The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition to a pathogen by the immune system. The level of MHC class II directly influences T lymphocyte activation. The aim of this study was to identify the possible mechanisms of the down-regulation of MHC class II expression by Zta during EBV lytic cycle. The data in the present study demonstrated that ectopic expression of Zta can strongly inhibit the constitutive expression of MHC class II and CIITA in Raji cells. The negative effect of Zta on the CIITA promoter activity was also observed. Scrutiny of the DNA sequence of CIITA promoter III revealed the presence of two Zta-response element (ZRE) motifs that have complete homology to ZREs in the DR and left-hand side duplicated sequence promoters of EBV. By chromatin immunoprecipitation assays, the binding of Zta to the ZRE221 in the CIITA promoter was verified. Site-directed mutagenesis of three conserved nucleotides of the ZRE221 substantially disrupted Zta-mediated inhibition of the CIITA promoter activity. Oligonucleotide pull-down assay showed that mutation of the ZRE221 dramatically abolished Zta binding. Analysis of the Zta mutant lacking DNA binding domain revealed that the DNA-binding activity of Zta is required for the trans repression of CIITA. The expression of HLA-DRα and CIITA was restored by Zta gene silencing. The data indicate that Zta may act as an inhibitor of the MHC class II pathway, suppressing CIITA transcription and thus interfering with the expression of MHC class II molecules.
Epstein-Barr virus is a ubiquitous human γ-herpesvirus found in nearly all humans worldwide (1). EBV infection in adolescence causes the syndrome of infectious mononucleosis. The persistence of EBV-infected cells is thought to be a major risk factor in pathogenesis of several malignancies of lymphoid and epithelial origin, particularly Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma, and posttransplant lymphoproliferative disease (2, 3). During primary infection, EBV initially undergoes lytic replication in the epithelial cells of the oropharynx and salivary glands. The virus subsequently infects trafficking B cells, where the virus establishes a lifelong persistence and proceeds periodic spontaneous reactivation, resulting in lytic replication, infectious virus production, and transmission (4).
Lytic replication of EBV is initiated by the expression of two immediate-early genes, BZLF1 and BRLF1. Zta (also called ZEBRA, BZLF1) encoded by BZLF1 is the control switch for expression of many early lytic genes and is also important in mediating viral DNA replication (5, 6, 7, 8). As a transcription factor, Zta activates its own expression and induces the lytic cascade of viral gene expression through interaction with a series of sequence-specific DNA binding sites termed Zta-response elements (ZREs)4 within their respective promoters (9). Recent studies indicate that Zta also regulates the transcription of cellular genes either positively or negatively by direct binding or via their associations with other transcription factors (9, 10, 11, 12). However, the mechanisms of regulating cellular genes by Zta have been poorly understood.
It has been suggested that Zta may have important immunomodulatory functions. It has been reported that the lytic cycle of EBV correlates with the diminution of cell surface MHC class I molecules, and down-regulation of surface MHC class I expression is maintained throughout the lytic cycle of EBV, which could conceivably have a significant effect on Ag presentation (13). In EBV-negative B cells, Zta completely inhibits the up-regulation of surface MHC class I expression induced, and also directly inhibits the constitutive activation of NF-κB (13, 14). In addition, it has recently been shown that the expression of the IFN-γ receptor gene is down-regulated at both the mRNA and protein levels following introduction of an adenovirus vector expressing Zta (15).
It has also been proposed that MHC class II molecules and the Ag-processing pathway may be targets of EBV for escaping CD4+ T lymphocyte immunosurveillance. The studies with human CMV (HCMV) and mouse CMV reveal that CMV blocks IFN-γ-inducible MHC class II transcription (16, 17). By inhibiting Jak1 expression and disrupting IFN-γ-stimulated signaling pathway, HCMV interferes with MHC class II transcription and CIITA activation. Other strategies for the down-modulation MHC class II surface expression by HCMV are promoting the proteasome-mediated degradation of DR-α and DM-α molecules by glycoprotein US2 (18, 19). In addition, US3 protein of HCMV competes with Ii for binding to MHC class II molecules, interfering with Ag presentation (19, 20). Only recently have analogous immune evasion strategies been described for EBV. It has been demonstrated that the EBV gp42 protein binds to HLA class II molecules at their various stages of maturation and impairs TCR-mediated activation of Ag-specific Th cells in an HLA class II-dependent manner (21). A truncated soluble EBV gp42 protein, generated by proteolytic cleavage of full-length gp42 in the endoplasmic reticulum during the EBV lytic infection, can mediate Ag-specific evasion from Th cell recognition by blocking the TCR-HLA class II-peptide association (22). It has also been reported that EBV lytic infection is associated with the reduction of MHC class II expression (13). However, the mechanism underlying modulation of MHC class II expression by EBV has not yet been established.
