EBV BILF1 Evolved To Downregulate Cell Surface Display of a Wide Range of HLA Class I Molecules through Their Cytoplasmic Tail This information is current as of October 2, 2021. Bryan D. Griffin, Anna M. Gram, Arend Mulder, Daphne Van Leeuwen, Frans H. J. Claas, Fred Wang, Maaike E. Ressing and Emmanuel Wiertz J Immunol 2013; 190:1672-1684; Prepublished online 11

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Supplementary http://www.jimmunol.org/content/suppl/2013/01/14/jimmunol.110246 http://www.jimmunol.org/ Material 2.DC1 References This article cites 63 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/190/4/1672.full#ref-list-1

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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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

EBV BILF1 Evolved To Downregulate Cell Surface Display of a Wide Range of HLA Class I Molecules through Their Cytoplasmic Tail

Bryan D. Griffin,*,†,1 Anna M. Gram,*,1 Arend Mulder,‡ Daphne Van Leeuwen,† Frans H. J. Claas,‡ Fred Wang,x Maaike E. Ressing,*,†,2 and Emmanuel Wiertz*,†,2

Coevolution of herpesviruses and their hosts has driven the development of both host antiviral mechanisms to detect and eliminate infected cells and viral ploys to escape immune surveillance. Among the immune-evasion strategies used by the lymphocryptovirus

(g1-herpesvirus) EBV is the downregulation of surface HLA class I expression by the virally encoded G –coupled receptor BILF1, thereby impeding presentation of viral Ags and cytotoxic recognition of the infected cell. In this study, we show EBV BILF1 to be expressed early in the viral lytic cycle. BILF1 targets a broad range of HLA class I molecules, including multiple HLA- A and -B types and HLA-E. In contrast, HLA-C was only marginally affected. We advance the mechanistic understanding of the Downloaded from process by showing that the cytoplasmic C-terminal tail of EBV BILF1 is required for reducing surface HLA class I expression. Susceptibility to BILF1-mediated downregulation, in turn, is conferred by specific residues in the intracellular tail of the HLA class I H chain. Finally, we explore the evolution of BILF1 within the lymphocryptovirus genus. Although the homolog of BILF1 encoded by the lymphocryptovirus infecting Old World rhesus primates shares the ability of EBV to downregulate cell surface HLA class I expression, this function is not possessed by New World marmoset lymphocryptovirus BILF1. Therefore, this study furthers our knowledge of the evolution of immunoevasive functions by the lymphocryptovirus genus of herpesviruses. The Journal of Immu- http://www.jimmunol.org/ nology, 2013, 190: 1672–1684.

ymphocryptoviruses (LCVs) are a genus of the g-her- individuals (5). This is partly due to the ability of EBV, like all pesvirus subfamily whose members are only found in herpesviruses, to enter a state of latency in which protein ex- L primates (1). The LCV targeting humans, EBV, is carried pression is minimized, thereby limiting viral Ag display by the by .90% of adults worldwide (2, 3). Although is usually infected cell. Yet, for EBV to spread to a new host, it must pro- asymptomatic, primary encounter with the can present as duce infectious virions by entering the replicative, or lytic, phase of its life cycle. In this phase, .80 EBV-encoded are

infectious mononucleosis. EBV infection is also strongly associ- by guest on October 2, 2021 ated with tumors of lymphoid and epithelial origin, reflecting the expressed in a temporal cascade, with initial production of im- tropism of the virus for B cells and epithelial cells and its potential mediate early (IE) transactivators triggering induction of early for oncogenic transformation (4). , including enzymes required for viral replication (2). This Despite the activation of a robust host T cell response upon is followed by expression of late genes encoding virion structural primary infection and the capacity for a memory T cell response components. Thus, the lytic phase creates ample viral Ags for thereafter, the virus persists for life even in immunocompetent proteasomal processing and HLA class I cell surface presentation, which can then recruit memory CD8+ T cells capable of elimi- nating the infected cell. However, millions of years of coevolution *Department of Medical Microbiology, University Medical Center Utrecht, 3584 CX with their hosts have seen herpesviruses acquire active immune- Utrecht, The Netherlands; †Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands; ‡Department of Immunohaema- evasion mechanisms to thwart this host response and permit suf- tology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, ficient time for the lytically infected cell to generate new virus The Netherlands; and xDepartment of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115 particles (6–8). In the case of EBV, several such immune-evasion strategies 1B.D.G. and A.M.G. contributed equally to this work. target the HLA class I Ag-processing and -presentation pathways 2M.E.R. and E.W. contributed equally to this work. (9). The EBV host shutoff protein BGLF5 degrades mRNA, thus Received for publication September 6, 2011. Accepted for publication December 11, 2012. obstructing synthesis of new HLA class I molecules (10, 11). Meanwhile, BNLF2a blocks entry of proteasome-generated pep- This work was supported by Dutch Cancer Foundation Grant UL 2005-3259 (to B.D.G. and E.W.), Netherlands Organisation of Scientific Research Vidi Grant 917.76.330 (to tides into the (ER) and subsequent loading M.E.R.), and National Institutes of Health Grant CA068051 (to F.W.). onto HLA class I molecules by inhibiting the heterodimeric TAP Address correspondence and reprint requests to Prof. Emmanuel Wiertz, Department complex (12, 13). TAP transport is also hindered by the viral che- of Medical Microbiology G04.614, University Medical Center Utrecht, P.O. Box mokine homolog, vIL-10, which reduces expression of the TAP1 85500, 3508 GA Utrecht, The Netherlands. E-mail address: [email protected] subunit (14). Another manner in which EBV can downregulate cell The online version of this article contains supplemental material. surface HLA class I expression and inhibit T cell recognition of Abbreviations used in this article: ER, endoplasmic reticulum; GPCR, G protein– coupled receptor; HC, H chain; IE, immediate early; IRES, internal ribosome entry infected cells is through BILF1, a viral G protein–coupled receptor site; LCV, lymphocryptovirus; NGFR, nerve growth factor receptor; PAA, phospho- (vGPCR) (15, 16). noacetic acid; trNGFR, truncated nerve growth factor receptor; vGPCR, viral G Several poxviruses and herpesviruses encode G protein–coupled protein–coupled receptor; wt, wild type. receptors (GPCRs) that were most likely pirated from their host Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 by retrotransposition (17–19). vGPCRs serve many functions, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102462 The Journal of Immunology 1673 including the scavenging of host chemokines (20), cell-to-cell ad- R.J. Lebbink, University Medical Center Utrecht, The Netherlands). The hesion (21), and the reprogramming of intracellular signaling net- pSicoR-EGFP backbone vector (Addgene) was altered by removing the U6 works to promote efficient viral replication (22). BILF1 is expressed promoter and replacing the CMV-EGFP cassette with the human EF1A promoter driving expression of the Zeocin resistance and the gene of during the EBV lytic cycle and was first identified as a potential interest. Zeocin and HLA-B8 genes were fused together by a self-cleaving vGPCR by the presence of seven membrane-spanning domains and 2A peptide derived from the porcine teschovirus-1 (P2A). The P2A peptide (limited) homology with known herpesviral GPCRs (23–25). Al- allows expression of both proteins from a single transcript. HLA-B8wt was though it displays both structural and functional similarities to amplified by PCR from the bidirectional lentiviral vector mentioned above; the cytoplasmic tail mutants were generated by using reverse primers that chemokine receptors, EBV BILF1 modulates intracellular signaling encoded the . pathways constitutively as an orphan receptor (24, 25). Restriction digests and sequence analysis verified the integrity of all gene Initial immune-evasion ability was ascribed to EBV BILF1 sequences. because it reduced phosphorylation of the dsRNA-dependent Cell lines protein kinase R (24). It was also found to heterodimerize with + human chemokine receptors (26). In the case of CXCR4, this The AKBM cell line is an EBV Burkitt’s lymphoma B cell line (Akata) results in impairment of ligand-induced receptor signaling (27). stably transfected with the pHEBO-BMRF1p-rCD2-GFP reporter plasmid (30). AKBM cells were maintained in RPMI 1640 medium (Invitrogen) Finally, EBV BILF1 decreases cell-surface levels of HLA class I supplemented with 10% FBS (PAA Laboratories, Pasching, Austria), 2 (15). This can be achieved by BILF1-mediated acceleration mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.3 of and subsequent lysosomal degradation of HLA class mg/ml hygromycin B. The MJS (HLA typing A*01, B*08) melanoma- I molecules from the cell surface or by diversion of newly syn- derived cell line (31), the monocytic U937 cell line (A*03, B*18, Cw*01), and primary fibroblasts obtained from a common marmoset (Callithrix thesized HLA class I molecules from the normal exocytic pathway

