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Bovine papular stomatitis virus encodes a functionally distinct VEGF that binds both VEGFR-1 and VEGFR-2

Virus Research Unit, Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand

Correspondence
Lyn M. Wise
lyn.wise{at}stonebow.otago.ac.nz

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bovine papular stomatitis virus (BPSV), a member of the genus Parapoxvirus, causes proliferative dermatitis in cattle and humans. Other species of the genus cause similar lesions, the nature of which has been attributed, at least in part, to a viral-encoded vascular endothelial growth factor (VEGF) that induces vascularization and dermal oedema through VEGF receptor-2 (VEGFR-2). The results of this study showed that BPSV strain V660 encodes a novel VEGF and that the predicted BPSV protein showed only 33–52 % amino acid identity to VEGFs encoded by the other species of the genus. BPSV VEGF showed higher identity to mammalian VEGF-A (51 %) than the other parapoxviral VEGFs (31–46 %). Assays of the purified BPSV VEGF (BPSV_V660VEGF) demonstrated that it was also functionally more similar to VEGF-A, as it showed significant binding to VEGFR-1 and induced monocyte migration. Like VEGF-A and the other viral VEGFs, BPSV_V660VEGF bound VEGFR-2 with high affinity. Sequence analysis and structural modelling of BPSV_V660VEGF revealed specific residues, outside the known receptor-binding face, that are predicted either to influence VEGF structure or to mediate binding directly to the VEGFRs. These results indicate that BPSV_V660VEGF is a biologically active member of the VEGF family and that, via its interaction with VEGFR-2, it is likely to contribute to the proliferative and highly vascularized nature of BPSV lesions. This is also the first example of a viral VEGF acting via VEGFR-1 and influencing haematopoietic cell function. These data suggest that BPSV_V660VEGF is an evolutionary and functional intermediate between VEGF-A and the other parapoxviral VEGFs.

The GenBank/EMBL/DDBJ accession number for the sequence of the VEGF-like gene of BPSV strain V660 reported in this paper is AY513237.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Vascular endothelial growth factor (VEGF) is a specific mitogen for endothelial cells and has been shown to induce vascular permeability (Ferrara, 2004; Senger et al., 1983). Members of the VEGF family of molecules play a critical role in vasculogenesis during embryonic development and in the adult during angiogenesis associated with wound healing and a number of pathological conditions including tumour formation and inflammatory conditions (Carmeliet, 2005; Ferrara, 2004; McColl et al., 2004). The mammalian VEGF family consists of five members: VEGF-A (also known as VEGF), VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF). All are secreted, homodimeric glycoproteins, sharing 30–45 % amino acid sequence identity (Stacker & Achen, 1999).

The VEGF family members exert their biological activity via a family of tyrosine kinase receptors, VEGF receptor-1 (VEGFR-1), VEGFR-2 and VEGFR-3 (Shibuya & Claesson-Welsh, 2006; Zachary, 2003). VEGFR-2 is the primary signalling receptor of VEGF-induced endothelial cell mitogenesis, angiogenesis and vascular permeability, and is bound by VEGF-A, VEGF-C and VEGF-D. VEGFR-1 is expressed on endothelial and haematopoietic cells and appears to play a role in their recruitment by VEGF-A, VEGF-B and PlGF and in the induction of pro-inflammatory gene expression. VEGFR-3 is involved in regulation of lymphangiogenesis by VEGF-C and VEGF-D. In addition, the neuronal cell guidance receptors neuropilin-1 (NP-1) and NP-2 have been shown to interact with VEGF-A, PlGF and VEGF-B, and VEGF-A, PlGF and VEGF-C, respectively, acting as co-receptors enhancing their binding to the VEGFRs.

Recently, we and others (Lyttle et al., 1994; Mercer et al., 2002; Meyer et al., 1999; Ogawa et al., 1998; Ueda et al., 2003; Wise et al., 1999, 2003) have characterized a group of poxvirus-derived homologues of VEGF, collectively designated VEGF-E, which are encoded by Orf virus (ORFV) and Pseudocowpox virus (PCPV). PCPV and ORFV belong to the genus Parapoxvirus, of which ORFV is the type species. ORFV and PCPV readily infect humans, but usually infect the muzzle or teats of sheep and goats, and cattle, respectively (Haig & Mercer, 1998). The resulting lesions are characterized by vascular dilation, dermal oedema and proliferation of endothelial cells (Groves et al., 1991). The presence of this VEGF homologue provides a probable explanation for the highly vascularized and proliferative nature of parapoxvirus lesions, and the disruption of this VEGF-like gene in ORFV causes a marked reduction in the vascularization and, surprisingly, epidermal proliferation and scab formation (Savory et al., 2000).

