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International Immunology Advance Access originally published online on December 16, 2005
International Immunology 2006 18(1):173-182; doi:10.1093/intimm/dxh362
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© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Subtle sequence variation among MHC class I locus products greatly influences sensitivity to HCMV US2- and US11-mediated degradation

Martine T. Barel1, Nathalie Pizzato2, Philippe Le Bouteiller2, Emmanuel J. H. J. Wiertz1 and Francoise Lenfant2,3

1 Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
2 INSERM U563, CPTP, Bat A, Hôpital Purpan, BP 3028, 31024 Toulouse cedex 3, France
3 Present address: INSERM U 589, Institut L. Bugnard, Hôpital Rangueil, BP 84225, 31 432 Toulouse cedex 4, France

Correspondence to: F. Lenfant; E-mail: lenfant{at}toulouse.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Human cytomegalovirus (HCMV) interferes with cellular immune responses by modulating surface expression of MHC class I molecules. Here, we focused on HCMV-encoded unique short (US) 2 and US11, which bind newly synthesized MHC class I heavy chains (HCs) and support their dislocation into the cytosol for subsequent degradation by proteasomes. Not all MHC class I locus products are equally sensitive to this down-modulation. The aim of this study was to identify which domains, and ultimately which residues, are responsible for the resistance or sensitivity of MHC class I molecules to US2- and US11-mediated down-regulation. We show that, besides endoplasmic reticulum–lumenal regions, the C-terminus of class I molecules represents an important determinant for allele specificity in US11-mediated degradation. HLA-E becomes sensitive to US11-mediated down-regulation when its cytoplasmic tail is extended. Interestingly, this only requires two additional residues, lysine and valine, at its C-terminus. For US2, the MHC class I allele specificity is largely determined by a small region at the junction of the {alpha}2/{alpha}3 domain of the HC. It is quite remarkable that minor changes, in only four residues, can completely revert the sensitivity of naturally US2-resistant HLA-E molecules. With this study we provide better insights into the features underlying the selectivity in MHC class I down-regulation by US2 and US11.

Keywords: cytomegalovirus, HLA-E, immune evasion, US


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Human cytomegalovirus (HCMV) establishes persistent infections in human populations worldwide and can give rise to serious disease in immunocompromised individuals. HCMV uses various defense mechanisms to elude the host immune system. Elimination of infected cells by CD8+ T cells can be prevented by down-regulation of viral antigen-presenting MHC class I molecules. In the course of HCMV infection, MHC class I surface expression is affected by several HCMV unique short (US) region-encoded proteins, which act at different levels of the antigen-processing and presentation pathway (1–5). Antigen presentation is prevented by blocking the supply of peptides through TAP inhibition (US6), by retaining newly synthesized MHC class I molecules in the endoplasmic reticulum (ER) compartment (US3) or by dislocating class I heavy chains (HCs) back into the cytosol for subsequent degradation by proteasomes (US2 and US11). Efficient down-regulation may very well require the concerted action of several of these US proteins.

A complete reduction of MHC class I expression could have serious consequences for the survival of the virus, as cells lacking MHC class I surface molecules are more susceptible to an NK-cell attack (6). Several proteins encoded within the unique long (UL) region of the HCMV genome (UL16, UL18, UL40, UL141) appear to protect infected cells against NK-cell lysis. They either block expression of ligands that activate NK cells (UL16, UL141) or allow expression of ligands that can inhibit NK-cell triggering (UL18, UL40) (7, 8).

In general, host cells express HLA-A, HLA-B, HLA-C and HLA-E alleles. The (surface) expression levels of the various locus products are differentially regulated (9, 10). HLA-A and HLA-B are generally most abundant at the cell surface. Between the various MHC class I locus products, most sequence variation is found in the region encompassing the antigen-binding groove (11). This generates different restrictions for peptide binding as well as for the variety of peptide display for each allele. MHC class I molecules can serve a dual role, as a ligand for both TCRs and NK-cell receptors, but their key task may be biased. HLA-A and HLA-B alleles are probably most powerful in the presentation of foreign peptides. This is supported by a high degree of polymorphism in the world population for these alleles, with 325 different HLA-A and 592 different HLA-B alleles reported to the IMGT/HLA database so far. Next comes HLA-C (175 alleles) and the lowest polymorphic is HLA-E (5 alleles) (11). Although there are reports showing that HLA-C and -E can present foreign antigens to TCRs, they may primarily serve as interaction partners for NK-cell receptors (7, 12–17).

