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Down-Regulation of MHC Class II
Expression through Inhibition of CIITA
Transcription by Lytic Transactivator Zta
during Epstein-Barr Virus Reactivation
Dan Li, Lu Qian, Changguo Chen, Ming Shi, Ming Yu,
Meiru Hu, Lun Song, Beifen Shen and Ning Guo
J Immunol 2009; 182:1799-1809; ;
doi: 10.4049/jimmunol.0802686
http://www.jimmunol.org/content/182/4/1799
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References
The Journal of Immunology
Down-Regulation of MHC Class II Expression through
Inhibition of CIITA Transcription by Lytic Transactivator Zta
during Epstein-Barr Virus Reactivation1
Dan Li,2*† Lu Qian,2* Changguo Chen,*‡ Ming Shi,* Ming Yu,* Meiru Hu,* Lun Song,*
Beifen Shen,* and Ning Guo3*
E
pstein-Barr virus is a ubiquitous human ␥-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 medi-
*Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing,
People’s Republic of China; †Department of Clinical Laboratory, 306th Hospital,
Beijing, People’s Republic of China; and ‡Department of Clinical Laboratory, Navy
General Hospital, Beijing, People’s Republic of China
Received for publication August 14, 2008. Accepted for publication November
25, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
ating viral DNA replication (5– 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 –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 EBVnegative B cells, Zta completely inhibits the up-regulation of surface MHC class I expression induced, and also directly inhibits the
constitutive activation of NF-␬B (13, 14). In addition, it has recently been shown that the expression of the IFN-␥ 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-␥-inducible MHC class II transcription (16, 17). By inhibiting Jak1
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: [email protected]
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802686
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-Otetradecanoylphorbol-13-acetate.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
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-DR␣ 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. The Journal of Immunology, 2009, 182: 1799 –1809.
1800
INHIBITION OF CIITA TRANSCRIPTION BY Zta
Table I. The primers useda
Application for
Construction of pZta and p⌬Zta
Amplification of ⫺1383/⫹44
region of CIITA PIII
Amplification of ⫺288/⫹44
region of CIITA PIII
Amplification of ⫺199/⫹44
region of CIITA PIII
Site-directed mutation of ZRE221
for pCIITA(⫺288/⫹44)Mut
Site-directed mutation at ZRE221
by overlap extension PCR for
pCIITA(⫺1383/⫹44)Mut
RT-PCR for HLA-DR␣
RT-PCR for CIITA
Amplification of a 221-bp fragment
spanning ZRE221 in ChIP
Amplification of a 155-bp fragment
spanning ZRE1287 in ChIP
P1
P2
P3
P4
P5
5⬘-GCGGCCGCCACCATGGACCCAAACTCGA-3⬘ (NotI)
5⬘-GGGCCCTCATTATTTATCGTCATCGTCTTTGTAGTCGAAATTTAAGAGATCCTCGTG-3⬘ (ApaI)
5⬘-CCGCTCGAGGCTGGATGTAGGAGAATGGC-3⬘ (XhoI)
5⬘-CCCAAGCTTAGCTCAGAAGCACACAGCCT-3⬘ (HindIII)
5⬘-CCGCTCGAGAGTGCGGTTCCATTGTGATC-3⬘ (XhoI)
P6
5⬘-CCCCTCGAGAAATTCAGTCCACAGTAAGGAAGT-3⬘ (XhoI)
P7
P8
P9
5⬘-CTGTCTTCACCAAATTCAGTCCA-3⬘
5⬘-AAAGATGAACCGAAAGTCTGTTGG-3⬘
5⬘-ACTTTCGGTTCATCTTTCTGTCTTCACCAA-3⬘
P10
5⬘-CAGAAAGATGAACCGAAAGTCTGTTGGGGG-3⬘
P11
P12
P13
P14
P15
P16
P17
P18
5⬘-CGAGTTCTATCTGAATCCTG-3⬘
5⬘-GTTCTGCTGCATTGCTTTTGC-3⬘
5⬘-GATTCCTACACAATGCGTTGCCTGG-3⬘
5⬘-CATACTGGTCCAGTTCCGCGATATTGG-3⬘
5⬘-AGTGCGGTTCCATTGTGATC-3⬘
5⬘-AAAACTCTCCCTGCAAGGTG-3⬘
5⬘-GCTGGATGTAGGAGAATGGC-3⬘
5⬘-CCTGCCACTATGTCCAGCTA-3⬘
Restriction enzyme sites are underlined.
expression and disrupting IFN-␥-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-␣ and DM-␣ 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 (SigmaAldrich) and 3 mM NaB (Sigma-Aldrich) for 36 h.
