Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18

Characterization of a highly glycosylated form of the humancytomegalovirus HLA class I homologue gpUL18

Cora Griffin1, Eddie C. Y. Wang1, Brian P. McSharry1, Carole Rickards1, Helena Browne2,Gavin W. G. Wilkinson1, and Peter Tomasec1

1Section of Infection and Immunity, Cardiff University, Tenovus Building, Heath Park, CardiffCF14 4XX, UK2Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP,UK

AbstractHuman cytomegalovirus (HCMV) gpUL18 is a HLA class I (HLA-I) homologue with highaffinity for the inhibitory receptor LIR-1/ILT2. The previously described 67 kDa form of gpUL18is shown here to be sensitive to endoglycosidase-H (EndoH). A novel form of gpUL18 with amolecular mass of ~160 kDa and resistance to EndoH was identified in cells infected with HCMVstrain AD169 or the low passage HCMV isolates Merlin and Toledo. The 67 kDa EndoH-sensitivegpUL18 glycoform was detected earlier in a productive infection (from 24 h post-infection) thanthe slower-migrating EndoH-resistant glycoform (from 72 h post-infection). Deletion of the US2–US11 region from the HCMV genome was associated with a substantial up-regulation ofendogenous HLA-I in infected cells, but had no obvious effect on the gpUL18 expression pattern.Vaccinia virus and adenovirus vectors were used to further analyse gpUL18 expression.Depending on the delivery vector system, differences in the electrophoretic motility of the EndoH-resistant >105 kDa form of gpUL18, but not the EndoH-sensitive 67 kDa form, were observed;post-translational modification of the higher molecular mass glycoform appears to be influencedby active virus infection and vector delivery. The EndoH-sensitive 67 kDa gpUL18 had a rapidturnover, while the maturation to the EndoH-resistant >105 kDa form was relatively slow andinefficient. However, synthesis of the EndoH-resistant >105 kDa form was enhanced withelevated levels of β2-microglobulin. When expressed by using an adenovirus vector, both theEndoH-sensitive 67 kDa and the EndoH-resistant >105 kDa gpUL18 forms could be detected onthe cell surface.

INTRODUCTIONHuman cytomegalovirus (HCMV) is a β-herpesvirus with the largest genome (~236 kb) ofany characterized human virus. Although its genetic content is not fully characterized, asubstantial proportion has been shown to, or is predicted to, encode functions capable ofmodulating the human immune system. HCMV encodes four genes (US2, US3, US6 andUS11) that down-regulate cell surface HLA class I (HLA-I) expression (Tortorella et al.,2000), three genes that modulate natural killer (NK) cell functions (UL16, UL40, UL141)(Cosman et al., 2001; Tomasec et al., 2000, 2005), an interleukin 10 (IL10) homologue(UL111A) (Kotenko et al., 2000), chemokine homologues (UL146, UL147) (Penfold et al.,

© 2005 SGM

Correspondence Gavin W. G. Wilkinson [email protected].

Four figures showing the mobility of highly glycosylated forms of UL18, UL18 sequence alignments and a time course of gpUL18surface exprssion in HCMV-infected cells are available as supplementary material in JGV Online.

Europe PMC Funders GroupAuthor ManuscriptJ Gen Virol. Author manuscript; available in PMC 2010 March 23.

Published in final edited form as:J Gen Virol. 2005 November ; 86(Pt 11): 2999–3008. doi:10.1099/vir.0.81126-0.

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1999), potential (UL33, UL78, US27) (Chee et al., 1990) or recognized (US28) chemokinereceptors (Neote et al., 1993), inhibitors of interferon (TRS1, IRS1, UL83) (Child et al.,2004), inhibitors of apoptosis (UL36, UL37, IE1, IE2) (Zhu et al., 1995) and a tumournecrosis factor receptor homologue (UL144) (Benedict et al., 1999). HCMV is ubiquitousworldwide, with the vast majority of primary infections being subclinical and followed bylifelong asymptomatic carriage of the virus. Thus, even though HCMV encodes animpressive array of immunomodulatory functions, infections are normally efficientlycontrolled by immune surveillance. Clinical disease is found primarily in individuals whoseimmune systems have not fully developed (congenital transmission resulting in cytomegalicinclusion disease) or are severely compromised, most notably transplant recipients andAIDS patients. Although the different arms of the immune system act in concert to controlinfection, individuals with genetic defects affecting NK cell function are particularlysusceptible to HCMV disease (Arase et al., 2002; Biron et al., 1989; Gazit et al., 2004). Invivo pathogenesis studies with murine CMV indicate that not only is the NK cell responsecrucial to controlling disease but also the virus-encoded genes that modulate NK cellfunctions are important virulence determinants (Abenes et al., 2004; Arase et al., 2002;Farrell et al., 1997).

