The Varicellovirus UL49.5 Protein Blocks the Transporter ... · PDF fileThe Varicellovirus...

The Varicellovirus UL49.5 Protein Blocks the TransporterAssociated with Antigen Processing (TAP) by InhibitingEssential Conformational Transitions in the 6�6Transmembrane TAP Core Complex1

Marieke C. Verweij,* Danijela Koppers-Lalic,2* Sandra Loch,† Florian Klauschies,†

Henri de la Salle,‡ Edwin Quinten,* Paul J. Lehner,§ Arend Mulder,¶ Michael R. Knittler,�

Robert Tampe,† Joachim Koch,3† Maaike E. Ressing,* and Emmanuel J. H. J. Wiertz4*

TAP translocates virus-derived peptides from the cytosol into the endoplasmic reticulum, where the peptides are loaded onto MHCclass I molecules. This process is crucial for the detection of virus-infected cells by CTL that recognize the MHC class I-peptidecomplexes at the cell surface. The varicellovirus bovine herpesvirus 1 encodes a protein, UL49.5, that acts as a potent inhibitorof TAP. UL49.5 acts in two ways, as follows: 1) by blocking conformational changes of TAP required for the translocation ofpeptides into the endoplasmic reticulum, and 2) by targeting TAP1 and TAP2 for proteasomal degradation. At present, it isunknown whether UL49.5 interacts with TAP1, TAP2, or both. The contribution of other members of the peptide-loading complexhas not been established. Using TAP-deficient cells reconstituted with wild-type and recombinant forms of TAP1 and TAP2, TAPwas defined as the prime target of UL49.5 within the peptide-loading complex. The presence of TAP1 and TAP2 was required forefficient interaction with UL49.5. Using deletion mutants of TAP1 and TAP2, the 6�6 transmembrane core complex of TAP wasshown to be sufficient for UL49.5 to interact with TAP and block its function. However, UL49.5-induced inhibition of peptidetransport was most efficient in cells expressing full-length TAP1 and TAP2. Inhibition of TAP by UL49.5 appeared to be inde-pendent of the presence of other peptide-loading complex components, including tapasin. These results demonstrate that UL49.5acts directly on the 6�6 transmembrane TAP core complex of TAP by blocking essential conformational transitions required forpeptide transport. The Journal of Immunology, 2008, 181: 4894–4907.

M ajor histocompatibility complex class I-mediated pep-tide presentation to the TCR on CD8� T cells is anessential process in the defense against intracellular

pathogens, like viruses. Upon infection, viral proteins are sub-jected to proteasomal degradation in the cytoplasm. The resultingpeptides are transported into the endoplasmic reticulum (ER)5 via

TAP (ABCB2/3). TAP is associated with several other proteins,including MHC class I molecules, tapasin, and ERp57, togetherconstituting the peptide-loading complex (PLC). These moleculescooperate to facilitate efficient loading of MHC class I moleculeswith peptides and guide the release of the MHC class I-peptidecomplexes from the ER (1–3).

The TAP heterodimer, consisting of the subunits TAP1 and TAP2,transports peptides from the cytosol into the ER in an ATP-dependentmanner (4, 5). TAP1 and TAP2 consist of 10 and 9 transmembrane(TM) helices, respectively, and a cytosolic nucleotide binding domainthat is required for ATP hydrolysis (6). TM helices 5–10 of TAP1 and4–9 of TAP2 together comprise the core of TAP. This 6�6 TMcomplex appeared to be sufficient for dimerization of the TAP sub-units and efficient binding and translocation of peptides (7). The as-sembly of MHC class I H chain-�2-microglobulin (�2m) complexesis assisted by the chaperone molecules calnexin, calreticulin, and, incooperation, ERp57 and tapasin (8–12). The ERp57-tapasin het-erodimer bridges TAP and the H chain/�2m complex to facilitateloading of the transported peptides into the peptide-binding groove (8,13, 14). In addition, tapasin is required for stable expression of TAP(15–18). The first N-terminal helix of TAP1 and TAP2 is essential forthe interaction with tapasin (19, 20).

Herpesviruses cause a lifelong latent infection in their host andreactivate occasionally. Specific T cell responses against herpes-viruses exist in (healthy) carriers. For the �-herpesvirus bovine

*Department of Medical Microbiology, Center of Infectious Diseases, Leiden Uni-versity Medical Center, Leiden, The Netherlands; †Institute of Biochemistry, Bio-center, Goethe-University Frankfurt, Frankfurt/Main, Germany; ‡Institut National dela Sante et de la Recherche Medicale Unite 725, Etablissement Francais du Sang-Alsace, Strasbourg, France; §Department of Medicine, Cambridge Institute of Med-ical Research, Cambridge, United Kingdom; ¶Department of Immunohematology andBlood Transfusion, Leiden University Medical Center, Leiden, The Netherlands; and�Institute of Immunology, Friedrich-Loeffler-Institute, Tuebingen, Germany

Received for publication January 4, 2008. Accepted for publication July 24, 2008.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Dutch Diabetes Research Foundation(to D.K.-L.), the Dutch Cancer Society (Grant UL 2005-3259, to M.E.R. andE.J.H.J.W.), the M.W. Beijerinck Virology Fund of the Royal Academy of Arts andSciences (to M.E.R.), and The Netherlands Organization for Scientific Research (VidiGrant 917.76.330, to M.E.R.).2 Current address: Department of Molecular Cell Biology, Leiden University MedicalCenter, 2300 RC, Leiden, The Netherlands.3 Current address: Georg-Speyer-Haus, Institute of Biomedical Research, Paul-Ehrlich-Strasse 42-44, D-60596, Frankfurt am Main, Germany.4 Address correspondence and reprint requests to Dr. Emmanuel J.H.J. Wiertz, De-partment of Medical Microbiology, Leiden University Medical Center, 2300 RC,Leiden, The Netherlands. E-mail address: [email protected] Abbreviations used in this paper: ER, endoplasmic reticulum; �N, N-terminallytruncated; �2m, �2-microglobulin; huTAP, human TAP; MDR, multiple drug resis-

tance; NGFR, nerve growth factor receptor; PLC, peptide-loading complex; TM,transmembrane.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

The Journal of Immunology

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herpesvirus 1 (BHV-1), strong CD8� T cell responses directedagainst various glycoproteins were found in BHV-1-infectedcalves (21). Following reactivation of varicella-zoster virus, highfrequencies of circulating T cells recognizing a variety of struc-tural and regulatory proteins were found (22, 23). Strong CD8� Tcell responses to the tegument phosphoprotein 65 and the imme-diate early protein 1 were found for the �-herpesvirus humanCMV (24, 25). In healthy carriers of the �-herpesvirus EBV, aconsiderable proportion of the peripheral T cell repertoire is di-rected against EBV-encoded Ags, predominantly derived from la-tent and immediate early viral proteins (26). Thus, despite the pres-ence of a fully competent immune system mounting a powerfulresponse against herpesvirus infections, these viruses fail to beeliminated. Most likely, this is related to the expression of specificviral immune evasion proteins, efficiently preventing detection andelimination of the virus-producing cells by the immune system.TAP appears to represent a favorite target for herpesvirus immuneevasion strategies. The �-herpesviruses HSV-1 and HSV-2 encodethe ICP47 protein, which blocks peptide binding to the cytosolicside of TAP (27–31). The US6 protein encoded by human CMVinhibits TAP by preventing ATP binding to TAP (32–36). TheEBV-encoded BNLF2a, which is unrelated to ICP47 and US6,inhibits TAP by blocking both ATP and peptide binding (37).

Koppers-Lalic et al. (38) identified the UL49.5 gene product ofBHV-1 as a powerful inhibitor of TAP. Although all herpesvirusesencode a homologue of the UL49.5 protein (also called glycopro-tein N), inhibition of TAP by UL49.5 appears to be restricted to asubset of the genus Varicelloviruses, including bovine herpesvirus1 (BHV-1), pseudorabies virus, equine herpesvirus 1 and 4, andcanine herpesvirus (38, 39). ICP47, US6, BNLF2a, and UL49.5are structurally unrelated, and the molecular mechanisms throughwhich they block TAP are different. The BHV-1-encoded UL49.5protein does not interfere with ATP or peptide binding to TAP.Instead, UL49.5 causes a conformational arrest of the TAP com-plex, thereby preventing translocation of peptides over the ERmembrane (38). Additionally, TAP1 and TAP2 are targeted fordegradation via the ubiquitin-proteasome system (38). The directinteraction partner of UL49.5 within the PLC has still to be de-fined. Tapasin may play a role in UL49.5-related instability ofTAP, because this molecule contributes essentially to the stabilityof TAP.

To identify the proteins within the PLC that are targeted byUL49.5, the function of the viral protein was evaluated in cellslacking various components of the PLC, including TAP1, TAP2,and tapasin. Efficient interaction with the PLC and inhibition ofpeptide transport required the presence of both TAP1 and TAP2;tapasin was dispensable. To study the molecular interaction be-tween TAP and UL49.5 in more detail, the TAP-deficient T2 cellswere reconstituted with recombinant TAP proteins carrying N-ter-minal deletions. The inhibition of TAP by UL49.5 appeared toinvolve the minimal functional entity of TAP, including the C-terminal 6 TM helices of TAP1 and TAP2. Inhibition was, how-ever, most efficient in the presence of full-length TAP2 next totruncated TAP1. The coexpression of UL49.5 with TAP1 andTAP2 in insect cells resulted in inhibition of peptide transport,indicating that all other constituents of the PLC were dispensablefor UL49.5-mediated inhibition of TAP.

Materials and MethodsDNA constructs

UL49.5 was amplified from viral DNA and cloned into the pLZRS vector,as described (38). The pLZRS vector information can be found atwww.stanford.edu/group/nolan/retroviral_systems/retsys.html.

Cell lines and recombinant viruses

The human melanoma cell line Mel JuSo has been transfected to stablyexpressed human TAP (huTAP)1-GFP (40), as described. The EBV-trans-formed lymphoblastoid cell line 721.220 lacks a functional tapasin protein(13, 41). The 721.220 cells transfected to stably express HLA-B44.05 (re-ferred to as .220) or to stably express HLA-B44.05 and human wild-typetapasin (referred to as .220 tapasin) were used in this study (cell lines wereprovided by A. Williams, Cancer Sciences Division, University ofSouthampton School of Medicine, Southampton, U.K.). T2 cells do notexpress TAP1, TAP2, and MHC class II due to a deletion within the regionof chromosome 6 carrying the loci for MHC class II and related genes (42).T2 cells were transfected to express TAP constructs of rat origin (20, 43).The cell lines were maintained in RPMI 1640 medium supplemented with10% FCS and antibiotics.

