Raji is a human EBV-positive BL cell line, and LCL-Ao, LCL-Ka and LCL-Ku are EBV-immortalized human B-cell lines generated from peripheral blood mononuclear cells of healthy volunteers. These lymphoid cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10 or 20% heat-inactivated fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin. Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin and 50 μg/ml streptomycin.

The expression of CD69 was analyzed by flow cytometry with phycoerythrin (PE)-labeled mouse monoclonal antibody against CD69 (clone TP1.55.3; Beckman Coulter, Fullerton, CA). Analyses were carried out with isotype-matched control antibody. The cells were incubated with the antibody for 30 min, washed with cell WASH (Becton-Dickinson Immunocytometry Systems, San Jose, CA) and then subjected to analysis in the cytometer.

Total RNA was prepared from various cell cultures by TRIzol (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer. First-strand cDNA was synthesized from 1 μg total cellular RNA using a PrimeScript RT-PCR kit (Takara Bio Inc., Otsu, Japan) with random primers. The primers used were 5′-CATAGCTCTCATTGCCTTATCAGT-3′ (forward) and 5′-CCTCTCTACCTGCGTATCGTTT-3′ (reverse) for CD69, 5′-GTGACTGGACTGGAGGAGCC-3′ (forward) and 5′-GAGGGAGTCATCGTGGTGGTG-3′ (reverse) for LMP-1, and 5′-GTGGGGCGCCCCAGGCACCA-3′ (forward) and 5′-CTCCTTAATGTCACGCACGATTTC-3′ (reverse) for β-actin. The length of the semiquantitative reverse transcription-PCR (RT-PCR) for each gene was: 30 cycles for CD69 and LMP-1, and 28 cycles for β-actin. The PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.

Luciferase assay was performed to confirm that LMP-1 induces the CD69 promoter activation. Approximately 3×10⁵ 293T cells per plate were transfected by the calcium phosphate DNA co-precipitation method. All transfections included appropriate reporter and effector plasmids. The expression plasmids pSG5-LMP-1, pSG5-LMP-1Δ187-351, pSG5-LMP-1Δ 349 and pSG5-LMP-1Δ194-386 were kindly provided by Dr Martin Rowe (University of Wales College of Medicine, Cardiff, UK) (18,19). The CD69 promoter-luciferase gene constructs have already been described (20,21). The single and combined internal deletion mutants of NF-κB sites were constructed by deletion of the NF-κB sites of the plasmid pAIM 255-LUC. The IκBαΔN- and IκBβΔN-dominant negative mutants are IκBα and IκBβ deletion mutants lacking the amino-terminal 36 and 23 amino acids, respectively (22,23). The dominant negative mutants of IKKα, IKKα (K44M), IKKβ, IKKβ (K44A), IKKγ, IKKγ (1–305) and NIK, NIK (KK429/430AA) have been described previously (24,25). Plasmids for truncated TRAF2 and TRAF5 proteins retaining only the TRAF domain, ΔTRAF2 and ΔTRAF5, have been described previously (26,27). In all cases, the reference plasmid phRL-TK, which contains the Renilla luciferase gene under the control of the herpes simplex virus thymidine kinase promoter, was cotransfected to correct for transfection efficiency. After 24 h, the transfected cells were collected by centrifugation, washed with phosphate-buffered saline and lysed in reporter lysis buffer (Promega, Madison, WI). Luciferase assays were conducted using the dual luciferase reporter system (Promega), in which the relative luciferase activity was calculated by normalizing transfection efficiency according to the Renilla luciferase activities.

