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1 Infectious Diseases, Animal Health Trust, Newmarket, Suffolk CB8 7UU, UK
2 The University of Queensland, Clinical Medical Virology Centre and Royal Children's Hospital, Sir Albert Sakzewski Virus Research Centre, QLD 4072, Australia
Correspondence
H. E. Farrell
h.farrell1{at}uq.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). In order to maintain a stable relationship with their host, the CMVs have evolved various mechanisms to manipulate normal cellular responses, in particular innate and adaptive immune responses (reviewed by Mocarski, 2002).
Among the viral gene products that are predicted to be linked to CMV dissemination are seven transmembrane receptors (7TMR) that signal via coupling to G proteins. Leukocyte trafficking is regulated by the concerted responses of multiple cellular 7TMR, both in response to infection and during normal cellular homeostasis (Le et al., 2004) and thus, the CMV 7TMR homologues have been implicated in usurping this role during infection to promote virus dissemination. Additional roles for CMV 7TMR include chemokine sequestration, membrane fusion and production of an intracellular environment favourable for virus replication (Vischer et al., 2006).
Human cytomegalovirus (HCMV) encodes four 7TMR: UL33, UL78, US27 and US28. Of these, US28 has been the most extensively characterized, both pharmacologically and with respect to its intracellular trafficking properties. US28 has been shown to bind both CC and CX3C chemokines with high affinity, resulting in the activation of multiple intracellular activation pathways (Kuhn et al., 1995). In addition to agonist-induced signalling, US28 exhibits constitutive signalling and endocytosis, which appear to be modulated by the binding of fractalkine (Casarosa et al., 2001). Unlike most cellular chemokine receptors, US28 is located predominantly in late and recycling endosomes, rather than at the cell surface, which support the suggestion that it plays a role in sequestration of cellular chemokines (Bodaghi et al., 1998; Fraile-Ramos et al., 2001). While constitutive endocytosis of US28 has been shown to occur independently of β-arrestin proteins, it is nevertheless internalized, at least in part, via a clathrin-mediated pathway (Fraile-Ramos et al., 2003). Studies with UL33 and US27 have demonstrated that, like US28, they co-localize with endocytic vesicles. Notably, the localization of UL33 and US27 in endocytic vesicles in HCMV-infected cells has been shown to overlap with intracellular membranes containing HCMV glycoproteins important for virion maturation, consistent with the incorporation of these viral 7TMR in the virion envelope (Fraile-Ramos et al., 2002).
It has been suggested that dimerization/oligomerization of 7TMR influences their intracellular trafficking. Homo- and hetero-dimerization has been reported for a variety of 7TMR, and has been shown to play an important role in 7TMR biogenesis and transport to the cell surface. While some reports have suggested that dimerization/oligomerization is induced at the cell surface by the binding of ligand, others have demonstrated oligomer formation early in the biosynthetic pathway, within the endoplasmic reticulum (Bulenger et al., 2005). Higher molecular mass species consistent with 7TMR dimers have been identified for HCMV US27, US28 and for UL33, suggesting that their biogenesis may be similar to that of their cellular 7TMR counterparts (Fraile-Ramos et al., 2001; Margulies & Gibson, 2007).
In contrast to US28, UL33 and US27, little is known about the biochemical and intracellular trafficking properties of the UL78 family members, which, of the betaherpesvirus 7TMR homologues, are the least conserved with chemokine receptors. UL78 counterparts in human herpesvirus (HHV)-6 and HHV-7, encoded by U51, have been shown to bind β-chemokines, resulting in stimulation or modulation of signal transduction (Milne et al., 2000; Tadagaki et al., 2005; Fitzsimons et al., 2006). Studies with HHV-6 U51 have shown that it promotes cell fusion mediated by the G protein of vesicular stomatitis virus, consistent with a possible role for U51 in promoting cell–cell spread of virus (Zhen et al., 2005). In contrast to U51, the ligand(s) for the CMV UL78 members have not yet been identified. Clues to the contribution of the UL78 gene family to the virus life cycle have come from studies of rodent CMV homologues, M78 of MCMV and R78 of rat cytomegalovirus. Gene knockout experiments have shown that both M78 and R78 contribute to efficient cell–cell spread of these viruses in vitro and replication in target organs during acute and persistent stages of infection in vivo (Beisser et al., 1999; Oliveira & Shenk, 2001). The in vitro effects of M78 on MCMV replication were linked to an increase in immediate-early (IE) mRNA, which was observed in cells infected with wild-type MCMV, but not by a MCMV mutant with M78 deleted (Oliveira & Shenk, 2001). As M78 was identified in semi-purified virions, it was further postulated that M78 delivered to the cell upon virus entry facilitates IE mRNA accumulation (Oliveira & Shenk, 2001). Additional characterization of this phenomenon, in particular whether M78 mediates this activity as a G-protein-coupled receptor (GPCR), has not been reported.