The expressed VEGF proteins from ORFV strains NZ2 (ORFV_NZ2VEGF) and NZ7 (ORFV_NZ7VEGF) and PCPV strain VR634 (PCPV_VR634VEGF) have been shown to be mitogenic for endothelial cells and capable of inducing vascular permeability (Meyer et al., 1999; Ogawa et al., 1998; Ueda et al., 2003; Wise et al., 1999, 2003). The viral VEGFs, however, differ from the mammalian VEGF family in their receptor-binding profile by binding and cross-linking VEGFR-2, but not VEGFR-1 or VEGFR-3 (Shibuya, 2003). The shared receptor specificities and biological activities are surprising given the extreme sequence divergence among these viral VEGFs (41–61 % amino acid identity to each other and only 25–35 % amino acid identity to VEGF-A; Ueda et al., 2003). Despite these sequence variations, structural predictions for the viral VEGFs are very similar to each other and to that of VEGF-A, suggesting that considerable variability in amino acid sequence can be tolerated whilst maintaining the ability to bind VEGFR-2 (Mercer et al., 2002).

Another member of the genus Parapoxvirus is Bovine papular stomatitis virus (BPSV) (Buller et al., 2005; Mercer & Haig, 1999). BPSV infects the muzzle, oral mucosa, tongue and udder of cattle of all ages, but clinically is seen most commonly in calves (Griesemer & Cole, 1960; Jolly & Daniel, 1966; Snider et al., 1982). BPSV also causes infections in humans, with lesions characterized by large nodules on the hands and sometimes face (Bowman et al., 1981; Carson & Kerr, 1967). BPSV infection causes proliferative lesions characterized by dermal oedema and scab formation (Nagington et al., 1967). The production of a functional VEGF homologue by BPSV would provide a likely explanation for these histological observations.

We report here the sequence and functional analysis of a new member of the parapoxviral VEGF group encoded by BPSV strain V660.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Cells and viruses.
Primary bovine testis (BT) cells were grown in Eagle's minimal essential medium containing 10 % FCS and 5 % lactalbumin hydrolysate. BPSV strain V660 (Gassmann et al., 1985) was propagated in BT cells, as described previously (Robinson et al., 1982). Murine BaF3 cell lines expressing chimeric VEGFR-1 or VEGFR-2 were kindly donated by Andrew MacKenzie (Amrad Corporation, Australia) (Makinen et al., 2001) and Steven Stacker (Ludwig Institute of Cancer Research, Australia) (Stacker et al., 1999), respectively. BaF3 cells were cultured in Dulbecco's modified Eagle's medium containing 10 % heat-inactivated FCS and 10 % WEHI-3-conditioned medium containing interleukin (IL)-3.

Purification of virus and DNA extraction.
Viral particles propagated in BT cells were purified in sodium diatrizoate gradients, as described previously (Robinson et al., 1982). Purified virions were treated with proteinase K and SDS, followed by isolation of viral DNA in guanidine HCl/CsCl gradients, as described previously (Mercer et al., 1987).

DNA cloning and sequence analysis.
A 10 kb fragment (BamHI E) generated by digestion of the BPSV genomic DNA with BamHI was mapped to the near left terminus of the BPSV genome (data not shown). A BamHI–KpnI 6 kb subfragment of the BamHI E fragment was cloned into an appropriate vector and a partial sequence of a 6 kb fragment was determined on an AB3700 automated DNA sequencer using the primer-walking method. Nucleotide and predicted amino acid sequences of BPSV were compared with the sequence of ORFV strain NZ2 (Mercer et al., 2006) using the BLAST suite of programs (http://www.ncbi.nlm.nih.gov/BLAST/).

