Both soluble and membrane-anchored forms of Felid herpesvirus 1 glycoprotein G function as a broad-spectrum chemokine-binding protein

B. Costes1, M. B. Ruiz-Argüello2,3, N. A. Bryant2, A. Alcami2,4 and A. Vanderplasschen1

1 Immunology-Vaccinology (B43b), Department of Infectious and Parasitic Diseases (B43b), Faculty of Veterinary Medicine, University of Liège, B-4000 Liège, Belgium
2 Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 2QQ, UK
3 Centro de Investigación en Sanidad Animal (INIA), Valdeolmos, 28130 Madrid, Spain
4 Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain

Correspondence
A. Vanderplasschen
A.vdplasschen{at}ulg.ac.be


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Recently, glycoprotein G (gG) of several alphaherpesviruses infecting large herbivores was shown to belong to a new family of chemokine-binding proteins (vCKBPs). In the present study, the function of Felid herpesvirus 1 (FeHV-1) gG as a vCKBP was investigated and the following conclusions were reached: (i) FeHV-1 secreted gG is a high-affinity broad-spectrum vCKBP that binds CC, CXC and C chemokines; (ii) gG is the only vCKBP expressed by FeHV-1 that binds CCL3 and CXCL1; (iii) secreted gG blocks chemokine activity by preventing their interaction with high-affinity cellular receptors; (iv) the membrane-anchored form of gG expressed on the surface of infected cells is also able to bind chemokines; and (v) the vCKBP activity is conserved among different field isolates of FeHV-1. Altogether, these data demonstrate that FeHV-1 gG is a new member of the vCKBP-4 family. Moreover, this study is the first to demonstrate that gG expressed at the surface of FeHV-1-infected cells can also bind chemokines.

The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this paper is AY740677.

Supplementary figures showing real-time binding data of human chemokines to purified soluble gG, and a schematic of the construction of a recombinant Felid herpesvirus 1 lacking gG and a derived revertant virus are available as supplementary material in JGV Online.


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The central role of chemokines in antiviral defence has been highlighted by the discovery that large DNA viruses (poxviruses and herpesviruses) and at least two RNA viruses (Human immunodeficiency virus 1 and Human respiratory syncytial virus) have developed strategies to modulate the host chemokine system (Alcami, 2003). These viruses use three known strategies for interacting with the chemokine system: virus-encoded chemokines, virus-encoded chemokine receptors and virus-encoded secreted chemokine-binding proteins (vCKBPs) (Lalani & McFadden, 1999; Murphy, 2001). So far, four families of vCKBPs (vCKBP-1–4) have been described. The description of the first three families led to the conclusion that this strategy was unique to poxviruses and gammaherpesviruses (Alcami, 2003). However, recently, the secreted form of glycoprotein G (gG) from some alphaherpesviruses infecting large herbivores was found to encode a fourth family of vCKBPs that bind with high affinity to a broad range of CXC, CC and C chemokines to prevent their interaction with both glycosaminoglycans (GAGs) and cellular receptors (Bryant et al., 2003).

gG orthologues have been described in several alphaherpesviruses as a minor non-essential glycoprotein (Baranowski et al., 1996). Based on the viral species, gG has been reported either as a structural or a non-structural protein (Drummer et al., 1998). However, gG orthologues exist under two different forms, a membrane-anchored form and a secreted form, the latter is generated by proteolytic cleavage of the former (Drummer et al., 1998).

Felid herpesvirus 1 (FeHV-1) is an alphaherpesvirus with a worldwide distribution in the cat population (Roizman & Pellett, 2001). It is the causative agent of feline viral rhinotracheitis, but has also been associated with other clinical disorders (Stiles, 2003).