We examined the cellular localization of MCMV M78 in both transfected and MCMV-infected cells to determine whether M78 shares features with US28 and cellular GPCRs. We produced anti-peptide antiserum specific for M78 and constructed N- and C-terminal tagged forms of M78, utilizing either green fluorescent protein (GFP) fused to the C-terminal (intracellular) domain of M78, or the influenza haemagglutinin (HA) epitope fused to the N-terminal (extracellular) domain. We demonstrated that, like US28, M78 was rapidly and constitutively endocytosed from the cell surface. Furthermore, both M78 and US28 were endocytosed by routes utilized by transferrin (Tfn) and cholera toxin subunit B (CTxB), markers for clathrin-dependent and lipid raft/caveolae-mediated pathways, respectively (Yamashiro et al., 1984; Pelkmans & Helenius, 2002). In MCMV-infected cells, the localization of M78 with markers of the secretory and early endosomal pathways at 5 h post-infection (p.i.) was markedly diminished at 16 h p.i., suggesting a temporal shift in M78 trafficking.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Virus.
The K181 (Perth) strain of MCMV was used in these studies. The virus titre was quantified on MEF as described previously (Allan & Shellam, 1984).
Receptor constructs.
M78 was PCR amplified from the HindIII ‘C’ fragment of a K181 (Perth) genomic library and was cloned into pcDNA3 via introduced EcoRI flanking sites. M78 was cloned in-frame into pEGFP-N1 and pCMV-HA (Clontech) to produce M78GFP and HA-M78, respectively. The fidelity of expression constructs was confirmed by sequencing. The CD4-US28 and US28GFP plasmids were provided by M. Marsh (UCL, UK) and have been described previously (Fraile-Ramos et al., 2001, 2002). The HA-CCR5 plasmid was purchased from the UMR cDNA Resource Centre, University of Missouri-Rolla, USA.
Antibodies.
The mouse anti-huCD63 was obtained from M. Marsh (UCL, UK) and has been described previously (Fraile-Ramos et al., 2001). Mouse monoclonal antibodies to paxillin, EEA-1 and GM130 were obtained from BD Transduction Laboratories. Polyclonal rabbit antisera against HA and GFP were obtained from Abcam. Mouse monoclonal antibodies (IgG1) against the HA epitope tag (designated HA7) and CD4 were obtained from Sigma. Mouse IgG1 isotype controls were obtained from Dako. Hoechst nucleic acid counterstain, Alexa Fluor488/594 conjugates of goat anti-mouse (GAM) or goat anti-rabbit (GAR) antibodies and Alexa Fluor594 conjugates of human Tfn and CTxB were obtained from Molecular Probes.
Rabbit antisera to M78 were produced to a mixture of the following M78 peptides: CAVDYSYPEVDAEHL, SRLFEMRYSARDGT and YSDGGEKEGVQGDEG, corresponding to segments of the N terminus, third intracellular loop and C terminus of M78, respectively. Pre-immunization serum was used for controls in Western blotting and immunofluorescence studies. Preliminary experiments established that the anti-M78 antiserum was able to detect M78 in permeabilized cells, but not in non-permeabilized cells.
Transient transfection.
COS-7 cells seeded in six-well dishes (2x105 cells per well) were transfected with M78 constructs and Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 24 h post-transfection, cells (from duplicate wells for each sample) were harvested and washed in PBS and cell pellets were stored at –80 °C before processing. Untransfected COS-7 cells were used as controls.
MCMV infection.
MEF seeded in six-well dishes (106 cells per well) were infected at the indicated m.o.i. In order to block late gene expression, MCMV infections were performed in the presence of 200 µg phosphonoacetic acid (PAA) ml–1 and the cells were harvested at 23 h p.i. All harvested cells were washed with PBS and the pellets were stored at –80 °C prior to Western blotting. Mock-infected cells were used as controls for both immunofluorescence and Western blotting.
Cell lysis.
Cell pellets were resuspended in 200 µl solubilization buffer [20 mM Tris-HCl, 150 mM NaCl, 1 % sodium deoxycholate, 1 % NP-40, 2 mM EDTA, 10 mM iodoacetamide, supplemented with a protease inhibitor cocktail (Roche)] at 4 °C and incubated for 20 min on ice. Samples were centrifuged at 17 900 g for 15 min at 4 °C and the supernatants were retained.