Construction of expression vectors.
The expression vectors for VEGF-A (murine VEGF isoform 164), VEGF-D (human VEGF-DNC) and ORFV_NZ2VEGF were derived from the pAPEX-3 vector (Evans et al., 1995) and have been described previously (Achen et al., 1998; Wise et al., 2003). A DNA fragment containing the VEGF-like gene of BPSV was amplified by PCR using a plasmid containing the BamHI E fragment of BPSV as template with the following primers: BPSV-VEGF5' (5'-ATCGGCGCGCCAGAAGTGCTTAATAGTATGCA-3', AscI site underlined) and BPSV-VEGF3' (5'-GGCCAAACGCGTTCGTCTGTGTGATTCCT-3', MluI site underlined). The PCR product was digested with AscI and MluI and ligated to a pAPEX-3-derived vector, pAPEX-mVEGF-A (Wise et al., 2003), from which the DNA sequence encoding VEGF-A but not the FLAG octapeptide (IBI/Kodak) had been removed by digestion of the vector with AscI. Protein synthesis from all of the expression vectors described above gave rise to secreted proteins that were tagged with the FLAG octapeptide at their C termini.

Recombinant protein production.
Recombinant FLAG-tagged BPSV_V660VEGF, VEGF-A, VEGF-D and ORFV_NZ2VEGF were expressed in 293-EBNA cells, purified and quantified, as previously described (Wise et al., 2003).

ELISA competitive displacement assay with soluble VEGFR-1 or VEGFR-2 extracellular domains.
Maxisorp 96-well immunoplates (Nunc) were incubated with 400 ng VEGF-A ml^–1 in coating buffer (15 mM Na₂CO₃, 35 mM NaHCO₃, pH 9.6) at 4 °C for 16 h and blocked with 1 % BSA and 0.02 % Tween 20 at 37 °C for 45 min. Plates were washed between steps with wash buffer (PBS with 0.02 % Tween 20). Samples of purified growth factors, serially diluted in binding buffer (PBS with 0.4 % BSA, 0.02 % Tween 20 and 2 µg heparin ml^–1 in VEGFR-2 assays only), were incubated with 300 ng human VEGFR-1-Ig or VEGFR-2-Ig fusion protein (R&D Systems) ml^–1 in non-absorbent plates at 25 °C for 1 h. The mixture was then transferred to plates coated with VEGF-A and incubated at 25 °C for 1 h to capture the unbound VEGFR–Ig fusion protein. The captured VEGFR–Ig fusion protein was detected by biotinylated anti-human Ig (Dako) and horseradish peroxidase-conjugated streptavidin (Sigma), developed with tetramethylbenzidine substrate reagent (BD Biosciences) and quantified by measuring the absorbance at 450 nm.

ELISA receptor-binding assay with soluble VEGFR-3 or NP-1 extracellular domains.
Maxisorp 96-well immunoplates (Nunc) were coated with a titration of purified VEGFs at 4 °C for 16 h and blocked with 0.5 % BSA and 0.02 % Tween 20 at 25 °C for 1 h. Plates were washed between steps with wash buffer. Immobilized VEGFs were then incubated with 1 µg purified human VEGFR-3-Ig or rat NP-1-Ig fusion protein (R&D Systems) ml^–1 at 25 °C for 2 h. Captured VEGFR–Ig fusion protein was detected and quantified as described above.

Bioassays for the binding and cross-linking of the extracellular domains of VEGFR-1 and VEGFR-2.
Bioassays for monitoring the binding and cross-linking of VEGFR-1 and VEGFR-2, using BaF3-derived cell lines expressing chimeric receptors consisting of the extracellular, ligand-binding domains of human VEGFR-1 or mouse VEGFR-2 and the transmembrane and cytoplasmic domains of the erythropoietin receptor, were carried out as described previously (Makinen et al., 2001; Stacker et al., 1999). Briefly, the bioassay cell lines were incubated with various concentrations of purified growth factors for 48 h at 37 °C. DNA synthesis was quantified by measuring [³H]thymidine incorporation during a further 4 h incubation.

