1 Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, UK
2 Princeton University, 301 Schultz Laboratory, Washington Road, Princeton, NJ 08544, USA
Correspondence
Colin M. Crump
cmc56{at}mole.bio.cam.ac.uk
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ABSTRACT |
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INTRODUCTION |
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gM is one of the few glycoproteins to be conserved throughout the entire family Herpesviridae. Despite this conservation, gM is classed as a non-essential protein in alphaherpesviruses because viruses with gM deletions are viable in cell culture. For members of all three herpesvirus subfamilies, gM has been shown to form a disulphide-linked complex with the gene product of UL49·5 (or homologues). In pseudorabies virus (PRV), human cytomegalovirus, EpsteinBarr virus (EBV) and human herpesvirus-8 (HHV-8), this gene product is glycosylated and is termed gN (Jons et al., 1996; Koyano et al., 2003
; Lake et al., 1998
; Mach et al., 2000
). In contrast, the corresponding UL49·5 gene products of HSV-1 (termed UL49A), bovine herpesvirus 1 (BoHV-1) and equine herpesvirus 1 (EHV-1) do not appear to be glycosylated and so are referred to by their gene names (UL49A or UL49·5; Adams et al., 1998
; Liang et al., 1996
; Rudolph et al., 2002
).
Even though the gM/N complex is defined as non-essential, reports in the literature suggest potentially important roles for gM/N in viral assembly and egress. Disruption of gM coding sequences in HSV-1, PRV, EHV-1, BoHV-1 and infectious laryngotracheitis virus (ILTV) have all been reported to reduce viral titres and plaque size (Baines & Roizman, 1991; Dijkstra et al., 1996
; Fuchs & Mettenleiter, 1999
; Konig et al., 2002
; MacLean et al., 1991
, 1993
; Osterrieder et al., 1996
). Furthermore, disruption of gN (or the relevant homologue) coding sequences in PRV, EHV-1, varicella-zoster virus and EBV have also been reported to reduce viral titres, penetration or assembly to varying degrees (Jons et al., 1998
; Lake & Hutt-Fletcher, 2000
; Ross et al., 1997
; Rudolph et al., 2002
). Interestingly, in both PRV and EHV-1 it has been shown that, even though deletion of gM alone causes only mild defects in virus production, very severe defects in secondary envelopment are observed when gM is deleted in combination with gE and gI (Brack et al., 1999
; Seyboldt et al., 2000
). Taken together, these data suggest that the gM/N complex may function, at least in a redundant fashion, in the final envelopment of herpesviruses.
Included in the set of glycoproteins that need to be incorporated into the final envelope of alphaherpesviruses are the essential glycoproteins gB, gD, gH and gL. It has previously been shown that expression of these essential envelope glycoproteins from HSV-1 are necessary and sufficient to induce membrane fusion in cell-culture systems (Turner et al., 1998). Interestingly, the gM or gM/N complexes from PRV, ILTV and EHV-1 have been shown to inhibit the membrane fusion mediated by the PRV essential glycoproteins in a transfection-based assay (Klupp et al., 2000
). Furthermore, gM/N complexes from HSV-1 and HHV-8 have been shown to inhibit membrane fusion caused by the HSV-1 essential glycoproteins in similar assays (Koyano et al., 2003
). PRV gM demonstrated somewhat different characteristics to the gM molecules from the other herpesviruses in that PRV gM was able to inhibit membrane fusion in the absence of gN, whereas gM from EHV-1, ILTV, HSV-1 and HHV-8 all required the presence of their respective gN homologues (Klupp et al., 2000
; Koyano et al., 2003
). The inhibitory activity of gM on membrane fusion activity appears to have broad specificity, as PRV gM inhibits bovine respiratory syncytial virus (BRSV) F protein-induced membrane fusion, and both HSV-1 and HHV-8 gM/N complexes inhibit Moloney murine leukaemia virus (MoMLV) Env protein-induced membrane fusion (Klupp et al., 2000
; Koyano et al., 2003
). Furthermore, expression of BRSV F protein from recombinant BoHV-1 virions did not lead to the formation of syncytia until the gM gene was disrupted in this virus, suggesting that BoHV-1 gM expression also caused inhibition of BRSV F protein-mediated fusion (Konig et al., 2002
).
