1 Section of Infection and Immunity, Cardiff University, Tenovus Building, Heath Park, Cardiff CF14 4XX, UK
2 Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK
Correspondence
Gavin W. G. Wilkinson
WilkinsonGW1{at}cf.ac.uk
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ABSTRACT |
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Four figures showing the mobility of highly glycosylated forms of UL18, UL18 sequence alignments and a time course of gpUL18 surface exprssion in HCMV-infected cells are available as supplementary material in JGV Online.
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INTRODUCTION |
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UL18 was identified as a HLA-I homologue during the analysis of the strain AD169 sequence (Beck & Barrell, 1988) and, like HLA-I, was found to form a trimeric complex with
2-microglobulin (
2m) and endogenous peptides (Browne et al., 1990
; Fahnestock et al., 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 then found 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-fold higher affinity for gpUL18 than for HLA-I molecules, thus even low levels of gpUL18 could be expected to compete efficiently for binding (Chapman et al., 1999
). The prediction that gpUL18 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
) by experimental data in published studies. LIR-1 expression is restricted to myeloid cells, B cells, and subpopulations of T and NK cells (Young et al., 2001
). Interestingly, the proportion 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 stimulated non-HLA-restricted killing by CD8+ cytotoxic T lymphocytes, via an interaction with LIR-1, although the mechanism responsible for enhanced recognition and its role in HCMV infection have yet to be elucidated.
UL18 is non-essential for HCMV replication in vitro (Browne et al., 1992), and is transcribed 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 detect gpUL18 in HCMV-infected cells, and a failure to generate stable cell lines expressing the UL18 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 expressed efficiently and was detected as a 67 kDa glycoprotein (Park et al., 2002
). In this study, we have further characterized gpUL18 in the context of productive HCMV infection, and also when expressed using vaccinia virus (v-UL18) and replication-deficient adenovirus (RAdUL18) vectors. The previously reported 67 kDa form of gpUL18 is shown here to be sensitive to digestion with endoglycosidase-H (EndoH), whereas a novel, EndoH-resistant form of gpUL18 migrating with an apparent molecular mass in excess of 105 kDa is described.
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METHODS |
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Immunofluorescence and flow cytometry.
Cells on glass coverslips were washed in PBS and fixed with 2 % paraformaldehyde for 15 min at room temperature. The cells were washed in PBS and then, to ensure sustained permeabilization 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 µg ml1) 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 the coverslips, 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 a Hamamatsu ORCA-ER camera and Improvision Openlab software.
One-colour flow cytometry was used to detect surface gpUL18 or HLA-I expression. Cells were washed twice with cold PBS, 2 % FCS and incubated for 30 min at 4 °C with monoclonal 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 used as a negative control. The cells were washed twice with cold PBS, 2 % FCS and then labelled with FITC-conjugated anti-mouse Fab fragments (Sigma) on ice for 30 min. Cells were washed twice with cold PBS, and fixed with 2 % paraformaldehyde. A total of 10 000 events were collected by the FACSCalibur cytometer and analysed with CellQuest Pro software (BD Biosciences).
Immunoblotting.
To prepare extracts, cells were first washed with PBS, before being resuspended in NP-40 lysis buffer (1 % NP-40, 150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 8) supplemented with P8340 protease inhibitor cocktail (Sigma). Cells were lysed during an incubation for 30 min, on ice, and clarified by centrifugation for 30 min at 13 000 r.p.m. in a Heraeus Biofuge Fresco at 4 °C. For glycosidase treatments, samples were adjusted to 0·05 % SDS, 0·1 % -mercaptoethanol, and protein denatured by heating at 100 °C for 10 min, before being digested 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 the manufacturer's instructions (NEB). Enzyme was omitted from mock-digested samples. All samples were boiled after the addition of SDS-PAGE loading buffer, and proteins separated 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 transfer at 100 V for 1 h using a Bio-Rad mini trans-blot system. The membrane was washed with Tris-buffered saline (TBS), blocked for 16 h at 4 °C in 5 % BSA, TBST, and for a further 8 h at 4 °C in Superblock (Pierce), before being incubated with mAbs specific for gpUL18 (M71; 2·5 µg ml1), HLA-I (HC10; 1 : 200 dilution) or HLA-E (1 : 200 dilution; Serotec) for 16 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 room temperature. Supersignal substrate (Pierce) was used to detect the HRP signal and the membrane exposed to Kodak BioMax MR film.
