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Journal of Virology, May 2009, p. 5269-5277, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00097-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

The LMP1 Promoter Can Be Transactivated Directly by NF-{kappa}B{triangledown}

Constantinos Demetriades1 and George Mosialos1,2*

School of Biology, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece,1 Institute of Immunology, Biomedical Sciences Research Center Al. Fleming, 34 Al. Fleming Str., 16672 Vari, Greece2

Received 15 January 2009/ Accepted 17 February 2009


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ABSTRACT
 
A bioinformatic analysis identified two putative NF-{kappa}B binding sites in the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) promoter. The ability of p65RelA to interact with the LMP1 promoter was shown by in vitro and in vivo assays. Using an EBV-transformed lymphoblastoid cell line as a reporter system for the activity of the +40/–328 LMP1 promoter region, the functional importance of NF-{kappa}B and other transcription factor binding sites was demonstrated. p65RelA could also induce LMP1 expression from the EBV genome in Daudi and P3HR1 Burkitt's lymphoma cell lines. Finally, it was shown that p65RelA could cooperate with EBNA2 or the aryl hydrocarbon receptor in the transactivation of the LMP1 promoter. Our study established the importance of NF-{kappa}B and several cis-acting elements in the regulation of the LMP1 promoter in a latency III environment and highlighted a complex interplay between NF-{kappa}B and other transcription factors in this process.


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INTRODUCTION
 
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a dominant oncoprotein that has been implicated in the development of most EBV-associated malignancies (reviewed in reference 28). LMP1 is an integral membrane protein that consists of a short amino-terminal cytoplasmic region, six transmembrane domains, and a 200-amino-acid cytoplasmic carboxyl-terminal tail (CCT). It signals continuously by virtue of its intrinsic ability to oligomerize and interact constitutively with intracellular signaling molecules (reviewed in reference 19). It mimics activated members of the tumor necrosis factor receptor family to transmit growth and antiapoptotic signals. These signals are conveyed to the nucleus through the activation of the canonical and noncanonical NF-{kappa}B activation pathways as well as mitogen-activated protein kinase pathways. The activation of NF-{kappa}B by LMP1 is of particular importance since it is essential for B-lymphocyte transformation by EBV (6, 7, 10). Therefore, the mechanism of NF-{kappa}B activation by LMP1 and its effects on specific cellular gene expression have been the subject of intense investigation. Two regions in the CCT of LMP1 mediate the activation of NF-{kappa}B (reviewed in reference 33). The membrane-proximal region CTAR1 mediates primarily the activation of the noncanonical NF-{kappa}B activation pathway and coincides with the principal transformation effector site (TES1). A second region (CTAR2), which is located near the carboxyl terminus of the CCT, mediates a powerful induction of the canonical NF-{kappa}B activation pathway and coincides with a second transformation effector site (TES2). The role of LMP1-induced NF-{kappa}B activation in cellular gene induction has been demonstrated in many cases. However, until recently, NF-{kappa}B has not been implicated in the transactivation of EBV genes.

The expression of LMP1 is suppressed in EBV-infected B lymphocytes of healthy carriers and EBV-positive Burkitt's lymphomas. Nevertheless, LMP1 expression is permissive in most EBV-associated malignancies, and for this reason, the mechanism of LMP1 transcription regulation has been the subject of intense investigation. Two promoters can drive the transcription of the LMP1 gene (reviewed in reference 19). A proximal promoter (ED-L1) is active in latency III, whereas a distal promoter (LT-R1) located in the terminal repeats mediates the expression of LMP1 in latency II. A number of viral and cellular transcription regulators have been implicated in the regulation of the ED-L1 LMP1 promoter (which will be referred to as the LMP1 promoter from this point on) using primarily EBV-negative B-cell lymphoma and epithelial cell lines as reporter systems. These experiments have shown a prominent role of the latent EBV antigens EBNA2 and EBNALP in the transactivation of the LMP1 gene, which depends on the RBPJ{kappa}, PU.1, POU-box, and AP2 binding sites of the LMP1 promoter (11, 14, 15, 26, 27, 31, 36, 39). The activity of the LMP1 promoter can be regulated also by the latent EBV antigens EBNA3A and EBNA3C (29). In addition, activating transcription factor/cyclic AMP response element (ATF/CRE), interferon regulatory factor (IRF), and STAT elements have been implicated in the transactivation of the LMP1 promoter (13, 24, 25, 32).

In the present report, bioinformatic tools, macromolecular interaction analyses, and transcription reporter assays identified a direct mechanism of regulation of LMP1 transcription by NF-{kappa}B and established the role of several other cis-regulatory elements of the LMP1 promoter in type III latent infection of B lymphocytes by EBV.