Construction of BZLF1 and CIITA promoter reporter plasmids
Total RNA was isolated from B95-8 cells using the TRIzol reagents (Invitrogen), according to the manufacturer’s instructions. Reverse transcription was performed with the Moloney murine leukemia virus reverse transcriptase (Promega). The BZLF1 cDNA was amplified by PCR with
Pyrobest DNA polymerase (Takara) using the primers P1 and P2 (Table I).
The PCR product was inserted into the plasmid pRc/CMV (Invitrogen) by
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
(p⌬Zta).
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 sitedirected 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⬘-CAA
CAGCTAGCAGACATTG-3⬘, 5⬘-GCTAGCAGACATTGGTGTT-3⬘, 5⬘GGACAACAGCTAGCAGACA-3⬘, and 5⬘-AGCAACTGCTGCAGCAC
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
a
Primers
The Journal of Immunology
TA-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 ␮g of DNA was electroporated into Raji cells (1 ⫻ 107 cells in 400
␮l of RPMI 1640) with an Electro square porator 830 (BTX) at 1100 V,
40 ␮s, twice pulse, with an interval of 1 min. For gene silencing by siRNA,
5 ␮l of 20 ␮M stock dsRNA oligonucleotides was electroporated into 2 ⫻
106 Raji cells treated with TPA/NaB in 100 ␮l of RPMI 1640 at 550 V, 40
␮s, 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.
Semiquantitative RT-PCR
Flow cytometry (FACS)
A total of 1 ⫻ 106 Raji cells was harvested. After washing in ice-cold PBS
containing 1% FCS and 0.05% sodium azide, staining of cell surface-expressed HLA-DR was conducted with PE-labeled mAb against human
HLA-DR (eBioscience; 12-9956) on ice for 30 min. Then the cells were
washed three times in PBS containing 1% FCS and 0.05% sodium azide,
and resuspended in 1% paraformaldehyde. For simultaneous staining of
HLA-DR and CD80, Raji cells, either transfected with pZta or control
empty plasmid, were incubated with PE-labeled anti-human HLA-DR mAb
and FITC-labeled anti-human CD80 (B7-1) Ab (eBioscience; 11-0809).
Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). The experiment was conducted in duplicate.
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 ␮l of cytosol extraction buffer A. The suspension was incubated on ice
for 10 min, then mixed with 30 ␮l 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 ␮l
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.
Western blot
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-DR␣ (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-2␣ using the anti-GAPDH (Kangchen; KC5G4) and anti-AP-2␣ (Santa Cruz Biotechnology; sc-184) Abs, respectively. Bands were visualized by SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).
Luciferase assay
Raji cells were cotransfected with 10 ␮g of reporter plasmid (with a luciferase gene driven by various CIITA promoters) and 10 ␮g of effector
plasmid (the Zta or ⌬Zta-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
The 5⬘-biotinylated double-stranded oligonucleotides (5⬘-CAACAGACTT
TCTGTGCAACTTTCTGTCTT-3⬘) corresponding to positions ⫺233 to
⫺204 of the CIITA PIII and containing the ZRE motif were synthesized by
Augct Biotechnology. The same double-stranded sequences that are not
biotinylated were used as the competitors. The biotinylated oligonucleotides containing a mutated ZRE (5⬘-CAACAGACTTTCgGTtCAtCTTTC
TGTCTT-3⬘), in which three conserved nucleotides of ZRE consensus are
replaced (in lowercase), and the biotinylated oligonucleotides (5⬘-GCTA
TGATACTGGCCCCATCCTGCAGAAGG-3⬘) corresponding to positions ⫺342 to ⫺313 of the CIITA PIII, which is lack of the ZRE, were also
synthesized. Nuclear protein extracts were prepared from Raji cells that
were treated with TPA/NaB. A total of 200 ␮g of nuclear extracts was
incubated at 4°C for 4 h with each pair of the oligonucleotides coupled
previously to Dynabeads M-280 (Invitrogen; 112.05D). The protein-DNA
complexes were separated with a Dynal magnet, denatured in SDS sample
buffer, and subjected to SDS-PAGE. Zta was detected by Western blot.