Nuclear proteins were extracted as described by Antalis and Godbolt (28) with some modifications, and NF-κB binding activity to the NF-κB element was examined by EMSA. Briefly, 5 μg of nuclear extracts were preincubated in a binding buffer containing 1 μg polydeoxyinosinic-deoxycytidylic acid (GE Healthcare Biosciences, Buckinghamshire, UK), followed by the addition of ³²P-labeled oligonucleotide probes containing the NF-κB element. The mixtures were incubated for 15 min at room temperature. The DNA protein complexes were separated on 4% polyacrylamide gels and visualized by autoradiography. The probes and competitors used were prepared by annealing the sense and antisense synthetic oligonucleotides as follows: the NF-κB element (κB1) of the CD69 gene (5′-GATCCAGACAACAGGGAAAACCCATACTTC-3′); the NF-κB element (κB2) of the CD69 gene (5′-GATCCAGAGTCTGGGAAAATCCCACTTTCC-3′); a typical NF-κB element from the IL-2 receptor α chain (IL-2Rα) gene (5′-GATCCGGCAGGGGAATCTCCCTCTC-3′) and an AP-1 element of the IL-8 gene (5′-GATCGTGATGACTCAGGTT-3′). The above underlined sequences represent the NF-κB and AP-1 binding sites, respectively.

We examined first whether CD69 upregulation is a general feature of EBV-infected B cells. All EBV-immortalized B-cell lines, LCL-Ao, LCL-Ka and LCL-Ku, and EBV-positive BL cell line, Raji, constitutively expressed CD69 on the cell surface (Fig. 1A). In contrast, CD69 was hardly expressed on normal peripheral blood mononuclear cells (data not shown).

EBV-immortalized B-cell lines and Raji cells constitutively express LMP-1 (29,30). Therefore, we analyzed the induction of CD69 expression by LMP-1. Transient expression assays using a mammalian expression vector for LMP-1 were performed in 293T cells. In 293T cells transfected with empty vector (pSG5), CD69 mRNA was not detectable as determined by RT-PCR analysis (Fig. 1B). In contrast, ectopic expression of LMP-1 induced the expression of CD69 mRNA in 293T cells. Next, we investigated whether LMP-1 also transcribed the endogenous CD69 gene. The expression of CD69 antigen on the cell surface of 293T cells increased after ectopic expression of LMP-1 in 293T cells, and such increase was time-dependent (Fig. 1C).

To determine whether LMP-1 regulates CD69 promoter activity, transient expression assays using the reporter plasmid pAIM 1.4-LUC and either an expression vector for LMP-1 or empty vector (pSG5) were performed in 293T cells. LMP-1 induced relative levels of CD69 promoter-directed luciferase expression in a dose-dependent manner, suggesting that LMP-1 functionally activates CD69 promoter (Fig. 2A). To map the region in the LMP-1 protein that mediates activation of CD69 promoter, LMP-1 mutants were expressed in 293T cells and their effect on CD69 promoter activity was investigated. The LMP-1 mutants used included LMP-1Δ187-351 (which contains only CTAR-2 in the carboxy-terminus), LMP-1Δ349 (which lacks CTAR-2), and LMP-1Δ194-386 (in which the entire carboxy-terminal cytoplasmic region is deleted) (Fig. 2B, left panel). In cells that expressed LMP-1Δ194-386, CD69 promoter activity was not significantly increased (Fig. 2B, right panel). In contrast, LMP-1Δ187-351 induced 50% of wild-type LMP-1 CD69 promoter activation. Furthermore, LMP-1Δ349 showed substantial impairment of CD69 promoter activation. These results suggest that LMP-1 activates CD69 expression via the cooperative activity of CTAR-1 and CTAR-2 signaling motifs.

CTAR-1 and CTAR-2 are known to activate the NF-κB signaling pathway (5–8). Based on the discussed background, we tested the ability of the dominant interfering mutants of IκBα, IκBβ, IKKγ, TRF2 and TRAF5, and kinase-deficient mutants of IKKα, IKKβ and NIK to inhibit LMP-1-mediated transactivation of CD69-driven reporter gene activity. Expression of each of these inhibitory mutants inhibited LMP-1-induced activation of CD69 promoter (Fig. 2C). These results demonstrated that the signaling components, TRAFs, NIK and IKKs, which are involved in the activation of NF-κB, are also necessary for LMP-1 transactivation of the CD69 promoter.

