Epstein-Barr virus LMP2A suppresses MHC class II

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Blood First Edition Paper, prepublished online January 28, 2015; DOI 10.1182/blood-2014-08-594689
Epstein-Barr virus LMP2A suppresses MHC class II expression by regulating
the B cell transcription factors E47 and PU.1
Jiun-Han Lin1, Ju-Yin Lin1, Ya-Ching Chou1, Mei-Ru Chen1, Te-Huei Yeh2,
Chung-Wu Lin3, Sue-Jane Lin4,5,6* and Ching-Hwa Tsai1*
1
Graduate Institute of Microbiology, College of Medicine, National Taiwan
University, Taipei, Taiwan; 2Department of Otolaryngology, National Taiwan
University Hospital, College of Medicine, National Taiwan University, Taipei,
Taiwan;3Department of Pathology, National Taiwan University Hospital, College of
Medicine, National Taiwan University, Taipei, Taiwan; 4Research Center for
Emerging Viral Infections, College of Medicine, Chang Gung University, Tao-Yuan,
Taiwan; 5Graduate institute of Medical Biotechnology, College of Medicine, Chang
Gung University, Tao-Yuan, Taiwan;6Department of Medical Biotechnology and
Laboratory Science, College of Medicine, Chang Gung University, Tao-Yuan,
Taiwan.
Short title: MHC class II repression by EBV LMP2A
Key words: EBV, LMP2A, MHC class II, CD74, E47, PU.1
*Co-corresponding authors
Addresses for Correspondence:
Sue-Jane Lin, PhD
Mailing address: No. 259, Wen-Hwa 1st Road, Research Center for Emerging Viral
Infections, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan 333,
Taiwan
Phone: 886-3-2118800, ext 3727
Fax: 886-3-2118700
E-mail:suejane.lin@mail.cgu.edu.tw
Ching-Hwa Tsai, PhD
Mailing address: Room 719, No. 1, 1st section, Jen-Ai Rd, Graduate Institute of
Microbiology, College of Medicine, National Taiwan University, Taipei 10051,
Taiwan.
Phone: 886-2-23123456, ext 88298
Fax: 886-2-23915293
E-mail: chtsai@ntu.edu.tw
1
Copyright © 2015 American Society of Hematology
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Key point:
EBV LMP2A alters B cell gene expression; E47 and PU.1 are repressed by LMP2A,
resulting in down-regulation of MHC class II expression.
Abstract
Oncogenic Epstein-Barr virus (EBV) utilizes various approaches to escape host
immune responses and persist in B cells. Such persistent infections may provide the
opportunity for this virus to initiate tumor formation. Using EBV-immortalized
lymphoblastoid cell lines (LCLs) as a model, we found that the expression of MHC
class II and CD74 in B cells is repressed post-EBV infection. CIITA is the master
regulator of MHC class II-related genes. As expected, CIITA was down-regulated in
LCLs. We showed that down-regulation of CIITA is caused by EBV LMP2A and
driven
by
the
CIITA-PIII
promoter.
Furthermore,
we
demonstrated
that
LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression.
Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was
critical for the repression of E47 and PU.1 promoter activity via Syk, Src and the
PI3K/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach
showed that the expression levels of E47, PU.1, CIITA, MHC class II and CD74 are
reversed. These data indicated that the LMP2A may reduce MHC class II expression
through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that
MHC class II may be detected in tonsils and EBV negative Hodgkin's disease (HD)
but not in EBV-associated post-transplant lymphoproliferative disease and HD.
2
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Introduction
During infection, viruses face the various challenges of the host immune response
and have evolved a number of immune evasion strategies to enable successful
infection of the host cells. Epstein-Barr virus (EBV) is a ubiquitous, human
gamma-herpesvirus that persistently infects over 90% of the human population1. EBV
has evolved several mechanisms to escape immune surveillance: EBV limits the
expression of its proteins and persists in immune B cells. EBV uses various strategies
to attenuate the first line of innate immunity; for example, latent membrane protein 1
(LMP1) negatively regulates the expression of the important sensor toll-like receptor
92, BCRF1 encoded vIL10 inhibits interferon production3 and the tegument protein
BPLF1 blocks toll-like receptor signaling4. In counteracting the adaptive immune
response, EBV interferes with the major histocompatibility complex (MHC) class I
and II antigen presentation, which are the key factors for adaptive immunity. MHC
antigens are critical in the cellular immune response, which is important for viral
clearance. Several studies have addressed the question how EBV down-regulates
MHC class I antigen expression. EBNA1 has a glycine-alanine repeat motif that
protects it from degradation and also inhibits its own synthesis5,6. The EBV lytic
protein BNLF2a interferes with both the peptide and ATP binding to transporter
associated with antigen processing (TAP)7. Like the HSV1 virion host shut off protein,
the EBV DNase BGLF5 inhibits MHC class I protein production8. The viral
G-coupled receptor protein BILF1 also down-regulates MHC class I expression by
promoting the degradation of its mRNA, increasing endocytosis and degradation9.
