taken together, our results demonstrate
that the NF-Y transcription factor is present both in Rael and cbc-Rael and that it interacts with the CCAAT box containing sequence in the
71/
63 Cp region.

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FIG. 6.
Identification of transcription factors that bind to the
80/ 55 region. A 32P-labeled double-stranded synthetic
oligonucleotide corresponding to the 80/ 55 region was incubated
with nuclear extracts from Rael (A) and cbc-Rael (B) cells. Lanes 1, binding pattern obtained with the nuclear extract; lanes 2 to 6, patterns obtained with a 400-fold excess of unlabeled competitors
indicated below the autoradiogram (in lanes 3 to 5, the competitors
include the mutations m10, m11, and m12, respectively [Table 1]);
lanes 7 to 10, patterns obtained after incubation with antibodies
against four different CCAAT box binding factors. The position of the
anti-NF-Y antibody-shifted complex is shown by an arrowhead. One band
that was not abolished by competition or antibody reactions is
indicated by a dashed arrow.
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Sequence elements involved in oriPI-EBNA1-induced
activation of Cp.
It is a well-established fact that
oriPI functions as a transcriptional enhancer of the Cp and
is necessary for detectable Cp activity in cells expressing a group III
phenotype (36, 40, 42, 55). The mechanism for the
interaction between the oriPI-EBNA1 complex and Cp is,
however, not understood at the molecular level. We have chosen a
strictly reductionistic approach to this presumably complex problem
with the intention to identify the minimal Cp promoter still possessing
the ability to be activated by oriPI-EBNA1 in the proper
cellular environment. To that end, an EBV DNA fragment containing the
oriPI family of repeats was inserted 5' of the Cp fragments
in the deletion series of reporter plasmids described above.
Transfection of the resulting reporter plasmids into Rael and cbc-Rael
cells revealed that the pgCp(oriPI/
111)CAT plasmid and
plasmids with longer Cp inserts were activated in both cell lines and
that the pgCp(oriPI/
55)CAT plasmid remained inactive (Fig.
7). The results suggested that the
minimal sequences necessary for oriPI-EBNA1-induced Cp
activation are located in the
111/+76 Cp region. To map this region
in greater detail, we inserted the oriPI fragment
immediately 5' of the
170 position in the previously described series
of reporter plasmids with point mutations in Cp (Table 1 and Fig. 3A)
and transfected the resulting plasmids into Rael and cbc-Rael cells.
Among plasmids with mutations in the
111/
55 Cp region, reporter
gene expression was abolished by the m7, m10, and m11 point mutations
and the Del-1 deletion both in Rael cells and in cbc-Rael cells (Fig.
8). Mutation m6 reduced the activity to
approximately 45% of that of the wild type. The data suggest that the
108/
92 and
71/
63 regions of Cp are important for the
oriPI-EBNA1-induced activation of the minimal Cp promoter.
Notably, these regions are the same as those that were found to be
essential for the oriPI-EBNA1-independent Cp stimulation
examined in Rael cells (Fig. 3B).

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FIG. 7.
The minimal oriP-responsive region of Cp
contains sequences between positions 111 and +76 relative to the Cp
transcription initiation site as shown by analysis of the
activity of pgCp(oriPI/...)CAT reporter plasmids in
the Rael and cbc-Rael cell lines. The values are means of at least two
independent transfection series with double samples. Error bars
indicate standard errors of the means.
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FIG. 8.
Regions required for oriPI-EBNA1-mediated
Cp activation in the context of the 170/+76 promoter regulatory
region determined by measuring activity of
pgCp(oriPI/ 170/...)CAT reporter plasmids with mutations
(as detailed in Table 1 and Fig. 3) in the Rael and cbc-Rael cell
lines. CAT activities are percentages of the activity obtained with the
wild-type construct, pgCp(oriPI/ 170)CAT. The 100% values
correspond to 18.7 and 13.3% acetylation, respectively. The values are
means of at least four independent transfection series. Error bars
indicate standard errors of the means.
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We also addressed the question of whether mutations in the
170/+76
fragment of Cp that abolished the activity of the reporter plasmids
also had an effect in the context of the full-length promoter region.
The point mutations with the lowest activity in the
170/+76 Cp
context (m4, m5, m7, and m11) were introduced into the
pgCp(
1024)CAT, pgCp(oriPI/
1024)CAT, and
pgCp(
3889)CAT plasmids, and the resulting plasmids were
transfected into Rael and cbc-Rael cells (Fig.