jacchus) (kindly provided by G. Koopman, Biomedical Primate Research Downloaded from that allows proteins to travel from the ER to the cell surface (28). Center, Rijswijk, The Netherlands) were both maintained in RPMI 1640 In turn, this leads to the inhibition of CD8+ T cell recognition of (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (European Union infected cells. approved; Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, and 100 In this study, we further investigate the ability of EBV BILF1 mg/ml streptomycin. 293T (A*02, B*07), 293-kB-luc (a gift from G.B. Lipford, Coley Pharmaceutical Group, New York, NY) (32), U373 to subvert the Ag-presentation pathway. We assess the ability of (A*0201, B*18), and HeLa (A*68, B*15) cells were maintained in DMEM BILF1 to target a range of specific HLA class I alleles and identify (Invitrogen) supplemented with 10% FBS (Invitrogen), 2 mM L-glutamine, the protein domain present in BILF1 and specific amino acid 100 U/ml penicillin, and 100 mg/ml streptomycin. 293-kB-luc cells were http://www.jimmunol.org/ residues in the HLA class I H chain (HC) that are required to cultured in the presence of 0.7 mg/ml Geneticin. facilitate downregulation of HLA class I from the cell surface. Replication-deficient lentiviruses and retroviruses Finally, we examine the evolution of BILF1 by comparing the Replication-deficient recombinant lentiviruses were generated by calcium immunoevasive ability of EBV BILF1 with that of homologs phosphate cotransfection of HEK 293T cells with a pLV-CMV-IRES-eGFP, expressed by LCVs infecting Old World rhesus and New World pCCLsin.PPT.pA.CTE.EF1A.hPGK.NGFR.pre, or pSicoR-EF1a-zeocin- marmoset primates. P2A lentiviral vector encoding the gene of interest, as well as pCMV- VSVG, pMDLg-RRE, and pRSV-REV (kindly provided by R. Hoeben, Leiden University Medical Center) (33). Replication-deficient recombinant Materials and Methods retroviruses were produced using the Phoenix amphotropic packaging by guest on October 2, 2021 DNA constructs system, as described previously (13). After 48–96 h, culture supernatants were harvested and frozen or filtered through a 0.45-mm-pore filter. MJS, BILF1 wild type (wt) and mutant-coding sequences were subcloned into the HeLa, 293T, U373, and U937 cell lines were infected with 1 ml lentivirus- PstI/XhoI sites upstream of the internal ribosomal entry site (IRES) in the containing medium in tissue culture dishes coated with 12 mg/ml Retro- lentiviral expression vector pLV-IRES-GFP (29). All were engineered Nectin. Transduction efficiency was examined by measuring GFP or sur- to contain an N-terminal FLAG-tag. The EBV BILF1wt sequence was face nerve growth factor receptor (NGFR) expression. In the case of U937 subcloned from pcDNA5-FLAG-BILF1, and the EBV BILF1-K122A se- cells transduced with pSicoR-EF1a-zeocin-P2A–based vectors, cells were quence was subcloned from pcDNA3.1/TOPO-FLAG EBV BILF1 K122A selected using Zeocin (400 mg/ml) to obtain pure populations. (both gifts from H. Vischer and M. Smit, Vrije Universiteit, Amsterdam, The Netherlands). The coding sequence of rhesus LCV BILF1 was am- Induction of EBV lytic cycle in AKBM cells, RNA isolation, and plified by PCR from pcDNA-Rhe LCV BILF1-IRES-GFP (a gift from RT-PCR M. Rowe, University of Birmingham, Birmingham, U.K.). The EBV BILF1 The EBV lytic phase was induced in AKBM cells by cross-linking surface C-terminal truncation mutant DC19 was generated by PCR amplification of IgG with 50 mg/ml goat F(ab9) fragments to human IgG (Cappel; MP Bio- the wt sequence with the introduction of an early stop codon. To derive the 2 medicals, Solon, OH). Discrimination between IE and late lytic phases was EBV BILF1 point mutants, EBV BILF1-V A and EBV BILF1-Y A, 299 303 achieved by inhibition of viral DNA replication and late lytic phase gene the QuikChange Site-Directed Mutagenesis Kit (Stratagene) was used expression using phosphonoacetic acid (PAA). PAA (pH 7.4 in 100 mM according to the manufacturer’s instructions. The chimeric gene encoding HEPES) at a final concentration of 300 mg/ml was added 1 h prior to EBV a protein with amino acid residues 1–285 of marmoset LCV BILF1 fused lytic phase induction. Total RNA was extracted using TRIzol reagent (Invi- to residues 293–312 of EBV BILF1 (BILF1 C-tail swap) was generated by trogen) and treated with DNase (TURBO DNA-free kit; Applied Biosystems), PCR amplification using a reverse primer that encoded the 19 most C- according to the manufacturer’s protocols. cDNA was synthesized using ran- terminal residues of the EBV BILF1 C-tail and annealed to nt 830–855 of dom hexamers and the Moloney murine leukemia virus reverse transcriptase the marmoset LCV BILF1 coding-sequence template. (Finnzymes) and used for amplification with Taq DNA Polymerase. EBV The construct containing the N-terminally HA-tagged EBV BILF1wt BILF1 expression was measured using the primers 59-GTATGGCGTTG- coding sequence in the retroviral expression vector pLZRS-IRES-GFP was GAGAAGACC-39 and 59-TAATCAGCAGGAGTACCAGACA-39,BZLF2/ a kind gift from J. Zuo and M. Rowe (University of Birmingham). gp42 expression was measured with the primers 59-ATTCTACCTGTGG- The HLA-B8wt sequence was amplified by PCR from pLZRS-HLA-B8- TAACTAGA-39 and 59-TTAGCTATTTGATCTTTG-39, and 18S rRNA ex- GFP (a gift from M. Heemskerk, Leiden University Medical Center) and pression was measured with the primers 59-GTAACCCGTTGAACCCCATT- subcloned into a lentiviral bidirectional vector, pCCLsin.PPT.pA.CTE.eGFP. 39 and 59-GATCCGAGGGCCTCACTAAAC-39. DNA fragments of the mCMV.hPGK.NGFR.pre (kindly provided by L. Naldini, San Raffaele Sci- expected length were visualized by 1% agarose gel electrophoresis and entific Institute and Vita-Salute University, Milano, Italy), in which the human ethidium bromide staining. EF1A promoter replaced the minimal CMV-eGFP cassette. The HLA-B8 short-truncation mutant was generated by PCR amplification of the wt Abs sequence with the introduction of an early stop codon. To express HLA-B8wt and C-tail mutants in U937 cells, cells were The mouse mAbs used in this study were W6/32, which detects HLA class transduced with replication-deficient lentiviruses generated from the I molecules (34); L243, which detects class II HLA-DR (American Type following lentiviral vector pSicoR-EF1a-Zeo-P2A (kindly provided by Culture Collection); anti-FLAG M2 (Sigma); and 3D12, detecting HLA-E 1674 LYMPHOCRYPTOVIRUS BILF1 DOWNREGULATION OF MHC CLASS I

