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Division of Virology, Department of Pathology, University of Cambridge, UK
Correspondence
Philip G. Stevenson
pgs27{at}cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Immunology–Vaccinology, Faculty of Veterinary Medicine, University of Liège, Liège, Belgium. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Much less is known of how herpesviruses evade pre-formed antibody. We are using murid herpesvirus 4 (MuHV-4) to define molecular mechanisms behind the epidemiologically evident resistance of herpesviruses to neutralization (Xu et al., 1996). One important factor may be antibody-coated virions exploiting host Fc receptors for uptake when normal cell binding is blocked (Rosa et al., 2007). This critically requires that viral membrane fusion remains intact. Thus, a key question is how membrane fusion avoids inhibition by antibody.
One possibility is that the fusion machinery remains hidden on cell-free virions, much as conformation changes in the human immunodeficiency virus gp120 restrict antibody access until after cell binding (Chen et al., 2005). Herpes simplex virus (HSV) entry is initiated by conformation changes in glycoprotein D (gD) (Fusco et al., 2005; Krummenacher et al., 2005), an alphaherpesvirus-specific addition to the core fusion complex (Spear & Longnecker, 2003). The inhibitory effects of gp150 on MuHV-4 infection of cells with low glycosaminoglycan (GAG) expression (de Lima et al., 2004) and of gp350 on Epstein–Barr virus infection of epithelial cells (Shannon-Lowe et al., 2006) suggest that gammaherpesvirus entry may be triggered similarly. However, gD, gp150 or gp350 could hardly protect the whole, multi-protein entry machinery. Their engagement is probably just the first of several conformation changes in virion glycoproteins that cumulate in membrane fusion. Understanding how each glycoprotein changes in the context of infection should tell us the limits imposed on antibody-mediated neutralization.
We have focused on gB, the most conserved component of herpesvirus membrane fusion (Turner et al., 1998). The HSV gB structure (Heldwein et al., 2006) provides a template for understanding these proteins as a whole. Comparison with vesicular stomatitis virus glycoprotein G (VSV-G) (Roche et al., 2006, 2007) suggests that herpesvirus gBs might adopt distinct conformations during entry, with the solved structure a downstream form.
Although gB is exposed on MuHV-4 virions (Lopes et al., 2004), it presents a very difficult neutralization target (Gillet et al., 2006). As with other herpesviruses (Ohlin et al., 1993; Holloway et al., 1998; Akula et al., 2002; Okazaki et al., 2006), the gB N terminus is a neutralization target for MuHV-4 (Gillet et al., 2006). However, this neutralization requires IgM monoclonal antibodies (mAbs), which are rare in MuHV-4 carriers, and even then remains incomplete. In order to understand how gB is exposed to antibody, we used conformation-specific mAbs to track its antigenicity during viral entry. By keeping to the context of infectious virions, we preserved important interactions between gB and other virion glycoproteins such as gH (Gillet & Stevenson, 2007a). We found evidence of a dramatic gB conformation shift that sheds new light on how herpesviruses resist neutralization.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were all grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 2 mM glutamine, 100 U penicillin ml–1, 100 µg streptomycin ml–1 and 10 % fetal calf serum (PAA Laboratories). 293T cells were transfected with the GPI-linked gB extracellular domain or domains derived from it (Gillet et al., 2006) by using FuGENE 6 (Roche Diagnostics). All viruses were derived from a cloned MuHV-4 bacterial artificial chromosome (Adler et al., 2000). Virions were harvested from infected BHK-21 cell supernatants by ultracentrifugation; infected-cell debris was removed by low-speed centrifugation (May et al., 2005a). Of note, rigorous removal of infected-cell debris was critical for clean immunofluorescence data. In some experiments, we purified virions further on Ficoll gradients, but this made no difference to the data obtained.
mAbs.
mAbs were derived from MuHV-4-infected BALB/c mice at least 3 months post-infection by fusion of spleen cells with NS0 cells (Köhler & Milstein, 1975). The mAbs used in this study are listed in Table 1. All glycoprotein-specific mAbs were selected first by their capacity to recognize virus-infected cells; each was then typed for its target glycoprotein (Gillet et al., 2007a).
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Immunofluorescence.