SDS-PAGE.
Cell lysates were mixed 1 : 1 with 2x reducing sample buffer [SB-R; 125 mM Tris-HCl, 20 % glycerol (v/v), 4 % SDS, 0.2 % bromophenol blue and 200 mM DTT] and loaded directly (without heating) onto 10 % or 4–15 % polyacrylamide gels (Bio-Rad). Equivalent volumes of diluted samples were loaded per lane and duplicate gels were stained with Coomassie blue for total protein detection.
Immunoblotting.
Following SDS-PAGE, protein was transferred to nitrocellulose, blocked with PBS-T (PBS/0.05 % Tween 20) containing 5 % BSA, and probed with the designated primary antibodies. Following extensive washing with PBS-T, membranes were probed with the designated horseradish peroxidase (HRP)-conjugated secondary antibodies; bound antibody was visualized with an enhanced chemiluminescence (ECL) kit, using ECL hyperfilm (Amersham Biosciences). Protein separation was visualized with rainbow pre-stained markers (Bio-Rad). Biotinylated protein markers (Bio-Rad) in conjunction with avidin-HRP (Bio-Rad) were used for size estimation on ECL blots.
Immunofluorescence detection of M78.
HeLa cells seeded in eight-well chamber slides (8x104 cells per well; Labtek) were transfected with the receptor constructs. At 24 h post-transfection, the cells were washed with PBS, fixed with 3 % paraformaldehyde and, where designated, permeabilized with 0.2 % (v/v) Triton X-100. For MCMV-infected cells, MEF seeded on glass coverslips (2x105 cells per well) were infected with MCMV at the indicated m.o.i. and processed as described above at the designated times p.i. Expression of M78GFP was visualized directly. For detection of HA-M78 and untagged M78, cells were blocked for 1 h at 37 °C with 5 % normal goat serum in PBS, and subsequently incubated at 37 °C for 1 h with mouse anti-HA or rabbit anti-M78, respectively. The cells were then washed and incubated with GAM/GAR antibodies conjugated to Alexa Fluor488 or Alexa Fluor594. Cell nuclei were counterstained with Hoechst reagent, and cells were mounted with Vectashield (Vector Laboratories).
To determine whether M78 was expressed at the cell surface, HA-M78-transfected cells were either permeabilized or left intact and labelled with rabbit anti-HA and an intracellular protein marker (mouse anti-paxillin) followed by GAR488 and GAM594. A lack of detection of paxillin in non-permeabilized cells was used to confirm cell membrane integrity.
M78 internalization studies.
At 24 h post-transfection, HeLa cells were washed with 4 °C chilled PBS, and incubated with mouse anti-HA in binding medium (BM; MEM containing 10 mM HEPES and 0.2 % BSA) for 2 h at 4 °C to label the cell surface pool of receptors. Subsequently, the cells were transferred to 37 °C for 0, 5, 10, 30 and 60 min to permit endocytosis. After each time point, cells were washed three times with 4 °C chilled PBS, prior to fixation and permeabilization as described above. Anti-HA-labelled receptors were subsequently visualized by detection with GAM488.
Co-localization studies.
HeLa cells were incubated with rabbit anti-HA in BM for 1 h at 37 °C at 24 h post-transfection to permit endocytosis. Cells were washed three times with 4 °C chilled PBS, prior to fixation and permeabilization as described above. Cells were subsequently incubated for 1 h at room temperature with mouse monoclonal antibodies to either early or late endosomal markers [EEA-1 (Patki et al., 1997) or CD63 (Metzelaar et al., 1991), respectively]. Bound primary antibodies were detected with GAR488 and GAM594. For experimental controls, transfected HeLa cells were incubated with normal rabbit serum (NRS) and isotype control mouse antibodies in place of the primary antibodies.
Co-localization of endocytosed HA-M78 and CD4-US28 with Tfn and CTxB, was determined by incubating HeLa cells with Tfn594 (50 µg ml–1) or CTxB594 (8 µg ml–1) together with either mouse anti-HA (for HA-M78) or mouse anti-CD4 (for CD4-US28) for 1 h at 37 °C prior to fixation and permeabilization. To deplete intracellular Tfn levels prior to the addition of Tfn594, cells were incubated for 30 min at 37 °C in BM. Anti-HA or anti-CD4 antibodies were detected with GAM488. For control samples, normal mouse serum was used in place of the primary antibody.