We were interested in the mechanisms by which gM inhibits transfection-based membrane fusion and whether such activities of gM could correlate with a potential role of gM in the final envelopment of herpesviruses. Here we present evidence that gM/N from HSV-1 and gM from PRV cause a relocalization of several membrane proteins from the plasma membrane to the TGN, including the herpesvirus envelope proteins gD and gH/L. The mechanism by which this relocalization occurs appears to involve internalization and suggests that gM can inhibit cellcell fusion by removing fusogenic proteins from the cell surface. These data also suggest that the gM/N complex may be involved in the correct localization of viral envelope proteins to sites of secondary envelopment.
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METHODS |
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Production of rabbit polyclonal antisera to PRV gM (Ab183) and HSV gM (Ab980).
The antigen for Ab183 was a combination of two GST fusion proteins: GSTgMtail (GST fused to aa 342393 of PRV Becker strain gM) and GSTgMlooptail (GST fused to aa 3676 followed by three glycines and aa 342393 of PRV gM). GSTgMtail and GSTgMlooptail were expressed in Escherichia coli, purified on glutathioneagarose using standard protocols, combined in a 2 : 1 ratio and injected into rabbits for the production of polyclonal antisera. The antigen for Ab980 was GST fused to the C-terminal 132 aa of HSV-1 strain F gM. Antigen injections and serum purification of Ab183 and Ab980 were performed by ProSci.
Construction of expression plasmids.
Plasmids expressing HSV-1 gB, gD, gH and gL and LacZ have been described previously (Harman et al., 2002). Plasmids encoding HVEM (pBEC10) and nectin-2
(pMW20) have been described previously (Montgomery et al., 1996
; Warner et al., 1998
). A pcDNA3 plasmid encoding CD8
was constructed by subcloning the CD8
coding sequence from the pS84 vector (from S. Munro, Laboratory of Molecular Biology, Cambridge, UK) into the HindIII/XbaI sites in pcDNA3. A pcDNA3 plasmid encoding influenza virus strain PR8 HA was constructed by subcloning the HA coding sequence from pJZ102 (Young et al., 1983
) into the HindIII site in pcDNA3. pcDNA3 plasmids encoding HRSV F protein (Long strain; pI17-F) and PRV gM (Kaplan strain) have been described previously (Bembridge et al., 2000
; Klupp et al., 2000
). A plasmid encoding GFPPRV gM was generated by excising the PRV gM coding sequence from pcDNA3-PRVgM using EcoRI/XhoI and ligating into pEGFPC3 (Clontech). A sequence containing the HSV-1 gM coding region was excised from the HSV-1 genome (strain 17; nt 23 09825 190) with PvuI, end repaired and ligated into pMV11 cut with SmaI. The entire gM coding region was excised from pMV11 by digestion with BamHI/EcoRI and ligated into pcDNA3. To construct a plasmid encoding GFPHSV-1 gM, a BglII restriction site was introduced upstream of the gM coding region (at a site corresponding to nt 23 112) by site-directed mutagenesis. The gM coding region was excised using BglII and ligated into pEGFPC1. A sequence containing the HSV-1 UL49A coding region was excised from the HSV-1 genome (strain 17; nt position 106378107023) with BsrBI/BamHI, end repaired and ligated into pRK19 cut with SmaI. The UL49A coding region was excised using BamHI/EcoRI and ligated into pcDNA3. A pcDNA3 plasmid encoding a dominant-negative form of AP180 (AP180-C; Ford et al., 2001
; Zhao et al., 2001
) was constructed by excising the myc epitope-tagged AP180-C coding region and promoter region from pCMVmyc-AP180-C (a gift from B. Nichols, LMB, Cambridge, UK) with FspI/NotI and ligating into NruI/NotI-cut pcDNA3.
Transfection-based fusion assay.
Fusion assays using HSV-1 gB, gD, gH and gL were performed as previously described (Harman et al., 2002). Expression plasmids encoding PRV gM, HSV-1 gM, HSV-1 UL49A or LacZ were co-transfected with the gB, gD, gH and gL expression plasmids as required. Fusion assays with HRSV F protein were performed in an identical manner but replacing expression plasmids for HSV-1 gB/D/H/L with a plasmid expressing HRSV F protein.
Fluorescence microscopy.
