Herpes simplex virus type 1 glycoprotein H binds to {alpha}v{beta}3 integrins

Christopher Parry{dagger}, Susanne Bell, Tony Minson and Helena Browne

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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Glycoprotein H (gH) homologues are found in all members of the herpes virus family, and gH is one of the virion envelope glycoproteins that is essential for virus entry. In this study, a recombinant soluble form of Herpes simplex virus type 1 (HSV-1) gH, in which the ectodomain is fused to the Fc-binding region of IgG, has been generated. This was expressed in mammalian cells together with gL and the resulting gHFc–gL heterodimer was purified using Protein A Sepharose. Low-affinity cell binding assays showed that gHFc–gL bound specifically to Vero cells and mutation of a potential integrin-binding motif, Arg-Gly-Asp (RGD), in gH abolished binding. CHO cells failed to bind in this assay. However, CHO cells expressing the human {alpha}v{beta}3 integrin bound efficiently to gHFc–gL, suggesting that HSV-1 gH can bind to cells using {alpha}v{beta}3 integrins and that this binding is mediated by the RGD motif in the gH ectodomain.

{dagger}Present address: Nestlé Research Centre, PO Box 44, CH-1000 Lausanne 26, Switzerland.


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The current view of Herpes simplex virus (HSV) entry is that it involves three steps. Initial adsorption of viruses to host cells is mediated by interaction with cell-surface glycosaminoglycans (GAGS). Glycoprotein (g) C is the major GAG-binding protein but gB also has GAG-binding activity (Herold et al., 1991). This is followed by stable binding of the virus at the cell surface and is achieved by the interaction of gD with one of several receptors, including the herpesvirus entry mediator, nectin molecules and modified forms of heparin sulphate (reviewed by Spear, 2004). This can be blocked by soluble gD but is irreversible, and receptor binding leads to substantial changes to the N-terminal region of gD (Carfi et al., 2001). The virus envelope then fuses with the plasma membrane. gB, gD and gHL are implicated in the fusion step since virions that lack any one of these proteins are devoid of infectivity (Cai et al., 1988; Forrester et al., 1992; Ligas & Johnson, 1988), and co-expression of this set of glycoproteins in transfected cells results in fusion with untransfected neighbours (Muggeridge, 2000; Turner et al., 1998).

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 {alpha}3{beta}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 Epstein–Barr 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 1–803) was fused in-frame at the N terminus of the Fc portion of human IgG1 under control of the {beta}-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 {beta}-actin promoter, and the gHFc–gL 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. 1a). 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 gHFc–gL 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 ml–1 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 gHFc–gL protein as compared with the number bound to spots of human IgG. As shown in Fig. 2(a), Vero cells bound specifically to gHFc–gL 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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Fig. 1. (a) Purified gHFc–gL (1 µg) was analysed by SDS-PAGE under reducing conditions followed by staining with Coomassie blue. Molecular mass standards are indicated in kDa, and the gHFc and gL polypeptides are indicated with arrows. (b) Serial double dilutions of purified gHFc–gL or a control protein gDFc (starting at 1 µg ml–1) were incubated on plates coated with anti-human IgG. Protein was detected with monoclonal antibody LP11 and anti-mouse alkaline phosphatase, followed by incubation with Sigmafast pNPP substrate and measurement of OD405. (c) ELISA assays (as described for Fig. 1b) were carried out on serial twofold dilutions of either gHFc–gL or gHRGEFc–gL. The first well in each case contained neat supernatant from cells expressing each molecule.

 


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Fig. 2. (a) Plastic dishes were coated with either gHFc–gL fusion protein or with human IgG1 and overlaid with either trypsinised Vero or CHO cells. After incubation and gentle washing the number of cells bound to each protein was counted. Counts were carried out in triplicate, and each histogram represents the mean values obtained, plus the standard error of the mean. (b) Plastic dishes coated with either wild-type gHFc–gL protein or with gHRGEFc–gL were overlaid with trypsinised Vero or CHO cells. The number of cells bound to each protein was counted in triplicate, mean values are plotted, and error bars represent the standard error of the mean.

 
Since HSV-1 gH contains an RGD motif it was of interest to test whether the binding of gHLFc to Vero cells was mediated by an interaction with cell surface integrins, so we repeated binding assays using soluble gHRGEFc–gL protein coated onto plastic dishes. As shown in Fig. 2(b), Vero cells failed to bind to this molecule, and no binding of either wild-type or mutated gH–gL fusion proteins to CHO cells was observed. These findings are consistent with the view that the binding of gHFc–gL to Vero cells occurs via an interaction between the RGD motif and a cellular integrin molecule.