9A and B). The results with Rael cells showed that every mutation decreased the activity of the three reporter
plasmids (Fig. 9A), although not quite to the same level as that
obtained with the corresponding mutated pgCp(
170)CAT derivatives. Mutations m10 and m11 diminished the activity of the
mutated pgCp(
1024)CAT to approximately 14% of that of the wild-type plasmid, and mutations m4 and m5 diminished it to
approximately 33%. The activity pattern was largely similar in the two
series of oriPI-containing constructs. In cbc-Rael cells,
the pgCp(
1024)CAT plasmid and its mutated derivatives were
inactive, since they lack oriPI (Fig. 9B). The
pgCp(oriPI/
1024)CAT plasmid was active, but the four mutated
derivatives were all heavily repressed, with a residual activity in the
range of 1 to 8%. Surprisingly, the wild-type pgCp(
3889)CAT
plasmid had a distinctly lower activity than the
pgCp(oriPI/
1024)CAT construct, although the relative effects of the mutations were similar. We speculated that this might be
either the effect of low transfection efficiency of this plasmid in
cbc-Rael cells because of its comparatively large size (8,765 bp) or
the result of the presence of negative transcription elements in the
3889/
1024 region of Cp. To test this hypothesis, an unrelated,
non-EBV fragment of 1,820 bp was inserted immediately 3' of the CAT
gene in pgCp(oriPI/
1024)CAT, resulting in a plasmid of
approximately the same size as pgCp(
3889)CAT. Transfection of
this construct into cbc-Rael cells showed that its activity was 16% of
that of the pgCp(oriPI/
1024)CAT plasmid, i.e., of about the
same order of magnitude as the activity obtained with pgCp(
3889)CAT (Fig. 9C). We conclude that the most likely
explanation for the low level of reporter gene activity measured after
transfection of pgCp(
3889)CAT in cbc-Rael cells compared with
that obtained with pgCp(oriPI/
1024)CAT was a low transfection
efficiency. Moreover, the effects of the m4, m5, m7, and m11 mutations
in Cp on oriPI-EBNA1-induced activation of the promoter seem
to be similar in the context of the short (
170/+76) and the native
(
3889/+76) promoter regulatory regions. m5 affected the C/EBP binding
site, m7 affected the promoter-proximal Sp binding site, and m10
affected the CCAAT box. We have also shown that m11 interfered with the
CCAAT box (Fig. 6). The low activity obtained with the m4 mutation in
both the
170/+76 and the full-length context is due to either
interference with the Sp binding site at position
141/
136 or
diminished binding of a factor not identified by us. We favor the
former explanation, since there were no additional complexes in the
EMSA of this region except from the Sp-containing complexes (Fig. 4 and
data not shown).

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FIG. 9.
Regions required for oriPI-EBNA1-mediated
Cp activation in the context of the native ( 3889/+76) promoter
regulatory region. Activity of pgCp(...)CAT reporter
plasmids with mutations (Table 1 and Fig. 3) in the Rael (A) and
cbc-Rael (B) cell lines. The values are means of at least four
independent transfection series. Error bars indicate standard errors of
the means. (C) The low activity of the pgCp( 3889)CAT reporter
plasmid in cbc-Rael is due to low transfection efficiency. An unrelated
non-EBV DNA fragment (ext.) was inserted 3' of the CAT gene in
pgCp(oriPI/ 1024)CAT. CAT activities of the indicated plasmids
were compared after transfection of cbc-Rael cells. Results are
percentages of the activity obtained with
pgCp(oriPI/ 1024)CAT. Error bars indicate standard errors of
the means.
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Transcription factors involved in oriP-EBNA1-induced
activation of Cp.