Cells were plated onto coverslips overnight, then exposed to MuHV-4 virions (3 p.f.u. per cell). After three washes in PBS to remove unbound virions, the cells were fixed in PBS with 4 % paraformaldehyde (30 min) and permeabilized with 0.1 % Triton X-100 (15 min). Viral glycoproteins were detected with murine mAbs plus either Alexa 488- or Alexa 568-conjugated goat anti-mouse IgG (Invitrogen) or a combination of Alexa 488- or Alexa 633-conjugated goat anti-mouse IgG1 and Alexa 568-conjugated goat anti-mouse IgG2a. None of the MuHV-4 mAbs used for immunofluorescence gave detectable staining of uninfected cells. Lysosome-associated membrane protein 1 (LAMP-1) was detected with the rat mAb 104B (BD Pharmingen) and Alexa 488- or Alexa 568-conjugated goat anti-rat IgG (Invitrogen). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole). Fluorescence was visualized with a Leica confocal microscope imaging single 1 µm sections, except for Figs 2 and 3, when we used an Olympus IX70 microscope plus a Retiga 2000R camera line (QImaging).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The relative efficiencies with which such mAbs recognized each form of gB varied (Fig. 1a). MG-2C10, a neutralizing mAb that recognizes a linear epitope near the MuHV-4 gB N terminus (Gillet et al., 2006), showed less difference between native and recombinant gB than did MG-4D11, which recognizes an epitope C-terminal to the gB furin-cleavage site (gB-C). BN-1A7 recognizes an epitope N-terminal to the gB furin-cleavage site (gB-N) (Fig. 2
) and was similar to MG-2C10. MG-1A12, which requires both gB-N and gB-C for recognition, was similar to MG-4D11. Thus, native and recombinant gB were antigenically distinct. BHK-21 cells infected with vaccinia virus expressing gB–GPI (Gillet et al., 2007b) showed staining similar to that of CHO-gB cells (data not shown). The BN-1A7 and MG-1A12 staining patterns were each observed with at least eight different gB-specific mAbs. All of those mapping to gB-N were similar to BN-1A7.
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), so we first bound the virions at 4 °C and then shifted the temperature to 37 °C to compare pre- and post-endocytosis (Fig. 1b). Single cells are shown for optimal resolution. In Fig. 1, as in subsequent figures, each cell shown is fully representative of at least 75 % of the total examined (n>100).
mAb MG-4D11 recognized gB both before and after endocytosis. In contrast, BN-1A7 recognized gB strongly at the cell surface and poorly after endocytosis, whilst MG-1A12 recognized endocytosed gB only. Virion gB therefore changed its antigenicity during entry into NMuMG cells. Other cell lines showed the same change (Fig. 3). It occurred after endocytosis, as BN-1A7 staining still co-localized with an invariant epitope defined by mAb MG-4A1 in peripheral endosomes, whereas MG-1A12 staining co-localized with MG-4A1 only in more central endosomes (Fig. 1c; see also Fig. 4b). This difference in co-localization made it clear that BN-1A7 and MG-1A12 recognize gB at different stages of entry. The complete shift in virion gB during entry from BN-1A7+MG-1A12– to BN-1A7–MG-1A12+ argued that these forms are mutually exclusive. Thus, gB-transfected and MuHV-4-infected cells, which were each recognized by both mAbs, must each express both forms, but in different proportions.
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). The domain II equivalent of the structurally analogous VSV-G (its domain III) occupies a comparable position in its post-fusion conformation (Roche et al., 2006), but is more exposed in the pre-fusion form, where it contributes much of the likely receptor-binding site (Roche et al., 2007). A similar pre-fusion exposure of gB domain II would be consistent with the cell binding of soluble gB-N (Gillet et al., 2007c) and with gB-N-specific mAbs, such as BN-1A7, preferentially recognizing pre-fusion gB (Fig. 1); the post-endocytic loss of BN-1A7 staining would reflect domain II moving to a more dependent position in post-fusion gB. It therefore seemed likely that gB has pre- and post-fusion conformations, much like VSV-G (we keep to the convention of ‘post-fusion’ being the fusion-competent form of a glycoprotein, although the VSV-G and gB crystal structures include neither ligand nor lipid and so are not strictly post-fusion). The main alternative explanation for one gB conformational epitope disppearing at the same time as another appears – an elaborate shift in an antigen-masking protein from one site on gB to another – seems very unlikely. The only MuHV-4 glycoproteins known to associate with gB are gH/gL and gp150 (Gillet & Stevenson, 2007a). Virions lacking gL, which display a markedly different gH conformation from the wild type (Gillet et al., 2007d), or lacking gp150 showed exactly the same changes in gB antigenicity (data not shown).
The gB conformation change occurs close to membrane fusion
How does the gB conformation change relate to membrane fusion? mAb MG-12B8 defines an MuHV-4 capsid epitope that is inaccessible on intact virions, but revealed once they have uncoated (Gillet et al., 2006). The MG-12B8 epitope appears when incoming virion glycoproteins reach the late endosomes/lysosomes marked by LAMP-1 (Marsh et al., 1987) (Fig. 4a). This coincides with the change in gB antigenicity (Fig. 4b): MG-1A12 staining co-localized with LAMP-1, whereas BN-1A7 staining was evident only in peripheral, LAMP-1– endosomes. The gB conformation change therefore occurs close in time and place to membrane fusion. This implied that BN-1A7+ gB engages in binding, whilst MG-1A12+ gB mediates fusion.