Co-localization of M78 in MCMV-infected MEF (3 p.f.u. per cell) was assessed with markers of the cis-Golgi (GM130; Nakamura et al., 1995) or early endosomes (EEA-1) on glass coverslip cultures at 5 or 16 h p.i. Cells were co-incubated with the rabbit anti-M78 and mouse monoclonal antibodies to either GM130 or EEA-1 for 1 h at 37 °C. Bound primary antibodies were detected with GAR488 and GAM594. To assess co-localization of M78 with Tfn594 or CTxB594 the infected cells were labelled with Tfn594 or CTxB594 as described above. The cells were then fixed, permeabilized and incubated with rabbit anti-M78 followed by GAR488. Uninfected MEF were included as controls. In addition, NRS and mouse isotype control antibodies were tested in MCMV-infected cells to confirm that the secondary antibody binding was specific.
Image capture.
An Axioskop microscope (Zeiss) was used for fluorescence studies. The microscope was equipped with a three-filter unit (supplied by Digital Scientific), for analysis of fluorescent proteins with red, green and blue emission spectra. Single-channel images were captured and assembled using SmartCapture2 digital analysis software (Digital Scientific).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and the duplicate set was stained with Coomassie blue to verify that similar total protein amounts were obtained from each set of transfected cells (data not shown). The predicted molecular masses of M78, HA-M78 and M78GFP, are 51.5, 52.8 and 78.4 kDa, respectively. The Western blots detected a major band for untagged M78 corresponding approximately to the predicted monomeric molecular mass. The HA-M78 band ran at a slightly slower mobility than the untagged form, consistent with the addition of the small 9 aa tag. In the case of M78GFP, the putative monomer ran at higher mobility than expected, corresponding to a molecular mass of 
60–65 kDa. It should be noted, however, that since the samples were not heated prior to loading, they may not have been fully denatured, which may have resulted in anomalous migration of particular protein species. Indeed, additional minor protein bands were detected on some blots, in particular for the M78GFP construct, which may have been due to incomplete denaturation or degradation products.
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). Bands corresponding to the putative monomers and dimers were observed in each of the singly transfected lysates. In the co-transfected lysate, an additional band, migrating at a position consistent with the expected mobility of M78–M78GFP dimers, was detected. This intermediate species was absent from the mixed lysate sample, suggesting that the M78–M78GFP dimers formed within the co-transfected cells, rather than as an artefact during sample preparation and SDS-PAGE. In the co-transfected sample, the band corresponding to the M78GFP–M78GFP dimer was only weakly detected, but whether this was a consequence of the formation, stability or detection of this species being less efficient than that of the M78–M78GFP dimer is not known. Western blot analysis of M78 from MCMV-infected cells demonstrated the detection of putative M78 monomers and dimers from MEF harvested as early as 6 h p.i. using the rabbit anti-M78 sera (Fig. 1c). These studies confirmed that the putative dimers were present in MCMV-infected cell lysates and therefore unlikely to be an artefact attributable to high level expression in transfected COS-7 cells. M78 was also detected in MEF infected with MCMV in the presence of PAA, confirming previous reports that M78 is expressed with early kinetics (Oliveira & Shenk, 2001). Further studies are required to confirm that M78 dimerization occurs within the cell and, if so, whether this has functional significance. The mobilities of the M78 protein species detected by Western blotting (transfected cells) were unaffected in the presence of tunicamycin, consistent with the absence of potential N-linked glycosylation sites for M78 (data not shown).
Intracellular localization of M78 in transfected cells
Most functional G protein-coupled 7TMR are located at the cell surface. Generally, following binding of ligand by the extracellular face and triggering of G-protein-mediated signalling, the GPCR is endocytosed and dissociated from ligand prior to recycling to the cell surface (Hanyaloglu & von Zastrow, 2008). Studies of several viral 7TMR have demonstrated variable degrees of cell-surface versus intracellular distribution; for example, in the absence of ligand HCMV US28 displays a predominantly intracellular distribution within endocytic organelles, in contrast to most cellular chemokine receptors (Fraile-Ramos et al., 2001).