The results obtained so far led us to conclude
that the
142/
92 and
71/
63 Cp regions are important for the
oriPI-EBNA1-mediated induction of the promoter in reporter
plasmids. Elements downstream of position
111 seem to be of
particular significance, since a reporter construct which, in addition
to oriPI, contained only the
111/+76 part of Cp was
induced in both Rael and cbc-Rael cells. The
71/
63 Cp region
contains a CCAAT box, and NF-Y was shown to be the factor involved in
binding to this region in both Rael and cbc-Rael cells, as described
above (Fig. 6). The
99/
95 Cp region contains overlapping Sp and
Egr-1 binding sites. Both Rael and cbc-Rael cells contain Sp1 and Sp3
transcription factors binding to this site. Moreover, cbc-Rael but not
Rael cells contain the Egr-1 factor, which binds to its recognition
site. The overlapping binding sites for Sp1/Sp3 and Egr-1 were both
affected by m7 in the series of point mutations in the
170/
55 Cp region. To determine the contribution to the regulation
of the reporter gene of each of the two factors, point mutations, which
would preferentially interfere with the binding of one or the other of
the two transcription factors, were introduced into the binding motifs
in EMSA probes in the
107/
84 Cp region. The probe with the Egr-1
mutation (m13) (Table 1) did not bind Egr-1 in cbc-Rael nuclear
extracts but bound the Sp factors (data not shown). The intensity of
the Sp-specific band was, however, lower with the Egr-1 mutated probe
than with the wild-type probe, and we infer that the Egr-1 mutation
also resulted in a slightly reduced Sp binding. The Sp mutation (m14) (Table 1), on the other hand, decreased the binding of the Sp factors
to the EMSA probe substantially, although not completely, but did not
affect the binding of Egr-1 in cbc-Rael extracts (data not shown). To
determine the effect of the mutated factor binding sites on promoter
activity, derivatives of pgCp(
170)CAT and
pgCp(oriPI/
170)CAT were made in which the mutations had been
included in the
170/+76 Cp fragment. The results of transfection of
the mutated reporter plasmids into Rael and cbc-Rael cells showed that
the Egr mutation (m13) decreased the Cp activity in the
oriPI-containing reporter plasmids to approximately 50% of
the activity of the wild-type construct (Fig.
10A and B). This reduction may be due
to the reduced binding of Sp1 to the Egr-1 mutated sequence, as
reported above. The Sp mutation (m14) was almost as effective as m7 in
abolishing Cp activity in both Rael and cbc-Rael cells.

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FIG. 10.
Effects of specific mutations of the overlapping Egr-1
and Sp binding sites on Cp activity in Rael cells. (A and B) Activity
of pgCp( 170/...)CAT and
pgCp(oriPI/ 170/...)CAT reporter plasmids with mutations
m7, m13, and m14 (Table 1) in the Rael (A) and cbc-Rael (B) cell lines.
CAT activities are percentages of the activity obtained with the
wild-type construct including oriPI,
pgCp(oriPI/ 170)CAT. The 100% values correspond to 71.4 and
41.6% acetylation, respectively. The values are means of at least four
independent transfection series. Error bars indicate standard errors of
the means. (C and D) Effects of Egr-1 on Cp activity without and with
oriPI. Increasing amounts of the Egr-1 expression vector
pCI/Egr-1 were cotransfected with pgCp( 170)CAT and
pgCp( 170/m13)CAT (C) and pgCp(oriPI/ 170)CAT and
pgCp(oriPI/ 170/m13)CAT (D). The total amount of expression
vector was adjusted with pCI empty vector to 3 pmol in each
transfection. The values are means of at least three independent
transfection series. Error bars indicate standard errors of the
means.
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Since Egr-1 gene expression is associated with latency III EBV gene
expression and is abolished in group I phenotype cells, an Egr-1
expression vector was created to investigate the effect of Egr-1 on the
pgCp(
170)CAT plasmid in the group I phenotype Rael
environment. The Egr-1 expression vector was based on cDNA from
cbc-Rael cells and was used for cotransfections in Rael cells. Results
showed that increasing amounts of Egr-1 decreased the activity of
pgCp(
170)CAT to background levels (Fig. 10C). This resembles
the situation in cbc-Rael cells, where the
170Cp construct was
inactive. The construct with oriPI,
pgCp(oriPI/
170)CAT, was also downregulated by cotransfection
with the Egr-1 expression vector but to a lesser extent (Fig. 10D).
However, a reporter construct in which the Egr site was mutated,
pgCp(
170/m13)CAT, was also downregulated to the background
level, and the activity of the corresponding construct with
oriPI, pgCp(oriPI/
170/m13)CAT, was decreased to
21% of the activity when no Egr-1 was added (Fig. 10C and D). Taken
together, the data indicate that Egr-1 represses Cp activity, but the
mechanism for this repression remains unclear and is discussed below.