(eBioscience). The rat mAb 3F10 (Roche) was used for detection of HA- tags. The human mAbs used were produced locally (35) and included VDK1D12, detecting HLA-A1; SN230G6, detecting HLA-A2; WIM8E5, detecting HLA-A68; VTM4D9, detecting HLA-B7; BVK 5B10, detecting HLA-B8; OUWF11, detecting HLA-B15; FVS4G4, detecting HLA-B18; and WK4C11, detecting HLA-C (Cw*01, Cw*03, Cw*08, Cw*12, and Cw*14). Additional Abs used were allophycocyanin-conjugated goat anti- mouse IgG (H+L) (Leinco Technologies), allophycocyanin-conjugated goat anti-human IgG + IgM (H+L) (Jackson ImmunoResearch), allophycocyanin- conjugated donkey anti-rat IgG F(ab9)2 (H+L) (Jackson ImmunoResearch), R-PE–conjugated goat anti-mouse Ig F(ab9)2 (Dako), PE-conjugated goat anti-human IgM F(ab9)2 (Southern Biotech), biotinylated goat anti-mouse Ig (Dako), and allophycocyanin-conjugated streptavidin (BD Pharmingen).

Flow cytometry Surface expression of specific molecules was determined using the in- dicated primary Abs. Bound Abs were detected using goat anti-mouse IgG-allophycocyanin; goat anti-human IgG + IgM-allophycocyanin was used for staining of specific HLA class I alleles. For HLA-E de- tection, a streptavidin-biotin–based three-step staining was performed. Double staining of marmoset fibroblasts, to examine coexpression of MHC class I and FLAG-BILF1 on single cells, was performed by con- FIGURE 1. The BILF1 gene is expressed early in the EBV lytic cycle. secutive incubation with anti-FLAG mAb, allophycocyanin-conjugated EBV lytic cycle gene expression was induced in AKBM cells by cross- Downloaded from anti-mouse Ab, and PE-conjugated W6/32. Stained cells were analyzed linking of the BCR with anti-IgG for the indicated time periods (hours) (A) on a FACSCalibur flow cytometer (Becton Dickinson) using FlowJo or for 16 h in the presence or absence of PAA (B). RNA was isolated, software (TreeStar). cDNA was generated, and expression levels of the indicated gene products were determined by PCR and ethidium bromide gel electrophoresis. Transient transfections and luciferase assays To examine activation of NF-kB by EBV BILF1wt, EBV BILF1-K122A, and EBV BILF1-DC19, 293-kB-luc cells were seeded in a 96-well plate at In a lentiviral vector, the EBV BILF1 gene was cloned upstream of http://www.jimmunol.org/ 5 a density of 2 3 10 cells/ml, 200 ml/well, 24 h before transfection with an IRES that is followed by the gene encoding enhanced GFP. Thus, Lipofectamine 2000, following the manufacturer’s instructions. Cells were transduced cells could be identified easily as a GFP+ population. In transfected with 70 ng phRL-TK (constitutively expressing Renilla lucif- erase) and 160 ng constructs encoding EBV BILF1wt or mutants, or vector addition, a FLAG-tag added to the BILF1 N terminus made it alone. NF-kB–induced firefly luciferase and Renilla luciferase activity possible to confirm surface expression of the viral protein. Cells were assayed using the Luciferase Assay Reagent and Renilla Luciferase were transduced with control GFP or EBV BILF1/GFP-encoding Assay System (both Promega, Madison, WI Promega), respectively, according lentivirus, and the presence of surface markers was analyzed by to the manufacturer’s instructions. Luminescence was measured with the LB940 Mithras Research II microplate reader (Berthold Technologies). For flow cytometry after 7 d. Prior to staining, transduced and untrans- ducedcellsweremixedtoallowcomparisoninasingleassay.