Immunoprecipitation.
HFFFs were infected with v-UL18 or v-Ctrl (m.o.i. of 10) for 6 h, then cultured in methionine-free medium for 30 min. Cells were labelled with 50 µCi (1·85 MBq) [35S]methionine ml1 (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 were resuspended in NP-40 lysis buffer (1 % NP-40, 1 % protease inhibitor (Sigma), 150 mM NaCl, 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 for 30 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 then washed, and the complexed proteins released by resuspending the beads in SDS denaturing buffer (NEB) and heating to 100 °C. Where indicated, precipitated proteins were digested with EndoH or PNGaseF, and then subjected to SDS-PAGE and fluorography.
Biotinylation of cell surface proteins.
Cells were washed in cold PBS then incubated according to the manufacturer's instructions for 45 min at 4 °C with 0·5 mg sulfo-NHS-biotin ml1 (Pierce). Cells were washed and lysed in NP-40 buffer, and the lysates cleared by centrifugation. Biotinylated proteins were captured with streptactin-sepharose (IBA), and washed with NP-40 and RIPA (150 mM NaCl, 50 mM Tris pH8, 0·5 % deoxycholate, 0·1 % SDS, 1 % NP-40) buffers. Bound proteins were eluted in SDS denaturing buffer and analysed by immunoblotting.
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RESULTS |
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gpUL18 expression during an HCMV infection
Analysis of UL18 during a productive HCMV infection is problematical due to a combination of extremely low gpUL18 levels and confounding antibody interactions with virus-encoded Fc receptors. Nevertheless, human fibroblasts infected with HCMV-AD169 or the AD169UL18 mutant (
UL18) for 96 h (m.o.i. of 10) were analysed by immunoblotting. gpUL18 levels were much lower in HCMV-infected cells than those observed with v-UL18- or RAdUL18-infected cells; however, both gpUL18 species, 67 kDa and
160 kDa, were evident (Fig. 6
a). To generate AD169
UL18, the Escherichia coli lacZ was inserted within the UL18-encoding gene (Browne et al., 1992
).
-Galactosidase was expressed at such high levels from the HCMV
2·7 early promoter (Spaete & Mocarski, 1987
) that it was detected by a non-specific antibody interaction (Fig. 6a
).
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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 (95·1100 % identity) between strains, with there being no sequence-predicted defect associated with strain AD169 to account for its low level of expression (see Supplementary Figs S2 and S3 available in JGV online). When assayed directly, levels of gpUL18 expression, gpUL18 migration on gels and the existence of dual forms of the protein were similar in cells infected with HCMV AD169, Toledo or Merlin (Fig. 6b
). When expressed from either strain AD169 or Toledo, the 67 kDa gpUL18 was again susceptible to EndoH digestion, whereas the
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 detected at 24 h post-infection (p.i.) and increased in abundance to reach a peak by 72 h p.i. The levels were then sustained through the late phase of infection. The
160 kDa form of gpUL18 appeared at 48 h p.i. and also reached peak levels by 72 h p.i (Fig. 7
a). gpUL18 was thus expressed most efficiently at a time when endogenous HLA-I had been effectively eliminated. At no point during this time-course assay were we able to detect gpUL18 on the cell surface of HCMV-infected cells (Fig. 7b
, Supplementary Fig. S4 available in JGV online). A major effort was taken to optimize staining conditions to enable detection of gpUL18 on the surface of cells undergoing lytic HCMV infection, as this was considered central 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 observed when using cells infected with the
UL18 mutant, and was thus consistent with an enhanced level of non-specific fluorescence associated with HCMV infection (Fig. 7b
).