STF1.169 cells are immortalized fibroblasts isolated from a TAP2-de-ficient patient (44). The cells were immortalized using a plasmid express-ing T and t SV40 Ags (StuI-BamHI fragment from the SV40 genome)under the control of the EIII promoter of adenovirus 2 (SmaI-HindIII frag-ment), human telomerase under the control of long terminal repeat of Abel-son leukemia virus (fragment NheI-VspI from pGRN45; provided byGeron), and a hygromycin resistance gene. Immortalized cells were se-lected using 25 �g/ml hygromycin. The STF1.169 cells were comple-mented with a TAP2 gene of human origin cloned into pIRES-neo (BDClontech). Transfected cells were selected with 250 �g/ml G418.STF1.169 cells and STF1.169 cells reconstituted with TAP2 (referred to asSTF1 and STF1-TAP2, respectively) were maintained in DMEM supple-mented with 10% FCS and antibiotics.

Recombinant viruses were made using the Phoenix amphotropic pack-aging system, as described before (www.stanford.edu/group/nolan/retroviral_systems/retsys.html). STF-1 cells, .220 cells, MJS TAP1-GFPcells, and the various T2 cell lines were transduced with recombinant ret-roviruses to generate the following stable cell lines: STF1 UL49.5 andSTF1-TAP2 UL49.5 (containing BHV-1 UL49.5, GFP�); MJS TAP1-GFPUL49.5 (containing BHV-1 UL49.5, � nerve growth factor receptor(NGFR)�); .220 UL49.5, .220 tapasin UL49.5, T2 rat TAP1 UL49.5, T2rat TAP1 rat TAP2 UL49.5, T2 rat TAP1 rat TAP2�N UL49.5, T2 ratTAP1�N rat TAP2 UL49.5, T2 tandem rat TAP1-rat TAP2�N UL49.5,and T2 tandem rat TAP1�N-rat TAP2�N UL49.5 (all containing BHV-1UL49.5KK/AA, GFP�). Transduced cells expressing GFP or �NGFR wereselected using a FACSVantage cell sorter (BD Biosciences). The T2 cellswere transduced with a retroviral vector encoding a mutant form ofUL49.5, in which the two lysine residues within the cytoplasmic tail werereplaced by alanines. This mutation did not affect UL49.5-mediated inhi-bition and degradation of TAP, but stabilized the UL49.5 protein (M. Ver-weij, D. Koppers-Lalic, and E. Wiertz, unpublished observation). More-over, exchange of both or individual lysines to arginines led to the samephenotype in Sf9 cells (S. Loch, J. Koch, and R. Tampe, unpublishedobservation). The enhanced expression of mutated UL49.5 is particularlyuseful in T2 cells, because these cells are difficult to transfect or transduce.

Insect cells (Spodoptera frugiperda, Sf9) were grown in Sf900II me-dium (Life Technologies). Sf9 cells were infected with recombinant bacu-loviruses following standard procedures to stably express TAP and UL49.5(45). For coinfections, a multiplicity of infection of 3 was used for thebaculoviruses encoding the TAP constructs and UL49.5. The cloning oftruncated TAP variants and the production of recombinant baculovirusesexpressing the TAP constructs have been described before (7).

Antibodies

The following Abs were used: anti-human MHC class I H chain mAbHC-10 (a gift from H. Ploegh, Whitehead Institute for Biomedical Re-search, Cambridge, MA), anti-human MHC class I complexes mAb W6/32(46), anti-HLA A2 mAb BB7.2 (47), human anti-HLA Cw1 mAb VP6G3(48), human anti-HLA B5 mAb GVK10H7 (the product of a human hy-bridoma established from the B lymphocytes of a multiparous woman withanti-HLA B5 serum Abs, by methodology described in Ref. 48), anti-GFP(49), anti-huTAP1 mAb 148.3 (50, 51), anti-huTAP2 mAb 435.3 (52) (pro-vided by P. van Endert, Faculte de Medecine Rene Descartes, Paris,France), goat anti-rat TAP1 M18 (Santa Cruz Biotechnology; used forWestern blotting), rabbit anti-rat TAP1 D90 (53) (used for immunopre-cipitation), anti-rat TAP2 mAb Mac394 (54) (used for Western blotting),sheep anti-rat TAP2 S635 (provided by S. Powis, Bute Medical School,University of St. Andrews, St. Andrews, United Kingdom; used for im-munoprecipitation), rat anti-human tapasin mAb 7F6 (7), rabbit anti-humanERp57 (a gift from S. High, Faculty of Life Sciences, University ofManchester, Manchester, U.K.), and anti-�2m BBM.1 (55) (provided by J.Neefjes, Department of Tumor Biology, The Netherlands Cancer Institute,Amsterdam, The Netherlands). In addition, the conjugated Abs W6/32-PE

4895The Journal of Immunology

(Serotec), anti-MHC class II L243-PE (BD Pharmingen), and anti-NGFR-biotin (BD Pharmingen) were used. For detection of UL49.5, a rabbit poly-clonal anti-serum (H11) was raised using a synthetic peptide derived fromthe N-terminal domain of BHV-1 UL49.5 as an Ag (56). mAbs againsthuman transferrin receptor (CD71; BD Biosciences) and human transferrinreceptor (H68.4; Roche Diagnostics) were used as controls.

Flow cytometry

Surface levels of MHC class I molecules and control proteins were deter-mined using the specific primary Abs indicated. Bound Abs, if not conju-gated to PE, were stained using rabbit anti-human Ig-PE or goat anti-mouseIg-allophycocyanin. NGFR-biotin was detected using streptavidin-allo-phycocyanin (BD Pharmingen). Stained cells were measured using aFACSCalibur (BD Biosciences) and analyzed using CellQuest software.

Immunoprecipitations and Western blotting

For immunoprecipitations, cells were dissolved in lysis buffer containing1% (w/v) digitonin, 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 150 mMNaCl, 1 mM leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluo-ride, and lysates were incubated with specific Abs, as indicated, and proteinG- and A-Sepharose beads (GE Healthcare) were used to isolate the im-mune complexes. Precipitated immune complexes and 1% Nonidet P-40lysates of the cells were separated by SDS-PAGE and transferred to poly-vinylidene difluoride membranes (GE Healthcare). UL49.5 was separatedusing 16.5%-tricine PAGE. The blots were incubated with the indicatedAbs, followed by HRP-conjugated secondary Abs (DakoCytomation andJackson ImmunoResearch Laboratories). Bound HRP-labeled Abs werevisualized using ECL Plus (GE Healthcare).

Peptide transport assay

Cells were permeabilized using Streptolysin-O (Murex Diagnostics) at37°C for 10 min. Permeabilized cells were incubated with the fluorescein-conjugated synthetic peptide CVNKTERAY (N-core glycosylation site un-derlined) in the presence or absence of ATP (10 mM final concentration)at 37°C for 10 min. Peptide translocation was terminated by adding 1 mlof ice-cold lysis buffer (1% Triton X-100, 500 nM NaCl, 2 mM MgCl2, and50 mM Tris-HCl (pH 8.0)). After lysis, cell debris was removed by cen-trifugation. Supernatants were collected and incubated with 100 �l of ConA-Sepharose beads (Amersham Pharmacia) at 4°C for 2 h to isolate gly-cosylated peptides. After extensive washing of the beads, peptides wereeluted with elution buffer (500 mM mannopyranoside, 10 mM EDTA, and50 mM Tris-HCl (pH 8.0)) by vigorous shaking at 25°C for 1 h. Thefluorescence intensity was measured using a fluorescence plate reader(CytoFluor, Applied Biosystems or Berthold Technologies; excitation 485nm/emission 530 nm). Peptide transport is expressed as percentage oftranslocation, relative to the translocation observed in control cells (set at100%).

Peptide transport in microsomes derived from Sf9 cells

Microsomes were prepared, as described (45), and incubated with the flu-orescein (F)-labeled peptide RRYQNSTCFL (N-linked glycosylation site isunderlined) in the presence or absence of ATP for 3 min at 32°C. Peptidetransport was terminated with stop buffer (PBS and 10 mM EDTA (pH7.0)) on ice. After centrifugation, membranes were lysed, and the super-natant was incubated with 100 �l of Con A-Sepharose beads (Sigma-Aldrich) overnight at 4°C. The samples were treated according to theprocedure described above. Fluorescence intensity was measured using afluorescence plate reader (Polarstar Galaxy; BMG Labtech).

ResultsConsequences of UL49.5 interaction with the MHC class I PLC

UL49.5 blocks the import of peptides into the ER by interferingwith essential conformational transitions of TAP required for

FIGURE 1. UL49.5 does not disintegrate the PLC. A, Surface expres-sion of MHC class I and class II molecules was assessed for MJS TAP1-GFP control cells (graph 2) and cells expressing UL49.5 (graph 3) usingthe MHC class I-specific Ab W6/32 and the MHC class II-specific L243.Graph 1, Background staining in the presence of secondary Ab only. B,UL49.5 does not alter the overall composition of the PLC. TAP1-GFP wasimmunoprecipitated (IP) from MJS cells using an anti-GFP Ab, and

coprecipitating proteins were analyzed by SDS-PAGE and Western blot-ting (WB) using Abs against TAP1, TAP2, MHC class I H chain (HC),�2m, tapasin, ERp57, and UL49.5. The relative amount of the PLC com-pounds detected in UL49.5-containing cells was expressed relative to theamount in control cells (set at 100%). C, UL49.5 was immunoprecipitatedfrom the MJS TAP1-GFP cells, and the resulting complexes were analyzedfor the presence of TAP1, TAP2, tapasin, and UL49.5 using specific Abs.One representative experiment of two independent experiments is shown.