The gB conformation change is pH-dependent
The delay in MuHV-4 capsid release until virions reach LAMP-1+ endosomes (Fig. 4a) suggests that membrane fusion requires a low pH. This was confirmed by inhibitors of lysosomal acidification blocking both infection (Fig. 5a) and capsid release (Fig. 5b). Virion glycoproteins (gN in Fig. 5b) were still endocytosed and reached LAMP-1+ endosomes, but the BN-1A7 gB epitope was preserved and now co-localized with LAMP-1, whereas the MG-1A12 epitope failed to appear. Thus, the gB conformation change was pH-dependent and again linked to membrane fusion. NH4Cl also blocked the conformation change but, like chlorpromazine, mainly blocked endocytosis (Fig. 6).
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). Thus, exposure to a pH of 4 increased MG-1A12 gB staining, but without much change in BN-1A7 staining. The number of gB molecules changing was evidently much lower than in normal infection (Fig. 1b). This argues that ligand engagement or another aspect of the endosomal environment contributes to the gB switch. Whilst not necessarily sufficient, low pH was clearly necessary.
Neutralization is limited to IgMs that recognize gB-N
MG-2C10 is representative of several IgM mAbs that recognize the gB N terminus and block infection at a post-binding step (Gillet et al., 2006). In three further fusions, we identified three more gB-specific neutralizing mAbs (in addition to re-isolating MG-2C10-like mAbs) (Fig. 7a). Again, all were IgMs. None of 85 gB-specific IgGs identified in the same fusions gave significant neutralization; Fig. 7(a) shows three examples. mAb reduction confirmed that a pentameric structure was crucial for neutralization (Fig. 7b). Neutralization was not a general property of IgMs, as gp70-specific IgMs failed to neutralize (Fig. 7a).
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). The new IgMs recognized a different epitope than MG-2C10. Thus, they reduced BN-1A7 binding much more than MG-2C10 did and had no effect on MG-15F6 binding, whereas MG-2C10 inhibited MG-15F6 markedly (Fig. 7d) (based on transfected gB truncation mutants, MG-15F6 recognition requires residues 13–30 after the predicted gB signal sequence cleavage, but not residues 2–13; MG-2C10 requires residues 2–6). The failure of gB-N-specific IgGs such as BN-1A7 and MG-15F6 to neutralize, even though their recognition sites overlapped with those of neutralizing IgMs, emphasized further that neutralization requires an IgM isotype. Indeed, the more abundant gB-N-specific IgGs in immune sera could conceivably outcompete gB-N-specific IgMs to impair neutralization.
The gB conformation change is difficult for antibodies to block
As with MG-2C10 (Gillet et al., 2006), BN-6E1, BH-6B5 and BH-8F4 blocked neither cell binding nor virion endocytosis. Fig. 7(e) shows data for BH-6B5; the other mAbs were equivalent. They partly inhibited the gB conformation switch – BH-6B5 was the most effective, retaining some BN-1A7+ gB. However, unlike bafilomycin or concanamycin A (Fig. 5b), they failed to block it completely – Fig. 7(e) shows that MG-1A12+ gB still appeared. Thus, once virions get to late endosomes, the gB conformation switch seems to be hard for antibodies to block. As gB-N incorporates the putative gB fusion loops (Heldwein et al., 2006), the gB-N-specific IgMs seemed more likely to neutralize by hindering fusion sterically. This would explain why they must be IgMs, as the much larger size of IgMs would vastly increase their scope for steric hindrance.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We have shown here that gB changes its antigenicity during viral entry, consistent with a shift between cell-binding and pro-fusion conformations. The gB N-terminal domains, which participate in cell binding (Gillet et al., 2007c), were more accessible on pre-fusion gB. Their reduced accessibility after endocytosis was consistent with the conformation change described for VSV-G, with the solved gB structure (Heldwein et al., 2006) being its ‘post-fusion’ form. The gB conformation switch probably precedes actual fusion, as GPI-linked gB readily adopted the ‘post-fusion’ form.