Taking into account the functions of the different MHC class I locus products, it is important to know which alleles are affected in HCMV-infected cells. Data from several laboratories have shown that there are allelic differences between MHC class I molecules with respect to sensitivity to US2, US3, US6 and US11, but the picture is still far from complete (18–23). In this study we aimed to characterize the precise regions in MHC class I alleles that determine sensitivity or resistance to these US proteins. This knowledge will also help to predict the down-modulatory effect of US2 and US11 for a broader range of MHC class I alleles.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell lines
J26 cells [H-2k murine Ltk– cells expressing human ß2-microglobulin (ß2m)] (24) and the Phoenix amphotropic retroviral producer cell line [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in DMEM (Invitrogen, Breda, The Netherlands), supplemented with 10% FCS (Greiner bv, Alphen aan den Rijn, The Netherlands), 100 U ml–1 penicillin and 100 µg ml–1 streptomycin and G418 (Invitrogen, Cergy-Pontoise, France). J26 cells expressing HLA-A2 (A*0201), HLA-B7 (B*070201), HLA-B27 (B*270502), HLA-Cw3 (Cw*0304, gift from B. van den Eynde, Brussels, Belgium), HLA-G (G*01011) and HLA-E (E*01033, gift from E. Weiss, Munich, Germany) were all described previously (19, 25).

Antibodies
The following anti-MHC class I mAbs were used for flow cytometry: W6/32 (anti-human MHC I complex) (26), BB7.2, MA2.1 (both anti-HLA-A2) (27), MEM-E/06 (anti-HLA-E; EXBIO Praha, Czech Republic), B1.23.2 (anti-HLA-B and -C) (28), BB7.1 (anti-HLA-B, ATCC) and Y-3 (murine MHC class I; ATCC). In most cases, primary mAbs were used in combination with PE-conjugated goat anti-mouse (g{alpha}m), IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). In some experiments biotinylated-g{alpha}m Ig was used as second antibody, in combination with streptavidin-conjugated PE as third antibody (PharMingen, Europe). The mAbs MEM-E/02 (denatured HLA-E; EXBIO Praha), H68.4 (transferrin receptor; Zymed Laboratories, San Francisco, CA, USA) and polyclonal antisera US2-N2 (US2) (19) and US11-N2 (US11) (29) were used for immunoprecipitations. A control experiment was performed with MEM-E/02 to exclude possible cross-reactivity with murine MHC class I. MEM-E/02 only precipitated MHC class I HCs in J26 cells transfected with HLA-E and no MHC class I in wild-type (wt) J26 cells (data not shown).

Construction of plasmids
Plasmid pLUMC9901 (encoding HLA-A*0201 cDNA) (29) was used as template for the construction of mutants HLA-A2LHLE (HLA-A2 with residues 180–183, QRTD, replaced by LHLE) and HLA-A2delCKV (HLA-A2 with a deletion of residues 340–342). pLUMC9901 and pcDNA-E (sigA2) (encoding cDNA of HLA-E*01033 with signal sequence of HLA-A2; kind gift of E. Weiss) (30) were used as templates to construct HLA-A2/E chimeras HLA-A21–184/E (residues 1–184 of HLA-A2 and rest of HLA-E) and its reverse HLA-E1–184/A2, HLA-E({alpha}3 + c A2) (HLA-E with {alpha}3 domain and connecting peptide region of HLA-A2), HLA-E(TM A2) (HLA-E with transmembrane domain of HLA-A2), HLA-E(tail A2) (HLA-E with cytoplasmic tail of HLA-A2) and HLA-A2(tail E) (HLA-A2 with cytoplasmic tail of HLA-E). pcDNA-E (sigA2) was also used to construct the mutants HLA-EQRTD (HLA-E with residues 180–183, LHLE, replaced by QRTD), HLA-E + ACKV, HLA-E + KV, HLA-E + K and HLA-E + V (HLA-E with tail extended with ACKV, KV, K or V residues, respectively). pCR-B7 [HLA-B*070201 in vector pCR3.1 (Invitrogen); kind gift of M. Heemskerk, Leiden, The Netherlands] was used as template to construct HLA-B7ETLQ (HLA-B7 with residues 177, 178, 180, DK-E, replaced by ET-Q, resulting in the sequence ETLQ at position 177–180). Amplifications were performed using Isis polymerase (Q.BIOgene, Illkirch, France). Mutations were introduced using the QuickChange XL site-directed mutagenesis kit and/or protocol (Stratagene, La Jolla, CA, USA) and chimeric constructs were generated applying the megaprimer method (31). All constructs were fully sequenced to verify the absence of unwanted mutations.

Transfection
J26 cells were transfected with the different MHC class I constructs using EffecteneTM Transfection Reagent (Qiagen, Courtaboeuf, France). After 48 h, stable transfectants were selected by adding 0.4 mg ml–1 G418 (Invitrogen). Cells were sorted by flow cytometry for expression of the introduced cDNA using MEM-E/06, W6/32, B1.23.2 or BB7.1 mAbs.

Production of retrovirus and transduction
US2 and US11 cDNA fragments were subcloned into the pLZRS-IRES-EGFP vector (19, 32, 33) and used for transfection of amphotropic Phoenix packaging cells to produce retrovirus, as described (19). Cells were transduced with retrovirus using Retronectin-coated (Takara Shuzo, Otsu, Japan) dishes.