4896 INHIBITION OF 6�6 TM TAP CORE COMPLEX BY UL49.5

translocation of peptides over the ER membrane (38). Addition-ally, both TAP subunits are degraded by the ubiquitin-proteasomesystem (38). In cells expressing a TAP1-GFP fusion protein, deg-radation of the TAP complex is not observed, but peptide transportis still blocked (38). To study the interactions of UL49.5 with thePLC, MJS cells expressing TAP1-GFP were used to preventUL49.5-mediated degradation of TAP1 and TAP2. The expressionof UL49.5 in the MJS TAP1-GFP cells resulted in inhibition ofpeptide transport and down-regulation of MHC class I moleculesat the cell surface (Fig. 1A, left panel). The expression of MHCclass II molecules was not affected (Fig. 1A, right panel). Thecomposition of the MHC class I PLC was evaluated in these cells.A proper composition of the PLC is essential for the stability of itsindividual components. For example, dissociation of TAP1 fromthe complex will result in destabilization of TAP2 (57). Alterna-tively, if UL49.5 would expel tapasin from the PLC, this woulddestabilize both TAP subunits (15–18). PLCs were isolated fromdigitonin-solubilized MJS TAP1-GFP cells using Abs againstGFP. The presence of TAP1, TAP2, MHC class I complexes, ta-pasin, ERp57, and UL49.5 was evaluated by Western blot analysis.Similar amounts of TAP1, TAP2, and ERp57 appeared to bepresent in PLCs from control cells and UL49.5-expressing cells(Fig. 1B). In the latter, UL49.5 was part of the PLCs. The levels of

MHC class I H chain and �2m molecules were �1.5 times higherin UL49.5-containing complexes compared with control com-plexes (Fig. 1B). This increase is probably resulting from the re-duction of peptide supply to MHC class I molecules caused byUL49.5, which leads to reduced maturation and prolonged asso-ciation of MHC class I complexes with the PLC. The levels oftapasin seemed slightly lower in the UL49.5-containing complexes(Fig. 1B, panel 5). This reduction was repeatedly observed. Therole of tapasin in UL49.5-induced inhibition of TAP is investigatedin more detail below. Theoretically, UL49.5 could coimmunopre-cipitate with incomplete PLCs, whereas the complete set of PLCcomponents could be coisolated with PLCs lacking UL49.5. Toexclude this possibility, UL49.5-containing PLCs were isolatedusing Abs directed against UL49.5 and probed for the presence ofPLC components. TAP1, TAP2, and tapasin were found to copre-cipitate with UL49.5 (Fig. 1C), confirming that UL49.5 does notdisintegrate the PLC in the TAP1-GFP cells.

Tapasin is not required for UL49.5-mediated inhibition of TAPfunction

Tapasin is an essential component of the PLC, facilitating the load-ing of MHC class I molecules with high affinity peptides and sta-bilizing the TAP complex (13, 14, 18, 41, 58). Based on this,

FIGURE 2. Tapasin stabilizes the TAP complex, but is not involved in UL49.5-mediated inhibition of TAP function. A, Surface MHC class I expressionon tapasin-deficient .220 and the tapasin-reconstituted .220 tapasin cells was assessed after staining with the HLA-CwI-specific Ab VP 6G3; .220 cellsincubated with secondary Ab only (graph 1), .220 control cells stained with VG 6G3 (graph 2), and UL49.5-expressing .220 cells stained with VG 6G3(graph 3). B, Transport activity of TAP was analyzed in .220 cells using the peptide CVNKTERAY, of which the cysteine was labeled with fluorescein.The assay was performed in the presence of 10 mM ATP (f) or EDTA (u). Peptide transport is expressed as percentage of translocation, relative to thetranslocation observed in control cells (set at 100%). C, Degradation of TAP is not observed in tapasin-deficient .220 cells. The influence of UL49.5 onexpression levels of TAP1, TAP2, and transferrin receptor (TfR) in .220 and .220 tapasin cells was assessed by SDS-PAGE and Western blotting usingspecific Abs. D, UL49.5 can interact with the TAP complex independent of the presence of tapasin. TAP1 was immunoprecipitated (IP) from both .220cell lines, and precipitated complexes were analyzed for the presence of TAP1 and UL49.5. One representative experiment of two independent experimentsis shown.


tapasin might play a role in the UL49.5-induced inhibition of TAPand, consequently, the down-regulation of surface MHC class Iexpression. To investigate this possibility, we made use of cellslacking a functional tapasin protein, the lymphoblastoid cell line721.220 (referred to as .220) (13, 41) and a .220 cell line recon-stituted with tapasin (referred to as .220 tapasin). Both .220 celllines were retrovirally transduced to express UL49.5, and the in-fluence of the viral protein on MHC class I surface expression wasassessed using flow cytometry. A clear UL49.5-induced reductionof MHC class I surface expression was detected (Fig. 2A). Inter-estingly, the MHC class I level on the .220 cells reconstituted withtapasin is visibly higher compared with the level on tapasin-defi-cient .220 cells (Fig. 2A; compare right and left panels). This prob-ably reflects tapasin-induced stabilization of TAP and, conse-quently, enhanced peptide-loading and cell surface expression ofMHC class I molecules. The influence of tapasin on the stabiliza-tion of TAP is reflected by enhanced TAP transport in .220 tapasincells (Fig. 2B). UL49.5 was found to block peptide transport inboth .220 and .220 tapasin cells (Fig. 2B). These results imply thattapasin is dispensable for UL49.5-mediated inhibition of TAPfunction.

To investigate whether UL49.5 mediates TAP degradation in theabsence of tapasin, steady-state levels of TAP1 and TAP2 wereassessed in .220 and .220 tapasin cells through Western blot anal-ysis. The presence of tapasin resulted in elevated levels of TAP1and TAP2 (Fig. 2C, panels 1 and 2; compare lanes 1 and 3). De-spite the apparent presence of UL49.5 (Fig. 2C, panel 3, lane 2),no degradation of TAP was detected in .220 cells (Fig. 2C, panels1 and 2; compare lanes 1 and 2). However, the UL49.5 protein didinduce TAP degradation in .220 tapasin cells (Fig. 2C, panels 1and 2; compare lanes 3 and 4). To control for equal loading, alllysates were stained for transferrin receptor (Fig. 2C, panel 4). Theabsence of TAP degradation in the UL49.5-expressing .220 cellslacking tapasin suggests that tapasin is needed for UL49.5 to in-duce degradation of TAP. However, the TAP levels in the .220cells are much lower than the TAP levels in .220 tapasin cells (Fig.2C, panels 1 and 2; compare lanes 1 and 3), because TAP is un-stable in the absence of tapasin. Thus, it is also possible that thedegradation of TAP occurring in tapasin-negative cells cannot befurther enhanced by the UL49.5 protein, and therefore, no differ-ence in TAP degradation is observed between UL49.5-expressingand wild-type .220 cells. Note that peptide transport is inhibited inthe wild-type .220 cells, despite the fact that UL49.5 does notaffect TAP steady-state levels in these cells. This is in agreementwith previous observations in cells expressing the TAP1-GFP fu-sion protein or UL49.5�tail, which does not cause degradation ofTAP, but still inhibits peptide transport (38).

To assess whether UL49.5 binds to TAP in the absence of func-tional tapasin, TAP complexes were isolated from .220 and .220tapasin cells solubilized in digitonin and stained for the presence ofUL49.5 (Fig. 2D). The viral protein was found to interact with theTAP complex in both cell lines, indicating that the interaction be-tween UL49.5 and TAP is independent of the presence of func-tional tapasin.

In conclusion, functional tapasin is dispensable for UL49.5-induced inhibition of peptide transport and the reduction ofMHC class I surface expression. Even though UL49.5 is stillable to interact with the TAP complex, degradation of TAP1and TAP2 is not accelerated in cells lacking tapasin.

The TAP1-TAP2 heterodimer is required for the association ofUL49.5 with the PLC

UL49.5 interacts with the PLC (Fig. 1) (38), but its interactionpartner within this multimeric complex has still to be identified. To

investigate whether UL49.5 interacts with one of the TAP sub-units, we made use of STF1 cells. STF1 cells are immortalizedfibroblasts isolated from an MHC class I-deficient patient (44). Thecells carry a mutation within the TAP2 gene, resulting in the de-letion of the C-terminal 413 aa residues. STF1 cells and TAP2-reconstituted STF1-TAP2 cells were retrovirally transduced to ex-press the UL49.5 protein. The influence of UL49.5 on MHC classI levels in these cells was evaluated using flow cytometry. In theretroviral vector encoding UL49.5, the viral protein was placed infront of an internal ribosomal entry site that is followed by en-hanced GFP. As a result, UL49.5-expressing cells can be detectedas a GFP-positive population. The UL49.5/GFP-expressing cellswere mixed with untransduced control cells. Thus, MHC class Isurface expression can be compared between UL49.5-expressingand control cells in one assay (Fig. 3A). Surface MHC class Iexpression on STF1 cells was significantly up-regulated when

FIGURE 3. The TAP1-TAP2 heterodimer is required for association ofUL49.5. A, UL49.5-induced down-regulation of surface MHC class I canonly be detected in cells expressing full-length TAP1 and TAP2. TheTAP2-deficient STF1 cells and the reconstituted STF1-TAP2 cells weretransduced with UL49.5/GFP-encoding retrovirus. The transduced cellswere mixed with untransduced, GFP-negative cells to facilitate comparisonof expression of MHC class I molecules. Surface MHC class I was stainedusing the Ab W6/32 and analyzed using flow cytometry. B, UL49.5 doesnot interact with TAP in STF1 cells lacking TAP2 expression. TAP1 orUL49.5 was immunoprecipitated (IP) from cell lysates, and coprecipitatingUL49.5 or TAP1 molecules were analyzed by SDS-PAGE and Westernblotting (WB) using the Abs indicated. One representative experiment oftwo independent experiments is shown.


