Specific Targeting of the EBV Lytic Phase Protein BNLF2a to the Transporter Associated with Antigen Processing Results in Impairment of HLA Class I-Restricted This information is current as Antigen Presentation of September 26, 2021. Daniëlle Horst, Daphne van Leeuwen, Nathan P. Croft, Malgorzata A. Garstka, Andrew D. Hislop, Elisabeth Kremmer, Alan B. Rickinson, Emmanuel J. H. J. Wiertz and Maaike E. Ressing Downloaded from J Immunol 2009; 182:2313-2324; ; doi: 10.4049/jimmunol.0803218 http://www.jimmunol.org/content/182/4/2313 http://www.jimmunol.org/ References This article cites 67 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/182/4/2313.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 26, 2021 • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Specific Targeting of the EBV Lytic Phase Protein BNLF2a to the Transporter Associated with Antigen Processing Results in Impairment of HLA Class I-Restricted Antigen Presentation1 Danie¨lle Horst,*§ Daphne van Leeuwen,* Nathan P. Croft,† Malgorzata A. Garstka,* Andrew D. Hislop,† Elisabeth Kremmer,‡ Alan B. Rickinson,† Emmanuel J. H. J. Wiertz,*§ and Maaike E. Ressing2*§ EBV persists for life in the human host while facing vigorous antiviral responses that are induced upon primary infection. This persistence supports the idea that herpesviruses have acquired dedicated functions to avoid immune elimination. The recently Downloaded from identified EBV gene product BNLF2a blocks TAP. As a result, reduced amounts of peptides are transported by TAP from the cytoplasm into the endoplasmic reticulum (ER) lumen for binding to newly synthesized HLA class I molecules. Thus, BNLF2a perturbs detection by cytotoxic T cells. The 60-aa-long BNLF2a protein prevents the binding of both peptides and ATP to TAP, yet further mechanistic insight is, to date, lacking. In this study, we report that EBV BNLF2a represents a membrane-associated protein that colocalizes with its target TAP in subcellular compartments, primarily the ER. In cells devoid of TAP, expression levels of BNLF2a protein are greatly diminished, while ER localization of the remaining BNLF2a is retained. For interactions of http://www.jimmunol.org/ BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Im- portantly, we now show that in B cells supporting EBV lytic replication, the BNLF2a protein is expressed early in infection, colocalizing and associating with the peptide-loading complex. These results imply that, during productive EBV infection, BNLF2a contributes to TAP inhibition and surface HLA class I down-regulation. In this way, EBV BNLF2a-mediated evasion from HLA class I-restricted T cell immunity contributes to creating a window for undetected virus production. The Journal of Immunology, 2009, 182: 2313–2324. ytotoxic T lymphocytes play an essential role in the con- minal domains for the binding of peptide substrates and ATP. To by guest on September 26, 2021 trol of many viral infections. Through their TCRs, CD8ϩ ensure efficient acquisition of optimal peptide cargo by nascent C CTLs scrutinize body cells for the presence of intracel- MHC class I complexes, TAP is part of the MHC class I peptide- lular foreign invaders reflected at the cell surface by virus-derived loading complex (PLC). Besides TAP1 and TAP2, the PLC com- ␤ ␤ peptides bound to host MHC class I molecules (HLA in humans). prises MHC class I H chains, 2-microglobulin ( 2m), tapasin, In general, these peptides result from protein degradation by cy- ERp57, and the chaperone calreticulin. Tapasin bridges TAP and tosolic proteasomes and are then transported into the endoplasmic MHC class I molecules and stabilizes TAP expression (1, 2). The reticulum (ER)3 lumen by the transporter associated with Ag pro- complex of tapasin and the thiol oxidoreductase ERp57 exerts a pep- cessing, TAP. TAP is a heterodimer composed of two multimem- tide-editing function (3, 4). Once newly synthesized MHC class I H ␤ brane-spanning subunits, TAP1 and TAP2, with cytosolic C-ter- chain/ 2m/peptide trimers are assembled, they are allowed to exit the ER and travel through the Golgi to the cell surface. Even in the face of functional antiviral immune responses, her- *Department of Medical Microbiology, Center of Infectious