Flow cytometry
Cell-surface expression of MHC class I molecules as well as enhanced green fluorescent protein (EGFP) expression in cells transduced with retrovirus were analyzed using flow cytometry as described (19). In cells expressing low levels of human MHC class I (e.g. HLA-E) and/or when indicated, cells were stained in three steps to intensify the MHC class I (PE) staining (first with specific anti-MHC class I antibody, then with biotinylated-g{alpha}m and finally with streptavidin-conjugated PE). Data are collected from several experiments (generally three), of which one representative experiment is shown.

Metabolic labeling, immunoprecipitation and SDS-PAGE
Metabolic labeling, immunoprecipitations on denatured samples and SDS-PAGE were performed as described (29). In brief, cells were starved in medium without methionine (Met) or cysteine (Cys) for ~1 h, labeled with promix (35S Met and Cys) and chased in medium with excess amounts of cold Met and Cys. Where indicated, media were supplemented with proteasome inhibitor carboxybenzyl-leucyl-leucyl-leucinal (ZL3H). Cells were lysed in a small volume of Nonidet-P40 lysis buffer, containing protease inhibitors. Cell debris was removed by centrifugation and the supernatant was transferred to a new tube to which one-tenth volume of 10% SDS and one-tenth volume of 0.1 M dithiothreitol was added. Samples were boiled for 5 min to further denature the proteins. Next, the volume was increased 10 times with non-denaturing buffer, supplemented with protease inhibitors and with 10 mM iodoacetic acid. Immunoprecipitations were performed ≥2 h on pre-cleared samples, with antibodies pre-coupled to protein A Sepharose beads. Samples were separated by SDS-PAGE and displayed via phosphor imaging.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Selectivity of US2 and US11 for MHC class I locus products
US2 and US11 can target specific MHC class I locus products for degradation while preserving surface expression of others (19, 21, 23, 34). In this study we aim to find more information on MHC class I allele specificity of US2 and US11.

Previously, we have found that HLA-A2, HLA-B27 and HLA-G were down-modulated by US2, while surface expression of HLA-B7, HLA-Cw3 and HLA-E was unaffected (32). For US11 it has been shown that it can down-modulate HLA-A2 and HLA-C molecules, but not HLA-G or HLA-E (19, 21, 23). Although HLA-B locus products are generally believed to be down-regulated by US11, as suggested by a destabilizing effect of US11 on general pools of MHC class I, formal proof is still lacking. To evaluate the stability of individual HLA-B locus products, we used murine J26 cells expressing one particular human MHC class I HC construct and transduced these cells with a retroviral vector encoding both US11 and EGFP. The EGFP expression is used as a marker for transduction (i.e. US11-positive cells). These J26 cells co-express human ß2m to allow proper MHC class I complex formation. In previous studies, we have shown that US2/US11 can efficiently target human MHC class I molecules for degradation in cells of murine origin (19, 32).

Figure 1(A) shows the effect of US11 (EGFP+ cells) on surface expression of HLA-B7 and HLA-B27 compared with the non-transduced (EGFP–) cell population, as analyzed by flow cytometry. It also includes data on HLA-C, HLA-E and murine alleles to provide a more complete overview in this experimental system. In the US11-expressing cells, surface expression of HLA-B7, HLA-B27, HLA-Cw3 and endogenous murine MHC class I molecules was reduced compared with the non-transduced cells, while HLA-E expression remained unaffected. Control wt EGFP-expressing retrovirus had no effect on MHC class I cell-surface expression (data not shown). Figure 1(B) shows an overview of the sensitivity to US2 and US11 for all the different MHC class I alleles that have been tested in this experimental system. Representative dot-plot data not shown here have been presented in our previous studies (18, 31). This overview shows that there are clear specificity differences between US2 and US11. US2 affects HLA-A2, HLA-B27 and HLA-G and not HLA-B7, HLA-Cw3, HLA-E or endogenous H-2k, while US11 affects all these alleles except HLA-E and HLA-G. HLA-E is the only allele that is not affected by either one of these two US proteins.



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  		Fig. 1. Selective down-regulation of HLA class I molecules by US2 and US11. (A) Murine J26 cells, transfected with different plasmids encoding HLA class I and transduced with US11-IRES-EGFP-encoding retrovirus, were analyzed using flow cytometry. The following mAbs were used to stain the different human MHC class I molecules with PE in a two- or three-step staining protocol (y-axis): HLA-B7, HLA-B27 and HLA-Cw3 (W6/32), HLA-E (MEM-E/06, three steps) or endogenous H-2k (Y-3). US11-positive cells are marked by EGFP expression (x-axis). (B) Overview of sensitivity of different MHC class I locus products to US2- and US11-mediated down-regulation, as evaluated in the same experimental system in this and in our previous studies (19, 32). The effect of US11 on surface staining of MHC class I was calculated by comparing mean PE fluorescence of EGFP-negative (defined as 100%) and EGFP-positive cells. The averages of calculations of generally three independent experiments are shown, with error bars.