In the present study, we investigated the function of FeHV-1 gG as a vCKBP. Firstly, the expression of a vCKBP in the supernatant of FeHV-1-infected cells was investigated by a cross-linking assay. Cell supernatants were prepared as follows: Crandell–Reese feline kidney cells (CRFK; ATCC CCL-94) were mock-infected or infected (1 p.f.u. per cell) with the FeHV-1 B927 strain (Gaskell, 1975). Cell supernatants were collected 2 days post-infection (p.i.) and submitted to three cycles of centrifugation (200 g for 5 min, 2000 g for 15 min and 100 000 g for 2 h). A cross-linking assay with human 125I-CCL3 (Amersham Biosciences) was performed on cell supernatants (equivalent to 5–7x104 cells) as described previously (Bryant et al., 2003). A complex of 125I-CCL3 with a soluble protein was observed with infected supernatants but not with control mock-infected cultures (Fig. 1a). The molecular mass of this complex was ~60 kDa, suggesting a vCKBP of 52 kDa. To test the specificity of the interaction observed, cross-linking experiments were repeated in the presence of increasing doses of unlabelled CCL3 (PeproTech) or heparin (Sigma) (Fig. 1a). The binding of vCKBP to 125I-CCL3 was inhibited by a 100-fold excess of unlabelled CCL3, demonstrating the specificity of the CCL3–vCKBP interaction. Pre-incubation of chemokine with heparin did not interfere with the formation of 125I-CCL3–vCKBP complex at doses up to 1000 µg ml–1. This result suggests that either vCKBP and heparin bind to distinct domains of the chemokine or that vCKBP binds to both receptor and GAG-binding domains with the property to displace pre-established low affinity chemokine–heparin interaction.



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Fig. 1. (a) FeHV-1 encodes a soluble chemokine-binding activity. Cross-linking assay of 125I-CCL3 to mock-infected or infected cell supernatants was performed in the absence (NC) or presence of increasing doses of unlabelled CCL3 (10-, 100-, 500- and 2000-fold excess) or heparin (0·001, 0·01, 0·1, 1, 10, 100 and 1000 µg ml–1). (b) Identification of FeHV-1 gG as a soluble vCKBP. Cross-linking assay of 125I-CCL3 to cell supernatants (left panel) or of 125I-CXCL1 to purified protein (right panel). (c) Expression of gG chemokine-binding activity among FeHV-1 field isolates. Cross-linking assay of 125I-CCL3 to mock-infected, B927-infected or field isolates-infected cell supernatants (lanes 1–5). Molecular masses in kDa and the positions of chemokines (free CK) and vCKBP–chemokine complexes (asterisk) are indicated.

 
Based on the results presented above, we hypothesized that FeHV-1 gG is a vCKBP. To study gG, a region of the FeHV-1 genome corresponding to this open reading frame (ORF) was sequenced (GenBank accession no. AY740677). The sequence revealed an ORF of 1305 bp encoding a 434 aa residue protein with a predicted molecular mass of 48·8 kDa and a type I membrane topology. Next, the transcript of the gG gene was characterized using a RT-PCR approach with the forward primer 5'-ATGGGAAATCGTATACATATTTTA-3' (nt 1–24 of gG ORF) and the reverse primer 5'-NotI-(dT)18-3' primer (First-Strand cDNA Synthesis kit; Amersham Biosciences) as described previously (Markine-Goriaynoff et al., 2004). A single 1300 bp PCR product was observed in infected cells, while no band was detected in mock-infected cells (data not shown). A Southern blot experiment hybridizing with a probe that corresponds to gG ORF did not reveal any other bands other than the 1·3 kb product. Altogether, these results indicate that the gG gene is transcribed as a single class of transcript encoding the full-length form of the protein.

To determine whether the secreted form of gG encodes chemokine-binding activity, gG was expressed using a baculovirus expression system (Bac-to-Bac; Invitrogen) as a full-length (BacgG.His) or a truncated form (BacgGTr.His) fused to a C-terminal six-His tag. The full-length form was produced by PCR using the forward primer 5'-CCGGATCCAATGGGAAATCGTATACATATTTTA-3', corresponding to BamHI site (underlined) and nt 1–24 of gG ORF, and the reverse primer 5'-GGCTCGAGTCAGTGATGGTGATGGTGATGTGGTTGGGGGTATCTTGTCAAC-3' corresponding to XhoI site (underlined), a six-His tag (bold) and nt 1281–1302 of gG ORF. Similarly, the truncated form was produced using the forward primer described above and the reverse primer 5'-GGCTCGAGTCAGTGATGGTGATGGTGATGTGGTTCGGTCGTAGGCTCG-3' corresponding to XhoI site (underlined), a six-His tag (bold) and nt 1092–1110 of gG ORF. Recombinant baculoviruses expressing full-length or truncated gG were then generated in Sf9 insect cells (ATCC CRL-1711) following the instructions of the manufacturer. Production of cell supernatants from baculovirus-infected Sf9 cell cultures for the chemokine cross-linking assay was performed as described previously (Bryant et al., 2003). The cross-linking assay with human 125I-CCL3 revealed a 125I-CCL3–vCKBP complex of ~55 kDa (Fig. 1b, left panel). This complex was not observed with either the supernatant of mock-infected cells or the supernatant of cells infected with a control baculovirus expressing {beta}-galactosidase (Bac{beta}gal). This result suggests that the full-length form of gG can be naturally cleaved by insect cells to produce a secreted active protein. Finally, the gG truncated form was expressed in Hi5 cells (Invitrogen) and purified as described elsewhere (Bryant et al., 2003). The chemokine-binding activity of the purified protein (gGTr.His; 150 ng) was demonstrated by cross-linking it to human 125I-CXCL1 (Amersham Biosciences) (Fig. 1b, right panel).