Regarding EBV and MHC class II antigen, the transactivator Zta can inhibit class II
antigen expression via direct down-regulation of the class II transactivator (CIITA) by
binding to its promoter or, indirectly, by inhibition of IFN-γ production10,11. In
3
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addition, the viral host protein shut off master BGLF5 directly blocks class II antigen
synthesis8. Furthermore, the EBV vIL10 also inhibits the production of TAP1 and the
proteasome subunit bi-LMP2 at the transcriptional level to prevent MHC I antigen
presentation and interferes with IFN-γ to block MHC class II induction12,13. In
addition, the BZLF2-encoded gp42 can associate with MHC class II molecules in the
endoplasmic reticulum and accompany the class II complexes to the cell surface. Then,
gp42 in the class II complex blocks the interaction with the receptors on CD4+ T
cells14. Taken together, EBV uses many strategies to prevent MHC antigen expression;
however, most involve lytic gene products. According to the evidence provided by Dr.
Thorley-Lawson, latent membrane protein 2A (LMP2A) may be the critical viral
protein expressed in the healthy EBV carrier15. So, we wonder how EBV escapes
immune surveillance in this situation.
It is interesting to consider the protein structure of LMP2A16. This latent protein
contains 12 transmembrane domains and has a short C-terminus and long, 119 a.a
N-terminus containing eight tyrosine residues. More impressively, the residues
tyrosine 74 and 85 can form a motif which mimics the immunoreceptor
tyrosine-based activation motif (ITAM) of the B cell receptor (BCR). So, the
biological functions of LMP2A are similar to the BCR in some ways17,18. A series of
studies from Dr. Longnecker’s lab revealed that LMP2A may provide survival
signaling to B cells in LMP2A-transgenic mice and also facilitate the oncogenicity of
c-myc19,20. Of interest, they demonstrated that LMP2A requires Notch1 to alter B cell
gene expression in a very similar manner to the gene expression in Reed-Sternberg
cells of EBV-associated Hodgkin's disease (HD)21. In addition, they speculated that
these cells can survive with an intact p5322, cell growth being directly promoted
through the c-myc pathway20. In our previous study, we showed that LMP2A can
4
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regulate c-Jun activity and so promote cell mobility23. Furthermore, we found that syk
can be activated by the ITAM of LMP2A and facilitate NPC metastasis24. Thus, we
address the question whether LMP2A plays any role in immune evasion, because it
may be the only EBV protein expressed in normal carriers.
In our preliminary cDNA array screening, we noted the down-regulation of MHC
class II antigen expression (data not shown). So, these results encouraged us to
investigate further the molecular mechanism how EBV gene products repress the
expression of MHC class II antigen. In this study, we found that LMP2A uses an
indirect strategy to down-regulate the expression of MHC class II proteins via
suppression of the B cell transcriptional factors PU.1 and E47. Moreover, MHC class
II expression is reduced in biopsies from EBV-associated post-transplant
lymphoproliferative disease (PTLD) and HD. The mechanism and significance of this
down-regulation will be discussed.
5
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Materials and Methods
B-cell purification and EBV infection
Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of
anonymous donors and CD19-positive B cells were purified using Dynabeads
(Invitrogen). Production of EBV virions (B95-8 strain) and infection of B cells by
EBV have been described previously25. Experiments involving human samples were
approved by the Institutional Review Boards (IRB) of National Taiwan University
Hospital (NTUH, Taipei, Taiwan).
Cell culture and inhibitors
Akata and BJAB are EBV-negative Burkitt’s lymphoma-derived cell lines. Raji cells
are an EBV-positive Burkitt’s lymphoma-derived cell line. LCLs were established
from EBV-infected PBMCs or purified B cells. All B-cell lines were cultured in
complete RPMI medium (containing 10% Fetal Calf Serum [FCS], 1mM glutamine,
100 U/mL penicillin and 100μg/mL streptomycin). Syk inhibitor (Piceatannol), Src
inhibitor (PP2), MEK inhibitor (U0126) and PI3K inhibitor (LY294002) were
purchased from Merck Millipore. SP1 inhibitor (Mithramycin) was purchased from
Calbiochem.
Construction of Plasmids
The EBNA1 plasmid pCEP4 (Invitrogen) carries the EBNA1 gene. BARF0 was
amplified from the genomic DNA of Akata cells by PCR then inserted into pcDNA3.
The EBER1and EBER2 were constructed into pLKO vector at the 5’ AgeI site and the
3’ EcoRI site. The pSIN-LMP1 was constructed by insertion of LMP1 cDNA into the
pSIN26. The pSIN-LMP2A was constructed by insertion of a full-length cDNA into
the pSIN at 5’ BamHI site and 3’ NotI site. The LMP2A-deleted 74-85 and Y112F
were constructed by site-directed mutagenesis. The LMP2A PY motif mutant
6
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constructs were kindly provided by Dr. Yao Chang (National Health Research
Institutes, Tainan, Taiwan)27. A series of luciferase reporter plasmids driven by the
E47 or PU.1 promoter were constructed in the pCDH-GL3-basic vector. The
pcDNA3-E47fD was kindly provided by Dr. Rudolf Grosschedl (Max-Planck Institute
of Immunobiology, Freiburg, Germany). The pLKO-FD-E47-puro was constructed by
insertion of the E47fd fragment from pcDNA3-E47fd into the pLKO-AS2-puro at the
NheI site. The pLKO-PU.1-neo was constructed by the insertion of PU.1 cDNA into
the pLKO-AS2-neo at the 5’ EcoRI site and the 3’ AscI site.