 
Residues around the junction of the {alpha}2/{alpha}3 domains of MHC class I alleles are critical for US2-mediated down-regulation
The summary in Fig. 1(B) clearly shows that individual human locus products differ in their sensitivity to US2-mediated down-regulation. Crystal structure data of a soluble HLA-A2–US2–ß2m complex have shown that at least a region in the {alpha}2/{alpha}3 domain of HLA-A2 is involved in the interaction with US2 (Fig. 2A) (35). These data cannot exclude the possibility that additional interactions with transmembrane or cytoplasmic tail regions contribute to sensitivity differences between alleles. So, before exploring the effect of sequence variation in this ER–lumenal region, we first addressed this question using chimeras consisting of US2-insensitive HLA-E and US2-sensitive HLA-A2 alleles. We tested the sensitivity of a chimeric molecule in which the ER–lumenal part comprising residues 1–184 is derived from HLA-A2, and the remainder from HLA-E (HLA-A21–184/E). This construct contains all the HLA-A2 residues that are implicated in US2 binding according to the crystal structure data. We also included a reciprocal version of this construct (HLA-E1–184/A2). Figure 2(B) shows that HLA-A21–184/E was almost equally sensitive to US2 as wt HLA-A2. Likewise, HLA-E1–184/A2 was as insensitive as wt HLA-E. This shows that the sensitivity differences of HLA-A2 and HLA-E are determined by something located between amino acids 1 and 184 of the ER–lumenal region.



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  		Fig. 2. Alterations in the {alpha}2/{alpha}3 domain of MHC class I alleles affect sensitivity to US2-mediated down-regulation. (A) Depicted is HLA-A2 showing ER–lumenal regions [{alpha}1–{alpha}3 and connecting peptide (c)], transmembrane domain (tm) and cytoplasmic tail, as well as an enlargment of the boxed regions (amino acids 101–110, 171–190) of HLA-A2 with residues directly involved in interaction with US2 marked in gray [according to crystal structure data from Gewurz et al. (35)]. The US2-binding site of HLA-A2 has been aligned with corresponding regions of other alleles, and their locus (sub)group consensus sequences obtained from the IMGT/HLA sequence database (11). (B) J26 cells were transfected with different HLA class I mutants, transduced with US2-IRES-EGFP-encoding retrovirus and analyzed using flow cytometry. The following mAbs were used to stain the different human MHC class I molecules with PE, in a three-step staining protocol (y-axis): HLA-A, HLA-A21–184/E (BB7.2), HLA-B, HLA-E1–184/A2 (W6/32), HLA-E (MEM-E/06). US2-positive cells are marked by EGFP expression (x-axis). (C) J26 cells expressing wt HLA-E or HLA-EQRTD or cells co-expressing US2 were metabolically labeled for 10 min and chased for 0 or 30 min in the absence or presence of proteasome inhibitor ZL3H. Immunoprecipitations were performed on denatured cell lysates using the following anti-sera: H68.4 [transferrin receptor (TfR)], MEM/02 (HLA-E) or US2-N2 (US2) antibodies. Deglycosylated degradation intermediates are marked by an asterisk.

 
A concordance can be found between US2-insensitive class I alleles (HLA-B7, HLA-E) and variation in the region that for HLA-A2 was shown to be involved in binding to US2 (E177, T178, Q180, R181, T182, D183). To test if sequence variation in this region can indeed account for the observed allelic sensitivity differences, several HLA class I mutants were evaluated for their sensitivity to US2. First, a mutant of HLA-A2 (HLA-A2LHLE) was tested that resembles US2-insensitive HLA-E for this region (see Fig. 2A). The alteration of only four amino acids rendered this mutant insensitive to US2-mediated down-regulation, in contrast to its wt equivalent (see Fig. 2B). Secondly, mutants of US2-insensitive HLA-B7 and HLA-E were constructed that resemble their US2-sensitive allelic counterparts (see Fig. 2A). Residues at positions 177, 178 and 180 of HLA-B7 were replaced with the corresponding residues E, T and Q of US2-sensitive HLA-B27. The flow cytometry data show that this HLA-B7ET(L)Q mutant had a reduced surface expression in the presence of US2 similar to that observed for HLA-B27, while wt HLA-B7 remained unaffected. Likewise, a clear sensitivity conversion was observed for an HLA-E mutant with residues 180–183 replaced by QRTD. Whereas wt HLA-E surface expression was unaffected in US2-expressing cells, surface expression of the mutant HLA-E was clearly reduced. The sensitivity shift was less dramatic, but still clearly visible, when only one mutation, H181R, was introduced into HLA-E.