UL18 was identified as a HLA-I homologue during the analysis of the strain AD169sequence (Beck & Barrell, 1988) and, like HLA-I, was found to form a trimeric complexwith β2-microglobulin (β2m) and endogenous peptides (Browne et al., 1990; Fahnestock etal., 1995). The leukocyte immunoglobulin-like receptor-1 (LIR-1, also referred to as ILT2)was initially identified by its interaction with gpUL18 (Cosman et al., 1997), and was thenfound to be an inhibitory receptor that recognized an exceptionally broad range of classical(HLA-A, HLA-B, HLA-C) and non-classical (HLA-E, HLA-F, HLA-G) HLA-I molecules(Chapman et al., 1999; Lepin et al., 2000; Shiroishi et al., 2003). LIR-1 has >1000-foldhigher affinity for gpUL18 than for HLA-I molecules, thus even low levels of gpUL18 couldbe expected to compete efficiently for binding (Chapman et al., 1999). The prediction thatgpUL18 would suppress NK cell recognition has been both supported (Kim et al., 2004;Reyburn et al., 1997) and questioned (Leong et al., 1998; Odeberg et al., 2002) byexperimental data in published studies. LIR-1 expression is restricted to myeloid cells, Bcells, and subpopulations of T and NK cells (Young et al., 2001). Interestingly, theproportion of both NK and T cells expressing LIR-1 increases in frequency in lung-transplant recipients with HCMV disease (Berg et al., 2003). On CD8+ T lymphocytes,LIR-1 is detected preferentially on highly differentiated activated and memory T cells(Young et al., 2001). Saverino et al. (2004) have reported that UL18 expression stimulatednon-HLA-restricted killing by CD8+ cytotoxic T lymphocytes, via an interaction withLIR-1, although the mechanism responsible for enhanced recognition and its role in HCMVinfection have yet to be elucidated.

UL18 is non-essential for HCMV replication in vitro (Browne et al., 1992), and istranscribed with late-phase kinetics in human fibroblasts infected with HCMV (Park et al.,2002). Analysis of gpUL18 function has been hampered by the apparent inability to detectgpUL18 in HCMV-infected cells, and a failure to generate stable cell lines expressing theUL18 open reading frame (ORF). When continuous cell lines stably transfected with US2,US3, US6 or US11 were infected with vaccinia virus UL18 vector, gpUL18 was expressedefficiently and was detected as a 67 kDa glycoprotein (Park et al., 2002). In this study, wehave further characterized gpUL18 in the context of productive HCMV infection, and alsowhen expressed using vaccinia virus (v-UL18) and replication-deficient adenovirus(RAdUL18) vectors. The previously reported 67 kDa form of gpUL18 is shown here to besensitive to digestion with endoglycosidase-H (EndoH), whereas a novel, EndoH-resistantform of gpUL18 migrating with an apparent molecular mass in excess of 105 kDa isdescribed.

Griffin et al. Page 2

J Gen Virol. Author manuscript; available in PMC 2010 March 23.

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METHODSCells and viruses

Human fetal foreskin fibroblasts (HFFFs) were cultured in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10 % fetal calf serum (FCS) and antibiotics(Invitrogen). Transporter associated with antigen processing (TAP)-deficient (NP)fibroblasts were derived from a skin biopsy of a patient with Bare Lymphocyte Syndrome, agenetic disease associated with severe down-regulation of surface MHC-I expression. NPcells, kindly provided by V. Cerundolo (University of Oxford, Oxford, UK), have a defect inTAP-1 expression (Tomasec et al., 2000). Unless otherwise indicated, cells were infectedwith HCMV (m.o.i. of 10) 96 h prior to analysis. HCMV infections were performed usingstrains AD169, Toledo, Merlin, the AD169 UL18 insertional mutant (△UL18) (Browne etal., 1992) or the AD169 US2–US11 deletion mutant (RV798) (Jones et al., 1995). Thevaccinia virus vector encoding UL18 (v-UL18) has been described previously (Browne etal., 1990). The UL18 ORF was amplified by PCR and inserted into a replication-deficienthuman adenovirus-5 vector (RAdUL18) under the control of the HCMV major IE promoter,as described by McGrory et al. (1988) and Wilkinson & Akrigg (1992). Unless otherwiseindicated, HFFFs were infected with RAdUL18 or the same vector without an insert(RAdCtrl) at an m.o.i. of 100 for 72 h prior to analysis.