Preparation and infection of lentiviruses
RNA interference fragments were purchased from the National RNAi Core Facility
(Academia Sinica, Taipei, Taiwan) and their sequences were shown in supplementary
Table 1. Briefly, plasmids p8.91, pMD2.G and pLKO.1-shLuc, pLKO.1-shPIK3CA,
pLKO.1-shPAX5, pLKO.1-shSP1, pLKO.1-shLMP2A, pSIN-LMP1, pSIN-LMP2A,
pSIN-Zta, pLKO-PU.1, pLKO-E47fd were co-transfected into HEK293T cells using
lipofectamineTM 2000 (Invitrogen). Infectious expressing lentiviruses were collected
at day 3 post-transfection and stored at -80oC. The method of production and infection
with lentiviruses was described previously25. For lentivirus infection, LCLs or BJAB
cells were infected with lentiviruses at a multiplicity of infection (MOI) of 1-2.
Electroporation
The method of electroporation was described previously26. Cells were electroporated
using a Neon kit (Invitrogen).
Analysis of reverse transcription (RT)-PCR and Quantitative polymerase chain
reaction (Q-PCR)
Total RNA was isolated from cells using TRIzol (Invitrogen). Synthesis of cDNA has
been described in our previous paper26. The cDNA was used as a template for PCR in
7
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the presence of specific primers shown as supplementary Table 1. Analysis of Q-PCR
was performed using the TaqMan primer/probe set (Pre-Developed Assay Reagents;
Applied Biosystems) in some experiments. Primers and probes for other transcripts
are shown as supplementary Table 1. The relative intensity fold of RT-PCR was
normalized with internal control and then standardized to the vector control.
Western blotting and antibodies (Abs)
Cells were lysed by buffer and western blotting was performed according to our
previous study25. Anti-E47, -PU.1, -CIITA, -HLA-DR, -CD74, -phospho-ERK
Thr202/Tyr204, -ERK and Akt Abs were purchased from Santa Cruz Biotechnology.
Anti-phospho-Akt Ser473 Ab was purchased from Cell Signaling Technology and
anti-β-actin Ab was purchased from Sigma-Aldrich. Anti-EBNA1(NPC47)28,
LMP1(S12)29 and LMP2A23 Abs were used according to previous studies. The
relative folds of the protein of interest were determined by normalizing the level of
each group to the corresponding β-actin intensity and then standardized with the
vector control.
Reporter assay
Cells were infected with the pCDH-GL3 promoter luciferase reporter lentiviruses at
an MOI of 1. On day 4 post-infection, the luciferase activities and GFP fluorescence
intensity were detected using the Bright-Glo Luciferase Assay System kit (Promega).
The relative fold induction of luciferase activity from each transfectant was
normalized to its GFP intensity and standardized with the vector control.
Immunohistochemistry (IHC) and in situ hybridization of EBER assays
Tonsil, PTLD and HD biopsies were obtained from the NTUH. Experiments
involving human samples were approved by the IRB of NTUH. IHC assays were
performed using the Super SensitiveTM Link-Label IHC Detection System (BioGenex),
8
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and in situ hybridization of EBER assays were described and performed according to
our previous paper26.
9
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Results
The expression of MHC class II and CD74 mRNAs decreases following EBV
infection
Regulation of MHC class II and its associated chaperone, CD74, has been reported
during virus infection30,31. To determine whether the expression of MHC class II and
CD74 is influenced by EBV infection, expression of HLA-DRA and HLA-DRB was
detected in primary B cells with or without EBV infection by RT-Q-PCR and western
blotting. As shown in Figure 1B, we demonstrated that down-regulation of MHC class
II and CD74 was observed when infected B cells expressed LMP2A at day 3
post-infection. After that, LMP2A was constitutively expressed in infected cells and
low level expression of MHC class II and CD74 was detected at the same time
(Figure 1B). In the EBV-infected primary B cell system, we concluded that EBV may
block viral antigen presentation by down-regulation of MHC class II and CD74.
EBV LMP2A is a critical factor in the down-regulation of MHC class II and
CD74
To identify which EBV gene product is responsible for the reduction of MHC class II
and CD74 expression, individual EBV genes were ectopically expressed in
EBV-negative BJAB cells. In Figure 1C, we observed that only LMP2A or Zta
down-regulates expression of MHC class II and CD74 transcripts, but not the other
viral gene products tested, including EBNA1, LMP1, EBER1, EBER2 and BARF0.