These flow cytometry data mainly provide information on alterations in surface expression levels in the presence of US2. To exclude the possibility of retention, rather than degradation, being the underlying mechanism for a reduced surface expression, we also evaluated the effect of US2 on the stability of the HLA-EQRTD mutant by pulse chase analysis (Fig. 2C). Like US11, US2 can mediate the retrotranslocation of newly synthesized class I HCs to the cytosol where they are first deprived of their N-linked glycan through the action of an N-glycanase and subsequently degraded by the proteasome (4, 5). This is a very rapid process (1–2 min), taking place from the start of pulse labeling as can be seen by the decrease of the intensity of the bands at time point zero. Figure 2(C) (left panel) shows that, in the absence of proteasome inhibitors, US2 has a strong destabilizing effect on HLA-EQRTD; only a small amount of HLA-EQRTD HCs could be immunoprecipitated in US2+ cells at the beginning of the chase and no HCs can be recovered after a 30-min chase, while HLA-EQRTD remained stable in the absence of US2. Equal amounts of transferrin receptor could be recovered over the chase course in US2+/– samples. In the presence of proteasome inhibitor (+ZL3H, middle and right panels), deglycosylated degradation intermediates could be recovered for HLA-EQRTD, which are absent in US2-negative cells and in US2-positive cells expressing wt HLA-E. It has to be noted that, in the presence of proteasome inhibitor, dislocated HCs remain targets for other cytosolic proteases.

Altogether, these results show that subtle changes in a small region around the junction of the {alpha}2/{alpha}3 domains of MHC class I can greatly affect their sensitivity to US2-mediated degradation.

In contrast to US2, US11 can discriminate between MHC class I locus products on the basis of their cytoplasmic tail sequences
For US11 it is still rather unclear which regions of MHC class I molecules determine allelic sensitivity differences to US11-mediated down-regulation. Chimeric HLA-A2/G molecules, consisting of US11-sensitive HLA-A2 and US11-resistant HLA-G alleles, showed that the length of the cytoplasmic tail was the most important determinant for the insensitivity of HLA-G (19). An extension of the short (6 amino acids) HLA-G tail with residues matching the relatively long (33 amino acids) tail of HLA-A2 made it very sensitive to US11-mediated degradation. However, this information cannot directly explain the insensitivity to US11-mediated down-regulation for HLA-E, which has a relatively long cytoplasmic tail of 29 residues. Chimeras of HLA-E and HLA-A2 were constructed to test which regions determine resistance or sensitivity to US11 (see Fig. 2A). As for US2, we first evaluated the effect of US11 on surface expression of the HLA-A21–184/E and HLA-E1–184/A2 chimeras. Figure 3(B) shows that the HLA-A2/E chimera consisting of residues 1–184 of HLA-A2 was somewhat down-regulated in US11-positive cells. This chimera was more sensitive to US11 compared with wt HLA-E, but less sensitive than wt HLA-A2. HLA-E1–184/A2 on the other hand, was as sensitive to US11 as wt HLA-A2, with an almost complete reduction of surface expression. We then tested what region of HLA-A2 was responsible for this down-regulation of HLA-E1–184/A2. Replacement of the {alpha}3 domain and connecting peptide region with the corresponding domains of HLA-A2 [HLA-E({alpha}3 + c A2)] caused a slight reduction in surface expression, observed in the highest EGFP (and US11)-expressing population only. Replacement of the transmembrane region [HLA-E(TM A2)] showed no effect. Exchanging the cytoplasmic tail [HLA-E(tail A2)] however, resulted in a remarkable increase in sensitivity to US11. This indicated once again that the cytoplasmic tail can be an important determinant for US11-mediated down-regulation.



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  		Fig. 3. Exchange of ER–lumenal domains or extension of the cytoplasmic tail of HLA-E by two residues alter their sensitivity to US11-mediated degradation. (A) Overview of HLA-A2/E chimeras showing regions that are exchanged or extended. (B) J26 cells transfected with the HLA class I chimeras shown in (A) and transduced with US11-IRES-EGFP-encoding retrovirus were analyzed using flow cytometry. HLA class I molecules were stained with PE in a three-step staining protocol (y-axis) using MEM-E/06 mAb, except for HLA-A2, HLA-A21–184/E (BB7.2), HLA-E1–184/A2 and HLA-E({alpha}3 + c A2), which were stained with W6/32 mAb. US11-positive cells are marked by EGFP expression (x-axis). (C) J26 cells expressing wt HLA-E or HLA-E + KV or cells co-expressing US11 were metabolically labeled for 10 min and chased for 0 or 30 min in the absence or presence of proteasome inhibitor ZL3H. Immunoprecipitations were performed on denatured cell lysates using the following anti-sera: H68.4 [transferrin receptor (TfR)], MEM/02 (HLA-E) or US11-N2 (US11) mAbs.

