From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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:[email protected] 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: [email protected] 1 Copyright © 2015 American Society of Hematology From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. and in situ hybridization of EBER assays were described and performed according to our previous paper26. 9 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 11 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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, 12 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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. 13 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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. 14 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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. 15 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 16 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 17 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. particular, PTLD and HD. 19 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. References 1. Rickinson AB, Kieff E. Epstein-Barr virus. In: Knipe DM, Howley PM, eds. Fields' virology Vol. 2: Lippincott Williams & Wilkins, Philadelphia, Pa 2007:2655-2700. 2. Fathallah I, Parroche P, Gruffat H, et al. EBV latent membrane protein 1 is a negative regulator of TLR9. J Immunol. 2010;185(11):6439-6447. 3. Nemoto Y, Otsuka T, Niiro H, et al. [Inhibitory effects of interleukin (IL) -10 and viral IL-10 (vIL-10) on the functions of monocytes/macrophages]. Nihon Rinsho Meneki Gakkai Kaishi. 1995;18(2):152-159. 4. van Gent M, Braem SG, de Jong A, et al. Epstein-Barr virus large tegument protein BPLF1 contributes to innate immune evasion through interference with toll-like receptor signaling. PLoS Pathog. 2014;10(2):e1003960. 5. Levitskaya J, Coram M, Levitsky V, et al. 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Neefjes J, Jongsma ML, Paul P, Bakke O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat Rev Immunol. 2011;11(12):823-836. 26 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 40. Long HM, Chagoury OL, Leese AM, et al. MHC II tetramers visualize human CD4+ T cell responses to Epstein-Barr virus infection and demonstrate atypical kinetics of the nuclear antigen EBNA1 response. J Exp Med. 2013;210(5):933-949. 41. Fogg MH, Wirth LJ, Posner M, Wang F. Decreased EBNA-1-specific CD8+ T cells in patients with Epstein-Barr virus-associated nasopharyngeal carcinoma. Proc Natl Acad Sci U S A. 2009;106(9):3318-3323. 42. Piriou E, van Dort K, Nanlohy NM, van Oers MH, Miedema F, van Baarle D. Loss of EBNA1-specific memory CD4+ and CD8+ T cells in HIV-infected patients progressing to AIDS-related non-Hodgkin lymphoma. Blood. 2005;106(9):3166-3174. 43. Gao J, De BP, Han Y, Choudhary S, Ransohoff R, Banerjee AK. 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Cai Q, Banerjee S, Cervini A, et al. IRF-4-mediated CIITA transcription is blocked by KSHV encoded LANA to inhibit MHC II presentation. PLoS Pathog. 2013;9(10):e1003751. 49. Kee BL. E and ID proteins branch out. Nat Rev Immunol. 2009;9(3):175-184. 50. Scott EW, Simon MC, Anastasi J, Singh H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science. 1994;265(5178):1573-1577. 51. McKercher SR, Torbett BE, Anderson KL, et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996;15(20):5647-5658. 52. Rosenbauer F, Wagner K, Kutok JL, et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat Genet. 2004;36(6):624-630. 53. Hertel CB, Zhou XG, Hamilton-Dutoit SJ, Junker S. Loss of B cell identity correlates with loss of B cell-specific transcription factors in Hodgkin/Reed-Sternberg 28 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. cells of classical Hodgkin lymphoma. Oncogene. 2002;21(32):4908-4920. 54. Jundt F, Kley K, Anagnostopoulos I, et al. Loss of PU.1 expression is associated with defective immunoglobulin transcription in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease. Blood. 2002;99(8):3060-3062. 55. Portis T, Longnecker R. Epstein-Barr virus LMP2A interferes with global transcription factor regulation when expressed during B-lymphocyte development. J Virol. 2003;77(1):105-114. 56. Li Y, Okuno Y, Zhang P, et al. Regulation of the PU.1 gene by distal elements. Blood. 2001;98(10):2958-2965. 57. Chen H, Zhang P, Radomska HS, Hetherington CJ, Zhang DE, Tenen DG. Octamer binding factors and their coactivator can activate the murine PU.1 (spi-1) promoter. J Biol Chem. 1996;271(26):15743-15752. 58. King AM, Van der Put E, Blomberg BB, Riley RL. Accelerated Notch-dependent degradation of E47 proteins in aged B cell precursors is associated with increased ERK MAPK activation. J Immunol. 2007;178(6):3521-3529. 59. Nie L, Xu M, Vladimirova A, Sun XH. Notch-induced E2A ubiquitination and degradation are controlled by MAP kinase activities. EMBO J. 2003;22(21):5780-5792. 60. Hata K, Mizuguchi J. Genomic organization and characterization of the promoter 29 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. for the E2A gene. Gene. 2004;325:53-61. 61. Yuki H, Ueno S, Tatetsu H, et al. PU.1 is a potent tumor suppressor in classical Hodgkin lymphoma cells. Blood. 2013;121(6):962-970. 62. Chu S. Transcriptional regulation by post-transcriptional modification--role of phosphorylation in Sp1 transcriptional activity. Gene. 2012;508(1):1-8. 63. Tsai PF, Lin SJ, Weng PL, et al. Interplay between PKCdelta and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr 2011;85(5):2373-2385. 30 virus reactivation. J Virol. From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 From www.bloodjournal.org by guest on February 6, 2015. For p From www.bloodjournal.org by guest on February 6, 2015. For personal use only. 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 Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). 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