Promoter-Proximal Regulatory Elements Involved in oriP-EBNA1-Independent and -Dependent Activation of the Epstein-Barr Virus C Promoter in B-Lymphoid Cell Lines

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: pΔC1CAT to pgCp(−55)CAT, pΔC2CAT to pgCp(−248)CAT, pΔC3CAT to pgCp(−1024)CAT, and pΔC4CAT 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 [γ-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/EBPα (sc-61X), C/EBPβ (sc-150X), C/EBPγ (sc-7659X), C/EBPδ (sc-636X), and C/EBPɛ (sc-158X) (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.).

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).

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.

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/EBPγ shifted the two fastest-moving complexes as well as the slowest-moving band. C/EBPβ 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/EBPγ could be detected by antibody supershift analysis (data not shown). We conclude that the C/EBPβ and C/EBPγ transcription factors are both present in Rael cells, in contrast to cbc-Rael cells, which contain only C/EBPγ. 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.

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.

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).

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).

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.

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).

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.