MHC class II is exquisitely controlled mainly at the level of transcription initiation by a highly conserved regulatory module. A very tight regulation of MHC class II expression is crucial for the control of the immune response. CIITA is the major regulator of MHC class II gene expression and functions as a non-DNA-binding transcriptional coactivator by interacting with almost all of the transcription regulatory proteins, forming a stable enhanceosome bound to the SXY regulatory module of MHC class II promoters and driving transcription of MHC class II genes (23, 24).
The present study aims to explore how MHC class II expression was down-regulated in EBV lytic cycle. We first observed that the expression of MHC class II was reduced in Raji cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (NaB) concomitantly with a significant decrease of CIITA expression. Computational analysis revealed two potential ZREs within the CIITA promoter. Experimental studies demonstrated that Zta inhibited the promoter activity and transcription of CIITA. The binding of Zta to the promoter of CIITA was confirmed both in vitro and in vivo. Furthermore, the expression of MHC class II and CIITA was restored by small-interfering (si)RNA-directed knockdown of Zta. Our data indicate that Zta-mediated suppression of CIITA transcription is a distinct mechanism of down-regulation of MHC class II expression during EBV reactivation.
Materials and Methods
Cells and induction of EBV reactivation
B95-8 is an EBV-secreting marmoset cell line and commonly used to obtain infectious virus (25, 26). Burkitt’s lymphoma-derived Raji cells are EBV-carrying nonproducer cells (27). Raji cells can be induced to enter an abortive viral replication cycle and to synthesize EBV early Ag by various agents, including TPA (28). Raji and B95-8 cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Yuanheng Biotech.) and maintained in a humidified atmosphere of 5% CO2 at 37°C. For induction of the lytic cycle, Raji cells were activated with 100 nM TPA (Sigma-Aldrich) and 3 mM NaB (Sigma-Aldrich) for 36 h.
Construction of BZLF1 and CIITA promoter reporter plasmids
⇓NotI and ApaI sites to generate the plasmid expressing Flag-tagged Zta (pZta). The plasmid containing the cDNA encoding a Zta mutant missing the DNA binding domain was also cloned into pRc/CMV by the same sites (pΔZta).BZLF1 cDNA was amplified by PCR with Pyrobest DNA polymerase (Takara) using the primers P1 and P2 (Table I
The sequence (nt −1383/+44 relative to transcription initiation site) of human CIITA promoter III (PIII) was amplified by PCR with Raji cell genomic DNA as a template using the primers P3 and P4 (Table I⇑) and cloned into the plasmid pGL3-basic vector (Promega), which expresses the firefly luciferase, by XhoI and HindIII sites, designated pCIITA(−1383/+44). The reporter plasmids with 5′ end deletion of CIITA PIII, pCIITA(−288/+44), and pCIITA(−199/+44) were constructed by inserting PCR-amplified CIITA promoter fragments using the primers P5, P6, and P4 (Table I⇑) into pGL3-basic vector at XhoI and HindIII sites. The site-directed mutation of the CIITA PIII (pCIITA(−288/+44)Mut) was generated with the primers P7 and P8 (Table I⇑) using TakaRa mutanBEST kit, as described by the manufacturer. The plasmid pCIITA(−1383/+44)Mut was produced by overlap extension PCR using primers P4/P9 and P3/P10 (Table I⇑). All constructs were verified by sequence analysis. The plasmid pRL-TK containing HSV thymidine kinase promoter driving Renilla luciferase gene (Dual Luciferase assay; Promega) was used as an internal control in all of the reporter plasmid transfections.