Immunofluorescence and flow cytometryCells on glass cover-slips were washed in PBS and fixed with 2 % paraformaldehyde for 15min at room temperature. The cells were washed in PBS and then, to ensure sustainedpermeabilization of cells, all subsequent treatments were done in PBS containing 3 % BSA,0·1 % saponin. Next, the cells were incubated with the gpUL18 antibody M71 (2 μgml−1)for 1 h at room temperature, washed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse immunoglobulin G (IgG) for 1 h at 37 °C. The cells, still on thecoverslips, were washed and mounted on slides with 2 % DABCO, 10 % (v/v) glycerol, PBS(Sigma). Fluorescence was visualized using a Leica DM IRBE microscope with aHamamatsu ORCA-ER camera and Improvision Openlab software.

One-colour flow cytometry was used to detect surface gpUL18 or HLA-I expression. Cellswere washed twice with cold PBS, 2 % FCS and incubated for 30 min at 4 °C withmonoclonal antibodies (mAbs) specific for gpUL18 (1 : 50 dilution; 10C7/CRL-2430)(Fahnestock et al., 1995) or HLA-I (1 : 100 dilution; W6/32). Normal mouse IgG was usedas a negative control. The cells were washed twice with cold PBS, 2 % FCS and thenlabelled with FITC-conjugated anti-mouse Fab fragments (Sigma) on ice for 30 min. Cellswere washed twice with cold PBS, and fixed with 2 % paraformaldehyde. A total of 10 000events were collected by the FACSCalibur cytometer and analysed with CellQuest Prosoftware (BD Biosciences).

ImmunoblottingTo prepare extracts, cells were first washed with PBS, before being resuspended in NP-40lysis buffer (1 % NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8) supplementedwith P8340 protease inhibitor cocktail (Sigma). Cells were lysed during an incubation for 30min, on ice, and clarified by centrifugation for 30 min at 13 000 r.p.m. in a Heraeus BiofugeFresco at 4 °C. For glycosidase treatments, samples were adjusted to 0·05 % SDS, 0·1% β-mercaptoethanol, and protein denatured by heating at 100 °C for 10 min, before beingdigested with either 1000 U Endo H in 5 mM sodium citrate (pH 5·5) or 500 U peptide: N-glycosidase F (PNGase F) in 5 mM sodium phosphate (pH 7) for 12 h according to themanufacturer’s instructions (NEB). Enzyme was omitted from mock-digested samples. Allsamples were boiled after the addition of SDS-PAGE loading buffer, and proteins separated



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by SDS-PAGE using a Mini Protean II gel apparatus (Bio-Rad). Following electrophoresis,protein was transferred to nitrocellulose membrane (Amersham Hybond-C), by wet transferat 100 V for 1 h using a Bio-Rad mini trans-blot system. The membrane was washed withTris-buffered saline (TBS), blocked for 16 h at 4 °C in 5 % BSA, TBST, and for a further 8h at 4 °C in Superblock (Pierce), before being incubated with mAbs specific for gpUL18(M71; 2·5 μgml−1), HLA-I (HC10; 1 : 200 dilution) or HLA-E (1 : 200 dilution; Serotec) for16 h in 10 % (v/v) Superblock, TBST. The membrane was washed in TBST, 5 % dried milk,then again in TBST, before being incubated with goat anti-mouse horseradish peroxidase(HRP) antibody (Bio-Rad) (1 : 1000 dilution) in 5 % dried milk, TBST for 1 h at roomtemperature. Supersignal substrate (Pierce) was used to detect the HRP signal and themembrane exposed to Kodak BioMax MR film.

ImmunoprecipitationHFFFs were infected with v-UL18 or v-Ctrl (m.o.i. of 10) for 6 h, then cultured inmethionine-free medium for 30 min. Cells were labelled with 50 μCi (1·85 MBq)[35S]methionine ml−1 (ICN) for 4 h prior to immunoprecipitation, or pulse-labelled with[35S]methionine for 15 min, then chased for the times indicated. When harvested, cells wereresuspended in NP-40 lysis buffer (1 % NP-40, 1 % protease inhibitor (Sigma), 150 mMNaCl, 50 mM Tris, pH 8) for 30 min at 4 °C, precleared by adding 100 μl protein G agarose(Pierce) for 45 min at 4 °C and centrifuged at 13 000 r.p.m. in a Heraeus Biofuge Fresco for30 min at 4 °C. Supernatants were then incubated with 10 μg M71 mAb overnight at 4 °C,then with 50 μl protein G agarose for 45 min at 4 °C. The protein G agarose was thenwashed, and the complexed proteins released by resuspending the beads in SDS denaturingbuffer (NEB) and heating to 100 °C. Where indicated, precipitated proteins were digestedwith EndoH or PNGaseF, and then subjected to SDS-PAGE and fluorography.