Analysis of M78 expressed in either transfected HeLa or MCMV-infected MEF indicated that the majority of the M78 protein was located intracellularly within the perinuclear region of permeabilized cells with punctate staining that is consistent with a vesicular distribution (Fig. 2a–d). Notably, a similar intracellular distribution was observed for untagged M78 (Fig. 2c, d) as for the N- or C-terminally tagged counterparts (Fig. 2a, b), suggesting that the tags did not interfere with the normal distribution of M78. A similar intracellular distribution was observed for US28GFP in transfected HeLa and MEF cells (Fig. 2e, f). In permeabilized cells, there was little detection of M78 near the cell-surface of transfected or infected cells (Fig. 2a–d and Fig. 3c). Nevertheless, HA-M78 was readily detected in non-permeabilized cells, which confirmed that M78 was competent for trafficking to the cell surface (Fig. 3d).
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; Signoret et al., 2000). As expected for HA-CCR5, internalization was negligible across the full incubation period (Fig. 4). This contrasted with the observations for HA-M78, where punctate intracellular staining of M78 was observed within 5 min of the 37 °C shift and by 10 min it appeared that most of the labelled surface pool was internalized. No staining was observed for untransfected controls (results not shown).
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). While M78 was shown to co-localize with the early endosomal marker EEA-1, co-localization with the late endocytic marker CD63, was not observed, suggesting that there was negligible transfer of M78 to late endosomes/lysosomes during the 1 h antibody feeding period (Fig. 5). Alternatively, it is possible that the HA-tagged M78 protein is masked in lysosomes, resulting in the lack of detection. Endocytosed M78 and US28 co-localized with Tfn594 consistent with the use of the clathrin-mediated pathway. However, both M78 and US28 also co-localized with CTxB594, suggestive of internalization via clathrin-independent mechanisms, although both M78+ and US28+ vesicles lacking the CTxB marker were also evident (Fig. 5). While co-localization with CTxB594 provides some indication that receptor internalization occurs through lipid raft/caveolae-mediated mechanisms, a degree of overlap of this marker with endosomes associated with clathrin-mediated endocytosis has been reported. It should be noted that internalization of CTxB has been shown to be mediated by lipid raft/caveolae in HeLa cells that bind high, rather than low levels of CTxB (Pang et al., 2004). For this reason, observations of co-localization with this marker were restricted to HeLa cells with high levels of CTxB594. For US28, inhibition of clathrin-mediated endocytosis using small interfering RNA (siRNA) has been shown to inhibit US28 internalization (Fraile-Ramos et al., 2003), although the isolation of US28 from detergent-resistant cellular membranes is suggestive of an endocytic route normally associated with lipid rafts or the caveolae-dependent pathway (Droese et al., 2004). While the results of the M78 and US28 co-localization studies presented here are consistent with the usage of both endocytic pathways, further studies using specific inhibitors of clathrin-dependent and -independent pathways are required to determine the endocytic itineraries of these receptors.
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) and may be delivered to cells upon virus entry. Any such virion-associated M78 was below the threshold for detection in our studies, since infected cells were negative for M78 immunofluorescence at 2 h p.i. (results not shown). At early times p.i., the cytoplasmic punctate distribution of M78 was accompanied by prominent perinuclear co-localization with GM130, consistent with high levels of M78 expression via the secretory pathway (Fig. 6). However, at late times p.i., the perinuclear distribution of M78 was much reduced. Similarly, co-localization of M78 with EEA-1 was prominent at 5 h p.i., although M78-containing vesicles that lacked these markers were also observed. However, at 16 h p.i., co-localization of M78 with EEA-1 was negligible. The reactivity of control rabbit serum or mouse isotype control (IgG1) antibodies with mock-infected and MCMV-infected MEF was negligible (results not shown). Co-localization of M78 with anti-CD63 was also investigated; however, the anti-CD63 IgG2b isotype was shown to non-specifically bind to MCMV-infected cells (results not shown). Taken together, these results suggest that M78 distribution in the cis-Golgi and early endosomal compartment is temporally affected, which may be due either to altered trafficking of M78 (for example, the incorporation of M78 into the virion envelope at sites of virion maturation at late times p.i.) or as an indirect consequence of potential perturbations to these organelles at late times in MCMV infection.
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) or in MEF infected for 5 h (results not shown) was negligible. At late times p.i. some co-localization of Tfn594 with M78 was observed (Fig. 7b). For CTxB594, uptake was observed in both infected and uninfected MEF (Fig. 7a) and minor co-localization with M78 was observed at both early (results not shown) and late times p.i. (Fig. 7b). These results demonstrate that the level of recycling endosomes was increased following MCMV-infection, and that M78 co-localized, at least in part, with these organelles. In comparison, CTxB594 uptake was unaffected by MCMV infection and M78 trafficking via vesicles positive for CTxB594 was demonstrated.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 12 June 2008;
accepted 10 September 2008.
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