Transfection with plasmid DNA or siRNA
Four dsRNA oligonucleotides targeting the BZLF1 sequences 5′-CAACAGCTAGCAGACATTG-3′, 5′-GCTAGCAGACATTGGTGTT-3′, 5′- GGACAACAGCTAGCAGACA-3′, and 5′-AGCAACTGCTGCAGCACTA-3′ were synthesized by Genechem. A positive control dsRNA oligonucleotide for GAPDH sequence 5′-GTGGATATTGTTGCCATCA-3′ and a nontargeting sequence 5′-UUCUCCGAACGUGUCACGU-3′, which was designed to minimize sequence homology to any known vertebrate transcript with similar length and germinal center content to BZLF1-specific siRNA, was also synthesized. For plasmid DNA transfection, 30 μg of DNA was electroporated into Raji cells (1 × 107 cells in 400 μl of RPMI 1640) with an Electro square porator 830 (BTX) at 1100 V, 40 μs, twice pulse, with an interval of 1 min. For gene silencing by siRNA, 5 μl of 20 μM stock dsRNA oligonucleotides was electroporated into 2 × 106 Raji cells treated with TPA/NaB in 100 μl of RPMI 1640 at 550 V, 40 μs, twice pulse, with an interval of 1 min. After 10 min of incubation on ice, RPMI 1640 medium supplemented with 15% FBS was added to the cells and incubated for 48 h.
Total RNA was isolated from Raji cells and quantified by spectrophotometry. cDNA was synthesized from 2 μg of total RNA using oligo(dT) and Moloney murine leukemia virus reverse transcriptase, and subsequently amplified by using specific primers P11 and P12 (Table I⇑) for a 644-bp HLA-DRα fragment and P13 and P14 for a 325-bp CIITA fragment. Amplification of β-actin gene was used as an internal control for cDNA loading. PCR was performed using serial dilutions of the template to verify that amplification was in the linear range for the assays. The product was analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The intensity of bands was quantified using LabWorks Image Acquisition and Analysis software (Ultraviolet Products). The experiment was conducted in triplicate.
Flow cytometry (FACS)
A total of 1 × 106
Preparation for nuclear extracts
The nuclear extracts of Raji cells were prepared by using a Nuclear-Cytosol Extraction Kit (Applygen Technologies; P1200), according to the manufacturer’s instructions. Briefly, ∼108 Raji cells treated with TPA/NaB were harvested by centrifugation. The pelleted cells were resuspended in 500 μl of cytosol extraction buffer A. The suspension was incubated on ice for 10 min, then mixed with 30 μl of cytosol extraction buffer B, and further incubated on ice for 1 min. The lysates were pelleted by centrifugation, washed with cytosol extraction buffer A, and resuspended in 100 μl of cold nuclear extraction buffer. After incubation at 4°C for 30 min with constant rotation, the suspension was spun at 12,000 × g at 4°C for 5 min to collect the nuclear extract in the supernatant fraction.
Raji cells were treated with or without TPA/NaB. After adding TPA/NaB for 36 h, cells were washed twice by ice-cold PBS, and the whole-cell lysates were prepared using radioimmunoprecipitation assay lysis buffer (Applygen Technologies; C1053), according to the manufacturer’s protocol. The preparation for nuclear extracts was as described above. Protein concentration of the whole-cell lysates and the nuclear extracts was determined by BCA Protein Assay Kit (Pierce; 23227). The proteins were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% milk and incubated with primary Abs against Flag M2 (Sigma-Aldrich; F3165), Zta (Santa Cruz Biotechnology; sc-53904), HLA-DRα (Santa Cruz Biotechnology; sc-55593), CIITA (Santa Cruz Biotechnology; sc-48797), and CD74 (Santa Cruz Biotechnology; sc-20082), followed by washing and incubation with HRP-conjugated secondary Abs (Dingguo). Equal loading was verified by detection of GAPDH or AP-2α using the anti-GAPDH (Kangchen; KC-5G4) and anti-AP-2α (Santa Cruz Biotechnology; sc-184) Abs, respectively. Bands were visualized by SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).
Raji cells were cotransfected with 10 μg of reporter plasmid (with a luciferase gene driven by various CIITA promoters) and 10 μg of effector plasmid (the Zta or ΔZta-expressing plasmid or the control empty plasmid). A total of 500 ng of pRL-TK was cotransfected along with the reporter plasmids to monitor the transfection efficiency. After 48 h, the cell lysates were used to assay for firefly luciferase and then Renilla luciferase, using a dual luciferase assay kit (Promega), according to the manufacturer’s instructions. Luciferase activity was calculated by dividing firefly luciferase activity by Renilla luciferase activity in each sample. Each assay was conducted in triplicate, and the experiments were performed three times independently.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed according to the protocol provided with the ChIP assay kit (Upstate Biotechnology; 17-295). Briefly, 3 × 106 Raji cells transfected with pZta were cross-linked with 1% formaldehyde at 37°C for 10 min, washed once with cold PBS containing protease inhibitors, and lysed in SDS lysis buffer. Chromatin was sheared by sonication to ∼800 bp. The lysates were immunoprecipitated with anti-Flag M2 mAbs or mouse IgG at 4°C overnight, followed by incubation with protein A-agarose/salmon sperm DNA at 4°C for 1 h with rotation. The immunoprecipitated complexes were washed with various complex wash buffers and TE buffer and eluted with elution buffer. The eluates were then treated with 5 M NaCl and digested with proteinase K. The DNA fragments were recovered by phenol-chloroform extraction, precipitated with ethanol, and analyzed by PCR using primers P15-P18 (Table I⇑) specific for a 221-bp region spanning positions −288 to −68 and a 155-bp region spanning positions −1333 to −1179 harboring two potential ZREs at positions −221 to −215 (ZRE221) and −1287 to −1281 (ZRE1287), respectively. Final products were resovled on a 1.5% agarose gel.