To study the effects of the Sp factors, we employed SL2 insect cells,
which do not express the Sp family of transcription factors. The
pgCp(oriPI/
170)CAT plasmid was cotransfected with expression
vectors for Sp1 and Sp3 into the SL2 cells. Results showed that Sp1
could activate Cp whereas Sp3 could not (Fig. 11A). When
pgCp(oriPI/
170/m14)CAT, where the proximal Sp site was
mutated, was introduced into cotransfection experiments, Sp1 still
could activate this construct, although not to the same level as the
wild type. The
170Cp fragment contains two more Sp sites, which might
contribute to the remaining activity of the mutated construct. To test
if the upstream Sp sites could compensate for the mutated proximal Sp
site in SL2 cells, additional Sp mutations were created,
rendering pgCp(oriPI/
170/m15)CAT, pgCp(oriPI/
170/m16)CAT, and
pgCp(oriPI/
170/m17)CAT (Table 1). pgCp(oriPI/
170/m3)CAT was also included in this series of
transfections, since mutation m3 affects the
141/
136 Sp site. The
results demonstrate that the different Sp sites to some extent could
compensate for each other when one Sp site was mutated and that
mutations of all three sites were necessary for a significant reduction
of Cp activity in SL2 cells (Fig. 11C). We conclude that Sp1 is an important factor for activation of Cp. To study Sp1 effects in conjunction with EBNA1 an EBNA1 expression vector for SL2 cells was
constructed. SL2 cells were cotransfected with the
pgCp(oriPI/
170)CAT reporter construct and various amounts of
the Sp1 and EBNA1 expression vectors (Fig.
12). The results presented in Fig. 12
indicate that EBNA1 in conjunction with increasing amounts of Sp1
generated a significant increase in Cp activation compared to the
effects of Sp1 alone. Notably, the results also show that EBNA1 alone failed to activate an oriPI-containing Cp reporter
construct. Sp1 thus seems to be an essential factor for the
transactivation functions of oriPI-EBNA1. The relatively low
transactivation efficiency by EBNA1 in these experiments could reflect
its inability to enter the nucleus or its low level of expression.
Another explanation would be that additional factors, not expressed in
SL2 cells, are important for efficient oriPI-EBNA1-induced
Cp activation. We favor the latter explanation, since intranuclear
EBNA1 expression was readily detected in 7% of the transfected SL2
cells (data not shown).

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FIG. 11.
Sp1 activates Cp. (A) The reporter plasmid
pgCp(oriPI/ 170)CAT was cotransfected with pPac (empty
vector), pPacSp1 (expression vector for Sp1), and pPacSp3 (expression
vector for Sp3) in SL2 cells. CAT activities are percentages of the
activity obtained with pgCp(oriPI/ 170)CAT cotransfected with
pPacSp1. (B) Schematic presentation of the three Sp sites in the
pgCp(oriPI/ 170)CAT reporter construct and the specific
mutations introduced, yielding the pgCp(oriPI/ 170/...)CAT
plasmid series (Table 1). (C) The reporter plasmids were cotransfected
with pPac and pPacSp1 in SL2 cells. CAT activities are percentages of
the activity obtained with the wild-type construct
pgCp(oriPI/ 170)CAT cotransfected with pPacSp1. The values are
means of at least three independent transfection series. Error bars
indicate standard errors of the means.
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FIG. 12.
EBNA1-induced transactivation of
pgCp(oriPI/ 170)CAT requires Sp1. (A) The reporter plasmid
pgCp(oriPI/ 170)CAT was cotransfected with increasing amounts
of pPacSp1 and a constant amount of pPacEBNA1 (1 µg, 0.16 pmol) or
pPac (empty vector). The total amount of expression vector was adjusted
with pPac to 0.64 pmol in each experiment. CAT activities are
percentages of the activity obtained with pgCp(oriPI/ 170)CAT
cotransfected with 1 µg of pPacSp1. The values are means of at least
three independent transfection series. Error bars indicate standard
errors of the means. (B) The reporter plasmid
pgCp(oriPI/ 170)CAT was cotransfected with increasing amounts
of the EBNA1 expression vector pPacEBNA1 and a constant amount of
pPacSp1 (1 µg, 0.16 pmol) or pPac (empty vector). The total amount of
expression vector was adjusted with pPac to 0.64 pmol in each
experiment. CAT activities are percentages of the activity obtained
with the pgCp(oriPI/ 170)CAT cotransfected with 1 µg of
pPacEBNA1. The values are means of at least three independent
transfection series. Error bars indicate standard errors of the
means.
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The results reported here are in apparent contradiction to a previous
report which states that the
245/
45 region of Cp does not encompass
elements important for Cp activity, except for the CCAAT box at
position
65 (40). However, in that study, a Cp mutant in
which the
245/
45 region was deleted retained most of the wild-type
activity. This was explained as being due to the concomitant
translocation of an upstream silent CCAAT box to the approximate
position of the active CCAAT box removed by the deletion. It should be
noted, however, that the deletion of the
245/
45 region not only
translocated the CCAAT box but also moved an upstream Sp element to
approximately the same position as that occupied by the essential
99/
93 site in the wild-type reporter plasmid. We hypothesized that
this translocation could compensate for the lost Sp sites in the
245/
45 deletion-containing construct. To test this hypothesis, we
made two new constructs based on the pgCp(
3889)CAT plasmid.