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MATERIALS AND METHODS
 
Cell lines and tissue culture reagents. 293FT (Invitrogen) is a human embryonic kidney cell line, and it was cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin. DG75 (4) is an EBV-negative Burkitt's lymphoma cell line, Daudi (21) and P3HR1 (22) are EBV-positive Burkitt's lymphoma cell lines, and WTLCL and LCL1 are EBV-transformed lymphoblastoid cell lines. The B-cell lymphoma cell lines were cultured in RPMI 1460 (Invitrogen) supplemented with 10% fetal bovine serum, glutamine, penicillin, and streptomycin.

Chromatin immunoprecipitation. The EBV-transformed lymphoblastoid cell line WTLCL was used for chromatin immunoprecipitation experiments. A total of 50 x 106 cells were fixed with 1% formaldehyde in medium for 10 min at room temperature. For the isolation of chromatin, cells were collected by centrifugation, washed twice with ice-cold phosphate-buffered saline, and lysed in lysis solution (50 mM HEPES [pH 7.9], 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 10% [vol/vol] glycerol, 0.5% [vol/vol] NP-40, 0.25% [vol/vol] Triton X-100, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) for 10 min on ice. Nuclei were collected by centrifugation, washed in wash buffer (10 mM Tris-Cl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, and 1 mM PMSF), resuspended in 2 ml RIPA buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 1% [vol/vol] Triton X-100, 0.1% Na-deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM PMSF) and subjected to sonication on ice (15 to 20 rounds of 30 s each) in order to obtain chromatin fragments of an average of 500 bp. A small portion of each sample was used for immunoprecipitation in RIPA buffer, following preclearing with protein A magnetic Dynabeads (Dynal Biotech ASA, Norway). Complexes were subsequently washed three times with each of the following buffers: RIPA buffer containing 140 mM NaCl, RIPA buffer containing 500 mM NaCl, LiCl buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% [vol/vol] NP-40, 0.1% Na-deoxycholate and 1 mM PMSF), and TE buffer containing 1% (vol/vol) Triton X-100 and 1 mM PMSF. Proteins were digested with proteinase K for 3 h at 56°C, and cross-linking was reversed at 65°C overnight. DNA fragments were extracted with phenol-chloroform twice and precipitated with glycogen. One twentieth of the samples was used for semiquantitative PCR analysis, with specific primers amplifying nucleotides +40 to –328 (+40 primer, 5'-GCGAAGCTTTCAGGGCAGTGTGTCAGGAG-3', and –328 primer, 5'-GCGCTCGAGCGCGCCTCTTTGTGCGGATT-3'), or +208 to +655 (+208 primer, 5'-GTTTTGGCTGTACATCGTTATG-3', and +655 primer, 5'-GCACCAAGTCGCCAGAGAATC-3') relative to the LMP1 gene transcription start site or the 3' untranslated region of the Dhfr gene (DHFR forward, 5'-CTGATGTCCAGGAGGAGAAAGG-3', and DHFR reverse, 5'-AGCCCGACAATGTCAAGGACTG-3'), which is a well-established genomic locus, widely used as a negative control for transcription factor binding in chromatin immunoprecipitation experiments. The appropriate number of amplification cycles was determined (30 to 35) and used to ensure that the PCR was in the linear phase of amplification.

Electrophoretic mobility shift assay (EMSA). 32P-labeled oligonucleotides (2 ng) were incubated with recombinant purified p50NF-{kappa}B1/p65RelA heterodimer (34) in binding buffer (10 mM Tris [pH 8.0], 15 mM HEPES [pH 7.9], 5 mM MgCl2, 5% glycerol, 0.1% NP-40, and 1 mg/ml bovine serum albumin) for 20 min at room temperature. For the competition experiments, 200 ng of unlabeled oligonucleotides were preincubated with probes, before the addition of protein to the mixture. For the supershift experiments, probes were mixed into protein as described above, antibodies were subsequently added and the reactions were incubated on ice for 30 min. In every case, protein-DNA complexes were resolved by electrophoresis in a 5% nondenaturing polyacrylamide gel containing 5% glycerol and visualized by autoradiography.

Plasmid construction. The +40/–328 and +40/–543 regions of the LMP1 promoter were amplified from B95-8 and P3HR1 genomic DNA preps, using primers containing appropriate restriction enzyme sequences and cloned into an XhoI/HindIII-digested pGL2-basic (Promega) plasmid. The primers (+40 and –328) used for the amplification of the +40/–328 region are described in "Chromatin immunoprecipitation." The +40/–543 region was amplified with the primers +40 and –543 (5'-GCGCTCGAGACACTCGCATACCCCACACC-3'). Reporter plasmids containing mutations in several major transcription factor binding sites were constructed, using site-specific PCR-directed mutagenesis. All constructs were verified by sequencing.