Promoter pull-down assays
Zta inhibits the expression of MHC class II
EBV reactivation in vitro can be triggered by treatment of the EBV nonproducer Burkitt’s lymphoma line Raji with TPA and NaB (29, 30). Increased expression of the key immediate-early gene BZLF1 is one of the first events that can be detected following the induction (29). The product of BZLF1, the lytic-cycle nuclear Ag Zta mediates the switch from latency to cytolytic replication of EBV. Previous reports suggested that Zta was responsible for the decreased cell surface expression of MHC class II in EBV-infected cells (13). To elucidate the molecular impact of Zta on MHC class II expression, Raji cells, which constitutively express high levels of all three isotypes of MHC class II proteins (31), were treated with or without TPA/NaB. The cell lysates were separated by SDS-PAGE and analyzed by Western blot using anti-HLA-DRα Ab. Fig. 1⇓A shows that the expression of Zta was efficiently induced by the treatment of TPA/NaB. A conspicuous decline of the expression of HLA-DRα molecules was observed in induced vs uninduced Raji cells, correlating with the observed increase in Zta protein (Fig. 1⇓A). Detection of GAPDH confirmed that equivalent proteins were assayed. Staining with PE-labeled mAb against HLA-DR revealed a distinct reduction of the surface expression of HLA-DR proteins on the Raji cells treated with TPA/NaB compared with the cells untreated (Fig. 1⇓B).
To ensure that the inhibition of MHC class II expression in Raji cells following TPA/NaB treatment was selectively and specifically mediated by Zta, pZta was constructed. Raji cells were transfected with 0, 5, 10, and 30 μg of pZta. Fig. 1⇑C shows that the HLA-DRα protein expression analyzed by Western blot was clearly decreased in the Raji cells transiently transfected with the Zta expressing plasmid in a dose-dependent manner. Ectopic expression of Zta also caused diminished HLA-DR on the surface of Raji cells as demonstrated by FACS (Fig. 1⇑, D–F). To examine whether Zta exerted an inhibitory effect on expression of CD80 molecule, a ligand for the CD28 costimulatory receptor on T cells, concomitant with the down-regulation of HLA-DR, Raji cells transfected with 30 μg of pZta were simultaneously stained for HLA-DR and CD80. Two-color FACS analysis was performed. In comparison with a significant decrease of HLA-DR expression, the level of CD80 on transfected Raji cells appeared to be unchanged (Fig. 1⇑, G and H). The result is consistent with that in a previous study, showing that CD80 expression was not inhibited in lytic Zta-positive lymphoblastoid B cell line Ag876 (13). The data indicated that down-regulation of MHC class II expression during EBV reactivation in Raji cells could be mediated by Zta.
Zta inhibits the expression of CIITA
CIITA is the master controller of constitutive and inducible MHC class II expression. Its expression pattern correlates with MHC class II gene expression qualitatively and quantitatively (23, 32). The results in Fig. 1⇑ demonstrate an apparent alteration in MHC class II expression in Raji cells treated with TPA/NaB or transfected with pZta. To determine whether CIITA expression was also affected by Zta, we then examined the CIITA protein level in Raji cells transiently transfected with 0, 5, 10, and 30 μg of pZta, respectively. After transfection for 48 h, the nuclear proteins were prepared and the CIITA expression was monitored by Western blot with anti-CIITA Ab. Detection of AP-2α was used as a loading control. As shown in Fig. 2⇓A, the transfection with increasing amounts of pZta resulted in decreasing expression of CIITA compared with either the cells transfected with empty plasmid or untransfected cells, which expressed significant levels of CIITA.
