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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

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^2,*,

^* Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands; Cancer Research UK Institute for Cancer Studies and Medical Research Council Centre for Immune Regulation, University of Birmingham, Edgbaston, Birmingham, United Kingdom; Institute of Molecular Immunology, Helmholtz Zentrum, Munich, Germany; and Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands

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 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 BNLF2a with the HLA class I peptide-loading complex, the presence of TAP2 is essential, whereas tapasin is dispensible. Importantly, 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 costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These studies have been financially supported by Dutch Cancer Foundation Grant RUL 2005-3259, Royal Dutch Academy of Sciences Beijerinck Premium 2005, and the Netherlands Scientific Organization (NWO) Vidi Grant 917.76.330.

² Address correspondence and reprint requests to Dr. Maaike E. Ressing, Department of Medical Microbiology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands. E-mail address: M.E.Ressing{at}lumc.nl

³ Abbreviations used in this paper: ER, endoplasmic reticulum; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; β₂m, β₂-microglobulin; BHV, bovine herpesvirus; HCMV, human CMV; IRES, internal ribosomal entry site; HA, hemagglutinin; KO, knockout; LCL, lymphoblastoid cell line; MCMV, murine CMV; MJS, MelJuSo; NGFR, truncated nerve growth factor receptor; PDI, protein disulfide isomerase; PLC, peptide-loading complex.