Transfections and reporter assays. A total of 2 x 106 WTLCL or LCL1 cells were mixed with 40 µg of firefly luciferase reporter plasmid and 10 µg PGK-βgal plasmid (12) in 0.2-mm cuvettes and electroporated at 140 V and 950 µF (exponential wave), using a Gene Pulser Xcell electroporator (Bio-Rad). Cells were harvested 48 h postelectroporation, lysed in passive lysis buffer (Promega), and used for the determination of luciferase and β-galactosidase activities, using a TD-20/20 luminometer (Turner Designs). The luciferase and β-galactosidase activities were determined by the luciferase assay system (Promega) and the Galacto-Light Plus reporter gene assay system (Tropix), respectively. DG75 cells were electroporated as described above at 120 V, using 40 µg of a luciferase reporter plasmid, 40 µg of effector plasmids, and 10 µg PGK-βgal plasmid. An empty pcDNA3 expression vector was used to equalize the amount of electroporated DNA among samples. Cells were harvested 48 h postelectroporation, and cell lysates were generated and used for the determination of luciferase and β-galactosidase activities as described above. Daudi and P3HR1 cells were electroporated as described above at 130 V, using 30 µg of each expression plasmid. Cells were harvested 48 h postelectroporation and lysed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer for the determination of protein expression or the TRI reagent (Ambion) for RNA extraction and cDNA preparation using the RevertAid M-MuLV H minus cDNA synthesis kit (Fermentas). In the reverse transcription-PCR experiments, the LMP1 cDNA was amplified using the +208 and +655 primers, whereas the interleukin-8 (IL-8) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNAs were amplified with previously described primers (1). The appropriate number of amplification cycles was determined and used to ensure that the PCR was in the linear phase of amplification.

For the transfection of 293FT cells, 4 x 105 cells/well were seeded in a 12-well plate 1 day before transfection. The 293FT cells were transfected with 250 ng of firefly and Renilla luciferase (pRLnull; Promega) reporter plasmids in the absence or presence of expression vectors, using the calcium phosphate method. Empty pcDNA3 expression vector was used to equalize the amount of transfected DNA among samples. The cells were harvested at 48 h posttransfection and lysed in passive lysis buffer (Promega), and the lysates were assayed with the dual luciferase assay kit (Promega).

Antibodies. The following antibodies and sera were used: anti-p65RelA rabbit polyclonal antibody (A; Santa Cruz Biotechnology, Inc.) and anti-RBPJ{kappa} rabbit polyclonal antibody (H-50; Santa Cruz Biotechnology, Inc., anti-green fluorescent protein (GFP) rabbit polyclonal antibody (FL; Santa Cruz Biotechnology, Inc.), anti-actin (C-4; Santa Cruz Biotechnology, Inc.), anti-Arnt 1 rabbit polyclonal antibody (H-172; Santa Cruz Biotechnology, Inc.), anti-LMP1 (S12), and anti-EBNA2 (PE2) mouse monoclonal antibodies.


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RESULTS
 
In order to identify previously uncharacterized elements that might regulate LMP1 transcription, the B95-8 LMP1 promoter sequence that spans nucleotides +40 to –543, relative to transcription start site, was analyzed bioinformatically using the TRANSFAC 7.0 platform. This effort identified two putative NF-{kappa}B binding sites, at positions –78/–87 (NF-{kappa}BA) and –486/–495 (NF-{kappa}BB), in addition to previously identified elements (Fig. 1A). Both the NF-{kappa}BA (GGGGATTTGC) and NF-{kappa}BB (GGGAATTTCA) sites differ from the consensus NF-{kappa}B binding site (GGGRNNYYCC) in one nucleotide.


Figure 1
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  		FIG. 1. NF-{kappa}B interacts with the LMP1 promoter in vivo. (A) The sequence of the region from position +40 to –543 of the LMP1 promoter is shown, along with the location of putative transcription factor binding sites that were identified using the TRANSFAC 7.0 platform. (B) Chromatin immunoprecipitation from WTLCL cells with the indicated antibodies. The presence of the LMP1 promoter region [LMP1p(+40/–328)], the LMP1 coding region [LMP1(+208/+655)] and the Dhfr 3' untranslated region (DHFR 3' untranslated region) in the immunoprecipitated material was detected by PCR.