The regulation of constitutive and IFN-γ-inducible CIITA gene expression is controlled predominantly at the level of transcription (33). In different B lymphocyte cell lines, a high level of CIITA type III mRNA is present (34). To investigate whether altered CIITA and HLA-DRα production was due to the reduction of mRNA levels, we therefore evaluated the effect of Zta on the expression of MHC class II and CIITA mRNA using semiquantitative RT-PCR. The various amounts of pZta were transfected into Raji cells. Total RNA was recovered from the transiently transfected Raji cells. Constant amounts of reverse-transcribed mRNA were amplified, and amplification of β-actin was used to normalize HLA-DRα and CIITA expression. Using the specific primers, an expected 644-bp PCR product of HLA-DRα and 325-bp product of CIITA were generated. A significant reduction of HLA-DRα and CIITA mRNA level was observed in Raji cells treated with TPA/NaB (Fig. 2⇑B). Transient transfection with increasing amounts of the pZta resulted in a dose-dependent decline of HLA-DRα expression. Likewise, the decreased CIITA mRNA expression was also detected. Fig. 2⇑B shows representative agarose gel electrophoresis of RT-PCR products. These results demonstrate that Zta is involved in the down-regulation of HLA-DRα and CIITA transcription in Raji cells.
HLA class II molecules are αβ heterodimers that associate in the endoplasmic reticulum with the invariant chain (Ii). The conserved cis-acting SXY elements, through which CIITA acts, are also found upstream of the Ii gene (35). The expression of Ii is typically coregulated with MHC class II proteins in many cell types (36). To investigate whether Ii expression is also affected by Zta, we analyzed Ii expression by Western blot using a rabbit polyclonal Ab against human CD74. As we expected, the expression of Ii was also significantly down-regulated in both TPA/NaB-induced and pZta-transfected Raji cells (Fig. 2⇑C).
Zta inhibits the activity of CIITA PIII
Zta is a member of the family of bZIP transcription factors (37). Above RT-PCR analysis indicates that ectopic expression of Zta affected CIITA transcription. It raises the hypothesis that Zta may down-regulate CIITA through cis-acting sequences in the CIITA promoter. Sequence analysis of CIITA PIII revealed the presence of two putative ZREs, Zta1287 and Zta221, which have not been reported, with a consensus sequence (T(G/T)(A/T)G(T/C)(G/C/A)A) at −1287 to −1281 (TGAGCCA) and −221 to −215 (TGTGCAA) regions of the CIITA PIII, respectively (Fig. 3⇓A). The TGTGCAA motif has been identified in EBV DR promoter and the divergent promoter in the left-hand side duplicated sequence (38), whose leftward transcript is known to be highly inducible in TPA-treated, latently infected B cells (39). ZRE221 has complete homology to this motif. In addition, ZRE221 is very similar to the ZRE in a strong EBV promoter, BHLF-1 (40), but differing by 1 nt (TGTGTAA) (Fig. 3⇓A). ZRE1287 is also perfectly homologous to the known ZRE in the BHLF-1 promoter region (40).
CIITA transcription is regulated via the differential usage of the four separate CIITA promoters, which are activated in a selective, tissue-specific manner, leading to the cellular and temporal diversity of CIITA mRNA expression (34). Constitutive transcription in dendritic cells and macrophages is mediated by the CIITA PI. The function of CIITA PII is unknown. CIITA PIII drives constitutive expression of CIITA in B cells and activated T cells. CIITA PIV is a principal IFN-γ-inducible promoter and controls the IFN-γ-inducible expression of CIITA in most MHC class II-negative cells (41). To clarify whether Zta affected CIITA transcription, we first examined the effect of Zta on the CIITA promoter activity. We constructed the plasmid containing a luciferase reporter gene driven by CIITA PIII, designated pCIITA(−1383/+44). Both putative ZREs are all present in this 1427-bp fragment (Fig. 3⇑B). To judge which of the potential ZREs is functionally important, the plasmids pCIITA(−288/+44) and pCIITA(−199/+44) were generated by removal of 1095-bp upstream region containing the ZRE1287 and 1184-bp upstream regions with the deletion of both ZREs, respectively (Fig. 3⇑B). The promoter reporter plasmids and pZta were cotransfected into Raji cells. The effect of Zta on the CIITA promoter activity was assessed by luciferase assay. In agreement with our hypothesis, CIITA promoter activities were remarkably decreased in Raji cells cotransfected with pCIITA(−1383/+44) and pZta (Fig. 3⇑C). Deletion of the region containing ZRE1287 did not cause prominent change of Zta-mediated inhibitory effect on CIITA PIII activity (Fig. 3⇑C). However, further removal of a 100-bp fragment that harbors the ZRE221 strongly abolished the inhibitory effect of Zta. The results suggest that Zta could inhibit CIITA promoter activities and the ZRE221 may act as a functional element through which Zta trans repress CIITA transcription.