1. control2. T23. T2 rTAP1 rTAP24. T2 rTAP1 rTAP2 UL49.5

HLA-A2 (BB7.2) HLA-ABC (W6/32)

control UL49.5

T2 rTAP1 rTAP2

+ ATP

- ATP

100

80

60

40

20

0

1 2 4 3 1 2 4 3

1 2 4 31 2,4 3 1 2,3,4

% o

f FL-

pept

ide

trans

port

HLA-B5 (GVK 10H7) HLA-Cw1 (VP 6G3) TFR (CD71)

WB: αTAP1

WB: αUL49.5

T2 rTAP1 T2 rTAP1 rTAP2

c UL49.5 c UL49.5

WB: αTAP1

WB: αUL49.5

IP: αTAP1

IP: αTAP1

WB: αtpsnIP: αTAP1

1 2 3 4


c UL49.5 c UL49.5

1 2 3 4


c UL49.5 c UL49.5

1 2 3 4

WB: αTAP2

WB: αUL49.5

A B

C E

D F T2 rTAP2 T2 rTAP1 rTAP2

c UL49.5 c UL49.5

WB: αTAP2

WB: αUL49.5

IP: αTAP2

IP: αTAP2

1 2 3 4

coun

tsco

unts

WB: αtpsn IP: αTAP2

ratio UL49.5/TAP - 7.8 - 100 ratio UL49.5/TAP - 15.8 - 100

FIGURE 4. UL49.5 inhibits the rat TAP complex expressed in T2 cells. A, Transport activity of TAP was assessed in T2 cells expressing rat TAP1 andrat TAP2, in the presence or absence of UL49.5. Translocation of the peptide CVNKTERAY, of which the cysteine was labeled with fluorescein, wasevaluated in the presence of 10 mM ATP (f) or EDTA (u). Peptide transport is expressed as percentage of translocation, relative to the translocationobserved in control cells (set as 100%). B, UL49.5-mediated down-regulation of MHC class I expression is not allele specific. T2 rat TAP1 rat TAP2 cells,with (graph 4) or without UL49.5 (graph 3), were stained for surface MHC class I molecules using the Abs indicated. Graph 2, MHC class I levels onuntransduced T2 cells. Graph 1, Background staining in the presence of secondary Ab only. C, The expression levels of rat TAP1 and UL49.5 in T2 cells.T2 cells expressing TAP1 or both TAP1 and TAP2, in the presence of absence of UL49.5, were analyzed for steady-state levels of TAP1 and UL49.5 bySDS-PAGE and Western blotting (WB) using specific Abs. D, UL49.5 interacts with rat TAP1 in the absence of TAP2. TAP1 was immunoprecipitated(IP) from the same cells, and coprecipitating proteins were analyzed for the presence of TAP1 and UL49.5. E, Steady-state levels of rat TAP2 and UL49.5in T2 cells. T2 cells expressing rat TAP2 or both rat TAP1 and rat TAP2, in the presence or absence of UL49.5, were lysed, and the levels of rat TAP1,rat TAP2, and UL49.5 were determined by SDS-PAGE and Western blotting (WB) using specific Abs. F, UL49.5 interacts with rat TAP2 in the absenceof TAP1. TAP2 was immunoprecipitated (IP) from cell lysates, and coprecipitating proteins were analyzed using specific Abs. For the cells expressingTAP2 in isolation, 4 times more cells were used for IP to compensate for lower expression levels of TAP2. The ratio between UL49.5 and TAP wasexpressed relative to the amount of UL49.5 coprecipitating with rat TAP1/rat TAP2 complexes (D and F, panel 4, set at 100%). Tpsn, Tapasin.


TAP2 was coexpressed (Fig. 3A; compare the GFP-negative cellpopulations in left and right diagrams). UL49.5-induced down-regulation of surface MHC class I was only detected on cells ex-pressing both TAP1 and TAP2 (Fig. 3A, right diagram).

To evaluate the ability of UL49.5 to interact with TAP, cellswere lysed in the presence of digitonin. TAP1 and UL49.5 wereimmunoprecipitated, and the immune complexes were probed forthe presence of UL49.5 or TAP1 by Western blot analysis. In theabsence of TAP2, TAP1 steady-state protein levels were similar incontrol and UL49.5-expressing STF1 cells (Fig. 3B, first panel,lanes 1 and 2). This indicates that UL49.5 does not destabilizeTAP1 in the absence of full-length TAP2. In STF1 cells expressingTAP1 and TAP2, TAP1 levels were considerably reduced in thepresence of UL49.5, reflecting UL49.5-induced degradation ofTAP (Fig. 3B, first panel, lanes 3 and 4). Despite the low levels ofTAP in the UL49.5-expressing STF1-TAP2 cells, an interactionbetween TAP and UL49.5 could be detected in these cells (Fig. 3B,second panel, lane 4). This interaction was absent in STF1 cells(Fig. 3B, second panel, lane 2). The lack of interaction was not dueto lower levels of UL49.5 in STF1 cells, because similar amountsof UL49.5 were detectable in STF1 and STF1-TAP2 cells (Fig. 3B,third panel). Again, an interaction between TAP1 and UL49.5 wasonly found in the STF1-TAP2 cells (Fig. 3B, fourth panel, lane 4).These data indicate that the presence of TAP1 and TAP2 is nec-essary for UL49.5 to efficiently interact with the TAP complex.

UL49.5 efficiently interacts with rat TAP and inhibits peptidetransport

Based on previous N-terminal truncations of huTAP1 andhuTAP2, leading to the identification of a functional 6�6 TM corecomplex (7), a unique collection of rat TAP proteins has beenconstructed that are very suitable to characterize the interactionsites for UL49.5 within the TAP complex (20, 59). The UL49.5protein is able to prevent peptide transport by murine TAP as wellas huTAP (38, 60). Therefore, it is likely that UL49.5 can alsoblock rat TAP. Indeed, expression of UL49.5 in a rat cell line(Rat-2) resulted in inhibition of peptide transport (data not shown).In agreement with this observation, UL49.5 also inhibited peptidetransport in T2 cells expressing rat TAP1 and rat TAP2 (Fig. 4A).The ability of UL49.5 to reduce MHC class I surface expressionwithin these cells was assessed by flow cytometry using variousMHC class I-specific Abs. Compared with control cells, UL49.5-expressing cells showed a clear down-regulation of MHC class Isurface expression, as detected by the mAb W6/32 (Fig. 4B, firstpanel). This Ab recognizes a wide range of MHC class I locusproducts. Because the withdrawal of peptides differentially affectsthe various MHC class I locus products, the cells were also stainedwith Abs specific for HLA-A2, HLA-B5, and HLA-Cw1. UL49.5reduced the cell surface expression of each of these MHC class Ihaplotypes (Fig. 4B). The down-regulation was specific, becausetransferrin receptor was expressed equally on all cell lines (Fig.4B, lower right panel). In conclusion, UL49.5 effectively inhibitsMHC class I surface expression by blocking peptide transport byTAP of rat origin.

Efficient interaction of UL49.5 with TAP requires both TAPsubunits

To investigate the ability of UL49.5 to bind to the individuallyexpressed TAP subunits, we made use of T2 cells stably express-ing rat TAP1, rat TAP2, or, as a control, both subunits. The ratTAP1 and UL49.5 steady-state protein levels were comparable inT2 rat TAP1 and T2 rat TAP1 rat TAP2 cells (Fig. 4C). Note thatthe rat TAP proteins are not degraded in the presence of UL49.5,again illustrating that peptide loading can be inhibited in the ab-

sence of TAP degradation. To study the interaction of UL49.5 withrat TAP1 in the absence of rat TAP2, the rat TAP1 protein wasimmunoprecipitated from digitonin-solubilized T2 rat TAP1 cells(Fig. 4D, upper panel). T2 cells expressing both TAP subunitswere included as a positive control. The resulting immune com-plexes were analyzed for the presence of UL49.5. Whereas similaramounts of rat TAP1 were precipitated from all cell lines, only asmall amount of UL49.5 was detectable in immunoprecipitatesfrom cells expressing rat TAP1 alone (Fig. 4D, middle panel; com-pare lanes 2 and 4; see also quantification). The immune com-plexes were also probed for the presence of tapasin. This moleculewas detectable at comparable levels in all rat TAP1 immunopre-cipitates, regardless of the presence of UL49.5 (Fig. 4D, lowerpanel). Thus, when rat TAP1 is expressed in the absence of ratTAP2, the interaction between UL49.5 and rat TAP1 is highlyinefficient.

huTAP2 is extremely unstable in the absence of TAP1. Conse-quently, huTAP2 cannot be detected when expressed in isolation(P. Lehner, M. Verweij, and E. Wiertz, unpublished observation).Interestingly, rat TAP2 appears less unstable when expressed inthe absence of rat TAP1. Thus, T2 cells expressing rat TAP2 couldbe used to study its interaction with UL49.5. Expression levels ofTAP2 and UL49.5 were verified by Western blotting. The ratTAP2 levels were much lower in T2 rat TAP2 cells compared withT2 cells expressing both TAP subunits (Fig. 4E, upper panel; com-pare lanes 1 and 2 with lanes 3 and 4). Despite these low levels ofTAP2 in the T2 rat TAP2 cells, a considerable amount of rat TAP2could be immunoprecipitated from these cells (Fig. 4F, upperpanel, lanes 1 and 2). UL49.5 was detectable in the rat TAP2immune precipitates from both cell lines (Fig. 4F, middle panel,lanes 2 and 4; see also quantification). These results indicate thatUL49.5 can interact with TAP2 alone, albeit with lower efficiency.Even though twice as much UL49.5 precipitated with TAP2 ex-pressed in isolation compared with TAP1, this does not necessarilyimply that UL49.5 binds better to TAP2. Due to different expres-sion levels of TAP1 and TAP2 and the use of different Abs forimmunoprecipitations and immunoblotting, it is very difficult toassess whether equal amounts of TAP were isolated from the T2rat TAP1 and T2 rat TAP2 lysates. The combined results show thatoptimal binding of UL49.5 to the TAP complex requires both TAPsubunits.

The N-terminal domains of TAP1 or TAP2 are nonessential forUL49.5-mediated inhibition of TAP

The N-terminal 4 TM helices of TAP1 and the first 3 TM he-lices of TAP2 are dispensable for peptide transport. The corecomplex containing the remaining 6 TM regions of TAP1 andTAP2 is still capable of translocating peptides across the ERmembrane (7). The N-terminal regions are, however, essentialfor the binding of tapasin to TAP1 and TAP2 (7, 19, 20). Toinvestigate whether the inhibitory activity of UL49.5 requiresthe N-terminal parts of TAP1 and/or TAP2, the viral proteinwas expressed in T2 cells transfected with various combinationsof full-length and N-terminally truncated (�N) TAP1 and TAP2proteins (depicted in Fig. 5A). First, the effect of deletion of thethree N-terminal TM helices of TAP2 (rat TAP1 rat TAP2�N)or the four N-terminal TM helices of TAP1 (rat TAP1�N ratTAP2) was analyzed. T2 cells expressing wild-type rat TAP1and rat TAP2 were taken along as a control. The cell lines weretransduced to express UL49.5 and subjected to Western blotanalysis to determine the steady-state levels of TAP1, TAP2,and UL49.5. These proteins appeared to be expressed at com-parable levels in all cell lines tested (Fig. 5B).


FIGURE 5. The N-terminal domains of TAP1 and TAP2 are dispensable for UL49.5-mediated inhibition of TAP. A, Schematic diagrams of TAPconstructs. NBD, nucleotide binding domain. B, Steady-state levels of TAP1, TAP2, and UL49.5 were assessed for T2 cells expressing wild-type ortruncated forms of rat TAP. Rat TAP1�N is deficient of the first four N-terminal TM domains of rat TAP1. Rat TAP2�N lacks the first three N-terminalTM domains of rat TAP2. C, UL49.5 inhibits heterodimers of N-terminal deletion mutants of TAP. MHC class I surface expression was assessed for T2TAP1 TAP2�N cells and T2 TAP1�N TAP2 cells, in the presence (graph 4) or absence (graph 3) of UL49.5, using the Abs indicated. Graph 1, Backgroundstaining in the presence of secondary Ab only; graph 2, untransduced T2 cells. D, Interaction between UL49.5 and TAP complexes deficient for the Nterminus of TAP1 or TAP2. TAP2 was immunoprecipitated (IP) from the cells, and the resulting complexes were stained for UL49.5 and tapasin. The ratiobetween UL49.5 and TAP was expressed relative to the amount of UL49.5 coprecipitating with rat TAP1/rat TAP2 complexes (D, panel 4, set at 100%).One representative experiment of three independent experiments is shown. Tpsn, Tapasin.