Transfected COS-7 cells were washed with PBS at 1624 h post-transfection and fixed with 4 % paraformaldehyde in PBS with 1 mM CaCl2, 1 mM MgCl2 for 20 min at room temperature. Cells were washed three times in immunofluorescence (IF) wash buffer [1 % (v/v) FCS, 0·1 % (v/v) Triton X-100 in PBS] followed by permeabilization in IF wash buffer supplemented with 0·5 % Triton X-100 for 10 min at room temperature. After additional washing, cells were incubated with appropriate primary antibodies in IF wash buffer, washed and incubated with the appropriate FITC- or Alexa Fluor 594-conjugated secondary antisera diluted in IF wash buffer. Following additional washing, coverslips were mounted on glass slides using Slowfade (Molecular Probes). Cells were examined using a Nikon Optiphot II epi-fluorescence microscope equipped with a Bio-Rad MRC 1024 confocal laser-scanning attachment and x100 objective. Images were processed using Adobe Photoshop software.
Antibody internalization assay.
The method used to assess the internalization of cell-surface proteins was similar to that described by Roquemore & Banting (1998). Briefly, 1624 h post-transfection, COS-7 cells were incubated with the appropriate primary antibody in PBS at 37 °C in a humidified chamber for 1 h. Cells were then washed three times in PBS and fixed with 100 % methanol at 20 °C for 5 min. Following additional washing, cells were incubated with appropriate fluorescently labelled secondary antisera in IF wash buffer. Coverslips were mounted on glass slides and cells examined by confocal microscopy as described above. Quantification of internalization assays were performed by counting fields of cells, with those showing internalized antisera in a TGN-like pattern scored as positive and cells showing only a cell-surface signal scored as negative.
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RESULTS |
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Topology, trafficking motifs and subcellular localization of gM
Herpesvirus gM is a highly hydrophobic protein that is predicted to contain eight transmembrane domains with cytoplasmic N and C termini, a potential N-glycosylation site within the first extracellular domain and a conserved cysteine residue within the same loop, which has been predicted to form a disulphide bond with the relevant gN homologue (Fig. 2; Dijkstra et al., 1996
; Jons et al., 1998
). The predicted C-terminal cytoplasmic domain of gM contains two classes of potential membrane-trafficking motifs: a tyrosine-based motif and an acidic cluster motif. Tyrosine-based motifs mediate the incorporation of membrane proteins into transport vesicles due to interaction with adaptor complexes AP-1, -2, -3 and -4 (Kirchhausen, 1999
), while acidic cluster motifs are known to interact with the connector protein PACS-1, which is involved in transport from endosomes to the TGN (Crump et al., 2001
; Wan et al., 1998
). The presence of these trafficking motifs in all gM sequences suggests that gM could have the capacity to traffic to and from the plasma membrane. To determine the subcellular distribution of gM in transfected cells, cells expressing PRV gM, HSV-1 gM or HSV-1 gM plus UL49A were analysed by immunofluorescence. Both PRV gM and HSV-1 gM localized to a juxtanuclear compartment where they showed significant co-localization with TGN46, a marker of the TGN (Fig. 3
). These data suggested that gM from both PRV and HSV-1 could localize to the TGN independently of other herpesvirus proteins and that HSV-1 gM localization was not affected by UL49A co-expression.
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DISCUSSION |
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In an effort to determine the mechanisms by which gM inhibits HSV-1 gB/D/H/L-mediated membrane fusion, we examined the effects of gM on the subcellular localization of these herpesvirus glycoproteins. Consistent with their membrane fusion inhibition activity, we observed that PRV gM and HSV-1 gM plus UL49A caused both gD and gH/L to be relocalized from the cell surface to an intracellular compartment, probably the TGN. This change in the localization of gD and gH/L was at least partly due to the internalization of these membrane proteins through clathrin-mediated endocytosis. Previous reports have suggested that binding of antisera to PRV gD can stimulate the internalization of this glycoprotein in PRV-infected cells (Favoreel et al., 2000). In the present study, it seemed highly unlikely that the observed internalization of gD and gH/L monoclonal antibodies was due to a similar antibody-induced endocytosis, as internalized signals were observed only in the presence of PRV gM or HSV-1 gM/UL49A and not in control cells. The other component of the fusion apparatus, HSV-1 gB, was found to be predominantly localized to an intracellular, juxtanuclear compartment, irrespective of gM expression (unpublished observations). These observations are unsurprising given the presence of internalization motifs within the cytoplasmic domain of gB and the known localization of herpesvirus gB isoforms to the TGN (Crump et al., 2003
; Fan et al., 2002
). The stoichiometry of gB, gD and gH/L required at the cell surface to stimulate membrane fusion is currently unknown, but taken together these data suggest that membrane fusion inhibition by gM/N involves perturbation of this stoichiometry by the removal of gD and gH/L from the cell surface and transport to an intracellular compartment, where they are no longer available to stimulate cellcell fusion.