 
A more detailed analysis was performed to determine which element of the HLA-A2 tail made the HLA-E(tail A2) chimera sensitive to US11. In a previous report, we have shown that MHC class I HCs are less sensitive to US11-mediated down-regulation when the cytosolic tail lacks the lysine and valine residues at the extreme end (19). HLA-E molecules have a slightly shorter tail than HLA-A2 as they lack C-terminal –ACKV residues. We tested whether addition of these residues could increase the sensitivity of HLA-E to US11-mediated down-regulation. Figure 3(B) shows that an extension of the cytoplasmic tail with –ACKV or –KV residues markedly reduced surface expression of these HLA-E mutants in US11-expressing cells. US11 had no effect on surface levels of HLA-E mutants with only one extra residue (HLA-E + K or HLA-E + V). Apparently, at least two extra residues are required for a down-modulatory effect.

We also evaluated the effect of US11 on the stability of HLA-E + KV in pulse chase experiments (Fig. 3C). In the absence of proteasome inhibitor (left panel), HLA-E + KV HCs remained stable over time in US11-negative cells, but were clearly unstable in the presence of US11. This effect of US11 is specific for class I HCs, as the amount of transferrin receptor in these cells was not reduced. In the presence of proteasome inhibitor (middle and right panels), a deglycosylated breakdown intermediate was observed only in cells expressing US11 and only for HLA-E with the –KV extension, but not for wt HLA-E.

From these results we can conclude that HLA-E, like HLA-G, becomes sensitive to US11-mediated degradation when its tail is extended with HLA-A2 tail residues. This indicates that the cytoplasmic tail can be an important determinant for sensitivity to US11. Interestingly, it only required two extra residues at its C-terminus for HLA-E to become completely sensitive to US11. Clearly, the tail is not the only determinant, as the {alpha}1/{alpha}2 region and, to a lesser extent, the {alpha}3/connecting peptide region also contribute to the efficiency of US11-mediated down-regulation of MHC class I molecules.

C-terminal lysine and valine residues influence efficiency, but are not essential for US11-mediated down-regulation of MHC class I molecules
When looking at the cytoplasmic tail sequences of MHC class I locus products, it becomes evident that other class I molecules besides HLA-G and HLA-E (e.g. HLA-B molecules) lack lysine and valine residues at their C-terminal tails (Fig. 4A). Figure 3 already showed that ER–lumenal regions influence the efficiency of down-modulation by US11. To evaluate if this region could also be sufficient for down-modulation of HLA-A2 molecules in the absence of lysine and valine residues, we tested two different HLA-A2 mutants. One mutant (HLA-A2delCKV) lacks the last three C-terminal residues, as do HLA-B alleles, and the other mutant has the same tail as HLA-E molecules [HLA-A2(tail E)]. Figure 4(B) shows that regions other than the cytoplasmic tail KV residues can mediate sufficient interactions for an US11-mediated reduction in surface expression, as HLA-A2 mutants without these residues were also down-regulated. Interestingly, the efficiency of down-regulation of class I molecules lacking (A)CKV residues seems to be somewhat lower than of those with these residues at the extreme end of the tail. In general, the level of down-modulation is lower in the low EGFP-positive cells and increases with rising EGFP (i.e. US11) levels. In cells expressing highly sensitive wt HLA-A2, effective reduction in surface expression can also be observed in cells expressing relatively low levels of EGFP.



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  		Fig. 4. Cytoplasmic tail residues of MHC class I molecules determine efficiency of US11-mediated down-regulation. (A) Alignment of amino acid sequences of the cytoplasmic tail region of different MHC class I locus products' consensus sequences, obtained from the IMGT/HLA sequence database (11). (B) J26 cells expressing wt HLA-A2, HLA-A2delCKV or HLA-A2(tail E) were transduced with US11-IRES-EGFP-encoding retrovirus and analyzed using flow cytometry. HLA class I molecules were stained with PE in a three-step staining protocol using BB7.2 as first mAb (y-axis). US11-positive cells are marked by EGFP expression (x-axis).

 
These results show that the C-terminal KV residues do not necessarily function as strong determinants for sensitivity to US11 for all locus products. As opposed to the crucial role of KV residues in US11-mediated down-regulation of HLA-E molecules, they are less important for down-regulation of other MHC class I locus products. This is shown for HLA-A2 molecules, where the (C)KV tail residues could be removed without completely losing sensitivity to US11. It can also explain the observed down-regulation of HLA-B molecules (Fig. 1A), which naturally lack CKV residues. However, the presence or absence of these KV residues can nevertheless influence the efficiency of down-modulation by US11, and can determine the levels of US11 that are required for sufficient modulation of antigen presentation by MHC class I.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
HCMV encodes several proteins that interfere with cross talk between infected host cells and host immune effector cells through modulation of surface expression of MHC class I molecules. The success of immune escape by HCMV through modulation of MHC class I surface expression is likely to be influenced by the efficiency, as well as by the specificity, of this down-modulation by the different US proteins. US3 mainly affects surface expression of tapasin-dependent MHC class I alleles (20). By blocking TAP, US6 prevents peptide transport into the ER and subsequent peptide loading. This affects surface expression of all MHC class I alleles (2, 3). In spite of this, surface expression of HLA-E molecules is preserved by supplying it with a TAP-independent peptide source (30, 36). In this study we focused on US2 and US11, both of which target different sets of newly synthesized MHC class I molecules for degradation. In this study we further clarify how, and to what extent, US2 and US11 can contribute to the efficiency and specificity of MHC class I down-regulation.