Biotinylation of cell surface proteinsCells were washed in cold PBS then incubated according to the manufacturer’s instructionsfor 45 min at 4 °C with 0·5 mg sulfo-NHS-biotin ml−1 (Pierce). Cells were washed and lysedin NP-40 buffer, and the lysates cleared by centrifugation. Biotinylated proteins werecaptured with streptactin-sepharose (IBA), and washed with NP-40 and RIPA (150 mMNaCl, 50 mM Tris pH8, 0·5 % deoxycholate, 0·1 % SDS, 1 % NP-40) buffers. Boundproteins were eluted in SDS denaturing buffer and analysed by immunoblotting.

RESULTSIdentification of a highly glycosylated form of gpUL18

The first analysis of the UL18 ORF using the vaccinia virus recombinant v-UL18demonstrated that the gene encoded a 67 kDa glycoprotein that formed a complex with β2m(Browne et al., 1990). In subsequent studies using v-UL18, an additional slower-migratingprotein with an apparent molecular mass >105 kDa was detected by immunoprecipitationwith a gpUL18-specific mAb (Fig. 1). This gpUL18 species was consistently observed as adiffuse band following denaturing SDS-PAGE, under reducing conditions. To analyse therate of gpUL18 synthesis and maturation, v-UL18-infected cells were pulsed with[35S]methionine, and labelled gpUL18 was then followed over time. Maximum levels of the67 kDa gpUL18 were detected immediately following the labelling, with levels decliningover time, indicating that this species had a relatively short half-life. In contrast, the >105kDa gpUL18 form was not apparent until 6 h after labelling, consistent with its probablesynthesis from the 67 kDa precursor and having an exceptionally slow rate of maturation(Fig. 2a). Endogenous HLA-I molecules were immunoprecipitated in parallel, demonstratingthat the stability and the kinetics of HLA-I maturation were normal, and thus under theseconditions not affected by the vaccinia virus vector (Figs 2a, b, respectively). Significantly,



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the 67 kDa gpUL18 was susceptible to EndoH digestion, whereas the >105 kDa form wasresistant, consistent with it being further post-translationally processed in the Golgiapparatus from the 67 kDa precursor. Levels of HLA-I heavy chain remained constant overthe duration of the time course, while gpUL18 levels decreased markedly. The endogenousHLA-I was thus more stable than gpUL18, while both the rate and efficiency with which itwas converted to an EndoH-resistant form exceeded that observed for gpUL18. Like HLA-I,gpUL18 forms a trimeric complex with β2m and peptide. Cell surface gpUL18 levels wereenhanced by co-expressing β2m from a second vaccinia virus recombinant (Browne et al.,1990). By its association with newly synthesized gpUL18, β2m may have acted to protect itfrom degradation and promote its maturation. In line with this model, we observed that co-infection of v-UL18 and v-β2m recombinant viruses enhanced the relative levels of theEndoH-resistant >105 kDa form of gpUL18 as determined by both immunoblotting (Fig. 3a)and immunoprecipitation (Fig. 3b) assays.

UL18 expression from an adenovirus vectorUsing the v-UL18 vector, transgene expression took place against a background of aproductive vaccinia-virus infection. To express gpUL18 efficiently yet free of lytic virusinfection, the UL18 gene was inserted into a replication-deficient human adenovirus-5vector, designated RAdUL18. Following infection of fibroblasts with RAdUL18, gpUL18immunofluorescence exhibited a predominantly intracellular staining that was excludedfrom the nucleus (Fig. 4a, b). In immunoblotting experiments, again, not only the 67 kDagpUL18 species could be detected, but also a slower migrating form with an apparentmolecular mass of ~110 kDa (Fig. 4C) could be detected. The 67 kDa protein was againfound to be EndoH sensitive, and the higher molecular mass form EndoH resistant (Fig. 4c).

In order to function effectively as a HLA-I mimic capable of interacting directly with itsreceptors on lymphoid cells, gpUL18 has to be present at the cell surface. To investigate thisaspect, HFFFs were infected with RAdUL18, and non-permeabilized cells analysed by flowcytometry. The fluorescence observed with RAdUL18-infected cells using the gpUL18-specific mAb 10C7 was significantly higher than for cells infected with an equivalent vectorwithout an insert (RAdCtrl) or when a control IgG was used (Fig. 5a). While gpUL18clearly reached the surface of RAdUL18-infected cells, flow cytometry was unable todifferentiate between the two forms of gpUL18 (Fig. 5b). We hypothesized that the ~110kDa species corresponded to a mature EndoH-resistant form of the protein, which would bepreferentially expressed on the cell surface. Cell surface proteins on RAdUL18-infectedcells were labelled with biotin and purified with streptactin beads. HSP40, being anintracellular protein associated with the endoplasmic reticulum, was used as a negativecontrol for this assay. HSP40 was not detected in the biotinylated fraction of cell surfaceproteins, although it was present in cell extracts (Fig. 5c). Both the 67 kDa and ~110 kDaforms of gpUL18 were clearly biotinylated, and thus both appear to be present on thesurface of RAdUL18-infected cells (Fig. 5d).