Chemotaxis assay.
Chemotaxis was assayed in 24-well plates containing Transwell inserts of 5 µm pore size (Corning Costar). THP-1 monocytes were washed twice in PBS, resuspended in RPMI 1640 containing 0.1 % BSA and then loaded into inserts at a concentration of 1.0x10⁵ cells per 100 µl for each well. Where indicated, monocytes were pre-incubated with 10 µg neutralizing antibody against VEGFR-1 (R&D Systems) ml^–1 for 16 h at 37 °C with 5 % CO₂. RPMI 1640 (600 µl) containing 0.1 % BSA and purified growth factor at various concentrations was placed in the bottom compartment. The monocytes were incubated for 3 h at 37 °C with 5 % CO₂. Non-migrated cells were then removed from the upper side of the filter membrane and the insert was washed twice with PBS. Adherent cells on the lower side of the filter membrane were fixed in 2.5 % glutaraldehyde for 15 min and stained using Gill's haematoxylin. For a quantitative assessment of migrated cells, a total of four fields of 40x magnification from two different wells was counted.

Statistical analysis.
Statistical analysis was performed using analysis of variance (single-factor ANOVA) with significant points of difference (P0.05) determined using Tukey's test.

Prediction of the tertiary structure of BPSV_V660VEGF.
The structure of BPSV_V660VEGF was modelled using SWISSMODEL and the Swiss-PdbViewer protein modelling program (version 3.7, http://www.expasy.ch/spdbv; Guex & Peitsch, 1997). The sequence of BPSV_V660VEGF was aligned against protein subunits A and B of human VEGF-A (PDB identifier 2VPF [PDB] ; Muller et al., 1997a) and ORFV_NZ2VEGF (PDB identifier 2GNN; Pieren et al., 2006). The Iterative Magic Fit function was used for energy minimization and the alignment was optimized manually. The Ramachandran plot was compared with VEGF-A and ORFV_NZ2VEGF to determine whether the predicted model contained residues that did not conform to acceptable and/or angles.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Identification of a VEGF-like gene in BPSV
Sequence analysis of the near-right terminus of the genome of BPSV strain V660 revealed homologues of ORFV genes 131 and 134 but not an equivalent of the intervening ORFV VEGF-like gene (gene 132). We therefore examined other regions of the genome. Analysis of the DNA sequence at each end of a 6 kb fragment derived from the left terminus of the genome revealed homologues of ORFV genes 001 and 007 (Fleming et al., 1995; Mercer et al., 1989, 2006). The corresponding region of the ORFV genome is only 2.5 kb, suggesting the presence of additional genes in the larger BPSV fragment. Sequencing of this 6 kb fragment revealed a VEGF-like gene between genes 005 and 007. An AT-rich early promoter-like sequence, which was similar to that of the ORFV and PCPV VEGF genes, was found in the non-coding region upstream of the VEGF-like gene of BPSV. An early transcriptional termination sequence (TTTTTGT) was located about 100 bp downstream of the stop codon of the VEGF-like gene in BPSV. The VEGF-like gene of V660 has a G+C content of only 44 mol%, despite the high G+C content of parapoxvirus genomes (approx. 65 mol%). This feature is also seen in the VEGF-like gene of all of the other parapoxviruses except for ORFV strain NZ2 (G+C content of 57 mol%).

After this analysis was completed, the genomic sequence of another strain (AR02) of BPSV was published (Delhon et al., 2004) that included a VEGF-like gene at an equivalent location to that of strain V660. The two BPSV VEGF genes have only 83 % nucleotide sequence identity, despite having 95 % identity within the 1.5 kb flanking the VEGF-like genes (data not shown). Although this score is higher than the nucleotide sequence identities of VEGF genes of ORFV strains NZ2 and NZ7 (47 %), it is lower than the inter-isolate variation between ORFV NZ2-like VEGF genes (93.5 %) (Mercer et al., 2002).

VEGF amino acid sequence comparisons
The predicted amino acid sequence encoded by the VEGF-like gene of BPSV strain V660 was compared with human VEGF-A (isoform 121) and the VEGFs from BPSV strain AR02 and ORFV strain NZ2 (Fig. 1a). Members of the mammalian and parapoxviral VEGF family share eight cysteine residues forming the cystine knot motif, which is located in the most highly conserved region of the protein, designated the VEGF homology domain (VHD) (Stacker & Achen, 1999; Wise et al., 2003). The cystine knot motif links the subunits of the anti-parallel homodimer. These eight cysteine residues are also conserved in the BPSV VEGFs (Fig. 1a). Analysis of the BPSV VEGFs revealed potential signal sequences, N-linked glycosylation site(s) and Thr/Pro-rich C-termini that contain putative O-linked glycosylation sites, similar to those described in the ORFV and PCPV VEGFs (Ueda et al., 2003; Wise et al., 1999, 2003) (Fig. 1a).