Integrin molecules exist as heterodimers comprised of {alpha} and {beta} subunits. Both subunits are type 1 transmembrane proteins and there are eight {beta} and 18 {alpha} 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 {alpha}- and {beta}-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 gHFc–gL protein. Fig. 3(a) shows that antibodies to {beta}1, {beta}2 and {alpha}IIb subunits failed to inhibit binding except when used at concentrations between 10–20 µg ml–1 where binding was reduced by approximately twofold. However, anti-{beta}3 antibodies at concentrations as low as 1 µg ml–1 reduced binding by 100-fold, and similarly pronounced inhibitory effects were seen with anti-{alpha}v antibodies at concentrations as low as 0·15 µg ml–1. 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 {alpha}v and a {beta}3 subunit, and this is a well-established integrin subunit combination. To confirm this, CHO cells expressing human {alpha}v{beta}3 integrin molecules (Mekrache et al., 2002) were tested in binding experiments with both wild-type gHFc–gL and the gHRGE mutant protein. These cells bound efficiently to gHFc–gL, 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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Fig. 3. (a) Vero cells were incubated with serial twofold dilutions of a panel of monoclonal antibodies to different integrin subunits, after which they were allowed to adhere to plates which had been coated with recombinant gHFc–gL. The number of cells bound was counted, and the mean values of four counts are shown together with the standard errors of the means. The highest concentrations of antibody used were 20 µg anti-{beta}1, {beta}2 and {alpha}v integrins ml–1, 10 µg anti-{alpha}IIb integrin ml–1 and 2 µg anti-{beta}3 integrin ml–1. The first column in each dataset represents the number of cells bound in the absence of antibody. The suppliers of the antibodies were Autogen ({alpha}v, {beta}1 and {beta}2), Chemicon ({beta}3) and DAKO ({alpha}IIb). (b) Suspensions of trypsinised CHO cells and CHO cells which express the human {alpha}v{beta}3 molecule were allowed to adhere to plates which had been coated with either gHFc–gL or gHRGEFc–gL and the number of cells bound was counted. Mean values of triplicate counts are shown together with the standard errors of the mean.

 
The observation that HSV-1 gH binds to {alpha}v{beta}3 integrins raises a number of questions as to its functional significance. Many viruses, including adenovirus, rotavirus, human parechovirus 1 and hantavirus use integrin molecules as entry receptors, and this is also true for HHV8 which binds to {alpha}3{beta}1 integrins via an RGD sequence present in gB (Akula et al., 2003; Wang et al., 2003a). This does not appear to be the case for HSV-1 since a recombinant virus in which the RGD motif in gH was mutated to RGE (Galdiero et al., 1997) showed no growth deficit in vitro, exhibited wild-type particle infectivity ratios, and entered cells at equivalent rates to wild-type virus. Furthermore, antibodies to {beta}3 and {alpha}v integrins failed to inhibit HSV-1 plaque formation (data not shown). This observation is consistent with data in the literature (Israel et al., 1998), which showed that RGD peptides also had no effect on inhibiting infection. Nevertheless, we cannot discount the possibility that there may be some elements of redundancy in terms of the receptor usage of HSV-1, nor can we rule out the fact that integrin binding by gH may be important for efficient entry into specific cell types in vivo. Furthermore, a consequence of the binding of many ligands to integrins is the induction of intracellular signalling pathways which lead to a wide spectrum of downstream events. These include activation of gene expression, promotion of cell survival and cell growth, alterations in calcium levels and cytoskeletal rearrangements (reviewed by Giancotti & Ruoslahti, 1999). It has recently been reported (Cheshenko et al., 2003) that HSV triggers activation of Ca2+ signalling pathways and phosphorylation of focal adhesion kinase (FAK), both of which are known downstream events that may occur as a result of the binding of a number of ligands to {alpha}v{beta}3 integrins. However, the phosphorylation of FAK was also induced when cells were incubated with virions containing the gHRGE mutation (B. Herold and N. Cheshenko, personal communication) and this may imply that different signalling pathways are triggered by the binding of HSV-1 gH to {alpha}v{beta}3 integrins.


   ACKNOWLEDGEMENTS
 
We wish to thank Dr N. Kieffer (CNRS, Luxembourg) for the CHO cells expressing human {alpha}v{beta}3 integrins. This work was funded by The Wellcome Trust and an MRC Co-operative Group Grant.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Akula, S. M., Naranatt, P. P., Walia, N. S., Wang, F. Z., Fegley, B. & Chandran, B. (2003). Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) infection of human fibroblast cells occurs through endocytosis. J Virol 77, 7978–7990.[Abstract/Free Full Text]

Buckmaster, E. A., Gompels, U. & Minson, A. C. (1984). Characterisation and physical mapping of an HSV-1 glycoprotein of approximately 115x103 molecular weight. Virology 139, 408–413.[CrossRef][Medline]

Cai, W. H., Gu, B. & Person, S. (1988). Role of glycoprotein B of herpes simplex virus type 1 in viral entry and cell fusion. J Virol 62, 2596–2604.[Medline]

Carfi, A., Willis, S. H., Whitbeck, J. C., Krummenacher, C., Cohen, G. H., Eisenberg, R. J. & Wiley, D. C. (2001). Herpes simplex virus glycoprotein D bound to the human receptor HveA. Mol Cell 8, 169–179.[Medline]

