Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
Correspondence
Helena Browne
hb100{at}mole.bio.cam.ac.uk
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ABSTRACT |
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Present address: Nestlé Research Centre, PO Box 44, CH-1000 Lausanne 26, Switzerland.
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MAIN TEXT |
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Although a number of molecules which bind to gD have been identified, there are as yet no reports of cellular receptors for either HSV-1 gB or gH. Nevertheless, the notion that a receptor for HSV-1 gH exists is supported by recent observations by Scanlan et al. (2003) who demonstrated that cells expressing gHL were resistant to virus infection. There is also evidence that gB and gH homologues in other herpesviruses interact with a number of different cellular proteins; gB molecules of human cytomegalovirus (HCMV) bind to the EGF receptor (Wang et al., 2003b
) and annexin II (Pietropaolo & Compton, 1997
), and human herpesvirus 8 (HHV8) gB binds to
3
1 integrins (Akula et al., 2003
). gH of human herpesvirus 6 binds to CD46 (Mori et al., 2003
; Santoro et al., 2003
), HCMV gH has been reported to bind to a 92·5 kDa cellular protein (Keay & Baldwin, 1991
) and in EpsteinBarr virus the gHL complex is associated with a further polypeptide, gp42, which reacts with HLA class II (Li et al., 1997
).
The predicted amino acid sequence of both the HSV-1 and HSV-2 gH ectodomains contains a potential integrin-binding motif, Arg-Gly-Asp (RGD), so to address the question as to whether HSV-1 gH can bind to cells, and if so, whether binding is mediated by an RGD-integrin interaction, we generated recombinant soluble forms of both wild-type gHL and mutated gHL, in which the RGD sequence was Arg-Gly-Glu (RGE), a mutation which ablates integrin-binding, and tested these molecules in cell binding assays.
The sequence encoding the predicted ectodomain of HSV-1 strain HFEM gH (aa 1803) was fused in-frame at the N terminus of the Fc portion of human IgG1 under control of the -actin promoter. A 6 aa residue linker connects the gH sequence to the natural hinge between the CH1 and CH2 domains of IgG, and the construct continues with the two immunoglobulin constant domains, CH2-CH3, which comprise the Fc-binding region of IgG. This hybrid molecule was expressed in Chinese hamster ovary (CHO) cells together with HSV-1 gL. The gL coding region was also cloned under the control of the
-actin promoter, and the gHFcgL fusion protein was purified from cell supernatants by affinity chromatography on a Protein A Sepharose column. SDS-PAGE analysis of the purified protein confirmed that the molecule was expressed as a heterodimer of the gHFc fusion polypeptide and gL (Fig. 1
a). The recombinant fusion protein was also recognized by the conformation-dependent neutralizing monoclonal antibody to gH, LP11 (Buckmaster et al., 1984
), as determined by ELISA (Fig. 1b
). Recognition of the LP11 epitope on HSV-1 gH is dependent on the presence of gL (Hutchinson et al., 1992
), so LP11 reactivity was considered to be a good indication that the recombinant molecules were conformationally authentic. A modified form of this protein, in which the RGD motif was mutated by site-directed mutagenesis to the non-integrin binding sequence, RGE was also produced and could also be detected in ELISA by LP11 (Fig. 1c
). To determine whether the gHFcgL fusion protein bound to cells, we carried out binding assays as described in Diamond et al. (1990)
. This is a method that is used to detect low-affinity binding interactions. A 50 µg ml1 solution of protein was coated as 50 µl spots onto a 20 cm2 plastic Petri dish and incubated at room temperature for 90 min. After blocking non-specific binding sites with 1 % BSA/PBS, the dishes were overlaid with 5x106 trypsinised Vero cells or CHO cells, and incubated at 37 °C for 1 h, after which time unbound cells were gently removed and the dishes were fixed in formal saline and stained with toluidine blue. Binding was quantified by counting the number of cells, per field of view, bound to spots of gHFcgL protein as compared with the number bound to spots of human IgG. As shown in Fig. 2
(a), Vero cells bound specifically to gHFcgL and not to the IgG control, while CHO cells failed to bind in this assay. This observation suggests that there may be a gH receptor on Vero cells which is resistant to trypsin digestion, while any gH-binding proteins on CHO cells must be sensitive to this treatment. If an interaction between gH and a cellular receptor is required for entry, then such receptors must be present on CHO cells, since CHO cells which express gD receptors can be infected with HSV (Montgomery et al., 1996
). Nevertheless, it remains a possibility that if gH binding to cells has additional functions, then CHO cells may not express the appropriate receptor.
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Integrin molecules exist as heterodimers comprised of and
subunits. Both subunits are type 1 transmembrane proteins and there are eight
and 18
subunits in mammalian cells, which are known to combine to form 24 distinct integrins. In an attempt to identify the nature of the integrin to which gH binds, binding assays were carried out using Vero cells that had been pre-treated with serial twofold dilutions of a panel of monoclonal antibodies to several different
- and
-integrin subunits, and the ability of these antibodies to inhibit binding was measured by counting the number of cells that adhered to spots of the gHFcgL protein. Fig. 3
(a) shows that antibodies to
1,
2 and
IIb subunits failed to inhibit binding except when used at concentrations between 1020 µg ml1 where binding was reduced by approximately twofold. However, anti-
3 antibodies at concentrations as low as 1 µg ml1 reduced binding by 100-fold, and similarly pronounced inhibitory effects were seen with anti-
v antibodies at concentrations as low as 0·15 µg ml1. Although these data do not formally prove the nature of the integrin that mediates binding of gH to Vero cells, they suggest that it may be comprised of an
v and a
3 subunit, and this is a well-established integrin subunit combination. To confirm this, CHO cells expressing human
v
3 integrin molecules (Mekrache et al., 2002
) were tested in binding experiments with both wild-type gHFcgL and the gHRGE mutant protein. These cells bound efficiently to gHFcgL, but did not bind to the form of gH in which the integrin-binding motif had been mutated to RGE (Fig. 3b
).
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ACKNOWLEDGEMENTS |
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Received 31 August 2004;
accepted 22 September 2004.