Zta binds to the CIITA promoter both in vitro and in vivo
To demonstrate that Zta could be recruited to the CIITA promoter in vivo, Raji cells were transfected with pZta. The sonicated chromatin containing ∼800-bp fragments of DNA was analyzed by ChIP. The PCR products selected for amplification extend from the −288 to −68 and −1333 to −1179 regions in the CIITA PIII, respectively. The immunoprecipitation with anti-Flag Ab followed by PCR with primers flanking the ZRE221 yielded an expected 221-bp band from the precipitants, but no band was amplified by PCR with the primers encompassing the ZRE1287 (Fig. 4⇓A). Immunoprecipitation using mouse IgG as a control did not give rise to the PCR products, demonstrating that the association of Zta with the CIITA promoter is specific, and Zta binds the ZRE211, but not ZRE1287.
To further characterize the interaction of Zta with the ZRE211, we performed a DNA affinity precipitation assay. After Raji cells were treated with TPA/NaB for 36 h, the nuclear extracts were prepared and incubated with the biotinylated DNA probes corresponding to −233 to −204 region of the CIITA PIII. Detailed sequence was shown in Fig. 4⇑B. The streptavidin-coated magnetic beads were used to precipitate biotin-labeled double-stranded oligonucleotides and associated DNA-binding proteins. The biotinylated oligonucleotides were incubated with 200 μg of the nuclear extracts for the pull-down assays. The binding of the nuclear proteins to the biotinylated oligonucleotide was analyzed in the presence of the double-stranded oligonucleotide competitor containing ZRE211 motif or absence of the competitor. The resulting DNA-protein complexes were resolved by SDS-PAGE, followed by Western blotting with anti-Zta Ab. The association of Zta with the oligonucleotides containing ZRE consensus sequences could be reproducibly detected. Competition with 15-fold amount of the specific competitors resulted in a remarkable decrease of the biotinylated DNA/protein complexes (Fig. 4⇑B). No binding of endogenous Zta to either the oligonucleotide containing a mutated ZRE or to nonspecific oligonucleotide control was detectable (Fig. 4⇑B). This experiment provides in vitro evidence to demonstrate that the binding of Zta to the ZRE211 is specific.
To validate the critical role of ZRE211 in Zta-mediated inhibition of CIITA promoter activity, we performed a site-directed mutagenesis of the ZRE211 in CIITA promoter, in which three conserved nucleotides of ZRE211 consensus are mutated. Two plasmids (pCIITA(−288/+44)Mut and pCIITA(−1383/+44)Mut), both of which contain a mutated ZRE211, were constructed. In pCIITA(−1383/+44)Mut, there is an additional wild-type ZRE1287. Raji cells were transiently cotransfected with pCIITA(−288/+44), pCIITA(−288/+44)Mut, pCIITA(−1383/+44), pCIITA(−1383/+44)Mut, and pZta. Luciferase assays were subsequently conducted. The data in Fig. 4⇑C clearly showed that disruption of the ZRE211 resulted in a conspicuous abrogation of Zta-mediated inhibition of the CIITA promoter activity in Raji cells, confirming that ZRE211 plays a functionally important role in the regulation of CIITA PIII activity by Zta, and three conserved nucleotides in ZRE211 motif are important for mediating this interaction.
CIITA and HLA-DRα expression was restored by specific siRNA-directed inhibition of Zta
Above data confirm that Zta inhibited the promoter activity of CIITA, indicating that Zta functions as a transcriptional repressor in the regulation of CIITA expression. To investigate whether blocking the endogenously expressed Zta could restore the expression of CIITA as well as HLA-DRα, Raji cells were pretreated with TPA/NaB for 36 h and then transfected with the siRNAs specific for four different BZLF1 target positions. Transfection with the siRNAs specifically knocked down Zta expression, whereas control siRNA, which does not cause specific degradation of any known cellular mRNA, did not interfere with Zta expression. Silencing by the siRNA targeting the first position on BZLF1 (siZta-1) appeared to be more effective to inhibit Zta expression (data not shown). Thus, siZta-1 was used in the following experiments. In the experiment shown in Fig. 5⇓A, a substantially larger amount of CIITA protein was detected in siZta-1-transfected cells than in either TPA/NaB-treated or control siRNA-transfected cells. HLA-DRα protein synthesis was also dramatically increased concomitant with the knockdown of Zta expression (Fig. 5⇓A). To determine whether the Zta gene silencing could restore the CIITA promoter activity, Raji cells were cotransfected with pCIITA(−288/+44) and siZta-1 or nonspecific siRNA. The luciferase assay was performed. A noticeable retrieve of CIITA PIII activity was obtained following Zta gene silencing (Fig. 5⇓B). These data prove that Zta affects the expression of HLA-DRα by interfering with CIITA transcription.