transfection of primary marmoset fibroblasts, cells were seeded in a six- by guest on October 2, 2021 well plate at a density of 2.5 3 105 per ml, 2 ml/well, 24 h before Melanoma-derived MJS cells (31), widely used in Ag-presenta- transfection with Lipofectamine 2000, according to the manufacturer’s tion studies because of their expression of both HLA class I and II, instructions. Cells were transfected with 4 mg DNA and analyzed for displayed strong cell surface BILF1 expression after transduction expression of GFP and surface proteins after 48 h. with BILF1/GFP lentivirus (Fig. 2A, top row). Total surface HLA class I expression was reduced in BILF1/GFP-expressing cells com- Results pared with untransduced cells and cells expressing GFP alone EBV BILF1 is an early lytic cycle gene (Fig. 2A, second row). However, BILF1 did not decrease cell Previous reports presented conflicting information on the expres- surface HLA class II expression (Fig. 2A, third row). Through the sion of BILF1 during the productive phase of EBV infection (24, 25, use of alloantigen-specific Abs, the effect of BILF1 on individual 36). Because the absence of a working anti-BILF1 Ab precluded the HLA class I types expressed by MJS cells was evaluated. BILF1 detection of BILF1 protein, we monitored the appearance of BILF1 reduced expression of HLA-A1 (Fig. 2A, fourth row) and medi- mRNA in an EBV+ Akata-derived B cell line following induction ated a particularly strong decrease in surface HLA-B8 expression of the viral lytic cycle by cross-linking of the BCR. BILF1 ex- (Fig. 2A, bottom row). pression was strongly induced by 4 h and remained elevated up to HeLa cells expressing EBV BILF1 after lentiviral transduction 16 h postinduction (Fig. 1A). The faint band observed for unin- also displayed reduced surface levels of W6/32-reactive HLA class duced cells may indicate a low level of BILF1 expression, as was I (Fig. 2B). Specific staining of both HLA-A68 and HLA-B15 described for other EBV+ B cell lines under strict latency (24). showed that expression of both was decreased in BILF1+ HeLa PAA inhibits the viral DNA polymerase and transcription of late cells. Similarly, surface HLA class I levels in BILF1+ HEK lytic genes and, thus, can be used to dissect the temporal profile of 293T cells were reduced compared with control cells (Fig. 2C). lytic gene expression. Although expression of the gp42-encoding Downregulation of HLA-B7 and a moderate decrease in HLA-A2 late gene BZLF2 was detectable after overnight lytic cycle in- levels contributed to this effect (Fig. 2C). Taking all of these data duction, it was completely blocked by prior addition of PAA (Fig. together, EBV BILF1 can target HLA-A and -B gene products, 1B, upper panel). In contrast, BILF1 mRNA was still induced in resulting in a reduction in expression of W6/32-reactive HLA the presence of PAA (Fig. 1B, middle panel). Together, these data class I molecules at the cell surface. show EBV BILF1 to be an early lytic cycle gene. We next examined whether expression of the nonclassical HLA- E is also affected by BILF1. U373 cells expressing endogenous EBV BILF1 can selectively modulate cell surface expression of HLA-E were lentivirally transduced to achieve BILF1 expression. HLA class I alleles BILF1 caused a decrease in cell surface levels of W6/32-reactive To examine its effect on individual HLA class I alleles, cells with HLA class I molecules (Fig. 2D). Interestingly, BILF1+ cells also different HLA haplotypes were transduced to express EBV BILF1. displayed reduced levels of HLA-E, demonstrating that EBV The Journal of Immunology 1675

FIGURE 2. EBV BILF1 reduces surface expression of a broad range of HLA class I alleles. (A) MJS cells

were transduced with GFP-encoding Downloaded from (control) or EBV BILF1/GFP-encod- ing (BILF1) replication-deficient len- tivirus. After 7 d, surface expression of FLAG-tagged EBV BILF1, total HLA class I, HLA class II, HLA-A1, and HLA-B8 was determined by flow cytometry. Transduced and untrans- http://www.jimmunol.org/ duced MJS cells were mixed before Ab staining to allow comparison in a single assay (left and middle pan- els). Surface levels of the indicated proteins were also compared between GFP+ control cells and BILF1/GFP+ cells (graphs; right panels). HeLa (B) 293T (C), and U373 (D) cells were transduced with GFP-encoding (con- trol) or EBV BILF1/GFP-encoding by guest on October 2, 2021 (BILF1) lentivirus. Surface expres- sion of FLAG-tagged EBV BILF1, total HLA class I, or the indicated HLA class I alleles was examined by flow cytometry after 7 d.

BILF1 can target a wide range of HLA class I molecules to bring following retroviral transduction (Fig. 3, top row), which led to about a reduction in cell surface HLA class I expression. a reduction in total surface HLA class I expression (Fig. 3, second Finally, we assessed whether EBV BILF1 can also downregulate row). Although surface expression of HLA-A3 (Fig. 3, third row) and cell surface levels of HLA-C. Although poorly expressed by many cell HLA-B18 (Fig. 3, fourth row) was strongly reduced by BILF1, HLA- types, HLA-C is expressed at the surface of U937 myeloid cells and C was only marginally downregulated (Fig. 3, bottom row). There- can be specifically detected using the human Ab WK4C11 (37). N- fore, EBV BILF1 can selectively decrease cell surface levels of terminally HA-tagged BILF1 was efficiently expressed in U937 cells HLA-A, -B and -E alleles, while only slightly affecting HLA-C. 1676 LYMPHOCRYPTOVIRUS BILF1 DOWNREGULATION OF MHC CLASS I

FIGURE 3. EBV BILF1 only marginally downregulates cell surface HLA-C expression. U937 cells were transduced with GFP-encod- ing (control) or EBV BILF1/GFP-encoding (BILF1) replication-deficient retrovirus. Sur- face expression of HA-tagged EBV BILF1, Downloaded from total HLA class I, HLA-A3, HLA-B18, and HLA-C was determined by flow cytometry. Surface levels of the indicated proteins were compared between BILF12/GFP2 cells and BILF1+/GFP+ cells (graphs; right panels). http://www.jimmunol.org/ by guest on October 2, 2021

The C-terminal tail of EBV BILF1 is required for terminal residues, is required to bring about the downregulation downregulation of surface HLA class I of HLA class I from the cell surface. The C-terminal domain of GPCRs is known to interact with in- Inspection of the sequence of the BILF1 C terminus identified tracellular proteins, including endocytic adaptors (38). To assess two potential internalization motifs (outlined in dashed line boxes whether the C-terminal domain of EBV BILF1 is involved in in Fig. 4A). The first was QVTV, a putative type II PDZ ligand downregulation of surface HLA class I molecules, we generated sequence conforming to the consensus sequence X-F-X-F, where a C-terminal–deletion mutant, BILF1-DC19, which lacks the 19 X is any amino acid, and F is a bulky hydrophobic residue (38, most C-terminally located amino acid residues (Fig. 4A). This C- 39). The second was a potential nonclassical tyrosine-based motif, terminal–deletion mutant retained some of the characteristics of YFRRV, conforming to the consensus sequence Y-X-X-X-F (40, its wt counterpart. EBV BILF1wt activates the transcription factor 41). Thus, we generated two independent BILF1 point mutant NF-kB in a manner dependent on the integrity of the EKT motif in proteins, substituting an alanine residue for either V299 of the its third transmembrane domain (15). BILF-DC19 expression also putative type II PDZ ligand sequence or Y303 of the proposed activated NF-kB in HEK 293-kB-luc cells following transient tyrosine-based motif. The resulting point mutants were both transfection (Fig. 4B). expressed at the cell surface. However, the ability to downregulate BILF1-DC19, containing an N-terminal FLAG-tag, was cloned HLA class I was retained in both cases, demonstrating that neither into a lentiviral expression vector. MJS cells were then transduced putative motif contributes to EBV BILF1–mediated downregula- with control, BILF1wt, or BILF1-DC19 lentivirus. Anti-FLAG tion of surface HLA class I (Supplemental Fig. 1). staining showed that similar levels of BILF1wt and BILF1-DC19 were expressed at the surface of transduced cells (Fig. 4C). The intracellular region of the HLA class I HC is required for However, although MJS cells expressing BILF1wt displayed downregulation by BILF1 a concomitant decrease in surface HLA-B8 expression, BILF1- Given the requirement for the C-terminal domain of BILF1 in DC19 was severely diminished in its ability to downregulate HLA- bringing about a reduction in surface HLA class I, we further B8. Meanwhile, neither BILF1wt nor BILF1-DC19 significantly hypothesized that the intracellular region of the HLA class I HC altered the surface levels of HLA class II. This indicates that the might also be necessary. Therefore, we generated a form of the C-terminal domain of EBV BILF1, specifically the 19 last C- HLA-B8 HC from which the 24 most C-terminal amino acid The Journal of Immunology 1677 Downloaded from

