Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK1
Author for correspondence: Helena Browne. Fax +44 1223 336926. e-mail hb100{at}mole.bio.cam.ac.uk
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The mechanistic details of this process remain to be defined, specifically in terms of how these four molecules interact with cell-surface receptors and with each other to mediate membrane fusion. A number of different molecules have, however, been identified that mediate the entry of alphaherpesviruses into cells by virtue of their ability to bind gD (Krummenacher et al., 1998 ; Nicola et al., 1998
; Whitbeck et al., 1997
). These are molecules of some structural diversity, including a member of the TNF/NGF receptor family (Hve A), members of the immunoglobulin superfamily, Hve B and Hve C (or nectin 2 and nectin 1, respectively), and a modified form of heparan sulphate (Shukla et al., 1999
). Previous studies have addressed the requirements for some of these gD-binding proteins for both cell-to-cell spread and syncytium formation. Terry-Allison et al. (1998)
, showed that Hve A participates in cellcell fusion as well as virus entry. In addition, Cocchi et al. (2000)
have reported that cell-to-cell spread of wild-type strains of HSV is mediated by nectin 1, while nectin 2 works for viruses with a mutation in gD. They also concluded that syncytium formation does not involve nectin 1. Cellular receptors for gB and gHL have yet to be identified. Although our understanding of the receptor requirements for fusion remains rudimentary, it has previously been reported that cell-surface glycosaminoglycans (GAGs) may play a role in the fusion process. Shieh & Spear (1994)
showed that cell fusion induced by a syncytial strain of HSV-1 is dependent on the presence of cell-surface GAGs, principally heparin sulphate, or on the addition of heparin to the medium, and it has been suggested that GAGs may alter the conformation of a viral heparin-binding protein required for the fusion event. This protein is presumably gB, since the other heparin-binding protein of HSV-1, gC, is not required for virus-induced membrane fusion (Davis-Poynter et al., 1994
; Schranz et al., 1989
). It has also been reported that a virus in which a lysine-rich heparin sulphate-binding domain of gB is deleted displays reduced penetration kinetics and reduced plaque size (Laquerre et al., 1998
), implying a potential role in fusion for gB interactions with GAGs.
It is also unclear whether the four glycoproteins required for fusion function as a complex, although it seems unlikely that they can function independently. Cross-linking studies on HSV virions (Handler et al., 1996a ) have shown the existence of very high molecular mass species containing gB, gD, gC and gHL, and these authors argued that they interact to form a functional complex. Furthermore, the cross-linking characteristics of these glycoproteins were altered during virus entry, suggesting that conformational changes may occur during fusion (Handler et al., 1996b
). Nevertheless, if such a complex exists, it must be able to form in the absence of each member of the complex, since the cross-linking patterns seen with wild-type virions are unaltered in virions that lack either gB, gD or gHL (Rodger et al., 2001
).
In order to examine the likely requirements for cell-surface GAGs and a gD receptor in a virus-free fusion system, we tested whether soluble heparin could inhibit fusion and whether target cells that lacked either GAGs or a gD receptor were able to undergo fusion with the plasma membranes of COS cells expressing gB, gD and gHL. In addition, with a view to determining whether these four glycoproteins might mediate fusion as a functional complex, we tested whether gB, gD and gHL can cooperate in trans to induce polykaryocyte formation in a system that lacks any other virus components.
COS 7 cells were transfected with plasmids expressing gB, gD and gHL (as described in Turner et al., 1998 ) and, after 2 days, the monolayers were overlaid with Vero cells and heparin was added to the medium at concentrations ranging from 10 to 200 µg/ml. Twenty-four h later, the cells were fixed and the number of nuclei that were recruited into polykaryocytes containing 11 or more nuclei was scored. The results of three independent experiments are shown in Fig. 1
. Despite some variation between experiments, we found that, at the higher concentrations tested (50200 µg/ml), heparin reduced fusion by between 42 and 80% of the untreated control values. At the lower concentrations (1020 µg/ml), the effect on fusion of adding soluble heparin was much less pronounced and was often negligible. One interpretation of these results is that, at high concentrations, heparin binds to gB on the surface of transfected COS 7 cells, thereby preventing its interaction with cell-surface proteoglycans on neighbouring Vero cells, and, by so doing, reduces the likelihood of membrane fusion occurring. Since it has been reported that cells lacking plasma-membrane GAGs fuse less efficiently than parental cell lines when infected with a syncytial strain of HSV-1 or when transfected with the genomic DNA of a syncytial virus (Shieh & Spear, 1994
), we examined the requirement for GAGs in the virus-free fusion system by testing whether effector cells expressing gB, gD and gHL could fuse with target cells that lacked plasma-membrane proteoglycans, either as a result of metabolic depletion or as a result of a genetic defect in GAG biosynthesis.
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In order to determine whether gB, gD and gHL can cooperate in trans to induce fusion, we transfected separate cultures of cells with plasmids expressing different combinations of the four glycoproteins, mixed the resulting cells and looked for evidence of fusion. COS 7 cells (105) were seeded in 10 cm2 dishes and transfected with a total of 1 µg DNA by using Fugene reagent (Boehringer), following the protocol recommended by the manufacturer. The transfection efficiency was determined by carrying out a parallel transfection with a plasmid expressing -galactosidase (pCDNA1.1ampl lacZ; Invitrogen) and counting the proportion of cells that stained blue in the presence of X-Gal. The efficiency in this experiment was estimated to be approximately 15%. After 48 h, the transfected cells were trypsinized, mixed with an equal number of cells expressing different combinations of glycoproteins or with cells expressing
-galactosidase and replated in 20 cm2 dishes. Twenty-four h later, the monolayers were fixed and the number of nuclei in syncytia containing 11 or more nuclei was counted. As shown in Fig. 3
, the only set of conditions that gave rise to significant levels of fusion was when cells expressing gB, gD and gHL were mixed with cells transfected with a LacZ-expressing plasmid. All other combinations tested, namely mixing cells expressing gB and gD with cells expressing gHL, mixing cells expressing gHL and gB with cells expressing gD or mixing cells expressing gHL and gD with cells expressing gB, failed to induce fusion above the levels observed when cells expressing gB were mixed with cells expressing gD or when untransfected cells were mixed with cells expressing
-galactosidase, suggesting that all four of the glycoproteins that are necessary and sufficient to mediate fusion must be present on the same membrane and appear to act in cis during the fusion process. This is also the case in the context of virus infection, since all four glycoproteins must be expressed on the same membrane in order for polykaryocyte formation induced by a syncytial strain of HSV-1 to occur (Davis-Poynter et al., 1994
).
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References |
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Received 16 November 2000;
accepted 27 January 2001.