We and others have found that US2 differentially affects surface levels of individual human MHC class I locus products (23, 32, 34). Based on crystal structure data of HLA-A2–ß2m–US2 and sequence alignments for the region of class I implicated in US2 binding, we hypothesized that allelic variation in the {alpha}2/{alpha}3 ER–lumenal region could form an explanation for the resistance of HLA-B7, HLA-Cw3 and HLA-E (35). In the present study, we tested this hypothesis to see if this would result in a more reliable prediction of US2 sensitivity of, as of yet, untested MHC class I alleles.

Using chimeras derived from US2-sensitive (HLA-A2) and -insensitive (HLA-E) alleles, we found that there are no other regions in HLA molecules, outside the ER–lumenal region implicated in US2 binding, that contribute to US2-mediated down-regulation. Sequence alignments of HLA-B27 and HLA-B7 also point to a role of the ER–lumenal region in selective down-regulation of only HLA-B27 and not HLA-B7, as there are no differences in the amino acid sequence outside the ER–lumenal region between these two alleles.

We then investigated if we could convert both the sensitivity of HLA-A2 as well as the resistance of HLA-B7 and HLA-E by replacing those residues that are assumed to allow/prohibit an interaction with US2, with corresponding residues found in US2-insensitive/sensitive alleles (as described in Fig. 2A). Indeed, an HLA-A2 mutant with four residues derived from HLA-E (HLA-A2LHLE) was not affected by the presence of US2, just like HLA-E itself. Besides HLA-A2LHLE, the HLA-B7ET(L)Q and HLA-EQRTD mutants showed that residues in this region are indeed important sensitivity determinants, as the alteration of only three or four residues clearly affected the surface expression of these mutants in the presence of US2. We showed for HLA-EQRTD that it is targeted for degradation, thereby excluding the possibility that retention is the underlying mechanism for the observed down-modulation.

We also tried to find an explanation for the resistance of HLA-Cw3 alleles to US2-mediated down-regulation. The presence of particular residues at positions 183 (E) and 184 (D) appeared not to be responsible for its resistance. This is supported by recent data showing that HLA-C molecules (HLA-Cw7 and HLA-Cw2), which have E183 and D184, can nevertheless be down-regulated by US2 (34; our unpublished results). Nonetheless, sequence variation at this site may still affect the efficiency of down-modulation, as US2-resistant HLA-Cw3 alleles became somewhat more sensitive when E183D and H184P mutations were introduced (our unpublished results). However, the presence of a positively charged lysine residue at position 173 is the most likely explanation for the resistance to US2-mediated down-regulation of HLA-Cw3, since US2-sensitive alleles, including the majority of HLA-C alleles, have a negatively charged glutamic acid at position 173.

For US11, the only MHC class I locus products completely insensitive to down-modulation were HLA-G and HLA-E (Fig. 1). Interestingly, all that is required to confer sensitivity to these two MHC class I locus products is an extension of their cytoplasmic tail. Previous studies have shown that the length of the class I cytoplasmic tail is very important. Tailless HLA-A2 molecules (with a tail shortened to either four or six amino acids) could no longer be targeted for degradation by US11 (19, 37). Conversely, HLA-G molecules naturally have a tail of six residues, and an extension of this tail with 27 HLA-A2 tail residues resulted in a very efficient degradation of these mutants in US11-positive cells (19). Interestingly, an extension of the tail of HLA-G with 25 HLA-A2 tail residues did not give this result. Apparently, the C-terminal lysine and valine residues were essential for degradation. HLA-E has a cytoplasmic tail that lacks only four residues compared with HLA-A molecules (see Fig. 4A). Interestingly, HLA-E required only two extra residues (lysine and valine) to become sensitive to US11. It was not necessary to make any additional changes in the luminal/extracellular domains of HLA-E. Furthermore, an extension of the HLA-E tail with only one residue, either valine or lysine, was not effective. This indicates that the length of the tail can be an important determinant for sensitivity differences among MHC class I locus products. HLA-B molecules, on the other hand, also lack the lysine and valine residues at their C-termini, but are nevertheless down-modulated by US11. We showed in Fig. 4 that the lysine and valine residues are not essential for down-regulation of all haplotypes, as HLA-A2 with a tail as long as that of HLA-B molecules (HLA-A2delCKV) or with the HLA-E tail, could still be down-modulated by US11. These residues can, however, determine the effectiveness or threshold for down-regulation, as HLA-A2delCKV and HLA-A2(tail E) seemed to require higher levels of US11 than wt HLA-A2 for a similar down-regulatory effect. At the same time, these data show that ER–lumenal residues also influence sensitivity to US11. This is further supported by our findings that the exchange of {alpha}1/{alpha}2 domains or of the {alpha}3 domain of HLA-E with those of HLA-A2 could, to some extent, also change the efficiency of down-modulation by US11.