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DISCUSSION |
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During productive HCMV infection, we were only able to demonstrate expression of gpUL18 by immunoblotting, at the detection limit of the assay. By flow cytometry, gpUL18 could not be detected on the cell surface. Higher levels of gpUL18 were obtained by the use of 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 enhanced levels of transcription. The synthesis and processing of the fully glycosylated gpUL18 appeared to be exceptionally slow and inefficient when compared to endogenous HLA-I. A requirement for limiting chaperone functions to stabilize the UL18 polypeptide undergoing glycosylation and maturation within the ER may have contributed towards its inefficient expression. Elevating the levels of 2m has previously been shown to promote cell surface expression of
2m and gpUL18 (Browne et al., 1990
), and we have shown here that the availability of
2m was a factor affecting the rate of synthesis of the >105 kDa form of gpUL18. In contrast to HLA-I, gpUL18 was expressed well in both TAP-1 positive or negative fibroblasts, suggesting that TAP-dependent peptide loading may not be essential for gpUL18 maturation, or sufficient levels of gpUL18-binding peptide can be scavenged independently of TAP. In this context, it is interesting to note that gpUL18 expression had previously been shown to be susceptible to down-regulation by HSV-1 ICP47 but not HCMV US6 (Park et al., 2002
), both genes being inhibitors of TAP function. Also, peptide binding is thought not to be required for the interaction with LIR-1 (Chapman et al., 1999
; Willcox et al., 2003
).
To act as a ligand for LIR-1 on NK or alternative target cells, gpUL18 must be present on the 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 kDa gpUL18, being a precursor of the >105 kDa EndoH-resistant form, would be preferentially expressed intracellularly, and the >105 kDa form would be preferentially targeted to the cell surface. However, Park et al. (2002)
had previously demonstrated that the 67 kDa form was present on the surface of cells infected with v-UL18. Additionally, labelling of cell surface proteins with biotin confirmed that both forms of gpUL18 were equally well targeted to the surface of RAdUL18-infected cells. As both forms of gpUL18 could be expressed at the cell surface, they could both be involved in intercellular signalling. The extensive and differential glycosylation of gpUL18 could also result in gains of function by promoting lectin interactions. For example, high mannose forms of HIV gp120 generated by growth in PBMC, but not macrophages, promote virion binding to DC-SIGN on dendritic cells and DC-SIGNR on endothelial cells (Lin et al., 2003
). Neither the 67 kDa nor the >105 kDa form 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 HCMV virions (Varnum et al., 2004
), consistent with it being a non-structural protein. Despite the results with viral vectors, surface expression of gpUL18 has yet to be demonstrated definitively in the context of HCMV infection. While some binding of UL18-specific mAb to the surface of HCMV-infected cells was detected, comparable binding was obtained using the
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-encoded antibody-binding proteins (Keller et al., 1976
), all hamper detection.
When using a vaccinia virus vector to express UL18, it was not down-regulated in continuous 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 US11 possibly pushing the equilibrium towards suppression of gpUL18. It was, therefore, important to investigate the effects of US2, US3, US6 and US11 on UL18 expression more cautiously, during a productive HCMV infection. While deletion of US2US11 in HCMV RV798 resulted in a typical up-regulation of endogenous classical HLA-I, relative to uninfected cells, it had no obvious effect on gpUL18 during the late phase of infection. Whilst US2US11 may yet prove capable of modulating gpUL18 expression, this gene cluster clearly preferentially targets endogenous classical HLA-I in fibroblasts. Great care should clearly be taken in using HCMV US2US11 deletion mutants in NK cell functional assays because of significant changes in both cellular classical HLA-I and non-classical HLA-E levels, uncharacteristic of conventional HCMV infections.
The migration of the EndoH-resistant gpUL18 form varied when expressed using different vector systems. The extreme level of gpUL18 glycosylation potentially makes it a sensitive indicator of cellular processes involved in modifying N-linked oligosaccharide side chains. While there is no indication that adenovirus, HCMV or vaccinia virus encode glycosyltransferases (Markine-Goriaynoff et al., 2004), HCMV infection has been shown to up-regulate transcription of multiple glycosyltransferases and the expression of the selectin ligand 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 be modified 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 a suppressive signal to an NK or CD8+ effector cell. Analysing the role of UL18 in either stimulating or suppressing NK or T cell recognition is not straightforward. HCMV encodes multiple genes that have the potential to impact on the cellular immune system. NK cells are a heterogeneous population expressing a complex and variable array of activating and inhibitory receptors. By using a replication-deficient adenovirus vector to express UL18 efficiently on the cell surface of human fibroblasts it should now be possible to identify and characterize NK cell clones that are either inhibited or activated by gpUL18, and test them back against HCMV-infected cells.
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ACKNOWLEDGEMENTS |
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Received 21 April 2005;
accepted 10 August 2005.
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