In one construct, we deleted the sequence between
247 and
57,
resulting in the pgCp(
3889/Del-2)CAT plasmid. In the other
variant, the upstream Sp consensus sequence was mutated in addition to
the deletion, resulting in the pgCp(
3889/Del-2/m18)CAT plasmid (Table 1). In our hands, Cp activities of the
247/
57 deletion-containing reporter plasmid in Rael and cbc-Rael cells were
only 15 and 25% of those of the wild-type construct, respectively (Fig. 13). Mutation of the translocated
upstream Sp site reduced the activity of the reporter plasmid even
further. This effect was most pronounced in Rael cells, in which the
activity of pgCp(
3889/Del-2/m18)CAT was completely abolished.
In cbc-Rael cells, the reporter showed a residual activity of 15% of
the activity of the wild-type construct. Taken together, these results
confirm that the Sp elements identified in this work are important for
both basal and oriPI-EBNA1-induced Cp activity.

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FIG. 13.
Effect on the activity of pgCp( 3889)CAT
reporter plasmid of a 247/ 57 deletion mutation alone (Del-2) and in
combination with a 3-bp mutation of an upstream Sp site (Del-2/m18) in
Rael and cbc-Rael cells. CAT activities are percentages of the activity
obtained with the wild-type construct, pgCp( 3889)CAT. The
100% values correspond to 356% (measured value times dilution factor)
and 10% acetylation for Rael and cbc-Rael, respectively. The values
are means of two and four independent transfection series,
respectively. Error bars indicate standard errors of the means.
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 |
DISCUSSION |
Although the patterns of EBV promoter usage in alternative forms
of virus latency in vitro have been well characterized, the cellular
controls of virus promoter activity are still poorly understood. The
identification of the cellular factors that control the transcription
regulatory activity of Cp is fundamental to the understanding of the
molecular mechanisms that control virus latent gene expression. In this
study we have made a detailed mutational analysis of the
promoter-proximal Cp region and identified transcriptional
elements important for oriPI-EBNA1-independent and
-dependent promoter stimulation. We confirm the previous report of the
dependence of Cp on a properly positioned CCAAT box (40), and we extend these results by demonstrating that the NF-Y
transcription factor interacts with the CCAAT box-containing sequence
in the
71/
63 Cp region. We also show that members of the C/EBP
transcription factor family interact with a C/EBP consensus sequence in
the
119/
112 region of Cp and that this interaction is important for
promoter activity. Our main finding, however, is the identification of
a GC-rich sequence in the
99/
91 Cp region that is essential for Cp
activity as well as for the transactivating properties of the
oriPI-EBNA1 complex. This region contains overlapping
binding sites for the Sp1 and Egr-1 transcription factors, and our
results indicate that Sp1 is a positive and Egr-1 is a negative
regulator of Cp activity. Furthermore, an EBV DNA segment, which in
addition to oriPI only contains the
111/+76 part of Cp, is
defined as a minimal Cp promoter, as it still possesses the ability to
be activated by EBNA1 in the proper cellular environment.
Previous studies in this field have demonstrated the presence of
relatively few positive cis-acting transcriptional elements upstream of Cp (Fig. 1). In 1989, Sugden and Warren showed that Cp is
dependent on oriP in cis and EBNA1 in
trans for its efficient expression (55). These
results were extended by our group and by Puglielli et al., who
demonstrated that oriP is essential for detectable
transcription from Cp in transient-transfection experiments in group
III BL cell lines and LCLs (36, 40). A positive GRE was
identified in the region approximately 850 bp upstream of Cp
(26), but its importance for transcription activation from Cp has been questioned, since deletion of this region did not affect
the activity of Cp (40). An EBNA2-responsive element that
contains one RBP-J
binding site was found at a position about
375 bp upstream of Cp (22, 56, 68). The significance of
this putative EBNA2-inducible enhancer has also been questioned, but a recent report strongly argues for an important role of this element in regulating Cp activity in a viral context (67).
Cp has also been shown to require a CCAAT box positioned at bp
65 for
activation of EBNA gene transcription (40). In addition, sequences downstream of Cp have been examined for promoter regulatory elements, and the results indicate that there may be a weak
Cp-activating cis element within the +2680 to +2880 Cp
region (39).
In a previous paper it was reported that the
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55 region of the
Cp promoter contains transcription regulatory elements that activate
reporter plasmids in group I phenotype cells. In addition, sequences in
this region seem to be important for oriPI-EBNA1-mediated upregulation of Cp activity (36). In the present study,
the individual cis elements in the
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