The results presented above revealed that the secreted form of gG binds to CCL3 and CXCL1. To determine the range of activity of gG, chemokine-binding specificity was investigated by surface plasmon resonance as described in Supplementary material available in JGV Online. A set of 29 human chemokines, encompassing members of the four chemokine subfamilies, was injected over a biosensor surface coupled with purified gG. According to the response at the end of injection (R), three levels of binding were arbitrarily defined: high (R>=30 % of Rmax), intermediate (R between 15 and 30 % of Rmax) and insignificant (R<=15 % of Rmax), where Rmax describes the binding capacity of the surface in terms of the response at saturation. Rmax was calculated by the formula: (MM analyte/MM ligand)ximmobilized amountxstoichiometric ratio. The results presented in Table 1 revealed that FeHV-1 gG exhibits a broad-binding activity, since most of the chemokines tested from CC, CXC and C subfamilies bound to some extent to gG. To measure the affinity of the interaction between gG and chemokines, kinetic parameters were also analysed for CXCL1, CXCL8, CCL5 and CCL3 chemokines. The sensograms obtained are available as Supplementary Fig. S1 in JGV Online. The results presented in Table 1 show that the four chemokines tested bind gG with rapid association kinetics (kass ranging from 0·56 to 5·77x106 M–1 s–1) and relatively slow dissociation kinetics (kdiss ranging from 0·26 to 9·46x10–3 s–1), yielding a calculated KD ranging from 260 pM to 1·64 nM. These affinities demonstrated a high affinity of binding, which is similar or two- to 20-fold higher than the affinity of most chemokines for their cellular receptors (Murphy, 1996). This observation suggests that, in vivo, gG may prevent the interaction of chemokines with their cellular receptors.


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Table 1. Chemokine-binding specificity and affinity of the gG soluble form immobilized on a biosensor chip

The results presented in this table were reproduced three times.

 
To test whether gG is the only vCKBP that binds CXCL1 encoded by FeHV-1, gG-deleted mutant (FeHV-1-{Delta}gG) and revertant (FeHV-1-RevgG) viruses were constructed using the FeHV-1 B927 strain as the parental strain (FeHV-1-WT). The procedure used to generate these recombinants is available as Supplementary Fig. S2 in JGV Online. Production of these recombinants demonstrates that gG is dispensable for FeHV-1 replication in vitro (data not shown). Supernatants from cells infected with wild type and recombinant viruses were tested in cross-linking assays with human 125I-CXCL1 (Fig. 2a). An 125I-CXCL1–vCKBP complex of ~60 kDa was detected in the supernatants from wild type and revertant viruses. No complex was found in infections with the deleted mutant, suggesting that gG is the only vCKBP that binds CXCL1 expressed by FeHV-1.



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Fig. 2. (a) Identification of gG as the only vCKBP that binds CXCL1 expressed by FeHV-1. Cross-linking assay of 125I-CXCL1 to mock-infected or recombinant-infected cell supernatants. Molecular masses in kDa and the positions of chemokines (free CK) and gG–chemokine complexes (asterisk) are indicated. (b) Inhibition of chemokine binding to cells by the gG secreted form. Binding assay of 125I-CCL3 to U-937 cells in the presence of increasing amounts of recombinant supernatants. The dose of supernatant corresponding to a number of cells (cell equivalent) is indicated. The results presented are the mean±SD of triplicate measures. (c) Membrane-anchored gG binds chemokines on FeHV-1-infected cells. Binding assay of 125I-CCL3 to recombinant-infected CRFK cells in the absence (black bars) or in the presence (white bars) of a 1000-fold excess of cold CXCL1. The results presented are the mean±SD of triplicate measures.