Results
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-DR␣
Ab. Fig. 1A shows that the expression of Zta was efficiently induced by the treatment of TPA/NaB. A conspicuous decline of the
expression of HLA-DR␣ molecules was observed in induced vs
uninduced Raji cells, correlating with the observed increase in Zta
protein (Fig. 1A). Detection of GAPDH confirmed that equivalent
proteins were assayed. Staining with PE-labeled mAb against
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Total RNA was isolated from Raji cells and quantified by spectrophotometry. cDNA was synthesized from 2 ␮g 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-DR␣ fragment and P13 and P14 for a 325-bp CIITA fragment. Amplification of ␤-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.
1801
1802
INHIBITION OF CIITA TRANSCRIPTION BY Zta
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. 1B).
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 ␮g of pZta. Fig. 1C shows that the
HLA-DR␣ 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 ␮g 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 ␮g of pZta,
respectively. After transfection for 48 h, the nuclear proteins were
prepared and the CIITA expression was monitored by Western blot
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FIGURE 1. Zta inhibits the expression of MHC class II. A, Raji cells were treated with or without 100 nM TPA and 3 mM NaB. The cell lysates were
separated by SDS-PAGE and analyzed by Western blot using anti-Zta and anti-HLA-DR␣ Abs. Detection of GAPDH was used as an internal control. B,
The surface expression of HLA-DR proteins on Raji cells was determined by FACS using PE-labeled mAb against HLA-DR␣. Filled histogram, negative
control; open histogram with thin line, the cells treated with TPA/NaB; thick line, untreated cells. C, Raji cells were transfected with 0, 5, 10, and 30 ␮g
of pZta. The expression of HLA-DR␣ protein was analyzed by Western blot. D–F, The expression of HLA-DR on the surface of transfected Raji cells was
evaluated by FACS. G and H, Two-color FACS assays of the expression of HLA-DR and CD80 on the surface of the transfected Raji cells with the empty
plasmid and pZta, respectively. Filled histogram, negative control; open histogram with thin line, the cells transfected with pZta; thick line, the cells
transfected with empty vector. D, The cells were transfected with 5 ␮g of pZta; E, 10 ␮g of pZta; and F, 30 ␮g of pZta.
The Journal of Immunology
1803
with anti-CIITA Ab. Detection of AP-2␣ was used as a loading
control. As shown in Fig. 2A, 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-␥-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-DR␣ 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 ␤-actin was used to normalize HLA-DR␣ and
CIITA expression. Using the specific primers, an expected 644-bp
PCR product of HLA-DR␣ and 325-bp product of CIITA were
generated. A significant reduction of HLA-DR␣ and CIITA
mRNA level was observed in Raji cells treated with TPA/NaB
(Fig. 2B). Transient transfection with increasing amounts of the
pZta resulted in a dose-dependent decline of HLA-DR␣ expression. Likewise, the decreased CIITA mRNA expression was also
detected. Fig. 2B shows representative agarose gel electrophoresis
of RT-PCR products. These results demonstrate that Zta is involved in the down-regulation of HLA-DR␣ and CIITA transcription in Raji cells.
HLA class II molecules are ␣␤ 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/NaBinduced and pZta-transfected Raji cells (Fig. 2C).
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. 3A). 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.
3A). 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-␥-inducible promoter and controls the IFN-␥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. 3B). 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. 3B). 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. 3C). Deletion of the region containing
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FIGURE 2. Zta inhibits the expression of CIITA. A, Raji cells were transiently transfected with 0, 5, 10, and 30 ␮g of the pZta, respectively. After transfection for 48 h,
the nuclear proteins were prepared and CIITA expression was analyzed by Western blot with anti-CIITA Ab. Detection of AP-2␣ was used as a loading control. B, Total
RNA was isolated from the transiently transfected Raji cells. HLA-DR␣ and CIITA mRNA expression was analyzed by semiquantitative RT-PCR. Amplification of
␤-actin was used to normalize HLA-DR␣ and CIITA expression. The intensity of bands was quantified using LabWorks Image Acquisition and Analysis software. C, The
expression of Ii was analyzed by Western blot with anti-CD74 Ab.
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INHIBITION OF CIITA TRANSCRIPTION BY Zta
ZRE1287 did not cause prominent change of Zta-mediated inhibitory effect on CIITA PIII activity (Fig. 3C). 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. 4A).