Chromatin immunoprecipitation experiments were performed in order to investigate the involvement of NF-{kappa}B in the regulation of the LMP1 promoter, using chromatin from WTLCL cells. The LMP1 promoter region was clearly more enriched in the immunoprecipitated material with the anti-p65RelA and anti-RBPJ{kappa} rabbit polyclonal antibodies than that in the immunoprecipitated material with an anti-GFP rabbit polyclonal antibody (Fig. 1B, top panel, compare lanes 2 and 3 with lane 1). The anti-p65RelA and anti-RBPJ{kappa} antibodies did not immunoprecipitate unrelated cellular or viral genomic sequences (Fig. 1B, middle and bottom panels). This experiment demonstrates that NF-{kappa}B interacts directly with the LMP1 promoter region in vivo.

In order to determine whether the putative NF-{kappa}B binding sites in the LMP1 promoter can interact in vitro with NF-{kappa}B subunits, an EMSA was used. A combination of recombinant p50NF-{kappa}B1 and p65RelA could readily interact and retard the mobility of a radioactively labeled oligonucleotide containing the NF-{kappa}BA sequence (Fig. 2A and B, lane 2). Two complexes were visible. The presence of p65RelA in the slower-migrating retarded complex was verified by supershifting this complex with an anti-p65RelA antibody but not a control anti-GFP antibody (Fig. 2B, compare lanes 2, 7, and 8). Although a direct demonstration of p50NF-{kappa}B1 homodimer binding to NF-{kappa}BA is not provided, the inability of the anti-p65RelA antibody to supershift the faster-migrating retarded complex suggests that this complex consists of p50NF-{kappa}B1 homodimers. This interpretation is also consistent with previous reports on the relative mobilities of p50NF-{kappa}B1 homodimers and p50NF-{kappa}B1/p65RelA heterodimers (2, 35). The interaction between NF-{kappa}B and the radioactive NF-{kappa}BA binding site-containing oligonucleotide was effectively competed with the nonradioactive B95-8 NF-{kappa}BA site-containing oligonucleotide, to a slightly lesser extent with the nonradioactive NF-{kappa}BB site-containing oligonucleotide, and to an even lesser extent with the nonradioactive P3HR1 NF-{kappa}BA site-containing oligonucleotide (Fig. 2A and B, compare lanes 2, 3, 4, and 6). However, this interaction was ineffectively competed with a nonradioactively labeled oligonucleotide containing a mutated B95-8 NF-{kappa}BA site (Fig. 2B, compare lanes 2 and 5). In accordance with these findings, comparison of the abilities of equivalent amounts of labeled oligonucleotides to interact with the same amount of recombinant p50NF-{kappa}B1 and p65RelA by EMSA showed that the B95-8 NF-{kappa}BA-containing oligonucleotide has a greater ability than the B95-8 NF-{kappa}BB-containing oligonucleotide, which in turn has a greater ability than the P3HR1-containing oligonucleotide to interact with NF-{kappa}B (data not shown). These experiments demonstrated the ability of NF-{kappa}B to interact directly with the LMP1 promoter in vitro via two previously unidentified NF-{kappa}B binding sites.


Figure 2
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  		FIG. 2. In vitro binding of NF-{kappa}B to two elements of the LMP1 promoter. (A) Nucleotide sequences of the probes used in the EMSAs shown in panel B. In each case, the putative wild-type or mutated NF-{kappa}B binding site is shown in capital letters. (B) EMSA analysis of the interaction between a mixture of recombinant p50NF-{kappa}B1 and p65RelA and the oligonucleotides shown in panel A. The electrophoretic mobility of the radioactively labeled B95-8 NF-{kappa}BA oligonucleotide was analyzed in the presence of recombinant p50NF-{kappa}B1 and p65RelA (p50+p65) with (+) or without (–) the indicated nonradioactively labeled competitor oligonucleotides and antibodies. The position of two retarded complexes is indicated by a bracket. The faster-migrating retarded complex is indicated by an arrow, and the antibody-supershifted complex is indicated by an arrowhead.