The EBV lytic cycle transactivator, Zta, functions as a repressor of MHC class II and
CD74 expression, which is consistent with a previous report31. In particular, we were
interested in LMP2A because it is expressed predominately during the viral latent
stage. We found that the protein levels of MHC class II and CD74 were
down-regulated in Akata and BJAB cells expressing ectopic LMP2A (Figure 1D). As
10
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shown in Figure 1E, MHC class II and CD74 proteins decreased in a dose dependent
manner following transduction of the cells with LMP2A-expressing lentiviruses.
LMP2A triggers the down-regulation of CIITA, E47 and PU.1
We speculated that down-regulation of MHC class II and CD74 possibly resulted
from LMP2A-triggered downstream signaling. Based on previous studies, CIITA is
the master regulator of MHC class II expression32, so the expression of CIITA was
measured in LMP2A-transduced Akata and BJAB cells. In Figure 2A, LMP2A
suppressed CIITA expression. In general, expression of CIITA is driven by three
independent promoter units PI, PIII, and PIV, which are cell type specific10. To
determine which promoter unit is regulated by LMP2A, the expression of CIITA-PI,
CIITA-PIII and CIITA-PIV transcripts was detected in Akata and BJAB cells
transduced with LMP2A-lentivirus. LMP2A suppressed the expression of CIITA-PIII
in this system, consistent with a previous study10 that showed CIITA-PIII mRNA is
specifically expressed in B cells (Figure 2B). According to previous studies, several
B-cell transcriptional factors are involved in the regulation of CIITA promoter III,
including E47, PU.1, IRF4 and IRF833. In Figure 2C, a significant reduction of E47
and PU.1 was seen in Akata and BJAB cells expressing LMP2A, but the expression of
IRF4 and IRF8 did not change. Protein expression levels of E47 and PU.1 were also
reduced in Akata and BJAB cells expressing ectopic LMP2A (Figure2D). In Li’s
study31, Zta-mediated suppression of MHC class II was via direct binding to the
CIITA promoter; our results indicated that Zta does not alter the expression of E47
and PU.1 expression (Figure2E). Taken together, it seems that EBV uses different
regulatory mechanisms to down-regulate MHC class II and CD74 during latency and
the lytic replication cycle.
LMP2A-mediated suppression of E47 and PU.1 is through the PI3K/Akt
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pathway
LMP2A contains 8 tyrosine residues and these are important for LMP2A function.
LMP2A is phosphorylated on tyrosine 112 through its interaction with Lyn34 and
interacts constitutively with Syk through the ITAM (tyrosine 74 and 85)35. In addition,
LMP2A PY motifs play an important role in protein stability and phosphorylation of
LMP2A-associated proteins36. To evaluate the potencies of the phosphorylated
tyrosine 112 and ITAM domain of LMP2A involved in the expression of E47, PU.1,
MHC class II and CD74, lentiviruses carrying Y112F mutated or Δ74-85 deleted
LMP2A were prepared. In Figure 3A, compared to the vector control, LMP2A-wt and
the Y112F mutant reduced the expression of E47, PU.1, MHC class II and CD74.
However, the LMP2A-Δ74-85 mutant had significantly lost the ability to suppress the
expression of E47 and PU.1. In addition, we addressed the involvement of PY
domains in LMP2A-mediated suppression of PU.1 and E47. In Figure 3B, expression
of PU.1 and E47 was not restored when cells express LMP2A with mutated PY
domains. Thus, downstream signaling molecules of the ITAM in LMP2A were
required for E47 and PU.1 suppression.
It is known that the ITAM of LMP2A mediates activation of Syk and Src, which
then activate the PI3K/Akt signaling pathway37,38. An inhibitor assay was used to
determine whether Syk and Src signaling pathways are involved in LMP2A-mediated
down-regulation. As shown in Figure 3C, blockage of activated Syk and Src by their
inhibitors, Piceatannol and PP2, respectively, resulted in decreased amounts of pAkt
and the expression of E47 and PU.1 was upregulated in LCL cells. Furthermore,
LMP2A-mediated suppression of E47, PU.1, MHC class II and CD74 was abolished
in the presence of an inhibitor of PI3K (LY294002). In contrast, LMP2A-mediated
suppression of these molecules was not altered in cells treated with the MEK inhibitor,
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U0126 (Figure 3D and 3E). Furthermore, the reduction of E47, PU.1, MHC class II
and CD74 was recovered when the p110 subunit of PI3K was knocked down by
shRNA (Figure 3F). Taken together, these data suggest that LMP2A down-regulates
the expression of E47 and PU.1 through the PI3K/Akt pathway. We then sought to
confirm whether the LMP2A-mediated down-regulation of MHC class II also was
through this pathway. Expression of MHC class II, CD74 and CIITA was measured in
cells expressing LMP2A, with or without overexpression of E47 and PU.1. In Figure
3G, co-expression of E47 and PU.1 significantly reversed the LMP2A-mediated
suppression of MHC class II and CD74.
LMP2A inhibits the promoter activities of E47 and PU.1
Luciferase promoter reporter assays were performed to understand more of the
mechanism of LMP2A-mediated repression of E47 and PU.1. The promoter activities
of PU.1 and E47 in Akata and BJAB cells were repressed by LMP2A and the
LMP2A-Y112F mutant but not by the LMP2A-Δ74-85 mutant (Figure 4A). In
addition, the promoter activities were restored by the PI3K inhibitor, LY294002
(Figure 4B). To further dissect which regions of the promoters are crucial for this
inhibition, Figure 5A and 5D illustrate the transcriptional factor binding sites in
schematic maps of the promoter regions of PU.1 (-470/+57) and E47 (-1000/+29).