Low pH was important for the gB conformation switch. This may explain why the MuHV-4 gB is a poor neutralization target (Gillet et al., 2006). A requirement for low pH would also explain why MuHV-4, like Kaposi's sarcoma-associated herpesvirus (KSHV) (Akula et al., 2003), fails to fuse with plasma membranes. Plasma-membrane fusion is possible with transfected KSHV glycoproteins (Pertel, 2002), and transfected MuHV-4 gB could adopt its pro-fusion form (Fig. 1a). However, this is a very different setting from infection. In the absence of other virion glycoproteins, transfected gB may switch its conformation before reaching the cell surface, perhaps in the acidic environment of the trans-Golgi network (Demaurex et al., 1998).
Although the cell-binding gB N-terminal domains are accessible on MuHV-4 virions, these virions readily escape from infected cells, suggesting that the binding site itself is hidden. Moreover, gB-N–Fc binds to a non-GAG ligand, but MuHV-4 cell binding is highly GAG-dependent (Gillet et al., 2007c). gp150 regulates MuHV-4 binding to a non-GAG cellular ligand (de Lima et al., 2004; Gillet et al., 2007c), as gp150 knockouts are much less GAG-dependent than the wild type (also, virions lacking gp150 do have a problem escaping from infected cells). The obvious explanation would be that gp150 covers the gB-N binding epitope until displaced by GAGs. Thus, gB has 2-fold protection: masking by gp150 and a post-endocytic conformation change. This protection allows efficient virion release and hides gB from antibody.
Many mechanisms of virion neutralization have been described (Klasse & Sattentau, 2002). By far the most common and certainly the most clearly defined is a block to receptor binding (Knossow & Skehel, 2006). However, the capacity of herpesviruses such as MuHV-4 to escape from endosomes and infect Fc receptor-positive cells productively (Rosa et al., 2007) forces neutralization to act at post-binding steps, such as membrane fusion. Such neutralization must cope with the dramatic conformation changes characteristic of viral fusion proteins (Knossow & Skehel, 2006). If antibody can bind only to the upstream form of a glycoprotein, because its switch is secluded, neutralization will be limited to steric hindrance – binding common epitopes close to the fusion loops if such exist – or to blocking the switch itself. Blocking the gB conformation switch was evidently hard, presumably because the pre-fusion conformation becomes highly unstable in late endosomes. Also, steric hindrance seems to require an IgM isotype, which is rare in steady-state B-cell responses. The gB conformation change therefore helps MuHV-4 to evade neutralization.
A post-endocytic gB conformation switch readily explains why gB-directed MuHV-4 neutralization is more difficult than for HSV (Bender et al., 2007) or human cytomegalovirus (Speckner et al., 1999). In the typical setting of neutralization assays, HSV and cytomegalovirus fuse at the plasma membrane (Compton et al., 1992; Browne et al., 2001); conformation-switched gB is therefore accessible. Neutralizing mAbs have accordingly been mapped to prominent positions on post-fusion gB (Speckner et al., 1999; Heldwein et al., 2006; Bender et al., 2007). In contrast, mAbs specific for post-fusion MuHV-4 gB, such as MG-1A12, never neutralized. HSV and cytomegalovirus can also infect via endocytosis (Clement et al., 2006; Ryckman et al., 2006) and this route may be harder for gB-specific mAbs to block. However, a more important consideration is where neutralization might act in the viral life cycle. Herpesviruses mostly spread within their hosts via cell–cell contacts (Peeters et al., 1993; Dingwell et al., 1994). These probably exclude antibody (Roth & Compans, 1980). Thus, antibody reduces the intra-host lytic spread of HSV, mainly by Fc receptor-dependent effector functions (Balachandran et al., 1982; Rector et al., 1982; Kohl et al., 1986); Fc inhibition, in turn, increases intra-host spread (Nagashunmugam et al., 1998). Neutralization is more relevant to cell-free virions, whose key role is inter-host spread. Virions are exposed to antibody when they exit an immune host. However, once they reach a new, naive host, the only antibody capable of blocking infection is that already attached. Epitopes revealed by conformation changes are therefore safe: as with endocytic uptake, they are effectively secluded from pre-formed antibody. Once transmission has occurred, cell–cell spread again takes over; the window for effective neutralization is shut.
Fusion-complex conformation changes do not act alone in antibody evasion. The highly immunogenic gp150 both drives Fc receptor-dependent infection and, through its immunodominance, suppresses the production of antibodies that might otherwise neutralize (Gillet et al., 2007a). The gB N terminus is protected by O-glycosylation and, in turn, protects a key epitope on gH/gL (Gillet & Stevenson, 2007b). Such mechanisms complement that described here by reducing the exposure of upstream forms of virion fusion proteins to antibody. For MuHV-4 at least, much of the complexity of herpesvirus entry makes sense as antibody evasion.
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ACKNOWLEDGEMENTS |
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Received 15 October 2007;
accepted 7 February 2008.
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