FIGURE 4. The EBV BILF1 C-terminal tail is required for HLA class I downregulation. (A) Schematic representation of the seven-transmembrane EBV

BILF1 vGPCR with the sequence of the 26 most C-terminally located amino acid residues described. The arrow indicates the point of truncation used to http://www.jimmunol.org/ generate EBV BILF1-DC19. Dashed boxes indicate putative endocytosis/sorting motifs. (B) EBV BILF1-DC19 activates intracellular signaling pathways. 293-NF-kB-luc cells were cotransfected with control expression vector or constructs encoding EBV BILF1wt, EBV BILF1-K122A, or EBV BILF1-DC19, as well as pGL3-Renilla (for normalizing transfection efficiency) luciferase. At 24 h posttransfection, cell lysates were assayed for firefly and Renilla luciferase activity. Data are presented as fold NF-kB induction relative to cells transfected with vector alone. Results are mean 6 SEM of a representative experiment performed in triplicate. (C) MJS cells were transduced with GFP-encoding (control), EBV BILF1wt/GFP-encoding (BILF1wt), or EBV BILF1- DC19/GFP-encoding (DC19) replication-deficient lentivirus. After 7 d, surface expression of FLAG-tagged EBV BILF1, HLA-B8, and HLA class II was determined by flow cytometry. by guest on October 2, 2021 residues were deleted, leaving only 6 intracellular residues (HLA- that discriminating differences exist at positions 344, 351, 358, B8 short; Fig. 5A). Full-length HLA-B8 and HLA-B8 short were and 361 of the HLA-B alleles, as indicated in Fig. 6A. Therefore, cloned into a lentiviral expression vector that coexpressed the we generated point mutations in HLA-B8 to examine whether the control protein truncated NGFR (trNGFR). The U373 cell line, replacement of HLA-B8 residues with those found in the corre- which is susceptible to the effects of BILF1 on HLA class I (Fig. sponding positions of the HLA-C intracellular tail would render 2D) and does not express endogenous HLA-B8 (Fig. 5B), was the mutant HLA protein resistant to the effects of BILF1. The C- then transduced with either HLA-B8wt/trNGFR or HLA-B8 short/ tail sequence of the HLA-B8 point mutants used is indicated in trNGFR lentivirus. Equivalent surface levels of trNGFR and of wt Fig. 6A (lower panel). and short HLA-B8 were observed in the respective U373 trans- U937 cells, which do not express endogenous HLA-B8 (data not ductants (Fig. 5B). shown), were transduced to express HLA-B8wt or HLA-B8 point The U373-HLA-B8wt and U373-HLA-B8 short cell lines were mutant proteins. Surface levels of endogenously expressed HLA- next transduced with either control or BILF1 lentivirus. BILF1 B18 were reduced by BILF1 to a similar extent in U937-HLA- functionality in both cell lines was confirmed by its ability to B8wt and U937-HLA-B8 point mutant cell lines (Fig. 6B). downregulate surface expression of the endogenously expressed However, although BILF1 downregulated cell surface HLA-B8wt, HLA-B18 allele to a similar extent (Fig. 5C). Interestingly, BILF1 it did not decrease cell surface HLA-B8 Y344C/D351N/V358E/T361I caused a decrease in HLA-B8wt levels in U373 cells, but it failed expression (Fig. 6B). This strongly indicated that one, some, or all to reduce surface levels of HLA-B8 short. Surface levels of of the HLA-B8 residues mutated in HLA-B8 Y344C/D351N/V358E/ trNGFR were not affected by BILF1, whereas surface expression T361I are required to facilitate targeting by BILF1. of BILF1 itself was equivalent in U373-HLA-B8wt and U373- In further analysis of HLA-B8 point mutants, surface expression HLA-B8 short cells (Supplemental Fig. 2). Therefore, these data of HLA-B8 T361I was efficiently decreased by BILF1, demon- show that, in addition to the C-terminal domain of BILF1, the in- strating that residue T361 is not essential for BILF1-mediated tracellular region of the HLA class I HC is required for BILF1- HLA-B8 downregulation. Indeed, as with HLA-B8 Y344C/D351N/ mediated downregulation of HLA class I surface expression. V358E/T361I, surface levels of HLA-B8 Y344C/D351N/V358E Having assessed the effect of BILF1 on a wide range of HLA remained unaffected by BILF1. Individual of residues class I alleles (Figs. 2, 3), we next examined whether the selective Y344, D351, and V358 has not allowed a single amino acid to be targeting of surface HLA-A, -B, and -E, but not HLA-C, mole- identified as being essential to targeting of HLA-B8 by BILF1 cules could help to pinpoint key residues in the HLA class I HC (data not shown). Rather, the possibility exists that each could tail that facilitate BILF1-mediated downregulation (Fig. 6A). play a contributory role. Comparing the amino acid sequence of the BILF1-sensitive HLA- In conclusion, the BILF1-mediated downregulation of HLA B intracellular tails with those of BILF1-resistant HLA-C showed class I molecules requires their cytoplasmic domains; the amino 1678 LYMPHOCRYPTOVIRUS BILF1 DOWNREGULATION OF MHC CLASS I