According to the results of serial deletions of the E47 and PU.1 promoters, the region
-121 to -52bp in the PU.1 promoter might be critical for LMP2A-medicated inhibition
(Figure 5A). There are five PAX5 and two PU.1 putative binding sites in this region.
Furthermore, the promoter activity of PU.1 was restored in cells overexpressing PU.1
(Figure 5B). However, the promoter activity of PU.1 was not affected in PAX5
knockdown cells (Figure 5C). These results demonstrated that PU.1 itself plays a key
role in regulating of LMP2A-mediated suppression of PU.1 promoter activity.
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However, we could not find a specific region on the E47 promoter which is involved
in LMP2A-mediated down-regulation (Figure 5D). There are many putative SP1 sites
on E47 promoter, so we tried to block the SP1-DNA binding activity. Mithramycin, an
SP1 inhibitor which blocks the binding between SP1 and GC riched DNA, was used
to test the involvement of SP1 in regulating E47 promoter activity. According to the
reporter assay results in Figure 5E and 5F, SP1 is involved in LMP2A-mediated
down-regulation of E47. These results reveal that LMP2A down-regulates E47 and
PU.1 promoter activity through ITAM-dependent activated PI3K/Akt signaling.
Meanwhile, PU.1 and SP1 were crucial for this regulation.
LMP2A mediation of MHC class II suppression via E47 and PU.1 was verified in
LCLs using an shRNA approach
We then investigated whether down-regulation of E47 and PU.1 in EBV-infected
primary B cells is a general phenomenon. Expression of the E47 and PU.1 proteins
was detected in four pairs of uninfected B cells and LCLs. EBV infection strongly
suppressed expression of the E47 and PU.1 proteins (Figure 6A). To validate the
importance of LMP2A-mediated reduction of the various molecules, endogenous
LMP2A was knocked down in LCLs (Figure 6B). As shown in Figure 6C-6H, the
expression of E47, PU.1, CIITA, HLA-DRA, HLA-DRB and CD74 transcripts was
restored in LMP2A knocked-down LCLs. In addition, compared to uninfected B cells,
knockdown of LMP2A restored approximately 60% of E47 and PU.1 mRNA levels
(data not shown). Furthermore, ectopic co-expression of E47 and PU.1 restored the
expression of MHC class II and CD74 in LCLs (Figure 6I). Collectively, LMP2A
potentially is the primary EBV-encoded product that contributes to the
down-regulation of MHC II molecules, which may be critical for EBV persistence in
healthy carriers.
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HLA-DR is expressed in tonsil biopsies but not in PTLD or EBV positive HD
biopsies.
The expression of HLA-DR was detected by IHC assay in 14 PTLD and 4 tonsil
biopsies (Supplementary Table 2-1). All PTLD samples were EBER positive and
HLA-DR negative (Supplementary Table 2-2). The IHC assay showed that HLA-DR
is expressed in four EBER-negative tonsil biopsies and HLA-DR is predominantly
expressed in the cytoplasm and at the cell membrane (Figure 7A). In addition, we
investigated 16 cases of HD. According to our results, 10 cases of EBER positive HD
were all negative for HLA-DR expression. However, 4 of 6 cases of EBER negative
HD were positive for HLA-DR expression (Figure 7B and supplementary Table 2-3).
Based on these results, it seems that EBV positive PTLD and HD specimens are
similar in terms of suppression of HLA-DR expression in vivo.
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Discussion
Recognition of antigens by CD4 T cells is through complexes of MHC class II
and viral peptides, which are endocytosed and digested by lysosomes39. However,
EBV is a pathogen that may persist for decades. In general, EBV is maintained in a
latent form in healthy individuals and escapes immune surveillance by expressing a
restricted set of viral proteins. As the only protein expressed in latency I, EBNA1 is
essential for maintaining the viral genome in an episomal form. Although it may
evade antigen presentation5, EBNA1 can be recognized as an endogenous antigen,
which is digested by autophagy for presentation by MHC class II molecules40, so that
CD4 T cells are generated against EBNA1 in infected healthy individuals. In healthy
carriers, the CD4 T cells consistently respond to EBV latent antigens, including
EBNA1 and EBNA2. In the latent stage of EBV infection, EBV-specific CD4 T cells
play a protective role, preventing reactivation of EBV. However, the numbers of CTLs
against EBNA1 are very low in individuals with EBV-associated malignancy41,42. To
avoid targeting by EBV-specific CD4 T cells, EBV has evolved several strategies to
hide itself in infected cells, including in B cells. In this study, we found that the EBV
latent protein, LMP2A, plays a critical role in down-regulating the expression of
MHC class II molecules in infected B cells.