In order to determine the functional importance of the NF-{kappa}B and other cis-acting elements for the activity of the LMP1 promoter, luciferase reporter constructs encompassing wild-type and mutated regions of the LMP1 promoter were analyzed in the WTLCL EBV-transformed lymphoblastoid cell line. A latency III background of EBV infection was chosen because the activity of most of the previously characterized cis-acting elements of the LMP1 promoter was analyzed in EBV-negative cell lines, which do not constitute a physiologically relevant environment. The P3HR1 LMP1 promoter region contains many alterations in comparison to the corresponding region of B95-8 (13). A functional comparison of the P3HR1 LMP1 promoter region spanning nucleotides +40 to –543 or +40 to –328 with the corresponding regions of B95-8 showed a substantially weaker activity by the P3HR1 promoter, in agreement with a previous study that used an EBV-negative B-lymphoma cell line for this analysis (Fig. 3B) (14). Two regions of the B95-8 LMP1 promoter encompassing nucleotides +40 to –543 or +40 to –328 were analyzed initially and showed comparable activities, with the latter region being slightly weaker than the former one (Fig. 3B). Therefore, the shorter region of the LMP1 promoter was chosen to evaluate the importance of cis-acting elements of the LMP1 promoter. A number of mutations that affect specific cis-elements and are shown in Fig. 3A were introduced in the +40/–328 LMP1 promoter region, and their effect was assessed by the electroporation of WTLCL with the corresponding luciferase reporter constructs. Mutation of the NF-{kappa}BA, AP-2, POU-box, PU.1, or RBPJ{kappa} had a dramatic negative effect on the activity of the LMP1 promoter (Fig. 3B). On the other hand, mutation of the E-box and the STAT sites did not affect the LMP1 promoter significantly (Fig. 3B). A mutation that inactivates the ISRE element increased the activity of the LMP1 promoter (Fig. 3B). The mutations of the NF-{kappa}BA, AP-2, RBPJ{kappa}, and ISRE sites were also evaluated in a different EBV-transformed lymphoblastoid cell line (LCL1), and they had a similar effect on the activity of the LMP1 promoter (data not shown). The role of the two NF-{kappa}B binding sites (NF-{kappa}BA and NF-{kappa}BB) was also analyzed in the context of the +40/–543 LMP1 promoter region in the WTLCL cell line. These experiments showed that mutation of the NF-{kappa}BA site reduced the activity of the +40/–543 LMP1 promoter by 60%, whereas mutation of the NF-{kappa}BB site resulted in a smaller but statistically significant reduction of the +40/–543 LMP1 promoter activity by 25% (data not shown). These experiments identify a major role of the NF-{kappa}BA and its overlapping AP-2 site in the regulation of the LMP1 promoter and establish the functional importance of the previously identified POU-box, PU.1, and RBPJ{kappa} sites in a latency III environment.


Figure 3
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  		FIG. 3. Functional characterization of LMP1 promoter elements in an EBV-transformed lymphoblastoid cell line. (A) The mutations introduced in the LMP1 promoter and analyzed in panel B are shown. In each case, the sequence of the cis-element (WT) and the corresponding mutated sequence (MUT), with the mutated nucleotides underlined, are shown in capital letters. (B) The activity of luciferase reporter constructs containing the indicated wild-type or mutated LMP1 promoter regions from the B95-8 or P3HR1 strain was evaluated following their introduction in the WTLCL lymphoblastoid cell line by electroporation along with a β-galactosidase expression plasmid, which was used for normalization of transfection efficiencies. The results are the means ± standard error (SE) of relative luciferase activity from at least three independent experiments. Statistical analysis was performed using Student's t test. Statistically significant differences from the activity of the wild-type B95-8 +40/–543 LMP1 promoter are indicated by # (P < 0.05) or ## (P < 0.01). Statistically significant differences from the activity of the wild-type B95-8 +40/–328 LMP1 promoter are shown by * (P < 0.05) or ** (P < 0.01).

To analyze further the role of NF-{kappa}B in the regulation of the LMP1 promoter, the effect of p65RelA on the activity of the +40/–328 LMP1 promoter region was investigated in 293FT epithelial cells (Invitrogen). p65RelA was capable of transactivating the +40/–328 LMP1 promoter in a dose-dependent manner (Fig. 4A). A mutation that abrogates the ability of NF-{kappa}B to interact with the NF-{kappa}BA site abolished the p65RelA-mediated transactivation of the LMP1 promoter. These experiments demonstrate that the NF-{kappa}BA site of the LMP1 promoter is necessary and sufficient for the transactivation of the +40/–328 LMP1 promoter by NF-{kappa}B. A number of cis-acting elements of the LMP1 promoter mediate its EBNA2 responsiveness. In order to determine whether the NF-{kappa}BA site is required for EBNA2-mediated transactivation of the LMP1 promoter, the EBNA2 responsiveness levels of wild-type and NF-{kappa}BA mutant luciferase reporters of the LMP1 promoter were compared in DG75 B-cell lymphoma cells. EBNA2 transactivated both reporters to a similar extent, showing that the NF-{kappa}BA site is not essential for the transactivation of the LMP1 promoter by EBNA2 (Fig. 4B). Since EBNA2 and p65RelA can independently transactivate the LMP1 promoter, the possible cooperation between the two transcription factors was investigated. Coexpression of EBNA2 and p65RelA led to a much higher activity of the LMP1 promoter than the one expected from the simple additive effect of the two transactivators (Fig. 4B). The cooperative activity between EBNA2 and p65RelA was abolished by a mutation that inactivated the NF-{kappa}BA site. This experiment shows that EBNA2 can cooperate with NF-{kappa}B for the transactivation of the LMP1 promoter.