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. 4B. 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 ␮g 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 DNAprotein 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. 4B). No binding of endogenous Zta to either the oligonucleotide containing a mutated ZRE
or to nonspecific oligonucleotide control was detectable (Fig. 4B).
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. 4C clearly showed that disruption of the
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FIGURE 3. Zta inhibits the activity of CIITA
PIII. A, comparison of ZRE consensus sequences
between CIITA PIII and EBV genes. B, Schematic
maps of the constructs used. C, The relative luciferase activity after cotransfection with the promoter
reporter plasmids and pZta.
The Journal of Immunology
1805
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-DR␣ 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-DR␣, 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. 5A, 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-DR␣ protein synthesis was also dramatically increased concomitant with the knockdown of Zta expression
(Fig. 5A). 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. 5B). These data prove that Zta affects the expression of HLADR␣ 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. 6A). 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 ⌬BZLF1 gene lacking exon 2, which
encodes the DNA binding domain of Zta (Fig. 6A) to test whether
the influence of Zta on CIITA transcription was dependent on the
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FIGURE 4. Zta binds to the CIITA
promoter both in vitro and in vivo.
A, A total of 3 ⫻ 106 Raji cells transfected with or without pZta was
cross-linked with 1% formaldehyde
and lysed in SDS lysis buffer. Chromatin was sheared by sonication to
⬃800 bp. The lysates were immunoprecipitated with anti-Flag Ab or
mouse IgG, followed by PCR using
primers specific for 221- and 155-bp
fragments of CIITA PIII harboring
ZRE221 and ZRE1287, respectively. B,
Nuclear protein extracts were prepared from Raji cells treated with or
without TPA/NaB. A total of 200 ␮g
of nuclear extracts was incubated
with 5⬘-biotinylated double-stranded
oligonucleotides coupled previously
to Dynabeads M-280. The proteinDNA complexes were separated with
a Dynal magnet, denatured in SDS
sample buffer, and subjected to SDSPAGE. Zta was detected by Western
blot. C, Raji cells were transiently cotransfected with pZta and pCIITA(⫺288/⫹44),
pCIITA(⫺288/
⫹44)Mut, pCIITA(⫺1383/⫹44), or
pCIITA(⫺1383/⫹44)Mut, and luciferase assays were conducted.
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INHIBITION OF CIITA TRANSCRIPTION BY Zta
FIGURE 5. The expression of CIITA and
HLA-DR␣ was restored by specific siRNAdirected inhibition of Zta. A, Raji cells were
pretreated with TPA/NaB for 36 h and then
transfected with the siRNAs. Whole-cell lysates and nuclear proteins were prepared
from the transfected cells. The expression of
HLA-DR␣ and CIITA was assayed by SDSPAGE and Western blot analysis. Detection
of GAPDH and AP-2␣ was used as the loading controls. B, Raji cells treated with or
without TPA/NaB were cotransfected with
pCIITA(⫺288/⫹44) and siZta-1 or nonspecific siRNA, and the luciferase assay was
performed.
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
FIGURE 6. Binding of Zta to the ZRE is required for inhibition of CIITA transcription. A, Schematic representation of the structures of BZLF1 gene,
Zta, and ⌬Zta lacking the DNA binding domain. B, The total RNAs were isolated from Raji cells transfected with pZta or p⌬Zta, and the expression of
CIITA and HLA-DR␣ mRNA was analyzed by semiquantitative RT-PCR. C, The HLA-DR␣ protein level in transfected Raji cells was analyzed by Western
blot. D, The effects of Zta and ⌬Zta on CIITA promoter activity were analyzed by luciferase assays.
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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-DR␣ expression both at mRNA and
The Journal of Immunology
1807
impaired ability to repress the CIITA promoter activity (Fig. 6D).
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.
Discussion
FIGURE 7. Zta down-regulates MHC class II expression through inhibition of CIITA transcription during EBV reactivation. CIITA interacts
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 (A). By binding to
the ZRE in the CIITA promoter Zta negatively regulates CIITA transcription, inhibiting the expression of MHC class II (B).
hibit 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-DR␣ 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␥-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).
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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-␥-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 –
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-␥-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-␤, and NF-␬B through a variety of
mechanisms of action, influencing certain cellular activities (14,
49 –51). A recent study revealed that the expression of the IFN-␥
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 in-
1808
Disclosures
The authors have no financial conflict of interest.
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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.
INHIBITION OF CIITA TRANSCRIPTION BY Zta
The Journal of Immunology
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