We also demonstrated that PRV gM and HSV-1 gM plus UL49A were able to cause relocalization of other non-herpesvirus glycoproteins, such as HRSV F protein, from the cell surface to a TGN-like compartment. These observations correlated with data presented here and by others showing that PRV gM and HHV-8 and HSV-1 gM/N can inhibit fusion induced by human or bovine RSV F protein and MoMLV Env protein (Klupp et al., 2000; Koyano et al., 2003
). Thus, the broad specificity exhibited by gM/N in terms of fusion inhibition is reflected in the ability of gM/N to remove a broad range of proteins from the cell surface.
The data presented in this report demonstrate that PRV gM and HSV-1 gM/UL49A are capable of altering the localization of a very broad range of membrane proteins. However, as the subcellular localization of CD8 or nectin-2
was unaffected by gM/N expression, the mechanism by which gM/N relocalizes glycoproteins is unlikely to be due to a global increase in membrane traffic events. How gM/N can stimulate changes in the subcellular localization of such a wide variety of membrane proteins is currently unclear. Given the presence of potential internalization motifs conserved within the C-terminal cytoplasmic domain of all herpesvirus gM sequences available to date, it is tempting to speculate that gM/N may physically interact with, for example, gD and cause subsequent internalization and intracellular targeting of the complex. If so, then it must be assumed that interactions would have no direct sequence requirements given that such a wide variety of proteins are affected. However, the possibility that gM could be stimulating the relocalization of such a diverse array of membrane proteins by an indirect mechanism cannot be discounted.
The ability of gM/N to cause localization of the herpesvirus envelope proteins gD and gH/L to the TGN could be part of the mechanism by which herpesviruses maintain sufficient concentrations of envelope proteins in the secondary envelopment compartment, thus allowing efficient assembly and viral egress. Such a possibility correlates well with observations showing that disruption of the gM gene in PRV and disruption of the gM or UL49·5 gene in EHV-1, together with the absence of the gE/I complex in these viruses, led to dramatic defects in secondary envelopment (Brack et al., 1999; Rudolph et al., 2002
). However, the redundant requirements of alphaherpesvirus envelope proteins for correct secondary envelopment is complicated by the observation that HSV-1 lacking gD and the gE/I complex also shows significant defects in secondary envelopment and egress (Farnsworth et al., 2003
). It has also been recently reported that the myristoylated tegument protein encoded by PRV UL11 is involved in secondary envelopment (Kopp et al., 2003
). The different roles played by the membrane proteins gM, gE, gI, gD, UL11 and possibly other viral proteins in controlling secondary envelopment in the various alphaherpesviruses await further study.
Herpesviruses have evolved many immune evasion mechanisms, including several aimed at inhibiting antigen presentation (Yewdell & Hill, 2002). It is conceivable that one of the advantages of gM-dependent removal of herpesvirus envelope proteins from the cell surface may be to avoid the recognition of infected cells by immune surveillance systems. Interestingly, deletion of gM in the RacH vaccine strain of EHV-1 has been shown to increase the immunogenicity of this vaccine in mice (Osterrieder et al., 2001
). Whether this increase in immunogenicity is due to an increase in cell-surface levels of viral glycoproteins in infected cells remains to be determined.
In summary, we have demonstrated that PRV gM and the HSV-1 gM/UL49A complex causes relocalization of the herpesvirus envelope proteins gD and gH/L to a TGN-like compartment, at least partially through endocytosis. These data correlate well with the ability of gM/N to inhibit membrane fusion in transfection-based assays and also the proposed role of gM/N in the secondary envelopment of herpesviruses. Furthermore, the specificity of gM/N for the relocalization of membrane proteins is very broad but not ubiquitous. The mechanisms by which gM/N may stimulate these membrane-trafficking events will pose intriguing questions for further studies.
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ACKNOWLEDGEMENTS |
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Received 14 June 2004;
accepted 25 August 2004.