To investigate the capacity of UL49.5 to interfere with pep-tide transport in cells expressing �N TAP1 or TAP2 proteins,the cells were screened for inhibition of MHC class I expressionby UL49.5. Reduced MHC class I surface staining was found onall UL49.5-expressing cells lacking the N-terminal domains ofrat TAP2 or rat TAP1 (Fig. 5C, upper and lower panels, re-spectively). The HLA-A, B, and C locus products were all sen-sitive to this effect. The MHC class I down-regulation observedin cells expressing truncated rat TAP2 was slightly less efficient(Fig. 5C, upper panel). To evaluate the effect of the N-terminal

deletions of TAP1 and TAP2 on UL49.5 interaction, coimmu-noprecipitation experiments were performed. TAP complexeswere isolated from digitonin-solubilized cells using anti-TAP2Abs (Fig. 5D, upper panel). The presence of UL49.5 in theimmunoprecipitates was evaluated by Western blotting. UL49.5was found to interact with all constructs (Fig. 5D, middlepanel). The amount of UL49.5 coprecipitating with the TAP com-plex was reduced by 56% when the N-terminal part of rat TAP2 waslacking (Fig. 5D, middle panel, lane 4). This is reflected by the resultsobtained in the flow cytometry experiments (Fig. 5C, upper panel).

FIGURE 6. UL49.5 interacts withthe 6�6 TM core complex of TAP. A,Schematic diagrams of TAP fusionproteins. NBD, nucleotide bindingdomain. B, The expression of TAP fu-sion constructs in T2 cells. T2 cellsexpressing TAP1 and TAP2�N, thefusion protein TAP1-TAP2�N, andthe fusion protein TAP1�N-TAP2�N, next to UL49.5, were ana-lyzed for steady-state levels of TAPand UL49.5 by SDS-PAGE and West-ern blotting (WB) using specific Abs.C, Interaction of UL49.5 with rat TAPfusion proteins. TAP was precipitatedfrom cell lysates using a TAP2-spe-cific Ab. Immune complexes wereseparated on SDS-PAGE and stainedfor TAP2 and UL49.5 by Westernblotting (WB). For the cells express-ing the fusion constructs TAP1-TAP2�N or TAP1�N-TAP2�N, 3–4times more material was loaded ongel for immunoblotting, to compen-sate for lower expression levels ofTAP. As a control for aspecific bind-ing of UL49.5 during immunoprecipi-tation (IP), transferrin receptor wasimmunoprecipitated from the cellsand resulting complexes were stainedfor UL49.5 by Western blotting(WB). The ratio between UL49.5 andTAP was expressed relative to theamount of UL49.5 coprecipitatingwith rat TAP1/rat TAP2 complexes(C, panel 5, set at 100%). One repre-sentative experiment of two indepen-dent experiments is shown. TFR,transferrin receptor. Tpsn, Tapasin.


The interaction between UL49.5 and the TAP1�N TAP2 complexwas reduced by 24% compared with the interaction betweenUL49.5 and the full-length TAP complex. However, this does notseem to affect the UL49.5-induced down-regulation of MHC classI surface expression (compare Fig. 5C, lower panel, with Fig. 4B).Tapasin was found to coprecipitate with TAP to a similar extent incontrol cells and UL49.5-expressing cells (Fig. 5D, lower panel,compare lane 3 with lane 4 and lane 5 with lane 6). Thus, theinteraction between tapasin and TAP was not affected by UL49.5.Taken together, UL49.5 is still able to cause down-regulation ofMHC class I surface expression and interact with the TAP com-plex in the absence of the N-terminal domains of TAP1 or TAP2.However, the deletion of the N-terminal part of TAP2 negativelyinfluences the binding of UL49.5 and the efficiency of TAPinhibition.

The 6�6 TM core of the TAP heterodimer is sufficient forUL49.5-mediated inhibition of TAP

To investigate whether the core complex of TAP comprising theC-terminal 6 TM helices and the cytoplasmic domain of TAP1 andTAP2 are sufficient for UL49.5 interaction, the ability of UL49.5to bind to the rat TAP1�N/rat TAP2�N heterodimer was evalu-ated. Separate expression of the two truncated subunits did notresult in sufficiently high expression. Therefore, the two subunitswere expressed as a fusion protein (20) (depicted in Fig. 6A). TheN terminus of TAP1 was linked to TM domain 4 of TAP2 in ahead-to-tail fashion, via the connector region of the murine mul-tidrug resistance protein, multiple drug resistance (MDR)1b (20).The functionality of the fusion proteins has been demonstrated inpeptide translocation assays (20). To control for the influence ofthis tandem formation, T2 cells coexpressing the individual full-length rat TAP1 and rat TAP2�N constructs and T2 cells express-ing the fused form rat TAP1-TAP2�N were compared. The cellswere transduced with retrovirus to express UL49.5. Steady-statelevels of the TAP subunits and UL49.5 were assessed by Westernblotting. UL49.5 was similarly expressed in all cell lines (Fig. 6B,lower panel). When the TAP subunits were fused, the expressionlevels were lower than that of free TAP2 (Fig. 6B, upper panel;compare lanes 1 and 2 with lanes 3 and 4 and 5 and 6). This wasparticularly obvious when the various TAP complexes were im-munoprecipitated from digitonin-solubilized cells (Fig. 6C, upperpanel). The presence of UL49.5 in the complexes was assessed byWestern blotting. A significant amount of UL49.5 was found tocoprecipitate with the TAP complex when its subunits were ex-pressed as separate proteins (Fig. 6C, second panel, lane 2). Whenthe same TAP subunits were expressed as a fusion protein, theamount of coprecipitating UL49.5 was much less (Fig. 6C, secondpanel; compare lanes 2 and 4; see also quantification). Apparently,the fusion of the TAP subunits and their lower expression levelsresulted in reduced detection of UL49.5. Yet, UL49.5 was found tointeract with the rat TAP1�N-rat TAP2�N fusion construct to asimilar extent (Fig. 6C, second panel, lanes 4 and 6). This resultindicates that UL49.5 is capable of interacting with the 6�6 TMcore complex of TAP as efficiently as with the TAP1 TAP2�Ncomplex. To control for nonspecific binding of UL49.5 during thecoimmunoprecipitation experiments, an irrelevant protein, trans-ferrin receptor, was immunoprecipitated from all cell lines. Theresulting immune complexes were stained for UL49.5. Nonspecificinteraction was not observed between the transferrin receptor andUL49.5 (Fig. 6C, third panel, lanes 1–6). When an aliquot of thecell lysate was loaded onto the gel directly, UL49.5 was readilydetectable (Fig. 6C, third panel, control lysate). Staining of theTAP2 immune precipitates for tapasin showed that the interactionof tapasin with TAP was not affected by the presence of UL49.5

(Fig. 6C, lower panel; compare lanes 1 and 2 and lanes 3 and 4).Tapasin was not detectable in the immunoprecipitates containingthe rat TAP1�N-rat TAP2�N fusion protein, confirming the 6�6TM core TAP complex to be devoid of tapasin binding sites (Fig.6C, lower panel, lanes 5 and 6) (7, 20).

Sf9 insect cells infected with recombinant baculoviruses toexpress TAP1 and TAP2 provide an ideal opportunity to studythe TAP heterodimer in the absence of other components of theMHC class I PLC (7). High, stable levels of TAP are well-known advantages of this system. In this study, Sf9 cells wereinfected with baculoviruses to coexpress UL49.5 with wild-typeand recombinant forms of huTAP (61). To investigate whetherUL49.5 was able to inhibit the function of the 6�6 TM huTAPcore complex, Sf9 cells expressing huTAP1�N and huTAP2�N

FIGURE 7. The 6�6 TM core complex of TAP is sufficient for UL49.5to block peptide transport. A, The interaction between UL49.5 and the 6�6TM core complex was studied in microsomes of Sf9 cells expressingTAP1�N and TAP2�N, and, as a control, wild-type TAP1 and TAP2. Theexpression levels of TAP1, TAP2, and UL49.5 were analyzed using SDS-PAGE and Western blotting (WB) with UL49.5- and TAP-specific Abs.The relative amount of TAP1 and TAP2 detected in UL49.5-containingcells was expressed relative to the amount in control cells (set at 100%). B,TAP was precipitated from Sf9 microsomes expressing wild-type TAPusing a TAP1-specific Ab and from Sf9 microsomes expressing the 6�6TM core complex using a TAP2-specific Ab. Precipitated complexes wereanalyzed on SDS-PAGE and stained for TAP1, TAP2, and UL49.5 byWestern blotting (WB). C, Peptide transport was assessed in Sf9 cells ex-pressing wild-type TAP or 6�6 TM core complex of TAP. Equal amounts ofmicrosomes from UL49.5-containing and control cells were used in peptidetranslocation assays, in the presence of 10 mM ATP (f) or apyrase (�).Peptide transport is expressed as percentage of translocation, relative to thetranslocation observed in control cells (set at 100%). One representative ex-periment of two independent experiments is shown.


or, as a control, full-length huTAP1 and huTAP2 were prepared(7). Expression of all constructs was verified by Western blotanalysis (Fig. 7A). In the presence of UL49.5, a reduction ofTAP levels was observed, reflecting degradation of huTAP byUL49.5 (Fig. 7A). However, the observed degradation is not aspronounced as in, for example, STF1 cells (Fig. 3B). This mightbe related to suboptimal degradation of huTAP by the insectubiquitin-proteasomal system. Subsequently, the ability ofUL49.5 to interact with full-length and truncated TAP com-plexes expressed in Sf9 cells was verified. TAP was isolatedfrom digitonin-solubilized cells using TAP-specific Abs, andthe resulting complexes were stained for TAP1, TAP2, andUL49.5. UL49.5 was found to interact with both TAP com-plexes. This confirms the results shown in Fig. 6C and provesthat UL49.5 is able to interact with the 6�6 TM core domain ofTAP. To assess whether this interaction leads to a functionalblock of TAP, transport activity of the full-length and truncatedTAP complex was evaluated in the absence or presence ofUL49.5. Peptide transport by the full-length TAP subunits wasfound to be inhibited efficiently by UL49.5 (Fig. 7C, left panel).Also, in cells expressing the human 6�6 TM core TAP com-plex, UL49.5 inhibited peptide transport, albeit less effectively(Fig. 7C, right panel).