 
To evaluate the biological relevance of the interaction of gG with chemokines, we investigated the ability of supernatants from FeHV-1-infected cultures to prevent the binding of 125I-CCL3 to U-937 cells (ATCC CRL-1593.2), using a procedure described elsewhere (Bryant et al., 2003). The binding of 125I-CCL3 to U-937 cells was inhibited in a dose-dependent manner by supernatants containing the gG protein (Fig. 2b). Inhibition began at 1·2 µl of medium (equivalent to the amount of gG synthesized by 10 000 cells) and was nearly complete in the presence of 12 µl of medium. Deletion of gG from FeHV-1 abolished the observed dose-dependent inhibition. The latter result suggests that gG is the only vCKBP expressed by FeHV-1 able to inhibit the binding of CCL3 to its cellular receptor.

To determine whether the membrane-anchored form of gG, expressed at the surface of FeHV-1-infected cells, could also bind chemokines, CRFK cells infected with the wild type, gG deleted or revertant viruses were tested for their ability to bind 125I-CCL3 as follows: at 2 days p.i. (m.o.i. of 2 p.f.u. per cell) cells were scraped and washed three times with binding medium. Cells in suspension (3·2x105 cells) were then incubated in a final volume of 150 µl with 160 pM 125I-CCL3 for 1 h at 4 °C. 125I-CCL3 bound to CRFK cells was then determined by phthalate oil (1·5 dibutyl phthalate/1 dioctyl phthalate; Sigma) centrifugation and {gamma}-counting as described elsewhere (Dower et al., 1985). Cells infected with wild type or revertant viruses bound significantly higher doses of 125I-CCL3 at their surface than cells infected with the recombinant virus lacking gG or mock-infected cells (Fig. 2c). A competition assay using a 1000-fold excess of unlabelled CXCL1 demonstrated the specificity of the observed binding. These results demonstrate that the membrane-anchored form of gG exhibits chemokine-binding properties.

In the last part of this study, we investigated the expression of the chemokine-binding activity detected in the laboratory strain B927 among field isolates. A cross-linking assay with human 125I-CCL3 was performed on supernatants from cultures infected with five FeHV-1 field isolates recovered independently throughout Belgium from cats with feline viral rhinotracheitis (Fig. 1c). A complex of 125I-CCL3 with a soluble protein was observed for all the strains tested, indicating that they all express a vCKBP.

The present study was devoted to investigate whether FeHV-1 gG acts as a vCKBP. Our results demonstrate that FeHV-1 gG belongs to the newly discovered vCKBP-4 family; binding with high affinity to a broad spectrum of CC, CXC and C chemokines. In comparison to the unique previous report on the role of gG as a vCKBP (Bryant et al., 2003), the present study leads to two main and original conclusions. Firstly, it demonstrates that not only the secreted form but also the membrane-anchored form of gG expressed at the surface of virus-infected cells binds chemokines. Secondly, it shows that the expression of a secreted vCKBP activity is a general property of field strains.

Both secreted and membrane-anchored forms of FeHV-1 gG function as chemokine-binding proteins. It is noteworthy to mention that the latter property is unique to gG among the vCKBP families because all members described to date are expressed exclusively as soluble proteins in the supernatant. All alphaherpesviruses encode a gG orthologue expressed both as a secreted protein and a membrane-anchored protein (Drummer et al., 1998) with the exception of Human herpesvirus 3 and Marek's disease virus. The only known exception to this rule is gG encoded by Human herpesvirus 1 that exists only as a truncated membrane-anchored protein (Richman et al., 1986). These observations suggest that both forms of gG play an important role in alphaherpesvirus biology. Concerning the secreted form, this study and the earlier report of Bryant et al. (2003) suggest that, in vivo, gG could prevent the interaction of chemokines with their cellular receptors, and consequently inhibits their biological activity. Concerning the membrane-anchored form, the present study suggests that gG expressed at the cell surface could act as a decoy receptor by preventing the interaction of chemokines with their cellular receptors, and consequently inhibits their biological activity. Alternatively, the membrane-anchored form of gG could transduce signalling induced by the binding of chemokines to cell surface gG. However, no observation available to date supports this hypothesis. The membrane form of gG, in addition to its expression at the surface of infected cells, has been reported as a structural protein for several alphaherpesviruses (Drummer et al., 1998). The function of gG present in alphaherpesvirus virions as a vCKBP is probable but it still needs to be formally demonstrated. If so, the chemokine-binding activity of gG present in virions could mediate the binding of virions to cell surfaces presenting chemokines bound to GAGs.