Viruses evade antiviral CD4 T cells responses via interference with MHC class
II molecule presentation. For example, human cytomegalovirus, human parainfluenza
virus type 3 and varicella zoster virus suppress IFNγ-induced MHC class II
expression43-45. Mechanistically, down-regulation of IFNγ-induced MHC class II
expression is through inhibition of activation of the JAK-STAT pathway, which
results in reduction of CIITA expression30. In EBV infection, most studies of
strategies of viral escape from the immune system have focused on lytic products. For
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example, BZLF1 down-regulates expression of MHC class I and II by binding
directly to the ZRE element on the CIITA promoters31. In addition, BGLF5 protein
induces the global degradation of mRNA, resulting in reduction of expression of
MHC class I and II molecules8.
It is well-documented that BCR-triggered signaling facilitates the formation of
complexes of MHC class II and viral peptides on the cell surface46. Functionally,
LMP2A
mimics
constitutively
activated
BCR
signaling;
however,
the
LMP2A-activated PI3K pathway mediates the suppression of MHC class II and CD74
in EBV-infected B cells. Previous studies have revealed that CIITA is a master
regulator of the expression of MHC class II molecules and CD7410. Here, we showed
that knockdown of LMP2A in LCLs rescues the expression of CIITA, MHC class II
and CD74 (Figure 6E-6H).
Expression of CIITA transcripts is controlled by three defined promoters,
depending on the cell type (promoters I and III) and IFNγ-inducible expression
(promoter IV) of CIITA. In B cells, expression of CIITA is regulated by promoter III,
producing type III transcripts which encode a 124 kDa CIITA protein47. The
transcription factors PU.1, E47 and IRF4 have been reported to bind to promoter III of
CIITA33. In this study, we demonstrated that LMP2A-mediated the reduction of CIITA
levels by down-regulation of PU.1 and E47 expression. In contrast, in KSHV
infection, LANA protein-mediated inhibition of MHC class II presentation is through
blocking the DNA binding activity of IRF4 on the CIITA promoter48.
E47, the bHLH transcription factor, has been shown to regulate many of the
processes involved in the development of B lymphocytes49. The transcription factor
PU.1 is required for the differentiation of both lymphoid and myeloid cells50,51.
Knockout of the PU.1 gene in B cells impairs cell differentiation and causes pre-B
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cell acute lymphoblastic leukemia52. Of interest, the expression of E47 and PU.1 is
reduced in Hodgkin lymphoma-derived cell lines and EBV positive RS cells53,54. In
LMP2A transgenic mice, both the E47 and PU.1 genes are specifically
down-regulated in the B cells55. In this study, we demonstrated that LMP2A reduces
the expression of E47 and PU.1 in EBV-infected B cells.
Regulation of PU.1 and E47 levels is commonly controlled by epigenetic
modification, such as promoter activity and phosphorylation56-60. For example, PU.1
is down-regulated in classical Hodgkin lymphoma cells through methylation of the
PU.1 promoter61. Phosphorylation of E47 through the MAPK pathway induces
degradation of the E47 protein58. In this study, we found that LMP2A inhibits the E47
and PU.1 promoter activities through its ITAM motif and the associated kinases, Syk
and Src. Thus, we speculate that the ITAM motif is critical for LMP2A-mediated
repression of cellular genes.
SP1 is a constitutively expressed transcription factor and can be a
transactivator or repressor of the promoter activities of viral and cellular genes,
depending on its mode of interaction62. In our previous study, we found that SP1 is
repressed by the Zta promoter when SP1 is associated with HDAC2. However, SP1 is
phosphorylated by PKC-δ and releases HDAC2 in the presence of an HDAC inhibitor,
which is an EBV reactivation factor63. In this study, SP1 acted as a repressor of the
promoter activity of E47. So far, phosphorylation of SP1 by the PI3K/Akt, PKCδ, and
MAPK pathways may affect the associated protein partners in regulating promoter
activity.
In summary, LMP2A-mediated reduction of E47 and PU.1 may down-regulate
the expression of MHC class II and CD74 in B lymphocytes. These data provide
novel insights into the roles of EBV and LMP2A in EBV-associated malignancies, in
18
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particular, PTLD and HD.
19
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Acknowledgments
The authors thank Dr Tim J. Harrison of UCL Medical School (London, United
Kingdom) for reviewing the manuscript critically. We thank that Taipei Blood Center
of Taiwan Blood Service Foundation for providing whole blood. This work was
supported by National Science Council and Ministry of Science and Technology
(grants: NSC 100-2320-B-002-100-MY3 and MOST 103-2320-B-002-038-MY3),
National
Health
Research
Institute
(grants:
NHRI-EX102-10031BI
and
NHRI-EX103-10306BI), Excellent Translational Medicine Research Projects of National
Taiwan University College of Medicine and National Taiwan University Hospital
(103C-101-A1) to Ching-Hwa Tsai, and by National Science Council and Chang Gung
Memorial Hospital (grants: MOST-103-2320-B-182-028-MY3 and CMRPD1D0011)
to Sue-Jane Lin
Authorship Contributions
J.-H.L designed experiments, performed experiments, analyzed the data and co-wrote
the manuscript; J.-Y.L designed experiments and performed experiments; Y.-C.C
performed experiments and analyzed the data; M.-R.C provided materials; T.-H.Y
provided materials; C.-W.L provided materials; S.-J.L designed experiments and
co-wrote the manuscript; and C.-H.T designed experiments and co-wrote the
manuscript.