The CD69 promoter contains three putative binding sites for NF-κB [positions −373 (κB3), −223 (κB2) and −160 (κB1)], a putative binding site for early growth response (EGR) at −69, a composite binding site for Sp1 and EGR at −56, a cyclic adenosine 3′,5′-monophosphate response element-binding protein (CREB) binding site at −46, and two putative AP-1 binding sites at −16 and +1 (Fig. 3A) (20). To study the role of NF-κB sites in the transcriptional induction of CD69 gene by LMP-1, we transfected 293T cells with several 5′-deletion fragments extending from position −1.4 k to −78 (Fig. 3A), and an expression vector for LMP-1. Progressive removal of 5′-sequence up to position −255 did not significantly inhibit the induced promoter activity, suggesting that the 271-bp fragment, spanning positions −255 to +16, contained the LMP-1-responsive elements (Fig. 3A). Further deletion of upstream sequences up to position −170 resulted in significant loss of responsiveness to LMP-1. Additional removal of the 5′-sequences up to position −78 further affected the inducible promoter activity.

Two potential NF-κB-binding sequences were identified at positions −160 (κB1) and −223 (κB2) spanning positions −255 to +16 bp. κB1 and κB2 were identical to those found in the gene promoters of c-myc and IL-6, respectively (31). To test the relative contribution of the NF-κB binding sites to the LMP-1-mediated activation of CD69, plasmids with internal deletion mutants of these sites in the CD69 promoter were transfected (Fig. 3B). Single deletion of the κB1 or κB2 site resulted in reduction of the inducible activity. Double deletions of the κB1 and κB2 sites further reduced Tax-mediated activation of this reporter construct. These results indicate that the two NF-κB binding sites in the CD69 promoter regulate LMP-1-induced upregulation of CD69.

Since both NF-κB binding sites of the CD69 promoter are required for LMP-1-mediated CD69 gene transcription, we studied the ability of LMP-1 to activate NF-κB binding to DNA. For this purpose, 293T cells were transfected with control plasmid (empty vector) or LMP-1 expression plasmid for 24 or 48 h, followed by extraction of nuclear proteins. EMSA demonstrated that LMP-1 increased the binding of NF-κB to ³²P-labeled oligonucleotides representing the κB1 and κB2 sites (Fig. 4A). The specificity of DNA-protein complex formation was determined by competition studies with unlabeled competitors. As expected, excess of cold CD69 κB1 or κB2 double-stranded oligonucleotide, or consensus NF-κB site from the IL-2Rα promoter, effectively competed with the labeled probes and eliminated the binding of nuclear extracts from 293T cells transfected with LMP-1 expression plasmid (Fig. 4B, lanes 2–5). In contrast, the unlabeled IL-8 AP-1 element did not compete with labeled probes (Fig. 4B, lanes 2 and 6). The nucleoproteins binding to both NF-κB sites were composed of p50, p65 and c-Rel subunits of the NF-κB family, as demonstrated by supershift experiments with specific antibodies (Fig. 4B, lanes 7–9).

Finally, to determine the role of LMP-1 on endogenous NF-κB binding to DNA, we measured NF-κB binding to respective NF-κB elements in the CD69 promoter in LMP-1- and CD69-expressing Raji cells. As expected, protein complexes bound to both κB1 and κB2 sites were detected in nuclear extracts from Raji cells (Fig. 4C, lane 1). The specificity of DNA-protein complexes in these extracts was determined by competition studies using unlabeled competitors. As observed in nuclear extracts from 293T cells transfected with LMP-1 expression plasmid, cold κB1 and κB2 oligonucleotides, and consensus NF-κB site from the IL-2Rα promoter, but not the IL-8 AP-1 element, efficiently competed with labeled probes (Fig. 4C, lanes 1–5). Antibodies against p50, p65, c-Rel and RelB induced a supershift of the DNA-protein complexes (Fig. 4C, lanes 6–8 and 10). Taken together, the results indicate that NF-κB proteins bind to both κB elements of the CD69 promoter in LMP-1- and CD69-expressing Raji cells.