During productive HCMV infection, US6 interferes with the TAP. As peptide binding isrequired for maturation of endogenous HLA-I molecules, US6 acts to down-regulate cellsurface expression of HLA-I. To test whether TAP-dependent peptide translocation wasrequired for efficient gpUL18 expression, NP fibroblasts were also infected with RAdUL18and analysed in a FACS experiment. NP fibroblasts have a genetic defect in TAP-1 function.gpUL18 was expressed efficiently on the cell surface in the absence of TAP-1 (Fig. 5e),albeit at a lower level than that observed in HFFFs when analysed in parallel (Fig. 5a).Significantly, both glycoforms of gpUL18 were detected in NP cells (Fig. 5f). TAP function,therefore, does not appear to be essential for normal expression of gpUL18.



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gpUL18 expression during an HCMV infectionAnalysis of UL18 during a productive HCMV infection is problematical due to acombination of extremely low gpUL18 levels and confounding antibody interactions withvirus-encoded Fc receptors. Nevertheless, human fibroblasts infected with HCMV-AD169or the AD169△UL18 mutant (△UL18) for 96 h (m.o.i. of 10) were analysed byimmunoblotting. gpUL18 levels were much lower in HCMV-infected cells than thoseobserved with v-UL18- or RAdUL18-infected cells; however, both gpUL18 species, 67 kDaand ~160 kDa, were evident (Fig. 6a). To generate AD169△UL18, the Escherichia coli lacZwas inserted within the UL18-encoding gene (Browne et al., 1992). β-Galactosidase wasexpressed at such high levels from the HCMV β2·7 early promoter (Spaete & Mocarski,1987) that it was detected by a non-specific antibody interaction (Fig. 6a).

It proved problematical to determine a precise molecular mass for the EndoH-resistantglycoform of gpUL18, as it was observed as a diffuse band on immunoblots, and itsmigration effectively depended on the virus vector used to infect cells. However, the mass ofgpUL18 expressed during productive HCMV infection was by far the greatest migratingclose to the 160 kDa molecular mass marker, while the mass of gpUL18 expressed fromRAdUL18 vector was the smallest and close to the 105 kDa marker (see Supplementary Fig.S1 available in JGV online). Vaccinia virus and HCMV infections are both recognized toexert gross effects on the host cells metabolism, and both may potentially influence thecellular glycosylation machinery.

HCMV strain AD169 has undergone genetic changes during passage in vitro (Akter et al.,2003); however, the UL18 amino acid sequence exhibits a high level of conservation (95·1–100 % identity) between strains, with there being no sequence-predicted defect associatedwith strain AD169 to account for its low level of expression (see Supplementary Figs S2 andS3 available in JGV online). When assayed directly, levels of gpUL18 expression, gpUL18migration on gels and the existence of dual forms of the protein were similar in cellsinfected with HCMV AD169, Toledo or Merlin (Fig. 6b). When expressed from either strainAD169 or Toledo, the 67 kDa gpUL18 was again susceptible to EndoH digestion, whereasthe ~160 kDa form was not affected by EndoH but was susceptible to PNGaseF treatment(not shown). Thus, the highly glycosylated, EndoH-resistant gpUL18 glycoform was typical,and not restricted to the laboratory strain AD169. The 67 kDa form of gpUL18 was detectedat 24 h post-infection (p.i.) and increased in abundance to reach a peak by 72 h p.i. Thelevels were then sustained through the late phase of infection. The ~160 kDa form ofgpUL18 appeared at 48 h p.i. and also reached peak levels by 72 h p.i (Fig. 7a). gpUL18 wasthus expressed most efficiently at a time when endogenous HLA-I had been effectivelyeliminated. At no point during this time-course assay were we able to detect gpUL18 on thecell surface of HCMV-infected cells (Fig. 7b, Supplementary Fig. S4 available in JGVonline). A major effort was taken to optimize staining conditions to enable detection ofgpUL18 on the surface of cells undergoing lytic HCMV infection, as this was consideredcentral to the investigation of its function. A small increase in staining with the UL18-specific mAb relative to control IgG was detected; however, a similar effect was observedwhen using cells infected with the △UL18 mutant, and was thus consistent with anenhanced level of non-specific fluorescence associated with HCMV infection (Fig. 7b).