Further studies are required to determine the role of alphaherpesvirus gG chemokine-binding activity in viral pathogenesis both as secreted and membrane-anchored forms. The identification of the chemokine-binding activity of FeHV-1 gG opens the possibility of using cat infection by FeHV-1 as a homologous animal model to determine the role of alphaherpesvirus gG chemokine-binding activity in viral pathogenesis.


   ACKNOWLEDGEMENTS
 
B. C. is a research fellow of the Belgian ‘Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture’. A. V. is a Senior Research Associate of the ‘Fonds National Belge de la Recherche Scientifique’ (FNRS). This work was supported by grants of the FNRS (grants ‘1.4.015.03F’ and ‘1.4.015.04F’) to A. V. and a Wellcome Trust Grant (051086/Z/97/Z) to A. A. N. A. B. was supported by a BBSRC studentship and M. B. R.-A. is supported by a Ramón y Cajal Fellowship (Spanish Ministry of Education and Science).


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Alcami, A. (2003). Viral mimicry of cytokines, chemokines and their receptors. Nat Rev Immunol 3, 36–50.[CrossRef][Medline]

Baranowski, E., Keil, G., Lyaku, J., Rijsewijk, F. A., van Oirschot, J. T., Pastoret, P. P. & Thiry, E. (1996). Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins. Vet Microbiol 53, 91–101.[CrossRef][Medline]

Bryant, N. A., Davis-Poynter, N., Vanderplasschen, A. & Alcami, A. (2003). Glycoprotein G isoforms from some alphaherpesviruses function as broad-spectrum chemokine binding proteins. EMBO J 22, 833–846.[Abstract/Free Full Text]

Dower, S. K., Kronheim, S. R., March, C. J., Conlon, P. J., Hopp, T. P., Gillis, S. & Urdal, D. L. (1985). Detection and characterization of high affinity plasma membrane receptors for human interleukin 1. J Exp Med 162, 501–515.[Abstract/Free Full Text]

Drummer, H. E., Studdert, M. J. & Crabb, B. S. (1998). Equine herpesvirus-4 glycoprotein G is secreted as a disulphide-linked homodimer and is present as two homodimeric species in the virion. J Gen Virol 79, 1205–1213.[Abstract]

Gaskell, R. (1975). Studies on Feline viral rhinotracheitis with particular reference to the carrier state. PhD thesis, University of Bristol, UK.

Lalani, A. S. & McFadden, G. (1999). Evasion and exploitation of chemokines by viruses. Cytokine Growth Factor Rev 10, 219–233.[CrossRef][Medline]

Markine-Goriaynoff, N., Gillet, L., Karlsen, O. A., Haarr, L., Minner, F., Pastoret, P. P., Fukuda, M. & Vanderplasschen, A. (2004). The core 2 {beta}-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins. J Gen Virol 85, 355–367.[Abstract/Free Full Text]

Murphy, P. M. (1996). Chemokine receptors: structure, function and role in microbial pathogenesis. Cytokine Growth Factor Rev 7, 47–64.[CrossRef][Medline]

Murphy, P. M. (2001). Viral exploitation and subversion of the immune system through chemokine mimicry. Nat Immunol 2, 116–122.[CrossRef][Medline]

Richman, D. D., Buckmaster, A., Bell, S., Hodgman, C. & Minson, A. C. (1986). Identification of a new glycoprotein of herpes simplex virus type 1 and genetic mapping of the gene that codes for it. J Virol 57, 647–655.[Medline]

Roizman, B. & Pellett, P. E. (2001). The family Herpesviridae: a brief introduction. In Fields Virology, 4th edn, pp. 2381–2397. Edited by P. M. Howley & D. M. Knipe. Philadelphia: Lippincott Williams & Wilkins.

Stiles, J. (2003). Feline herpesvirus. Clin Tech Small Anim Pract 18, 178–185.[CrossRef][Medline]

Received 27 July 2005; accepted 29 August 2005.



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