Cheshenko, N., Del Rosario, B., Woda, C., Marcellino, D., Satlin, L. M. & Herold, B. C. (2003). Herpes simplex virus triggers activation of calcium-signaling pathways. J Cell Biol 163, 283–293.[Abstract/Free Full Text]

Diamond, M. S., Staunton, D. E., de Fougerolles, A. R., Stacker, S. A., Garcia-Aguilar, J., Hibbs, M. L. & Springer, T. A. (1990). ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J Cell Biol 111, 3129–3139.[Abstract]

Forrester, A., Farrell, H., Wilkinson, G., Kaye, J., Davis-Poynter, N. & Minson, T. (1992). Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted. J Virol 66, 341–348.[Abstract]

Galdiero, M., Whiteley, A., Bruun, B., Bell, S., Minson, T. & Browne, H. (1997). Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H. J Virol 71, 2163–2170.[Abstract]

Giancotti, F. G. & Ruoslahti, E. (1999). Integrin signaling. Science 285, 1028–1032.[Abstract/Free Full Text]

Herold, B. C., WuDunn, D., Soltys, N. & Spear, P. G. (1991). Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. J Virol 65, 1090–1098.[Medline]

Hutchinson, L., Browne, H., Wargent, V., Davis-Poynter, N., Primorac, S., Goldsmith, K., Minson, A. C. & Johnson, D. C. (1992). A novel herpes simplex virus glycoprotein, gL, forms a complex with glycoprotein H (gH) and affects normal folding and cell surface expression of gH. J Virol 66, 2240–2250.[Abstract]

Israel, B. A., Schultz, K. T. & Murphy, C. J. (1998). Lack of detectable interaction between HSV-1 and integrins or tachykinins. Intervirology 41, 132–134.[CrossRef][Medline]

Keay, S. & Baldwin, B. (1991). Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment. J Virol 65, 5124–5128.[Medline]

Li, Q., Spriggs, M. K., Kovats, S., Turk, S. M., Comeau, M. R., Nepom, B. & Hutt-Fletcher, L. M. (1997). Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes. J Virol 71, 4657–4662.[Abstract]

Ligas, M. W. & Johnson, D. C. (1988). A herpes simplex virus mutant in which glycoprotein D sequences are replaced by beta-galactosidase sequences binds to but is unable to penetrate into cells. J Virol 62, 1486–1494.[Medline]

Mekrache, M., Kieffer, N. & Baruch, D. (2002). Activation of recombinant {alpha}IIb{beta}3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to {alpha}IIb{beta}3. Br J Haematol 116, 636–644.[CrossRef][Medline]

Montgomery, R. I., Warner, M. S., Lum, B. J. & Spear, P. J. (1996). Herpes simplex virus-1 entry into cells mediated by a novel member of the TNF/NGF receptor family. Cell 87, 427–436.[Medline]

Mori, Y., Yang, X., Akkapaiboon, P., Okuno, T. & Yamanishi, K. (2003). Human herpesvirus 6 variant A glycoprotein H-glycoprotein L-glycoprotein Q complex associates with human CD46. J Virol 77, 4992–4999.[Abstract/Free Full Text]

Muggeridge, M. I. (2000). Characterization of cell-cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells. J Gen Virol 81, 2017–2027.[Abstract/Free Full Text]

Pietropaolo, R. L. & Compton, T. (1997). Direct interaction between human cytomegalovirus glycoprotein B and cellular annexin II. J Virol 71, 9803–9807.[Abstract]

Santoro, F., Greenstone, H. L., Insinga, A., Liszewski, M. K., Atkinson, J. P., Lusso, P. & Berger, E. A. (2003). Interaction of glycoprotein H of human herpesvirus 6 with the cellular receptor CD46. J Biol Chem 278, 25964–25969.[Abstract/Free Full Text]

Scanlan, P. M., Tiwari, V., Bommireddy, S. & Shukla, D. (2003). Cellular expression of gH confers resistance to herpes simplex virus type-1 entry. Virology 312, 14–24.[CrossRef][Medline]

Spear, P. G. (2004). Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol 6, 401–410.[CrossRef][Medline]

Turner, A., Bruun, B., Minson, T. & Browne, H. (1998). Glycoproteins gB, gD and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J Virol 72, 873–875.[Abstract/Free Full Text]

Wang, F. Z., Akula, S. M., Sharma-Walia, N., Zeng, L. & Chandran, B. (2003a). Human herpesvirus 8 envelope glycoprotein B mediates cell adhesion via its RGD sequence. J Virol 77, 3131–3147.[Abstract/Free Full Text]

Wang, X., Huong, S. M., Chiu, M. L., Raab-Traub, N. & Huang, E. S. (2003b). Epidermal growth factor receptor is a cellular receptor for human cytomegalovirus. Nature 424, 456–461.[CrossRef][Medline]

Received 31 August 2004; accepted 22 September 2004.