Given the low levels of gpUL18 expression during lytic HCMV infection, it still remainedpossible that one or more of the HLA-I down-regulatory genes (US2, US3, US6 or US11)were suppressing, but not eliminating gpUL18 expression. To address this issue directly inthe context of HCMV infection, HFFFs were infected with HCMV AD169, △UL18 or theAD169 mutant RV798 deleted in sequences encoding US2–US11, and then analysed 96 hp.i. by immunoblotting (Fig. 8). At this late phase of infection, similar levels of the twogpUL18 forms were consistently detected in cells infected with strain AD169 or the strain



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AD169 US2–US11 deletion mutant (Fig. 8a). This was at a time when infection with AD169had efficiently down-regulated HLA-I expression, while the infection with HCMV RV798markedly stimulated HLA-I expression both at the cell surface (Fig. 8b) and overall (Fig.8c), relative to mock-infected controls. While US2–US11 genes efficiently down-regulatedHLA-I, they did not appear to affect gpUL18 expression during the late phase of aproductive infection in fibroblasts. HLA-E has been reported to be resistant to US2 andUS11, when co-expressed in isolation (Llano et al., 2003). In agreement with theseobservations, total cellular levels of the non-classical HLA-E increased during HCMV strainAD169 infection, in contrast to classical HLA-I, but not during infection with HCMVRV798 (Fig. 8d).

DISCUSSIONThe study of UL18 function has been frustrated by issues both of expression and detection.UL18 constitutive expression is not well-tolerated in long-term stable cell lines (Davis et al.,1997; Kim et al., 2004; Reyburn et al., 1997) and, to our knowledge, there has previouslybeen no definitive demonstration of gpUL18 expression during HCMV infection. The UL18ORF encodes 13 consensus N-linked glycosylation sites, whereas classical HLA-I moleculestypically have a single functional site. When treated with PNGaseF, the deglycosylatedUL18 protein exhibited an apparent molecular mass of 35 kDa as estimated by SDS-PAGE,compared with 41·7 kDa predicted from the sequence. Glycosylation increased themolecular mass to 67 kDa for the EndoH-sensitive protein, which further increased to >105kDa for the EndoH-resistant, PNGaseF-sensitive form, size being dependent on theexpression system. A similar pattern of gpUL18 expression was observed in cells infectedwith a range of low and high passage HCMV strains, with the EndoH-resistant glycoformconsistently estimated to have a molecular mass of ~160 kDa, a result consistent with theUL18 gene coding sequence being relatively conserved (Dolan et al., 2004; Valés-Gómez etal., 2005; Supplementary Figs S2 and S3 available in JGV online). The majority of theapparent mass of the fully glycosylated gpUL18, therefore, appears to be derived fromcarbohydrates added during post-translational modifications.

During productive HCMV infection, we were only able to demonstrate expression ofgpUL18 by immunoblotting, at the detection limit of the assay. By flow cytometry, gpUL18could not be detected on the cell surface. Higher levels of gpUL18 were obtained by the useof other viral vectors, which then resulted in detection of gpUL18 on the cell surface,suggesting the constraints of expression could at least be partially overcome by enhancedlevels of transcription. The synthesis and processing of the fully glycosylated gpUL18appeared to be exceptionally slow and inefficient when compared to endogenous HLA-I. Arequirement for limiting chaperone functions to stabilize the UL18 polypeptide undergoingglycosylation and maturation within the ER may have contributed towards its inefficientexpression. Elevating the levels of β2m has previously been shown to promote cell surfaceexpression of β2m and gpUL18 (Browne et al., 1990), and we have shown here that theavailability of β2m was a factor affecting the rate of synthesis of the >105 kDa form ofgpUL18. In contrast to HLA-I, gpUL18 was expressed well in both TAP-1 positive ornegative fibroblasts, suggesting that TAP-dependent peptide loading may not be essentialfor gpUL18 maturation, or sufficient levels of gpUL18-binding peptide can be scavengedindependently of TAP. In this context, it is interesting to note that gpUL18 expression hadpreviously been shown to be susceptible to down-regulation by HSV-1 ICP47 but notHCMV US6 (Park et al., 2002), both genes being inhibitors of TAP function. Also, peptidebinding is thought not to be required for the interaction with LIR-1 (Chapman et al., 1999;Willcox et al., 2003).