Figure 4
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  		FIG. 4. NF-{kappa}B transactivates the LMP1 promoter. (A) Luciferase reporter assay analysis (top panel) of the wild-type and mutated LMP1 promoter in 293FT epithelial cells. The activity of luciferase reporter constructs containing the wild-type (WT) or mutated + 40/–328 LMP1 promoter luciferase reporter plasmids was evaluated following their cotransfection in 293FT cells along with a Renilla luciferase expression plasmid, which was used for the normalization of transfection efficiencies in the absence (–) or presence of 1 (+), 2 (++), or 3 (+++) µg of a p65RelA expression vector (pCMV-p65), as shown. The results are the means ± SE of relative luciferase activity from at least three independent experiments. Representative Western blots (bottom panel) showing the expression of p65RelA (p65) and β-actin in the cell lysates analyzed in the top panel. (B) The role of NF-{kappa}B in EBNA2-mediated transactivation of the LMP1 promoter. The activity of luciferase reporter constructs containing the indicated wild-type or mutated LMP1 promoter regions was evaluated following their introduction into DG75 cells by electroporation along with a β-galactosidase expression plasmid, which was used for normalization of transfection efficiencies in the absence (–) or presence (+) of EBNA2 (pSG5-EBNA2) and/or p65RelA (pCMV-p65) expression vectors. The results are the means ± SE of relative luciferase activity from three independent experiments. Representative Western blots (bottom panels) showing the expression of p65RelA (p65; bottom left panel), EBNA2 (bottom right panel) and the corresponding β-actin in the cell lysates analyzed in the top panel. (C) Semiquantitative reverse transcription-PCR (RT-PCR) analysis of LMP1 and IL-8 mRNA expression normalized against GAPDH mRNA expression in Daudi cells electroporated with an empty expression vector (–) or a p65RelA (pCMV-p65) expression vector (+). The results from one representative experiment out of two are shown. (D) Western blot analysis of LMP1 expression in Daudi cells following transfection of a p65RelA (pCMV-p65) and/or an EBNA2 (pSG5-EBNA2) expression vector. The expression of p65RelA and β-actin is shown as well. The results from one representative experiment out of two are shown.

The previous experiments demonstrated the ability of NF-{kappa}B to transactivate the LMP1 promoter in reporter constructs. In order to investigate whether this is also possible in the context of EBV genome, the effect of p65RelA on LMP1 expression was examined in the EBV-positive Burkitt's lymphoma cell line Daudi. In this cell line, LMP1 expression is defective due to a deletion in the EBNA2 gene (17). Exogenous p65RelA expression readily induced the expression of LMP1 mRNA and protein (Fig. 4C and D). The induction of LMP1 mRNA by p65RelA in Daudi cells paralleled the induction of IL-8 mRNA, which is a known target of NF-{kappa}B (Fig. 4C). LMP1 protein was also induced by exogenous coexpression of p50NF-{kappa}B1 and p65RelA in P3HR1 cells, which, similarly to Daudi, lack EBNA2 expression (data not shown). Furthermore, p65RelA clearly cooperated with EBNA2 in the induction of LMP1 protein expression in Daudi cells (Fig. 4D, compare lanes 2, 3, and 4), establishing further the interplay between the two transcription factors in the regulation of the LMP1 promoter.

The bioinformatic analysis of the LMP1 promoter predicted a number of putative binding sites for the aryl hydrocarbon receptor (AhR) at positions –51 to –56 (E-box), –175 to –185, and –213 to –218, relative to the LMP1 transcription start site. In the absence of xenobiotics such as dioxins AhR is located in the cytoplasm as a complex with Hsp90 (reviewed in references 3 and 5). In the presence of xenobiotics, AhR translocates into the nucleus, where it forms heterodimers with the aryl hydrocarbon receptor nuclear translocator (ARNT) and activates a number of cellular genes. An interaction between AhR and NF-{kappa}B in the regulation of gene expression has been reported on several occasions (8, 20, 30, 37, 38). These findings prompted an investigation into the possible role of AhR in the transactivation of the LMP1 promoter. A constitutively active form of AhR (CAAhR) (23) or ARNT alone did not affect the +40/–328 LMP1 promoter region, whereas their combination caused a low (twofold)-level transactivation of the LMP1 promoter (Fig. 5). Interestingly, the coexpression of constitutively active AhR and/or ARNT enhanced the p65RelA transactivation of the LMP1 promoter beyond the level of an additive effect, suggesting a possible cooperation between AhR and NF-{kappa}B in the transactivation of the LMP1 promoter. This effect was abolished by a mutation that abrogates binding of NF-{kappa}B on the LMP1 promoter, revealing that the interplay between AhR and NF-{kappa}B depends on the NF-{kappa}BA element. These results suggest an involvement of AhR in the regulation of the LMP1 promoter.