FIGURE 5. The cytoplasmic region of HLA class I HC is required for BILF1- mediated cell surface downregulation. (A) Sequence of the HLA-B8 C terminus. The arrow denotes the point of truncation used to generate HLA-B8 short. (B) U373 cells were transduced with HLA-B8wt/trNGFR– encoding or HLA-B8 short/trNGFR-encod- ing replication-deficient lentivirus. Surface expression of HLA-B8 and trNGFR on transduced and untransduced cells was de- termined by flow cytometry. (C) U373-HLA- Downloaded from B8wt and U373-HLA-B8 short cells were further transduced with GFP-encoding (control) or EBV BILF1/GFP-encoding (BILF1) replication-deficient lentivirus. After 7 d, surface expression of HLA-B8 and the endogenous HLA-B18 allele was measured by flow cytometry. http://www.jimmunol.org/ by guest on October 2, 2021 acid residues at positions 344, 351, and 358 appear to contribute the viral protein could be functional in this respect in marmoset essentially to the observed phenotype in the case of HLA-B8. cells. Thus, we transiently transfected primary marmoset fibro- blasts with a vector offering coexpression of FLAG-tagged mar- Marmoset LCV BILF1 fails to downregulate cell surface MHC moset LCV BILF1 and GFP. Marmoset MHC class I on these cells class I was detectable using the W6/32 mAb. At 48 h posttransfection, BILF1 homologs are encoded by rhesus LCV (80.4% amino acid transfected cells were identified by GFP expression. No decrease identity to EBV BILF1 in a National Center for Biotechnology In- in surface MHC class I was evident in cells transfected with the formation blastp two-sequence comparison) and marmoset LCV marmoset LCV BILF1-IRES-GFP vector (Fig. 7D, upper left (41% identity to EBV BILF1) (Fig. 7A). Rhesus LCV BILF1 was panel). Because a population of GFP+ cells failed to express de- shown to mediate MHC class I downregulation (15), but the tectable levels of marmoset LCV BILF1 at the surface (Fig. 7D, functionality of marmoset LCV BILF1 in this regard is unknown. upper middle panel), a double-staining procedure was performed Cloning the rhesus and marmoset LCV BILF1 genes into a lenti- to more precisely assess MHC class I levels in cells clearly viral expression vector allowed us to directly compare the ability expressing marmoset LCV BILF1. This confirmed that GFP+ of the three LCV BILF1 homologs to downregulate surface HLA marmoset cells containing high surface levels of FLAG-tagged class I in MJS cells. Both EBV BILF1 and rhesus LCV BILF1 marmoset LCV BILF1 did not undergo a downregulation of strongly decreased surface HLA-B8 in lentivirally transduced MJS MHC class I (Fig. 7D, upper right panel). EBV BILF1 also failed cells, but marmoset LCV BILF1 failed to do so (Fig. 7B). HLA to reduce surface expression of marmoset MHC class I (Fig. 7D, class II was not affected by expression of any of the BILF1 pro- lower panels). This indicates that a species restriction exists with teins (data not shown). All three BILF1 homologs contained N- regard to EBV BILF1 function and suggests that a sufficient level terminal FLAG-tags, allowing their surface expression to be assessed of homology with the host protein(s) required for EBV BILF1– (Fig. 7B). Marmoset LCV BILF1 was efficiently expressed at the mediated MHC class I downregulation may not be present in cell surface, albeit to a lesser extent than its EBV and rhesus LCV marmoset cells. counterparts. Further analysis showed HLA-B8 levels to be re- Given the importance of the C-terminal domain of EBV BILF1 duced in the population of MJS cells expressing EBV or rhesus in mediating its effect toward HLA class I, we examined finally LCV BILF1 at an equivalent level to that obtained with marmoset whether this domain was itself sufficient to confer the ability to LCV BILF1 (Fig. 7C). downregulate HLA class I to a heterotypic protein. Therefore, we Although marmoset LCV BILF1 failed to reduce surface ex- generated a chimeric BILF1 protein in which the 19 most C- pression of MHC class I in human cells, it remained possible that terminal amino acids of marmoset LCV BILF1 were removed The Journal of Immunology 1679 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 6. EBV BILF1–mediated HLA-B8 downregulation requires amino acid residues in the cytoplasmic region of the HLA-B8 molecule. (A) Upper panel, Amino acid sequence alignment of selected HLA class I HC C-terminal tails. Alignments were generated using ClustalW2. An asterisk (*) indicates positions that have a single, fully conserved residue. A colon (:) indicates conservation between groups of strongly similar properties. A full stop (.) indicates conservation between groups of weakly similar properties. Sequences of the following subtypes were selected from the IMGT/HLA Database: A*01:01:01:01 (HLA-A1), A*02:01:01:01 (HLA-A2), A*03:01:01:01 (HLA-A3), A*68:01:01:01 (HLA-A68), B*07:02:01 (HLA-B7), B*08:01:01 (HLA- B8), B*15:03:01 (HLA-B15), B*18:01:01:01 (HLA-B18), C*01:02:01 (HLA-C), and E*01:01:01:01 (HLA-E). The dashed box indicates a putative YXXA internalization motif present in HLA-A, -B, and -E alleles but not HLA-C. Lower panel, C-terminal tail amino acid sequences of HLA-B8 point mutants. Amino acids in the dashed boxes were subject to substitution. The numbers above the dashed boxes indicate the position of the residue in the HLA-B8 protein. (B) U937 cells transduced to express HLA-B8wt, HLA-B8 Y344C/D351N/V358E/T361I, HLA-B8 T361I, or HLA-B8 Y344C/D351N/V358E were transduced with GFP-encoding (control) or EBV BILF1/GFP-encoding (BILF1) replication-deficient retrovirus. Surface expression of HLA-B8 and HLA- B18 was determined by flow cytometry. Surface levels of the indicated proteins were compared between BILF12/GFP2 cells and BILF1+/GFP+ cells (graphs). 1680 LYMPHOCRYPTOVIRUS BILF1 DOWNREGULATION OF MHC CLASS I Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 7. MHC class I is not downregulated by the marmoset LCV BILF1 homolog. (A) Amino acid sequence alignment of the BILF1 homologs encoded by EBV, rhesus LCV, and marmoset LCV. Alignments were constructed using ClustalW2 and displayed using BOXSHADE version 3.21. (B) MJS cells were transduced with GFP- (control), EBV BILF1/GFP- (EBV), rhesus LCV BILF1/GFP- (Rhe), or marmoset LCV BILF1 (Mar)-encoding replication-deficient lentivirus. After 7 d, surface expression of FLAG-tagged EBV BILF1 and HLA-B8 was determined by flow cytometry. Transduced and untransduced MJS cells were mixed before Ab staining to allow comparison in a single assay. (C) Populations of transduced MJS cells expressing equivalent surface levels of FLAG-tagged EBV, rhesus LCV, and marmoset LCV BILF1 were further analyzed to examine their HLA-B8 surface ex- pression levels. (D) Marmoset primary fibroblasts were transfected with EBV or marmoset LCV FLAG-BILF1 genes in the pLV-IRES-GFP bicistronic vector. Forty-eight hours posttransfection, cells were stained with PE-conjugated W6/32 mAb or with anti-FLAG and allophycocyanin-conjugated anti- mouse Ab. Double staining of samples was performed to determine surface MHC class I expression