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To act as a ligand for LIR-1 on NK or alternative target cells, gpUL18 must be present onthe cell surface, and such was observed in experiments using either v-UL18 (Browne et al.,1990; Park et al., 2002) or RAdUL18-infected fibroblasts. We anticipated that the 67 kDagpUL18, being a precursor of the >105 kDa EndoH-resistant form, would be preferentiallyexpressed intracellularly, and the >105 kDa form would be preferentially targeted to the cellsurface. However, Park et al. (2002) had previously demonstrated that the 67 kDa form waspresent on the surface of cells infected with v-UL18. Additionally, labelling of cell surfaceproteins with biotin confirmed that both forms of gpUL18 were equally well targeted to thesurface of RAdUL18-infected cells. As both forms of gpUL18 could be expressed at the cellsurface, they could both be involved in intercellular signalling. The extensive anddifferential glycosylation of gpUL18 could also result in gains of function by promotinglectin interactions. For example, high mannose forms of HIV gp120 generated by growth inPBMC, but not macrophages, promote virion binding to DC-SIGN on dendritic cells andDC-SIGNR on endothelial cells (Lin et al., 2003). Neither the 67 kDa nor the >105 kDaform of gpUL18 was found to be secreted or in purified HCMV virions (not shown).Furthermore, gpUL18 was not identified in a systematic proteomic analysis of HCMVvirions (Varnum et al., 2004), consistent with it being a non-structural protein. Despite theresults with viral vectors, surface expression of gpUL18 has yet to be demonstrateddefinitively in the context of HCMV infection. While some binding of UL18-specific mAbto the surface of HCMV-infected cells was detected, comparable binding was obtained usingthe △UL18 mutant in parallel. While gpUL18 may be expressed on the surface of HCMV-infected cells, its low level, weak immunogenicity and the co-expression of HCMV-encodedantibody-binding proteins (Keller et al., 1976), all hamper detection.

When using a vaccinia virus vector to express UL18, it was not down-regulated incontinuous cell lines stably transfected with US2, US3, US6 or US11 genes (Park et al.,2002). However, when both UL18 and US11 were co-expressed using adenovirus vectors,UL18 expression was clearly suppressed by US11 (not shown), the higher levels of US11possibly pushing the equilibrium towards suppression of gpUL18. It was, therefore,important to investigate the effects of US2, US3, US6 and US11 on UL18 expression morecautiously, during a productive HCMV infection. While deletion of US2–US11 in HCMVRV798 resulted in a typical up-regulation of endogenous classical HLA-I, relative touninfected cells, it had no obvious effect on gpUL18 during the late phase of infection.Whilst US2–US11 may yet prove capable of modulating gpUL18 expression, this genecluster clearly preferentially targets endogenous classical HLA-I in fibroblasts. Great careshould clearly be taken in using HCMV US2–US11 deletion mutants in NK cell functionalassays because of significant changes in both cellular classical HLA-I and non-classicalHLA-E levels, uncharacteristic of conventional HCMV infections.

The migration of the EndoH-resistant gpUL18 form varied when expressed using differentvector systems. The extreme level of gpUL18 glycosylation potentially makes it a sensitiveindicator of cellular processes involved in modifying N-linked oligosaccharide side chains.While there is no indication that adenovirus, HCMV or vaccinia virus encodeglycosyltransferases (Markine-Goriaynoff et al., 2004), HCMV infection has been shown toup-regulate transcription of multiple glycosyltransferases and the expression of the selectinligand component of the Lewis and sialyl-Lewis antigen in endothelial cells (Cebulla et al.,2000). Furthermore, glycosylation of PVR is modified in HCMV-infected fibroblasts(Tomasec et al., 2005). We speculate that the host cell’s glycosylation systems may bemodified during lytic infection with vaccinia virus and HCMV.

If gpUL18 can get to the surface it should be able to bind LIR-1 in trans and impart asuppressive signal to an NK or CD8+ effector cell. Analysing the role of UL18 in eitherstimulating or suppressing NK or T cell recognition is not straightforward. HCMV encodes



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multiple genes that have the potential to impact on the cellular immune system. NK cells area heterogeneous population expressing a complex and variable array of activating andinhibitory receptors. By using a replication-deficient adenovirus vector to express UL18efficiently on the cell surface of human fibroblasts it should now be possible to identify andcharacterize NK cell clones that are either inhibited or activated by gpUL18, and test themback against HCMV-infected cells.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThe authors are most grateful to the Amgen Corporation, Seattle, for generously providing the mAb M71, TomJones (Wyeth Research, Pearl River, New York) for providing the HCMV mutant RV978 and Lynne Neale (DeptMedical Microbiology, Cardiff) for low passage HCMV clinical isolates. Technical support was kindly provided byS. Llewellyn-Lacey (MRC Co-operative Tissue Culture Facility). This work was funded by a Division of HospitalSpecialities PhD Studentship and the Wellcome Trust.