In conclusion, these experiments show that the 6�6 TM core com-plex of TAP is sufficient for UL49.5 interaction and for inhibition ofpeptide transport, in the absence of other components of the PLC.

DiscussionThe results presented in this study show that the BHV-1-encodedUL49.5 protein acts directly on the TAP heterodimer to inhibitloading of peptides onto MHC class I molecules. The interaction ofUL49.5 with the TAP complex appears to be most efficient whenboth TAP1 and TAP2 are present. The N-terminal domains ofTAP1 and TAP2 are dispensable for UL49.5 binding and inhibi-tion of TAP function, thereby identifying the 6�6 TM TAP corecomplex as the primary target for UL49.5. However, completeinhibition of TAP by UL49.5 is only observed when full-lengthTAP2 is present in addition to TAP1�N.

To investigate whether UL49.5 binds to TAP1 or TAP2, theinteraction with UL49.5 was studied in cells lacking one of theTAP subunits. In the TAP2-deficient STF1 cells, interaction ofUL49.5 with TAP1 was not detectable (Fig. 3B). In T2 cellsexpressing either rat TAP1 or rat TAP2, a weak interaction ofUL49.5 with the individual TAP subunits was observed (Fig. 4,D and F). However, huTAP1 expressed individually in insectcells showed a clear association with UL49.5 (61). Most likely,the relatively high expression levels of the individual proteinsin Sf9 insect cells promote interactions between UL49.5 and theTAP subunits. In addition, overexpression of the individualTAP subunits in the T2 cells or insect cells might result in theformation of homodimers adopting a conformation similar tothat of TAP heterodimers, thereby creating a structure that fa-cilitates the interaction with UL49.5. In MJS TAP1-GFP cells,endogenous TAP1 has been found to interact with TAP1-GFP(M. Verweij, D. Koppers-Lalic, and E. Wiertz, unpublished ob-servation), showing the possibility of homodimer formation.Homodimerization of the individual TAP1 and TAP2 subunitshas also been observed in the presence of chemical cross-linkers(62) or in a system in which TAP subunits were overexpressed(63). In case of the STF-1 cells, homodimers of TAP1 might notexist because of lower TAP1 levels compared with the recom-binant T2 or Sf9 cells. Additionally, the presence in the STF-1cells of an incomplete TAP2 gene product, comprising its N-terminal 5 TM helices, might interfere with TAP1 homodimer

formation in these cells. This might explain the inability ofUL49.5 to interact with TAP1 in the STF1 cells.

The �-2 herpesvirus 68-encoded mK3 protein causes evasionof CTL recognition by inducing the degradation of ER-residentMHC class I molecules via ubiquitination. mK3 requires aninteraction with TAP and tapasin to target MHC class I Hchains and to preserve its own stability. The protein appears tobind to TAP1 alone, but the interaction with both TAP1 andTAP2 is needed for mK3 to function properly (64 –71). Theobservation that UL49.5 needs both TAP1 and TAP2 to effi-ciently block peptide transport resembles the reciprocity ofmK3 with the PLC. Both proteins are likely to interact withboth subunits, but heterodimerization is needed for optimalbinding and interference with MHC class I loading.

TAP1 and TAP2 carry 10 and 9 TM helices, respectively. The6�6 TM core complex containing TM helices 5–10 of TAP1and TM helices 4 –9 of TAP2 appeared to be sufficient fordimerization of the complex and efficient binding and translo-cation of peptides (7). Using T2 cells expressing �N forms ofrat TAP1 and TAP2, UL49.5 was found to interact with the 6�6TM core complex of TAP. Accordingly, TAP function was in-hibited by UL49.5 in Sf9 cells expressing the minimal TAPheterodimer. The inhibition was not as efficient as in Sf9 cellsexpressing full-length TAP1 and TAP2. This finding is in agree-ment with the results obtained in T2 cells expressing core TAP2(TAP2�N) next to full-length TAP1. UL49.5-mediated TAP in-hibition was found to be less pronounced in these cells com-pared with cells expressing full-length TAP2 and TAP1. Theseresults indicate that the N terminus of TAP2 contributes to theefficiency of UL49.5 binding and function. Probably, UL49.5primarily targets the 6�6 TM core of TAP, but optimal inhi-bition of TAP also requires the N-terminal region of TAP2,presumably because it stabilizes the TAP complex in a confor-mation that favors UL49.5 interaction.

The results discussed raise the question as to how a type Imembrane protein of only 9 kDa can block TAP function andultimately induce the degradation of this large heterodimerictransporter complex. Based on the results obtained, the follow-ing model can be proposed. The interaction of the ER-luminaland TM domains of UL49.5 with the 6�6 TM core complexmay cause a conformational arrest of the transporter that resultsin the inhibition of peptide transport (38). Both the TM helixand the ER-luminal domain of UL49.5 are required for thiseffect (38, 61). Insertion of the TM domain of UL49.5 withinthe core complex of TAP might prevent essential rearrange-ments of the TAP TM helices and may thus freeze TAP in atranslocation-incompetent state. At the same time, the ER-lu-minal part of UL49.5 might bend over the ER-luminal regionsof the transporter. The amino acid proline is known to allow astable flexure in a protein structure. The N-terminal part ofUL49.5 contains three prolines at positions close to the mem-brane that might facilitate the positioning of the ER-luminaldomain of UL49.5 over the luminally exposed regions of TAP.The interaction between the ER-exposed domains of UL49.5and TAP may further obstruct TAP function. Data from ourlaboratories indicate that UL49.5 occurs in the TAP complex asa disulfide-linked homodimer (61). Therefore, the interferencewith TAP function described above might be conducted by twoUL49.5 molecules simultaneously, with one UL49.5 moleculeinteracting with TAP1 and one with TAP2. This might explainthe observation that UL49.5 is able to interact with both TAP1and TAP2. Interestingly, a UL49.5 recombinant lacking thisER-luminal cysteine appeared to be equally active (61).


In addition to the conformational arrest of TAP induced by theER-luminal and TM parts of UL49.5, the cytoplasmic C-terminaldomain of the viral protein mediates proteasomal degradation ofUL49.5 and TAP (38). A recombinant form of UL49.5 lacking itscytoplasmic domain is incapable of inducing degradation of TAP(38). The cytoplasmic domain of UL49.5 contains two lysine res-idues representing potential targets for ubiquitination (72). How-ever, substitution of these lysines for alanines did not prevent deg-radation of TAP (M. Verweij, D. Koppers-Lalic, and E. Wiertz,unpublished observation). In addition to lysine residues, cysteine,threonine, and serine residues may serve as acceptors for ubiquitinmoieties (73, 74). Thus, the two serine residues within the cyto-plasmic domain of UL49.5 represent alternative ubiquitinationsites and may as well be involved in UL49.5-induced degradationof TAP.

Remarkably, degradation of rat TAP1 and TAP2 was not ob-served in T2 cells expressing UL49.5. Rat TAP can be targeted fordegradation by UL49.5, as seen in the rat embryo-derived fibro-blast cell line Rat2 (M. Verweij, D. Koppers-Lalic, and E. Wiertz,unpublished observation). Degradation of huTAP by UL49.5 wasclearly observed in T2 cells. The lack of degradation of rat TAP inT2 cells expressing UL49.5 might result from an incompatibilitybetween human components of the ubiquitin-proteasome pathwayand the rat TAP proteins. Because of the absence of UL49.5-in-duced degradation of rat TAP in T2 cells, we cannot draw anyconclusions on the importance of the N-terminal parts of TAP1and TAP2 for this mode of action.

Tapasin is an essential component of the PLC, facilitating theloading of MHC class I molecules with high affinity peptides. Inaddition, tapasin is required for stable TAP expression. Spleno-cytes derived from mice deficient for tapasin showed a 100-foldup-regulation of steady-state levels of TAP upon tapasin transfec-tion (15, 17). In addition, in .220 cells transfected to express full-length tapasin, TAP levels were enhanced 3- to 10-fold (16, 18).Therefore, UL49.5-induced degradation of TAP might involve in-terference with the stabilizing function of tapasin. Although thepresence of UL49.5 did affect the levels of tapasin within the PLCto some extent (Fig. 1B), this effect seems too modest to explainthe complete degradation of the TAP complex in UL49.5-express-ing cells. Moreover, if UL49.5 would expel tapasin moleculesfrom one of the N-terminal parts of the TAP complex, a clearreduction in tapasin levels should have been observed in eitherTAP1 TAP2�N or TAP1�N TAP2-containing PLCs (Fig. 5D).This, however, has not been found. Surprisingly, UL49.5-induceddestabilization of TAP was not observed in .220 cells deficient oftapasin. This could indicate that tapasin is required for the degra-dation of TAP by UL49.5. Alternatively, this finding may be re-lated to the fact that TAP is already very unstable in the .220 cells.Possibly, degradation cannot be increased further by UL49.5.UL49.5 did destabilize TAP1 and TAP2 in Sf9 insect cells ex-pressing TAP1, TAP2, and UL49.5 in the absence of tapasin (Fig.7B). The combined results favor the conclusion that tapasin is dis-pensable for UL49.5-mediated TAP inhibition and destabilization,and identify the TAP core complex as the functional entity targetedby UL49.5.

In summary, this study has identified the TAP1-TAP2 het-erodimer as the prime target of UL49.5. The 6�6 TM core com-plex of TAP1 and TAP2 is sufficient for UL49.5 to mediate itsinhibitory activity. These findings are particularly interesting inview of the fact that TAP forms part of a large family of trans-porters to which also the MDR proteins belong. Some members ofthis MDR transporter family confer drug resistance to malignantcells and microbial organisms (75, 76). Inhibitory molecules tar-

geting the core domain of such transporters might help to over-come MDR of these tumor cells and microorganisms.

In addition, UL49.5 can be exploited as an immunosuppressiveagent, for example, in HLA-mismatched transplantations and au-toimmune diseases. The expression of UL49.5 by transplantedcells will inhibit loading of MHC class I molecules with self Ags,resulting in reduced priming of autoreactive CTLs. The feasibilityof this approach has been shown in vitro for minor histocompat-ibility Ags (77). Finally, the expression of UL49.5 has been shownto elicit an unusual T cell repertoire, directed against T cellepitopes independent of peptide processing, demonstrating cyto-lytic activity against tumor cells (60, 78). A profound understand-ing of the mechanism of TAP inhibition by UL49.5 will be instru-mental in the development of novel therapies exploiting this viralimmune evasion protein.