Conflict of Interest Disclosures
The authors declare no competing financial interests.
20
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Figure legends
Figure 1. Down-regulation of the expression of MHC class II and CD74 in LCLs.
CD19-positive B cells were seeded in a 12-well plate at the density of 1 x 106 cells
per well and infected with EBV. RNA and proteins were harvested at the time points
indicated. (A) The expression of HLA-DRA, HLA-DRB1, and CD74 transcripts was
measured by RT-Q-PCR. The relative fold of the transcripts was normalized to
uninfected B cells with the corresponding β-actin mRNA. This is a representative
result from six independent experiments from anonymous donors. (B) Protein
expression of MHC class II, CD74, EBNA1, LMP2A and β-actin was detected by
western blotting. Detection of β-actin served as an internal control. (C) BJAB cells
were transfected with EBNA1 or BARF0 expression plasmids or infected with
EBER1, EBER2, LMP1, LMP2A or Zta lentiviruses. Expression of MHC class II,
CD74, EBNA1, EBER1, EBER2, BARF0, LMP1, LMP2A and Zta transcripts in the
transfectants and lentiviruses-infected cells were analyzed by RT-PCR. β-actin was
detected as an internal control. (D) Akata and BJAB cells were infected with
LMP2A-expressing lentiviruses. Cell lysates were harvested and the expression of
MHC class II, CD74 and LMP2A was detected by western blotting. β-actin was
detected as an internal control. (E) BJAB cells were infected with various doses of
LMP2A-expressing lentiviruses. At day 5 post-infection, cell lysates were harvested
for the detection of MHC class II, CD74, PU.1 and LMP2A by western blot analysis.
β-actin was detected as an internal control. The experiment was performed three times
and one representative is shown.
Figure 2. LMP2A down-regulates the expression of CIITA, E47 and PU.1.
(A) Akata and BJAB cells were infected with LMP2A-expressing lentiviruses.
Expression of CIITA and LMP2A was detected by western blotting. (B) The
31
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expression of the CIITA-PI, CIITA-PIII, CIITA-PIV and LMP2A transcripts was
measured by RT-PCR analysis. Raji cells were used as a positive control for the
CIITA promoters. (C) Expression of E47, PU.1, IRF4, IRF8 and LMP2A was
measured by RT-PCR analysis. (D) Expression of E47, PU.1 and LMP2A was
detected by western blotting. (E) Akata and BJAB cells were infected with
Zta-expressing lentiviruses. Expression of E47, PU.1 and Zta was detected by western
blotting. β-actin was detected as an internal control. Each experiment was performed
three times and one representative is shown.
Figure 3. LMP2A down-regulates the expression of E47 and PU.1 through the
PI3K/Akt pathway. (A) BJAB cells were infected with LMP2A, LMP2A-Δ74-85 or
LMP2A-Y112F-expressing lentiviruses. Expression of LMP2A, E47, PU.1, CD74,
and MHC class II was analyzed by western blotting. β-actin was detected as an
internal control. (B) BJAB cells were transfected with wild type LMP2A or LMP2A
with mutated PY motif expression plasmids, and the transfectants were subjected to
western analysis of LMP2A, E47 and PU.1. β-actin was detected as an internal
control. (C) LCLs were cultured in the presence of 25 μM Piceatannol or 10 μM PP2
at the indicated time. Expression of phospho-Akt (pAkt), total Akt, E47, PU.1 was
analyzed by western blotting. β-actin was detected as an internal control. Experiments
of 3B and 3C were performed twice and one representative is shown. (D-E) BJAB
cells were infected with LMP2A-expressing lentiviruses or vector control. After 5
days, the cells were cultured in the presence of 20 μM LY294002 (D) or 20 μM
U0126 (E) paired to DMSO control for 48 hr. Cell lysates were subjected to western
analysis of E47, PU.1 CD74, MHC class II and LMP2A. β-actin was detected as an
internal control. Special detection of pAkt and total Akt was in (D) and detection of
phosphoERK1/2 (pERK1/2) and total ERK in (E). (F) LCLs were infected with
32
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sh-Luc or sh-PIK3CA expressing lentivirus and the cell lysates were subjected to
western analysis of E47, PU.1, CD74 and MHC class II. β-actin was detected as an
internal
control.(G)
BJAB
cells
were
infected
with
LMP2A,
PU.1
or
forced-dimered-E47 (FD-E47) lentiviruses and the transfectants were subjected to
western analysis of FD-E47, endogenous E47 (endo-E47), PU.1, CD74, MHC class II
and LMP2A. β-actin was detected as an internal control. Each experiment was
performed three times and one representative is shown.
Figure 4. LMP2A inhibited the E47 and PU.1 promoter activity through the
ITAM motif. (A) Akata and BJAB cells were infected with pSIN, LMP2A,
LMP2A-Y112F and LMP2A-Δ74-85 expressing lentiviruses. After 3 days, the cells
were infected with pCDH-GL3, E47 and PU.1 reporter expressing lentiviruses.