on cells displaying cell (Figure legend continues) The Journal of Immunology 1681 and replaced by those of EBV BILF1 (marmoset LCV-EBV C-tail important for the presentation of viral peptides, a fact reflected in swap BILF1). This LCV BILF1 fusion gene was cloned into a their high level of polymorphism. Surface expression of all HLA- lentiviral expression vector, and human cells were transduced with A and -B alleles tested was reduced by BILF1. Interestingly, the control, EBV BILF1wt, marmoset LCV BILF1wt, or C-tail swap HLA-A2, -B7, and -B8 molecules shown in this study to undergo BILF1 lentivirus. C-tail swap BILF1 was expressed at the cell BILF1-mediated surface downregulation were found to present surface at a similar level to marmoset LCV BILF1wt in MJS cells epitopes derived from lytic cycle proteins, expressed by vaccinia but did not mediate downregulation of HLA class I (Fig. 7E). This virus infection of B cells, to EBV-specific CD8+ T cells (49). indicates that the intracellular C-terminal domain of EBV BILF1 Because some of the epitopes were from IE Ags, this provides is necessary, but not sufficient by itself, to bring about a reduction further support for the idea that EBV BILF1 may target pre-existing in surface HLA class I levels. HLA class I molecules for endocytosis upon its synthesis ∼4 h after lytic cycle induction. Discussion Additionally, it was proposed that the repertoire of peptides Central to the execution of host antiviral immunity is the action of bound by HLA-A2, -B7, and -B8 include those that do not require CD8+ T lymphocytes in detecting and eliminating virally infected TAP for transport into the ER (50). The EBV transmembrane protein cells. Herpesviruses have counterevolved several strategies to LMP2 contains HLA-A2–restricted TAP-independent CD8+ Tcell thwart this system of immune surveillance by inhibiting the dis- epitopes (51), whereas an HLA-B7–restricted T cell clone specific play of MHC class I–viral peptide complexes at the cell surface for the EBV lytic cycle protein BMRF1 has been isolated from (7–9). EBV inhibition of the HLA class I Ag-presentation pathway a TAP-deficient individual (52). Therefore, it is possible that HLA during the viral lytic cycle is mediated through the concerted class I molecules loaded with peptide in the presence of BNLF2a- action of BGLF5 (10, 11), BNLF2a (12, 13), BILF1 (15, 28), and mediated TAP inhibition during the lytic cycle may still be Downloaded from vIL-10 (14). In understanding the dynamic process of multigenic targeted by BILF1. immune evasion during the productive phase of the EBV life Different HLA class I molecules possess distinct peptide- cycle, it is important to ascertain the temporal profile of individual binding specificity and, thus, will present a unique spectrum of gene expression. Previous studies showed both BNLF2a and viral peptides to CD8+ T cells. A mechanism of immune escape BGLF5 protein to be detectable ∼3 h after lytic cycle induction confined to a narrow subset of HLA-A and -B alleles would not (11, 42). In contrast, the vIL-10–encoding BCRF1 gene is provide a selective advantage to the virus in transmission to a wide http://www.jimmunol.org/ expressed late in the lytic cycle (14). Conflicting reports exist on range of hosts. BILF1 likely mediates a broad-spectrum inhibition the temporal expression of EBV BILF1 (24, 25, 36). In this study, of HLA-A and -B surface expression that is particularly useful in we show BILF1 to be an early EBV lytic cycle gene, with tran- a virus targeting a population with such high HLA-A and -B scripts first detectable by 4 h postinduction. Although this suggests polymorphism. that BILF1 protein is first expressed in conditions where BGLF5- HLA-C molecules are predominantly involved in regulating NK mediated host-shutoff and BNLF2a-mediated TAP inhibition have cell function. HLA-C interacts with NK cell inhibitory receptors to been established, BILF1 may function to remove pre-existing prevent NK cell–mediated lysis. Interestingly, although a range of HLA class I molecules from the cell surface. Furthermore, HLA-A and -B alleles was downregulated from the cell surface by by guest on October 2, 2021 BNLF2a protein expression is transient, whereas the detection of EBV BILF1, HLA-C was only slightly affected. Through selective BILF1 transcripts 16 h postinduction suggests that BILF1 is able modulation of classical HLA class I molecules, BILF1 could to target HLA class I molecules presenting viral Ags late in the target HLA-A and -B alleles presenting viral peptides to CD8+ lytic cycle. T lymphocytes, while allowing the virally infected cell to retain Although we previously showed EBV BILF1 to downregulate the inhibitory effect of HLA-C on NK cells. In this way, BILF1 HLA class I in human cells (15), the use of the pan–HLA class could assist EBV evasion of both adaptive and innate immune I–reactive mAb W6/32 could have masked selective targeting of mechanisms. specific HLA class I molecules. Other herpesvirus proteins tar- HLA-E is also best known for its role in regulating NK cell geting HLA class I for degradation were found to demonstrate function. By presenting peptides derived from the HLA-A, -B, and HLA class I type specificity. The Kaposi’s sarcoma–associated -C signal sequences and acting as a ligand for the CD94/NKG2A herpesvirus E3 ligases K3 and K5 mediate ubiquitination of cell NK cell inhibitory receptor complex, HLA-E prevents killing by surface HLA class I molecules, which are subsequently endocy- NK cells. However, EBV BILF1 did decrease surface expression of tosed and degraded by the (43–46). Although K3 is HLA-E. Reducing surface HLA-E expression and removal of this broadly reactive toward HLA-A, -B, -C, and -E, K5 is selective for inhibitory signal from the virally infected cell may appear detri- HLA-A and -B (46). Meanwhile, HCMV US2 induces dislocation mental to virus survival. However, HLA-E was also shown to bind of newly synthesized HLA class I molecules from the ER to the viral Ags, including a BZLF1-derived peptide (53). HLA-E–viral for proteasomal degradation (47). US2 targets HLA-A and peptide complexes can then be recognized by CD8+ T cells (54). certain HLA-B types, whereas surface expression of HLA-C, Thus, it may be advantageous for EBV to target HLA-E through HLA-E, and other HLA-B alleles is not affected (48). BILF1, with the increased risk for NK cell attack being offset by We used a panel of alloantigen-specific mAbs to examine for the other NK cell immune-evasion tactics, such as the retention of first time, to our knowledge, the effect of BILF1 on expression of HLA-C expression, and the action of miRNA BART-2 in reducing individual HLA class I alleles in different cell types. Most cells expression of the NK cell–activating ligand MICB (55). express HLA-A and -B alleles, along with low levels of HLA-E, Previous studies showed BILF1 to accelerate internalization of and, in some cases, HLA-C. HLA-A and -B molecules are most HLA class I molecules, which are subsequently targeted for ly-