Diseases, Leiden Uni- pesviruses are capable of establishing lifelong persistent infections versity Medical Center, Leiden, The Netherlands; †Cancer Research UK Institute for in their hosts. This hallmark is shared by all members of the her- Cancer Studies and Medical Research Council Centre for Immune Regulation, Uni- pesvirus family, classified into ␣-, ␤-, and ␥-subfamilies. The past versity of Birmingham, Edgbaston, Birmingham, United Kingdom; ‡Institute of Mo- lecular Immunology, Helmholtz Zentrum, Munich, Germany; and §Department of decade has provided ample evidence that herpesviruses have ac- Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands quired gene products that thwart host antiviral responses (5–8). Received for publication September 25, 2008. Accepted for publication December Prominent among these are viral proteins that interfere with MHC 10, 2008. class I-restricted Ag presentation at various locations along the Ag The costs of publication of this article were defrayed in part by the payment of page processing route. For example, MHC class I molecules are retained charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. intracellularly in cells infected with both human and murine CMV ␤ 1 These studies have been financially supported by Dutch Cancer Foundation Grant (HCMV and MCMV, respectively; both -herpesviruses). To RUL 2005-3259, Royal Dutch Academy of Sciences Beijerinck Premium 2005, and achieve this, the HCMV US3 protein prevents the exit of MHC the Netherlands Scientific Organization (NWO) Vidi Grant 917.76.330. class I molecules from the ER in a tapasin-dependent manner (9, 2 Address correspondence and reprint requests to Dr. Maaike E. Ressing, Department 10), and in MCMV m152/gp40 is responsible for the retention of of Medical Microbiology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands. E-mail address: [email protected] 3 Abbreviations used in this paper: ER, endoplasmic reticulum; AEBSF, 4-(2-amin- NGFR, truncated nerve growth factor receptor; PDI, protein disulfide isomerase; PLC, ␤ ␤ oethyl)benzenesulfonyl fluoride; 2m, 2-microglobulin; BHV, bovine herpesvirus; peptide-loading complex. HCMV, human CMV; IRES, internal ribosomal entry site; HA, hemagglutinin; KO, knockout; LCL, lymphoblastoid cell line; MCMV, murine CMV; MJS, MelJuSo; Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803218 2314 TAP INHIBITION BY THE EBV-ENCODED LYTIC PHASE PROTEIN BNLF2a MHC class I in the ER-Golgi intermediate compartment (11). An B cells when compared with cells harboring a latent EBV infec- alternative strategy of MHC class I immune evasion adopted by tion, and this coincides with a block in TAP-mediated peptide several viruses is the acceleration of MHC class I turnover. In cells transport (41). Subsequently, we identified BNLF2a as an EBV expressing the HCMV US2 or US11 proteins, MHC class I mol- lytic cycle protein capable of blocking TAP function by interfering ecules are degraded rapidly upon synthesis and, interestingly, this with binding of both peptides and of ATP to TAP (42). Functional occurs by proteasomes following the dislocation of MHC class I homologues of EBV BNLF2a were found in Old World primate ␥ ␥ molecules into the cytosol (12, 13). Increased degradation of MHC 1-herpesviruses. Thus, -herpesviruses also appear to encode im- class I molecules is also achieved by viral E3 ubiquitin ligases mune evasion molecules specifically targeting peptide transport encoded by two members of the ␥-herpesvirus subfamily. The mu- by TAP. rine ␥-herpesvirus MHV68 encodes mK3, the expression of which In this article, we report novel mechanistic aspects of the EBV- leads to proteasomal degradation of MHC class I as well as other encoded BNLF2a protein, representing the first dedicated inhibitor components of the PLC (14–18). The human Kaposi sarcoma- of TAP in ␥-herpesviruses, and we provide evidence for its role associated herpesvirus expresses two proteins, K3 and K5, that during natural EBV infection. target MHC class I molecules for proteolysis at a different location compared with mK3, i.e., in lysosomes following endocytosis Materials and Methods (19–21). Enhanced lysosomal degradation is also effected by two Antibodies ␤-herpesvirus