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Fig. 1.Immunoblot showing expression of gpUL18 using a vaccinia virus vector (v-UL18). HFFFswere infected with v-Ctrl or v-UL18, or mock infected. At 4 h p.i. the cells were labelledwith [35S]methionine for 6 h. Cell lysates were divided into three equal parts andimmunoprecipitated with mouse IgG (mIg), the gpUL18 specific mAb M71 (M71) orwithout primary antibody (–). Both a fast- (single arrowhead) and slow-migrating (doublearrowhead) form of gpUL18 were apparent.



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Fig. 2.Immunoblot showing a time course of the expression of gpUL18 using a vaccinia virusconstruct (v-UL18). (a) HFFFs were infected with v-Ctrl or v-UL18. At 4 h p.i. the cellswere starved in methionine-free medium, pulse-labelled for 15 min with [35S]methionineand chased with cold methionine for the indicated times. Immunoprecipitations wereperformed using anti-UL18 mAb M71 or anti-HLA-I mAb W6/32. (b) One half of eachsample was also treated with EndoH.



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Fig. 3.Expression of the >105 kDa gpUL18 form was enhanced by β2m. (a) Immunoblot of celllysates prepared from HFFFs infected with v-UL18, or co-infected with v-UL18 and v-β2mas indicated. Cell lysates were divided into three equal parts and either left mock treated (–),or digested with EndoH (+E) or PNGaseF (+P). gpUL18 was detected using the mAb M71.(b) HFFFs were infected with v-UL18 or v-Ctrl alone, or co-infected with v-UL18 and v-β2m as indicated. Immunoprecipitation was performed using the mAb M71, one half of eachsample was then treated with EndoH.



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Fig. 4.UL18 expression using a recombinant adenoviral vector (RAdUL18). Immunofluorescenceof human fibroblasts infected with RAdUL18 when labelled with (a) control murinepolyclonal antibody or (b) the gpUL18-specific mAb M71. (c) Immunoblotting experimentusing extracts of HFFFs infected with either RAdCtrl or RAdUL18. Extracts were dividedinto three equal parts and either left mock treated (–), or digested with EndoH (+E) orPNGaseF (+P). gpUL18 was detected using the mAb M71.



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Fig. 5.gpUL18 expression on the cell surface. (a) HFFFs were infected with RAdUL18 orRAdCtrl, and at 72 h p.i. stained using the gpUL18 mAb 10C7. The flow cytometryhistogram shows expression of gpUL18 on the surface of RAdUL18 infected cells. MouseIgG was used as a negative control. (b) A proportion of cells was also analysed byimmunoblotting, using the mAb M71, showing the expression of gpUL18. Cell surfaceproteins of RAdUL18- or RadCtrl-infected fibroblasts were biotinylated and purified withstreptactin-sepharose; total cell extracts were prepared simultaneously. Extracts wereanalysed by immunoblotting using (c) anti-HSP40 antibody or (d) the gpUL18-specific mAbM71. (e) Histogram showing expression of gpUL18; NP human fibroblasts were analysedexactly as in (a). (f) Immunoblotting analysis showing expression of gpUL18; NP cells wereanalysed exactly as in (b).



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Fig. 6.gpUL18 expression in HCMV-infected cells. (a) Immunoblot of HFFFs infected withHCMV AD169 or △UL18 at 96 h p.i., using mAb M71. (b) Immunoblot of HFFFs infectedwith HCMV AD169, Merlin or Toledo at 96 h p.i., using mAb M71.



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Fig. 7.gpUL18 expression in HCMV-infected cells. (a) HFFFs were mock infected or infected withHCMV AD169, and extracts were prepared at the times (h p.i.) indicated. Immunoblot usingmAb M71 showing the time course of gpUL18 expression during productive infection. (b)At no time after infection, was it possible to detect gpUL18 on the surface of HCMV-infected cells. The histogram shows the 96 h time point and illustrates the non-specificstaining of mAb 10C7 to the infected cells. Grey shading, HCMV AD169.



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Fig. 8.The effect of HCMV US2, US3, US6 and US11 on gpUL18 and HLA-I expression. HFFFswere infected with HCMV AD169, △UL18 or RV798 (△US2–US11). (a) Immunoblotusing mAb M71 demonstrating that US2–US11 expression during productive HCMVinfection had no apparent effect upon gpUL18 expression. (b) Cell surface staining, usingmAbs W632, and (c) immunoblot assays, using mAb mAbs W632, showing US2–US11expression during productive HCMV infection had a significant effect upon cell surfaceHLA-I. (d) Immunoblot, using the mAb MEM-E/02, showing HLA-E synthesis wassignificantly increased following infection with HCMV AD169, but not the US2–US11mutant RV798.



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Characterization of a highly glycosylated form of the human cytomegalovirus HLA class I homologue gpUL18