AcknowledgmentsWe thank H. L. Ploegh, H. Hengel, and F. Momburg for generously sharingreagents; A. D. Lipinska for the production of the UL49.5-specific Ab;A. Williams for providing the .220 and .220 tapasin cells; W. E. Benckhuij-sen and J. W. Drijfhout for peptide synthesis and purification; andG. M. de Roo and M. A. W. G. van der Hoorn at the FlowcytometryResearch Unit (Leiden University Medical Center) for technical support.

DisclosuresThe authors have no financial conflict of interest.

References1. Lehner, P. J., and J. Trowsdale. 1998. Antigen presentation: coming out grace-

fully. Curr. Biol. 8: R605–R608.2. Wright, C. A., P. Kozik, M. Zacharias, and S. Springer. 2004. Tapasin and other

chaperones: models of the MHC class I loading complex. Biol. Chem. 385:763–778.

3. Cresswell, P., A. L. Ackerman, A. Giodini, D. R. Peaper, and P. A. Wearsch.2005. Mechanisms of MHC class I-restricted antigen processing and cross-pre-sentation. Immunol. Rev. 207: 145–157.

4. Knittler, M. R., P. Alberts, E. V. Deverson, and J. C. Howard. 1999. Nucleotidebinding by TAP mediates association with peptide and release of assembledMHC class I molecules. Curr. Biol. 9: 999–1008.

5. Neefjes, J. J., F. Momburg, and G. J. Hammerling. 1993. Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter. Science261: 769–771.

6. Van Endert, P. M., L. Saveanu, E. W. Hewitt, and P. Lehner. 2002. Powering thepeptide pump: TAP crosstalk with energetic nucleotides. Trends Biochem. Sci.27: 454–461.

7. Koch, J., R. Guntrum, S. Heintke, C. Kyritsis, and R. Tampe. 2004. Functionaldissection of the transmembrane domains of the transporter associated with an-tigen processing (TAP). J. Biol. Chem. 279: 10142–10147.

8. Wearsch, P. A., and P. Cresswell. 2007. Selective loading of high-affinity pep-tides onto major histocompatibility complex class I molecules by the tapasin-ERp57 heterodimer. Nat. Immunol. 8: 873–881.

9. Oliver, J. D., H. L. Roderick, D. H. Llewellyn, and S. High. 1999. ERp57 func-tions as a subunit of specific complexes formed with the ER lectins calreticulinand calnexin. Mol. Biol. Cell 10: 2573–2582.

10. Peh, C. A., N. Laham, S. R. Burrows, Y. Zhu, and J. McCluskey. 2000. Distinctfunctions of tapasin revealed by polymorphism in MHC class I peptide loading.J. Immunol. 164: 292–299.

11. Grandea, A. G., III, and L. Van Kaer. 2001. Tapasin: an ER chaperone thatcontrols MHC class I assembly with peptide. Trends Immunol. 22: 194–199.

12. Peaper, D. R., P. A. Wearsch, and P. Cresswell. 2005. Tapasin and ERp57 forma stable disulfide-linked dimer within the MHC class I peptide-loading complex.EMBO J. 24: 3613–3623.

13. Grandea, A. G., III, M. J. Androlewicz, R. S. Athwal, D. E. Geraghty, andT. Spies. 1995. Dependence of peptide binding by MHC class I molecules ontheir interaction with TAP. Science 270: 105–108.

14. Bangia, N., P. J. Lehner, E. A. Hughes, M. Surman, and P. Cresswell. 1999. TheN-terminal region of tapasin is required to stabilize the MHC class I loadingcomplex. Eur. J. Immunol. 29: 1858–1870.

15. Garbi, N., N. Tiwari, F. Momburg, and G. J. Hammerling. 2003. A major role fortapasin as a stabilizer of the TAP peptide transporter and consequences for MHCclass I expression. Eur. J. Immunol. 33: 264–273.

16. Lehner, P. J., M. J. Surman, and P. Cresswell. 1998. Soluble tapasin restoresMHC class I expression and function in the tapasin-negative cell line. 220. Im-munity 8: 221–231.

17. Papadopoulos, M., and F. Momburg. 2007. Multiple residues in the transmem-brane helix and connecting peptide of mouse tapasin stabilize the transporterassociated with the antigen-processing TAP2 subunit. J. Biol. Chem. 282:9401–9410.


18. Tan, P., H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hammerling, andF. Momburg. 2002. Recruitment of MHC class I molecules by tapasin into thetransporter associated with antigen processing-associated complex is essential foroptimal peptide loading. J. Immunol. 168: 1950–1960.

19. Koch, J., R. Guntrum, and R. Tampe. 2006. The first N-terminal transmembranehelix of each subunit of the antigenic peptide transporter TAP is essential forindependent tapasin binding. FEBS Lett. 580: 4091–4096.

20. Leonhardt, R. M., K. Keusekotten, C. Bekpen, and M. R. Knittler. 2005. Criticalrole for the tapasin-docking site of TAP2 in the functional integrity of the MHCclass I-peptide-loading complex. J. Immunol. 175: 5104–5114.

21. Denis, M., E. Hanon, F. A. Rijsewijk, M. J. Kaashoek, J. T. van Oirschot,E. Thiry, and P. P. Pastoret. 1996. The role of glycoproteins gC, gE, gI, and gGin the induction of cell-mediated immune responses to bovine herpesvirus 1. Vet.Microbiol. 53: 121–132.

22. Abendroth, A., and A. Arvin. 1999. Varicella-zoster virus immune evasion. Im-munol. Rev. 168: 143–156.

23. Arvin, A. M., M. Sharp, M. Moir, P. R. Kinchington, M. Sadeghi-Zadeh,W. T. Ruyechan, and J. Hay. 2002. Memory cytotoxic T cell responses to viraltegument and regulatory proteins encoded by open reading frames 4, 10, 29, and62 of varicella-zoster virus. Viral Immunol. 15: 507–516.

24. Vescovini, R., C. Biasini, F. F. Fagnoni, A. R. Telera, L. Zanlari, M. Pedrazzoni,L. Bucci, D. Monti, M. C. Medici, C. Chezzi, et al. 2007. Massive load offunctional effector CD4� and CD8� T cells against cytomegalovirus in very oldsubjects. J. Immunol. 179: 4283–4291.

25. Slezak, S. L., M. Bettinotti, S. Selleri, S. Adams, F. M. Marincola, andD. F. Stroncek. 2007. CMV pp65 and IE-1 T cell epitopes recognized by healthysubjects. J. Transl. Med. 5: 17.

26. Pudney, V. A., A. M. Leese, A. B. Rickinson, and A. D. Hislop. 2005. CD8�

immunodominance among Epstein-Barr virus lytic cycle antigens directly reflectsthe efficiency of antigen presentation in lytically infected cells. J. Exp. Med. 201:349–360.

27. Fruh, K., K. Ahn, H. Djaballah, P. Sempe, P. M. van Endert, R. Tampe,P. A. Peterson, and Y. Yang. 1995. A viral inhibitor of peptide transporters forantigen presentation. Nature 375: 415–418.

28. Hill, A., P. Jugovic, I. York, G. Russ, J. Bennink, J. Yewdell, H. Ploegh, andD. Johnson. 1995. Herpes simplex virus turns off the TAP to evade host immu-nity. Nature 375: 411–415.

29. Ahn, K., T. H. Meyer, S. Uebel, P. Sempe, H. Djaballah, Y. Yang, P. A. Peterson,K. Fruh, and R. Tampe. 1996. Molecular mechanism and species specificity ofTAP inhibition by herpes simplex virus ICP47. EMBO J. 15: 3247–3255.

30. Tomazin, R., A. B. Hill, P. Jugovic, I. York, P. van Endert, H. L. Ploegh,D. W. Andrews, and D. C. Johnson. 1996. Stable binding of the herpes simplexvirus ICP47 protein to the peptide binding site of TAP. EMBO J. 15: 3256–3266.

31. Aisenbrey, C., C. Sizun, J. Koch, M. Herget, R. Abele, B. Bechinger, andR. Tampe. 2006. Structure and dynamics of membrane-associated ICP47, a viralinhibitor of the MHC I antigen-processing machinery. J. Biol. Chem. 281:30365–30372.

32. Ahn, K., A. Gruhler, B. Galocha, T. R. Jones, E. J. Wiertz, H. L. Ploegh,P. A. Peterson, Y. Yang, and K. Fruh. 1997. The ER-luminal domain of theHCMV glycoprotein US6 inhibits peptide translocation by TAP. Immunity 6:613–621.

33. Hewitt, E. W., S. S. Gupta, and P. J. Lehner. 2001. The human cytomegalovirusgene product US6 inhibits ATP binding by TAP. EMBO J. 20: 387–396.

34. Lehner, P. J., J. T. Karttunen, G. W. Wilkinson, and P. Cresswell. 1997. Thehuman cytomegalovirus US6 glycoprotein inhibits transporter associated withantigen processing-dependent peptide translocation. Proc. Natl. Acad. Sci. USA94: 6904–6909.

35. Hengel, H., J. O. Koopmann, T. Flohr, W. Muranyi, E. Goulmy,G. J. Hammerling, U. H. Koszinowski, and F. Momburg. 1997. A viral ER-resident glycoprotein inactivates the MHC-encoded peptide transporter. Immunity6: 623–632.

36. Kyritsis, C., S. Gorbulev, S. Hutschenreiter, K. Pawlitschko, R. Abele, andR. Tampe. 2001. Molecular mechanism and structural aspects of transporter as-sociated with antigen processing inhibition by the cytomegalovirus protein US6.J. Biol. Chem. 276: 48031–48039.

37. Hislop, A. D., M. E. Ressing, D. van Leeuwen, V. A. Pudney, D. Horst,D. Koppers-Lalic, N. P. Croft, J. J. Neefjes, A. B. Rickinson, and E. J. Wiertz.2007. A CD8� T cell immune evasion protein specific to Epstein-Barr virus andits close relatives in Old World primates. J. Exp. Med. 204: 1863–1873.

38. Koppers-Lalic, D., E. A. Reits, M. E. Ressing, A. D. Lipinska, R. Abele, J. Koch,R. M. Marcondes, P. Admiraal, D. van Leeuwen, K. Bienkowska-Szewczyk,et al. 2005. Varicelloviruses avoid T cell recognition by UL49.5-mediated inac-tivation of the transporter associated with antigen processing. Proc. Natl. Acad.Sci. USA 102: 5144–5149.