Figure 5
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  		FIG. 5. The NF-{kappa}B p65 and AhR-ARNT cooperatively transactivate the LMP1 promoter. The activity of luciferase reporter constructs containing the wild-type (WT) or mutated +40/–328 LMP1 promoter luciferase reporter plasmids was evaluated following their cotransfection in 293FT cells along with a Renilla luciferase expression plasmid, which was used for the normalization of transfection efficiencies in the absence (–) or presence (+) of vectors expressing p65RelA (pCMV-p65), constitutively active AhR (pCAAhR/TRE), and ARNT (pSport-ARNT). The results are the means ± SE of relative luciferase activity from at least three independent experiments (top panel). The expression of p65RelA (p65), ARNT, and β-actin in the cell lysates was determined by Western blotting (bottom panel).


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DISCUSSION
 
A detailed mutational analysis of consensus cis-acting elements in the LMP1 promoter established the functional importance of the ISRE, POU-box, PU.1, RBPJ{kappa}, NF-{kappa}BA, and NF-{kappa}BA-overlapping AP-2 sites for the activity of the LMP1 promoter in type III latent EBV infection of human B lymphocytes. These findings are of particular importance since previous studies indicated a possible requirement of some of these elements for LMP1 expression in latent EBV infection, yet the analysis was performed largely in EBV-negative cell lines which do not offer a physiologically relevant background. In addition to a comprehensive analysis of the role of NF-{kappa}B in the regulation of the LMP1 promoter, our study demonstrated for the first time the functional importance of the NF-{kappa}BA-overlapping AP-2 site. A different consensus AP-2 binding site located at the position –95/–103 was identified as an important functional element by Jansson et al. in EBV-negative B-cell lymphoma cells (14). Our results taken together with those of Jansson et al. reinforce the possibility of an important functional role of a transcription factor that interacts with the AP-2 sites of the LMP1 promoter. Additional reports showed that ISRE mediates the positive effect of IRF7 on the LMP1 promoter which is antagonized by an IRF5-IRF7 complex (24, 25). The mutation that we introduced in the ISRE element upregulates the LMP1 promoter in EBV-transformed lymphoblastoid cell lines, suggesting that it alleviates a negative regulatory effect that may involve an IRF molecule.

While this report was being written, Johansson et al. (16) also reported a direct functional role of NF-{kappa}B in the transactivation of the LMP1 promoter in agreement with our findings. Johansson et al. showed that the LMP1 promoter can be transactivated not only by p65RelA homodimers or heterodimers with p50NF-{kappa}B1 but also by p50NF-{kappa}B1 alone. We were unable to demonstrate a transactivating effect by p50NF-{kappa}B1 alone on LMP1 expression in Daudi or P3HR1 cells. It is possible that this is due to the inadequate expression of p50 NF-{kappa}B1 in our experiments. Nevertheless, our findings and those of Johansson et al. are consistent with a positive NF-{kappa}B-dependent autoregulatory loop in LMP1 expression, since LMP1 is a potent activator of NF-{kappa}B. This mechanism would favor the establishment of latency III in EBV-infected B lymphocytes since even a low level of LMP1 expression could possibly augment the activity of its own promoter. Notably, previous studies have shown that STATs and IRFs mediate the autoregulation of LMP1 transcription. The present study broadens the network of autoregulatory factors of LMP1 promoter, highlighting the complexity of the mechanism of LMP1 expression.

Our study investigated the relationship between NF-{kappa}B and EBNA2, which is a major transactivator of the LMP1 promoter. We have demonstrated that NF-{kappa}B can transactivate the LMP1 promoter in the absence of EBNA2, in agreement with the results of Johansson et al. (16). This finding raises the possibility of triggering LMP1 transcription by inducers of the NF-{kappa}B pathway in latently infected cells that do not express EBNA2 and LMP1. Furthermore, our results support a positive interplay between EBNA2 and NF-{kappa}B for the optimal transactivation of the LMP1 promoter. The molecular nature of this cooperation is not known, but it may involve biochemical interactions between EBNA2 and NF-{kappa}B that favor the dislocation of repressor molecules and the concomitant recruitment of activators in the LMP1 promoter. Alternatively, the coordinated activation of the LMP1 promoter by EBNA2 and NF-{kappa}B may take place without a direct interaction between these two transcription factors.