Whereas specificity of US2-mediated down-modulation seems to rely mostly on a region at the junction of the {alpha}2/{alpha}3 domain, these data indicate that the conditions are different and more complicated for US11. Although ER–lumenal residues do play a role, replacement of residues LHLE in HLA-E by QRTD did not affect its sensitivity to US11 (our unpublished results). Also, US2 does not require MHC class I tail residues, but US11-mediated down-modulation depends largely on this region. In principal, all MHC class I cytoplasmic domains, with the exception of HLA-G, bear the essential residues necessary for US11 to target them for degradation. Our data indicate that a minimum of 29 class I tail residues have to go with either a favorable ER–lumenal region or with lysine and valine tail residues in order to see sufficient down-modulation. A favorable ER–lumenal region may bypass the function of the KV residues through a prolonged and/or stronger interaction with US11, thereby increasing the chances of dislocation and subsequent degradation.

The function of the KV, as well as of the other tail residues is still unclear. Enhancement of down-regulation by the KV residues may rely on the lysine, functioning as a potential ubiquitination site. It is known that ubiquitination is essential for US11-mediated targeting for dislocation and subsequent degradation of MHC class I molecules (29, 38). Although a study by Shamu et al. (39) showed that lysine residues in the tail of HLA-A2 are not essential for US11-mediated degradation, elimination of HLA-A2 without lysine residues in the tail seemed to be retarded compared with wt HLA-A2. Alternatively, the KV residues may merely facilitate access of components of the dislocation/degradation machinery to essential residues residing within another region of the tail. Phosphorylation can be an important signal for docking of E3 ligases, which in turn can ubiquitinate their substrates (40). The tail of MHC class I molecules encodes several potential phosphorylation sites, one of which, S335, is a known phosphorylation site that can be found in all class I locus products (41, 42). We have mutated residue S335 in HLA-A2, as well as two other potential phosphorylation sites close by (S328, S332), and replaced them with alanine. However, these mutants did not behave any differently from wt HLA-A2 in the presence of US11 (our unpublished results). More research will be required to unravel why the tail is essential for US11 to mark MHC class I molecules as substrate for the ubiquitination machinery.

All in all, we showed here that sequence variation around the region comprising residues 176–183 accounts for sensitivity differences of MHC class I locus products to US2-mediated degradation. This knowledge provides a valuable tool to predict the effect of US2 on a broader range of HLA class I molecules. For US11, we showed that not only ER–lumenal regions but also cytosolic tail residues are important determinants for the outcome of the down-modulatory effect of US11 on different class I locus products. The length of the tail can explain the insensitivity of HLA-G and HLA-E, and may also determine the efficiency by which other HLA class I molecules are down-modulated. More research is still required to define which regions in the {alpha}1, {alpha}2 and {alpha}3 domains of MHC class I molecules are also playing a role.

It is remarkable that the difference between complete resistance and full sensitivity of HLA-E alleles to US2- and US11-mediated degradation relies on as few as two to four residues. It is known that a preserved surface expression of HLA-E supports immune escape from NK-cell attack of HCMV-infected cells (34, 36, 43). From this point of view, it would be beneficial for the host to reduce its HLA-E surface levels in HCMV-infected cells. As mentioned before, it requires only small modifications within HLA-E to render this molecule sensitive to US2 or US11. Despite a long co-evolution of virus and host, HLA-E demonstrates limited polymorphism. This may imply that the residues determining resistance to these viral proteins are essential for interactions of HLA-E with components of the antigen presentation pathway and/or its biological function.


   Acknowledgements
 
We would like to thank M. Aguerre-Girr and V. Putanier for their technical assistance and F. L'Faqihi for her support in cytometry analysis. This work was supported by the Council for Medical Research from the Netherlands Organisation for Scientific Research (grant no. 901-02-218, M.T.B.), a fellowship from Ministère de la Recherche (N.P.) and grants from Sidaction and ASUPS (F.L.), Ligue Régionale Midi-Pyrénées and ANRS (P.L.B.).


   Abbreviations
 
EGFP   enhanced green fluorescent protein
ER   endoplasmic reticulum
g{alpha}m   goat anti-mouse
HC   heavy chain
HCMV   human cytomegalovirus
ß2m   ß2-microglobulin
TAP   transporter associated with antigen processing
UL   unique long
US   unique short
wt   wild type
ZL3H   carboxybenzyl-leucyl-leucyl-leucinal

   Notes
 
Transmitting editor: J. Borst

Received 23 March 2005, accepted 28 October 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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