39. Koppers-Lalic, D., M. C. Verweij, A. D. Lipinska, Y. Wang, E. Quinten,E. A. Reits, J. Koch, S. Loch, M. M. Rezende, F. Daus, et al. 2008. VaricellovirusUL 49.5 proteins differentially affect the function of the transporter associatedwith antigen processing, TAP. PLoS Pathog. 4: e1000080.

40. Reits, E. A., J. C. Vos, M. Gromme, and J. Neefjes. 2000. The major substratesfor TAP in vivo are derived from newly synthesized proteins. Nature 404:774–778.

41. Copeman, J., N. Bangia, J. C. Cross, and P. Cresswell. 1998. Elucidation of thegenetic basis of the antigen presentation defects in the mutant cell line: 220reveals polymorphism and alternative splicing of the tapasin gene. Eur. J. Im-munol. 28: 3783–3791.

42. Salter, R. D., D. N. Howell, and P. Cresswell. 1985. Genes regulating HLA classI antigen expression in T-B lymphoblast hybrids. Immunogenetics 21: 235–246.

43. Momburg, F., V. Ortiz-Navarrete, J. Neefjes, E. Goulmy, Y. van de Wal,H. Spits, S. J. Powis, G. W. Butcher, J. C. Howard, and P. Walden. 1992. Pro-teasome subunits encoded by the major histocompatibility complex are not es-sential for antigen presentation. Nature 360: 174–177.

44. De la Salle, H., D. Hanau, D. Fricker, A. Urlacher, A. Kelly, J. Salamero,S. H. Powis, L. Donato, H. Bausinger, and M. Laforet. 1994. Homozygous humanTAP peptide transporter mutation in HLA class I deficiency. Science 265:237–241.

45. Meyer, T. H., P. M. van Endert, S. Uebel, B. Ehring, and R. Tampe. 1994.Functional expression and purification of the ABC transporter complex associ-ated with antigen processing (TAP) in insect cells. FEBS Lett. 351: 443–447.

46. Barnstable, C. J., W. F. Bodmer, G. Brown, G. Galfre, C. Milstein,A. F. Williams, and A. Ziegler. 1978. Production of monoclonal antibodies togroup A erythrocytes, HLA and other human cell surface antigens: new tools forgenetic analysis. Cell 14: 9–20.

47. Parham, P., and F. M. Brodsky. 1981. Partial purification and some properties ofBB7.2: a cytotoxic monoclonal antibody with specificity for HLA-A2 and a vari-ant of HLA-A28. Hum. Immunol. 3: 277–299.

48. Mulder, A., M. J. Kardol, C. M. Uit het Broek, J. Tanke-Visser, N. T. Young, andF. H. Claas. 1998. A human monoclonal antibody against HLA-Cw1 and a humanmonoclonal antibody against an HLA-A locus determinant derived from a singleuniparous female. Tissue Antigens 52: 393–396.

49. Van den Born, E., C. C. Posthuma, K. Knoops, and E. J. Snijder. 2007. Aninfectious recombinant equine arteritis virus expressing green fluorescent proteinfrom its replicase gene. J. Gen. Virol. 88: 1196–1205.

50. Meyer, T. H., P. M. van Endert, S. Uebel, B. Ehring, and R. Tampe. 1994.Functional expression and purification of the ABC transporter complex associ-ated with antigen processing (TAP) in insect cells. FEBS Lett. 351: 443–447.

51. Plewnia, G., K. Schulze, C. Hunte, R. Tampe, and J. Koch. 2007. Modulation ofthe antigenic peptide transporter TAP by recombinant antibodies binding to thelast five residues of TAP1. J. Mol. Biol. 369: 95–107.

52. Van Endert, P. M., R. Tampe, T. H. Meyer, R. Tisch, J. F. Bach, andH. O. McDevitt. 1994. A sequential model for peptide binding and transport bythe transporters associated with antigen processing. Immunity 1: 491–500.

53. Powis, S. J., A. R. Townsend, E. V. Deverson, J. Bastin, G. W. Butcher, andJ. C. Howard. 1991. Restoration of antigen presentation to the mutant cell lineRMA-S by an MHC-linked transporter. Nature 354: 528–531.

54. Knittler, M. R., K. Gulow, A. Seelig, and J. C. Howard. 1998. MHC class Imolecules compete in the endoplasmic reticulum for access to transporter asso-ciated with antigen processing. J. Immunol. 161: 5967–5977.

55. Brodsky, F. M., P. Parham, C. J. Barnstable, M. J. Crumpton, and W. F. Bodmer.1979. Monoclonal antibodies for analysis of the HLA system. Immunol. Rev. 47:3–61.

56. Lipinska, A. D., D. Koppers-Lalic, M. Rychlowski, P. Admiraal, F. A. Rijsewijk,K. Bienkowska-Szewczyk, and E. J. Wiertz. 2006. Bovine herpesvirus 1 UL49.5protein inhibits the transporter associated with antigen processing despite com-plex formation with glycoprotein M. J. Virol. 80: 5822–5832.

57. Seliger, B., U. Ritz, R. Abele, M. Bock, R. Tampe, G. Sutter, I. Drexler,C. Huber, and S. Ferrone. 2001. Immune escape of melanoma: first evidence ofstructural alterations in two distinct components of the MHC class I antigenprocessing pathway. Cancer Res. 61: 8647–8650.

58. Williams, A. P., C. A. Peh, A. W. Purcell, J. McCluskey, and T. Elliott. 2002.Optimization of the MHC class I peptide cargo is dependent on tapasin. Immunity16: 509–520.

59. Bouabe, H., and M. R. Knittler. 2003. The distinct nucleotide binding states of thetransporter associated with antigen processing (TAP) are regulated by the non-homologous C-terminal tails of TAP1 and TAP2. Eur. J. Biochem. 270:4531–4546.

60. Van Hall, T., S. Laban, D. Koppers-Lalic, J. Koch, C. Precup, P. Asmawidjaja,R. Offringa, and E. J. H. J. Wiertz. 2007. The varicellovirus-encoded TAP in-hibitor UL49.5 regulates the presentation of CTL epitopes by Qa-1. J. Immunol.178: 657–662.

61. Loch, S., F. Klauschies, C. Scholz, M. C. Verweij, E. J. Wiertz, J. Koch, andR. Tampe. 2008. Signaling of a varicelloviral factor across the endoplasmic re-ticulum membrane induces destruction of the peptide-loading complex and im-mune evasion. J. Biol. Chem. 283: 13428–13436.

62. Antoniou, A. N., S. Ford, E. S. Pilley, N. Blake, and S. J. Powis. 2002. Interac-tions formed by individually expressed TAP1 and TAP2 polypeptide subunits.Immunology 106: 182–189.

63. Lapinski, P. E., G. G. Miller, R. Tampe, and M. Raghavan. 2000. Pairing of thenucleotide binding domains of the transporter associated with antigen processing.J. Biol. Chem. 275: 6831–6840.

64. Boname, J. M., J. S. May, and P. G. Stevenson. 2005. The murine �-herpesvi-rus-68 MK3 protein causes TAP degradation independent of MHC class I heavychain degradation. Eur. J. Immunol. 35: 171–179.

65. Boname, J. M., B. D. de Lima, P. J. Lehner, and P. G. Stevenson. 2004. Viraldegradation of the MHC class I peptide loading complex. Immunity 20: 305–317.

66. Boname, J. M., and P. G. Stevenson. 2001. MHC class I ubiquitination by a viralPHD/LAP finger protein. Immunity 15: 627–636.

67. Stevenson, P. G., S. Efstathiou, P. C. Doherty, and P. J. Lehner. 2000. Inhibitionof MHC class I-restricted antigen presentation by �2-herpesviruses. Proc. Natl.Acad. Sci. USA 97: 8455–8460.

68. Wang, X., R. Connors, M. R. Harris, T. H. Hansen, and L. Lybarger. 2005.Requirements for the selective degradation of endoplasmic reticulum-residentmajor histocompatibility complex class I proteins by the viral immune evasionmolecule mK3. J. Virol. 79: 4099–4108.


69. Wang, X., L. Lybarger, R. Connors, M. R. Harris, and T. H. Hansen. 2004. Modelfor the interaction of �herpesvirus 68 RING-CH finger protein mK3 with majorhistocompatibility complex class I and the peptide-loading complex. J. Virol. 78:8673–8686.

70. Lybarger, L., X. Wang, M. R. Harris, H. W. Virgin, and T. H. Hansen. 2003.Virus subversion of the MHC class I peptide-loading complex. Immunity 18:121–130.

71. Stevenson, P. G., J. S. May, X. G. Smith, S. Marques, H. Adler,U. H. Koszinowski, J. P. Simas, and S. Efstathiou. 2002. K3-mediated evasion ofCD8� T cells aids amplification of a latent �-herpesvirus. Nat. Immunol. 3:733–740.

72. Pickart, C. M. 2001. Mechanisms underlying ubiquitination. Annu. Rev. Biochem.70: 503–533.

73. Cadwell, K., and L. Coscoy. 2005. Ubiquitination on nonlysine residues by a viralE3 ubiquitin ligase. Science 309: 127–130.

74. Wang, X., R. A. Herr, W. J. Chua, L. Lybarger, E. J. Wiertz, and T. H. Hansen.2007. Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic

tail can induce ERAD of MHC-I by viral E3 ligase mK3. J. Cell Biol. 177:613–624.

75. Glavinas, H., P. Krajcsi, J. Cserepes, and B. Sarkadi. 2004. The role of ABCtransporters in drug resistance, metabolism and toxicity. Curr. Drug Deliv. 1:27–42.

76. Gillet, J. P., T. Efferth, and J. Remacle. 2007. Chemotherapy-induced resistanceby ATP-binding cassette transporter genes. Biochim. Biophys. Acta 1775:237–262.

77. Oosten, L. E., D. Koppers-Lalic, E. Blokland, A. Mulder, M. E. Ressing,T. Mutis, A. G. van Halteren, E. J. Wiertz, and E. Goulmy. 2007. TAP-inhibitingproteins US6, ICP47 and UL49.5 differentially affect minor and major histocom-patibility antigen-specific recognition by cytotoxic T lymphocytes. Int. Immunol.19: 1115–1122.

78. Chambers, B., P. Grufman, V. Fredriksson, K. Andersson, M. Roseboom,S. Laban, M. Camps, E. Z. Wolpert, E. J. Wiertz, R. Offringa, et al. 2007. In-duction of protective CTL immunity against peptide transporter TAP-deficienttumors through dendritic cell vaccination. Cancer Res. 67: 8450–8455.


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