surface FLAG-BILF1. (E) MJS cells were transduced with GFP- (control), EBV BILF1wt/GFP- (EBV), marmoset LCV BILF1wt- (Mar), or marmoset LCV-EBV C-tail swap (C-tail swap)-encoding replication-deficient lentivirus. After 7 d, surface expression of FLAG-tagged EBV BILF1 and HLA class I was determined by flow cytometry. Transduced and untransduced MJS cells were mixed before Ab staining to allow comparison in a single assay. 1682 LYMPHOCRYPTOVIRUS BILF1 DOWNREGULATION OF MHC CLASS I sosomal degradation (15). In an effort to gain further mechanistic be identified, our findings advance the molecular understanding of insight into EBV BILF1–mediated HLA class I downregulation, the process by identifying a critical domain in the viral effector wefocusedontheroleofBILF1C-terminaltail,becausethis protein and key amino acid residues in the host target proteins. region of GPCRs is often involved in intracellular sorting and In addition to EBV, other members of the LCV genus include interaction with endocytic adaptor proteins (56). Although the C- Callithricine herpesvirus 3 (marmoset LCV) and Macacine her- tail is required for cell surface expression in the case of some pesvirus 4 (rhesus LCV) (1). These LCVs are prototypes for the GPCRs (57), our EBV BILF1 truncation mutant lacking the 19 New World and Old World primate LCVs, respectively, and display most C-terminal residues was still detectable at the plasma broadly similar biological properties to EBV. They can induce B . The BILF1 deletion mutant was still functional with growth transformation in vitro, possess inherent oncogenic potential respect to modulation of intracellular signaling, because it retained in vivo, and persistently infect their hosts. Rhesus LCV has a genetic the capacity to activate NF-kB. However, BILF1-DC19 displayed repertoire that is identical to EBV, even though they are separated a substantially abrogated ability to downregulate HLA class I by an evolutionary distance ∼25 million years (61). All EBV open surface expression relative to BILF1wt. Using a BILF1-deletion reading frames are accounted for in rhesus LCVand vice versa, with mutant lacking the 21 most C-terminal amino acid residues, a similar relative genomic position. In contrast, marmoset LCV, a study by Zuo et al. (28) indicated that the BILF1 C-tail is re- which is estimated to have evolved 35 million years before EBV, has quired for directing endocytosed HLA class I molecules to lyso- a similar, but less complete, genetic repertoire to EBV and rhesus somes. Therefore, the results of the current study are in agreement LCV, suggesting a different evolutionary path (62, 63). with those of Zuo et al. (28) in identifying a critical role for the Notably, marmoset LCV lacks 14 genes encoded by the LCVs BILF1 C-tail in reducing HLA class I levels. infecting higher-order primates (61–63). None of these 14 genes is Because the C-tail of EBV BILF1 likely contains motifs in- known to be essential for viral replication or B cell immortaliza- Downloaded from volved in the targeting of HLA class I to , we scanned tion, but they do include the immunomodulatory genes BNLF2a, this domain for the short, linear amino acid sequences that typically vIL-10, the CSF receptor homolog BARF1 (64), and the EBERs mediate trafficking from the cell surface and sorting to lysosomes. (65). Thus, the viral genes acquired during LCV evolution from Two candidate putative motifs related to protein endocytosis and New World hosts to Old World and human hosts may be less intracellular sorting were identified. One, a type II PDZ ligand involved with intrinsic viral-replication pathways during latent or sequence, can be involved in regulating endocytosis, in addition to lytic LCV infection and may have evolved to survive in hosts with http://www.jimmunol.org/ protein recycling (38). The second, a nonclassical tyrosine-based increasingly sophisticated host immunity (e.g., a more diverse signal, differs from the more common consensus motif by con- MHC in humans and Old World versus New World hosts). Al- taining an extra residue between the tyrosine and hydrophobic though a BILF1 homolog is encoded by marmoset LCV, our residue (38, 41). Tyrosine-based motifs are recognized by clathrin results suggest the ability to downregulate cell surface MHC class adaptor proteins and can direct both protein endocytosis and ly- I expression functionally evolved within the same related gene sosomal targeting (40). In the case of both motifs, substitution of present in all LCVs. Thus, LCVs have made a concerted effort to a key amino acid residue by alanine was shown to functionally target the host Ag-presentation pathway and downregulate MHC inactivate the signal (41, 58). However, the failure of such point by multiple evolutionary mechanisms, including creation of novel by guest on October 2, 2021 mutations to block the effect of BILF1 on HLA class I surface immunomodulatory proteins (BNLF2a), acquisition of cellular expression indicates that other structural determinants with the homologs (vIL-10), and adaptation of novel functions into existing vGPCR C-tail are involved. gene products (BILF1). In addition to demonstrating the importance of the BILF1 C-tail, we found the intracellular portion of the HLA class I HC to be Acknowledgments essential for BILF1-mediated downregulation. Other viral immu- We thank Jianmin Zuo and Martin Rowe (School of Cancer Sciences, Uni- noevasins targeting the class I HC display a similar requirement. versity of Birmingham, Birmingham, U.K.), Martine Smit and Henry For instance, Kaposi’s sarcoma–associated herpesvirus K3 cata- Vischer (Department of Chemistry and Pharmaceutical Sciences, Vrije lyzes ubiquitination of lysine residues in the HC C-tail, thereby Universiteit, Amsterdam, The Netherlands), and Patrick Beisser (Depart- tagging the protein for internalization and degradation (59). ment of Medical Microbiology, Maastricht University, Maastricht, The However, the fact that BILF1 expression does not trigger ubiq- Netherlands) for helpful discussions and for generously sharing reagents. uitination of class I HC (15) points toward another basis for this dependence in the case of the EBV immunoevasin. Disclosures We identified three amino acid residues in the HLA-B8 intra- The authors have no financial conflicts of interest. cellular tail that may be essential for BILF1-mediated downreg- ulation. One of these, Y344, forms part of the tyrosine-based YXXA internalization motif, which is conserved and required for References constitutive endocytosis in the case of HLA-B27 (60). This motif is 1. Davison, A. J., R. Eberle, B. Ehlers, G. S. Hayward, D. J. McGeoch, A. C. Minson, P. E. Pellett, B. Roizman, M. J. Studdert, and E. Thiry. 2009. The present in all alleles tested in this study that were downregulated order Herpesvirales. Arch. Virol. 154: 171–177. by BILF1, but it was absent from HLA-C (Fig. 6A) and the HLA- 2. Kieff, E., and A. B. Rickinson. 2007. Epstein-Barr Virus and its replication. In Field’s Virology. D. M. Knipe and P. M. Howley eds. Lippincott Williams & B8 short mutant (Fig. 5A). 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