Binding of Zta to the ZRE is required for inhibition of CIITA transcription
The structure of Zta is modular in nature, containing a trans activation domain (aa 1–167), a putative regulatory domain (aa 168–177), a basic DNA binding domain (aa 178–194), a coiled-coil domain (aa 195–227), and an accessory activation domain (aa 228–245) (42) (Fig. 6⇓A). Previous study showed that site-directed mutations introduced into the DNA binding domain of Zta attenuated DNA binding, but retained the ability to activate the BZLF1 promoter (43). To specifically address the role for the DNA-binding activity of Zta in the repression of CIITA PIII, we used an alternatively spliced variant ΔBZLF1 gene lacking exon 2, which encodes the DNA binding domain of Zta (Fig. 6⇓A) to test whether the influence of Zta on CIITA transcription was dependent on the DNA-binding activity of Zta. Compared with the wild-type protein, the Zta mutant showed distinct impairment in the ability to reduce CIITA as well as HLA-DRα expression both at mRNA and protein levels (Fig. 6⇓, B and C). Wild-type Zta exhibited markedly negative effect on CIITA promoter activity, as analyzed by luciferase assays. In contrast, the Zta mutant displayed a significant impaired ability to repress the CIITA promoter activity (Fig. 6⇓D). The responsiveness to Zta was completely abolished, when Raji cells were cotransfected with pZta and pCIITA(−199/+41) containing the CIITA PIII fragment lacking the ZRE. The results indicate that the DNA-binding ability of Zta is essential for the trans repression of CIITA expression. Together, these findings provide the evidence that both Zta and the ZRE221 motif play the important roles in transcriptional repression of CIITA PIII in Raji cells.
The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition to a pathogen by the immune system. The level of MHC class II directly influences T lymphocyte activation. CIITA is the most important controller for MHC class II transcription. The expression of CIITA precisely parallels MHC class II synthesis. The regulation of both constitutive and IFN-γ-inducible CIITA gene expression is controlled predominantly at the transcriptional level and directly related to MHC class II gene regulation.
Although the peptides presented by MHC class II are mainly derived from extracellular proteins, there is increasing evidence for accumulation of viral proteins in endosomal compartments (44, 45, 46). The final stages of virion assembly of herpesviruses occur in endosomal compartments with extensive concomitant targeting of viral proteins to endosomes, suggesting that these endogenous viral Ags can efficiently enter MHC class II pathway (47). Interestingly, the members of the Herpesviridae family have evolved elaborate strategies to disrupt MHC class II-mediated T cell responses by inhibiting MHC class II expression. Inhibition of MHC class II-mediated presentation of endogenous viral Ags by HCMV US2 protein has been documented. The mechanism of interference with IFN-γ-induced MHC class II expression has been studied for HCMV, mouse CMV, and adenovirus (16, 17, 48). The interference effect is primarily at the level of mRNA transcription.
Zta is a potent transcriptional activator during EBV reactivation. Through binding to the ZRE in the target promoters, Zta can stimulate the transcription of many of the EBV early lytic genes. Once reactivated, a large array of EBV proteins is expressed and exposed to the immune system. However, lytically infected cells can evade host immune response, implying that certain mechanisms exist for EBV surviving during lytic cycle replication in vivo.
Emerging evidence suggests that Zta may have important immunomodulatory functions by directly regulating cellular genes whose promoters contain ZRE or interacting with a number of cellular proteins to modulate the expression of immune related cellular genes. For example, Zta affects many important cellular targets, including c-Fos, TGF-β, and NF-κB through a variety of mechanisms of action, influencing certain cellular activities (14, 49, 50, 51). A recent study revealed that the expression of the IFN-γ receptor is down-regulated at both the RNA and protein levels by introduction of an adenovirus vector expressing Zta (15). Zta can also activate transcription of the human IL-10, IL-8, and early growth response-1 genes by binding directly to specific DNA sequences in the corresponding promoters (11, 12, 52). These data point out the critical role of Zta in the modulation of immune microenvironment, interference with Ag recognition, and immunosuppression. Characterization of cellular Zta-responsive genes is important to fully understand EBV-host cell interactions.