NF-{kappa}B may play an important role in determining differential LMP1 expression among different EBV isolates. Significant variations in LMP1 expression among cell lines and latency types have been demonstrated (reviewed in reference 19). Interestingly the present study and previous reports have shown that the activity of the LMP1 promoter from the P3HR1 strain is lower than the activity of the corresponding region of the B95-8 strain, suggesting that the transcription factor binding site alterations in the P3HR1 promoter affect its strength (14). These alterations affect only the ATF/CRE and the NF-{kappa}BA sites among known transcription factor binding sites in the LMP1 promoter. We have shown that a one-base difference between the P3HR1 (5'-GGGGAGTTGC-3') and B95-8 (5'-GGGGATTTGC-3') NF-{kappa}BA sites weakens the interaction of p65RelA and p50NF-{kappa}B1 with the P3HR1 NF-{kappa}BA site. This happens despite the fact that this one-base change does not introduce an additional deviation from the consensus NF-{kappa}B binding site (5'-GGGRNNYYCC-3'). Therefore NF-{kappa}B, along with ATF1 and CREB1, may play a pivotal role in fine tuning LMP1 expression in latently infected human B lymphocytes.

A previous study concluded that NF-{kappa}B plays a negative role in the activity of the LMP1 promoter, based on the negative role of a nondegradable I{kappa}B molecule in LMP1-mediated transactivation of the LMP1 promoter (9). In the same study, p65RelA overexpression failed to transactivate the LMP1 promoter in HEK293 cells. The discrepancy between our study and the study by Goormachtigh et al. (9) cannot be easily reconciled. One possibility is that p65RelA was expressed in much higher levels in the study of Goormachtigh et al. (9) than in the present study, which resulted in a squelching effect. On the other hand, overexpression of the nondegradable I{kappa}B molecule might have non-NF-{kappa}B-dependent effects.

The presence of putative AhR binding sites in the LMP1 promoter and the previous functional association between AhR and NF-{kappa}B complexes led to the investigation of a possible involvement of AhR in the regulation of the LMP1 promoter. We have demonstrated that AhR can coordinately transactivate the LMP1 promoter with p65RelA in a manner that depends on the integrity of the NF-{kappa}BA site. AhR-containing complexes may be recruited onto the LMP1 promoter through direct interactions with cis-acting elements. Alternatively, p65RelA may mediate the recruitment of AhR complexes to the LMP1 promoter, as shown previously for the promoter of c-myc (20). Interestingly, a previous report has demonstrated that the latent EBV antigen EBNA3A can facilitate the ligand-dependent activation of AhR (18). EBNA3A can access the LMP1 promoter via an interaction with RBPJ{kappa} (29). Therefore, an additional possibility for the recruitment of AhR to the LMP1 promoter is through its ability to interact with EBNA3A. Taken together, these findings raise the possibility that environmental pollutants, such as dioxins, may induce LMP1 expression and contribute to EBV-associated carcinogenesis indirectly.

Our study revealed novel aspects of the regulation of LMP1 expression that involve highly complex interactions among cellular and viral transcription factor networks. This level of complexity would be consistent with the requirement for a broad range of finely tuned expression since LMP1 has both proliferative and antiproliferative functions.


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ACKNOWLEDGMENTS
 
The expression vectors pCAAhR/TRE, pSport-ARNT, and pCMV-p65 were kind gifts from Christoph Koehle (University of Tuebingen), Chris Bradfield (University of Wisconsin—Madison), and Dimitris Thanos (Biomedical Research Foundation, Academy of Athens), respectively. P3HR1 and Daudi cells were kindly provided by Eva Klein (Karolinska Institute). The PE2 monoclonal antibody was kindly provided by Paul Farrell (Imperial College London).

This work was supported in part by the Hellenic Secretariat of Research and Technology (Program {Pi}ENE{Delta} 2003) and in part by the Sixth Research Framework Programme of the European Union, Project INCA (LSHC-CT-2005-018704). G.M. is a Leukemia and Lymphoma Society Scholar.


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FOOTNOTES
 
* Corresponding author. Mailing address: School of Biology, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece. Phone and fax: 302310998907. E-mail: gmosialo{at}bio.auth.gr Back

{triangledown} Published ahead of print on 11 March 2009. Back


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Journal of Virology, May 2009, p. 5269-5277, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.00097-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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