Luciferase activities were normalized with the GFP intensities of each transfectant.
The activated fold was calculated by normalizing luciferase activities for the
transfectant versus that for the pSIN with pCDH-GL3 vector control. (B) BJAB cells
were infected with pSIN and LMP2A expressing lentiviruses. After 3 days, the cells
were infected with pCDH-GL3, E47 and PU.1 expressing lentiviruses and incubated
with DMSO or 20 μM LY294002 for other 48 hr. Luciferase activities from each
transfectant were normalized with the GFP intensities. The activated fold for each
reporter was calculated by normalizing luciferase activities for the transfectant versus
that for the pSIN with pCDH-GL3 vector control. These data are a composite of three
independent experiments (mean ±SD).
Figure 5. LMP2A inhibited the E47 and PU.1 promoter activity.
(A) Schematic maps of the promoter regions of PU.1(-470/+57). Deletion constructs
derived from the construct (PU.1 -470/+57) were subcloned into the pCDH-GL3
luciferase reporter vector. BJAB cells were infected with pSIN and LMP2A
33
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expressing lentiviruses. After 3 days, the cells were infected with the reporter
lentiviruses indicated. After 4 days, luciferase activities from each transfectant were
normalized with the GFP intensities. (B) BJAB cells were transduced with pLKO or
PU.1 lentiviruses. After 5 days, infected BJAB cells were transduced with PU.1
(-470/+57), pSIN or LMP2A-expressing lentiviruses and then the luciferase activities
were measured and normalized to GFP activity at day 5 post-infection. (C) BJAB
cells were transduced with shLuc or shPAX5 lentiviruses. After 5 days, infected
BJAB
cells
were
further
transduced
with
PU.1
(-470/+57),
pSIN
or
LMP2A-expressing lentiviruses. At day 5 post-infection, the luciferase activities were
measured and normalized to GFP activity. (D) Schematic maps of the promoter
regions of E47 (-1000/+29). Deletion constructs derived from the E47 construct
(-1000/+29) were subcloned into the pCDH-GL3 luciferase reporter vector. 293T cells
were transfected with pSG5 and LMP2A-expressing plasmids. After 3 days, the
luciferase activities were measured and normalized to GFP activity. (E) 293T cells
were transfected with pCDH-GL3-E47 (-1000/+29), pSG5 or LMP2A-expressing
plasmids and treated with 500 nM mithramycin. At day 3 post-infection, the luciferase
activities were measured and normalized to GFP activity. (F) 293T cells were infected
with shLuc or shSP1 lentiviruses. After 5 days, infected 293T cells were transfected
with pCDH-GL3-E47(-1000/+29), pSG5 or LMP2A-expressing plasmids. At day 3
post-infection, the luciferase activities were measured and normalized to GFP activity.
Each experiment was performed three times and one representative is shown.
Figure 6. LMP2A is the key factor of EBV inhibiting the expression of E47 and
PU.1 and their downstream genes
(A) Expression of EBNA1, LMP1, LMP2A, E47, PU.1 and β-actin in various pairs
of uninfected primary B cells and EBV-immortalized LCLs was detected by western
34
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blotting. β-actin served as an internal control. (B-H) The LCL lines B36, B37, B44,
and B47 were infected with shLuc or shLMP2A lentiviruses. After 5 days, the RNA
and protein were harvested. Expression levels of the LMP2A (B), E47 (C), PU.1 (D),
CIITA (E), HLA-DRA (F), HLA-DRB (G), and CD74 (H) transcripts were measured
by RT-Q-PCR. (I) LCL B47 cells were infected with PU.1 or FD-E47 expression
lentiviruses. Expression of E47, PU.1, CD74, MHC class II and LMP2A was analyzed
by western blotting. β-actin was detected as an internal control. (Paired t test,
*indicates p<0.05; **indicates p<0.01). Each experiment was performed three times
and one representative is shown.
Figure 7. HLA-DR is not expressed in PTLD biopsies compared to tonsil
biopsies.
PTLD, tonsils, HD sections were used for IHC assays and the nuclei counterstained
with hematoxylin. (A) Positive signals of HLA-DR were observed as a brown color in
tonsil biopsies but not in PTLD biopsies. (B) Positive signals of HLA-DR were
observed as a brown color in EBER(-) biopsy (case 11) but not in EBER(+) biopsy
(case 1). The nuclei are stained blue. Original magnification, x200. Scale bar indicates
50μm.
35
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Prepublished online January 28, 2015;
doi:10.1182/blood-2014-08-594689
Epstein-Barr virus LMP2A suppresses MHC class II expression by
regulating the B cell transcription factors E47 and PU.1
Jiun-Han Lin, Ju-Yin Lin, Ya-Ching Chou, Mei-Ru Chen, Te-Huei Yeh, Chung-Wu Lin, Sue-Jane Lin and
Ching-Hwa Tsai
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
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Copyright 2011 by The American Society of Hematology; all rights reserved.