Several reports suggested that Zta was responsible for suppression of MHC class II expression. The aim of this study was to identify the possible mechanisms of the down-regulation of MHC class II expression by Zta during the lytic cycle. The data in the present study demonstrated that ectopic expression of Zta can inhibit the constitutive expression of MHC class II and CIITA in Raji cells. The negative effect of Zta on the CIITA promoter activity was also observed, suggesting that Zta may interfere with the MHC class II expression through down-regulation of CIITA transcription. It has been postulated that the reduction of MHC class II expression may contribute to the decline of MHC class II Ag presentation in EBV-infected cells and interference with MHC class II-restricted T cell recognition, promoting viral infection in the host. Scrutiny of the DNA sequence revealed the presence of two ZRE motifs that have complete homology to ZREs in the DR and left-hand side duplicated sequence promoters of EBV. It is well documented that Zta can activate its target promoters through direct binding to DNA sequences that match or slightly deviate from the ZRE consensus. By ChIP assays, we verified the binding of Zta to the ZRE221 in the CIITA promoter. Site-directed mutagenesis of three conserved nucleotides of the ZRE221 substantially disrupted Zta-mediated inhibition of the CIITA promoter activity. Oligonucleotide pull-down assay showed that mutation of the ZRE221 dramatically abolished Zta binding. Analysis of the Zta mutant lacking DNA binding domain confirmed that the DNA-binding activity was required for the trans repression of CIITA. The expression of HLA-DRα and CIITA could be restored following Zta gene silencing. It could be envisaged that Zta affects CIITA PIII (maybe also PIV), interferes with the transcription of constitutive and IFN-γ-inducible MHC class II, and actively blocks the presentation of EBV Ags to CD4+ T cells, and hence, the activation of Th cells required to initiate an immune response during EBV reactivation (Fig. 7⇓).
However, little is known about presentation of EBV antigenic peptides to CD4+ cells during the lytic cycle. It has been difficult to identify experimentally the functional significance of the reduced MHC class II expression for EBV lytic Ag recognition. Addressing the potential importance has been hampered directly by the practical problem associated with lacking of a fully permissive culture system for the virus. It has been demonstrated that the reduced MHC expression in the lytic cycle is sufficient to impair the sensitivity of the B lymphoblastoid cells to CTL lysis in MLRs (53). The underlying mechanism remains obscure.
Taken together, the present study has identified a novel role of Zta in down-regulation of CIITA expression and a critical ZRE in the CIITA PIII. By binding to this element, Zta may act as a viral inhibitor of the MHC class II pathway, negatively regulating CIITA transcription, and thus inhibiting the expression of MHC class II molecules. This property may be important for EBV replication in APCs. It provides a significant circumvention of the immune response for survival of EBV, especially after reactivation. CIITA activates MHC class II transcription through protein-protein interactions with the ubiquitously expressed MHC class II promoter binding proteins RFX, CREB, and NF-Y. It has been demonstrated that Zta can inhibit CREB trans activation by direct interaction or via indirect interaction with CREB-binding protein (11). It is possible that Zta may influence more cellular genes that are involved in MHC class II pathway, intervening in the recognition of infected cells by the immune system. However, further investigation is still needed for the explication of the detailed molecular mechanism for the inhibition of MHC class II expression by Zta.
The authors have no financial conflict of interest.
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↵1 This work was supported by National Natural Science Foundation of China (Grant 30771981), National Basic Research Program of China (973 Program, Grant 2006CB504305), and National High-Tech Research and Development Plan (863 Program, Grant 2006AA02A245).
↵2 D.L. and L.Q. are co-first authors.
↵3 Address correspondence and reprint requests to Dr. Ning Guo, Department of Molecular Immunology, Institute of Basic Medical Sciences, Taiping Road 27, Beijing 100850, P.R. China. E-mail address:
↵4 Abbreviations used in this paper: ZRE, Zta-response element; ChIP, chromatin immunoprecipitation; HCMV, human CMV; Ii, invariant chain; NaB, sodium butyrate; pZta, plasmid expressing Flag-tagged Zta; si, small interfering; TPA, 12-O- tetradecanoylphorbol-13-acetate.
- Received August 14, 2008.
- Accepted November 25, 2008.
- Copyright © 2009 by The American Association of Immunologists, Inc.