View larger version (57K): [in this window] [in a new window]   Fig. 1. Sequence and phylogenetic analysis of parapoxviral VEGFs. (a) Amino acid comparisons of BPSVV660VEGF and representative mammalian and parapoxviral VEGFs. The deduced amino acid sequence of BPSVV660VEGF was compared with human VEGF-A (isoform 121; GenBank accession no. AAF19659) and the VEGFs from BPSV strain AR02 (GenBank accession no. AAR98363) and ORFV strain NZ2 (GenBank accession no. B49530). The alignment was prepared using CLUSTAL W with default settings. The signal sequence of BPSVV660VEGF and the other VEGFs, as predicted by SignalP 3.0 (Bendtsen et al., 2004), are boxed. The borders of the VHD are indicated by arrows below the alignment. The conserved cysteine residues that form disulphide bonds within the cystine knot motif are shaded dark grey. Residues of BPSVV660VEGF with potential N-linked or O-linked glycosylation (Julenius et al., 2005) are indicated below the alignment by a bracket or dashes, respectively. Residues showing identity to BPSVV660VEGF are shaded grey. Secondary structural elements of the VHD are indicated and numbered above the alignment by light grey boxes, black boxes and black lines representing -helices, -strands and variable loops, respectively. (b) VEGF sequence comparisons. The percentage of amino acid sequence identities of BPSVV660VEGF with the VEGFs from BPSV AR02, ORFV NZ2 and NZ7 (GenBank accession no. P52585) and PCPV VR634 (GenBank accession no. AAO16216), and with human VEGF-A, VEGF-B (GenBank accession no. AAC50721), VEGF-C (GenBank accession no. P49767), VEGF-D (GenBank accession no. O43915) and PlGF (GenBank accession no. CAA38698), were calculated from an alignment of the VHDs prepared using CLUSTAL W with default settings. (c) Phylogenetic tree of the parapoxviral VEGFs and members of the human VEGF family. The alignment prepared in (b) was used to construct a phylogenetic tree using PHYLIP version 3.6 (distributed by J. Felsenstein, University of Washington, Seattle, WA, USA). The genetic distance for each pair was estimated with the Jones–Taylor–Thornton model of the program PROTDIST. Resulting distance matrix data were used to estimate phylogenies by the Fitch–Margoliash method of FITCH. These programs, in addition to SEQBOOT and CONSENSE, were used in bootstrap analysis of 1000 resampled datasets. Phylogenetic trees were visualized by TreeViewPPC version 1.6.6 (Page, 1996). The number on each branch represents the percentage bootstrap support.

Expression and purification of the VEGF protein from BPSV stain V660 (BPSV_V660VEGF)
BPSV_V660VEGF, with a C-terminal FLAG octapeptide, was expressed and purified. SDS-PAGE followed by Western blot analysis revealed bands at 48–50 and 27–28 kDa under non-reducing and reducing conditions, respectively (data not shown). The bands detected were consistent with BPSV_V660VEGF being a disulphide-linked homodimer. Deglycosylation treatment with the enzymes N-glycosidase or sialidase and O-glycosidase reduced the monomeric size of BPSV_V660VEGF by 2–3 kDa and 2–4 kDa, respectively (data not shown), indicating that BPSV_V660VEGF conserves the N-linked and O-linked glycosylation seen in other viral VEGFs (Ueda et al., 2003; Wise et al., 1999, 2003).

BPSV_V660VEGF binds soluble VEGFR-1 and VEGFR-2 extracellular domains
To determine the receptor specificity of BPSV_V660VEGF, we examined its ability to bind to soluble dimerized Ig fusion proteins containing the extracellular domains of human VEGFR-1 or VEGFR-2 using a competitive displacement ELISA.

Pre-incubation of soluble VEGF-A was able to inhibit significantly the binding of VEGFR-1 to immobilized VEGF-A at all of the concentrations tested (200 ng ml^–1, P0.05) (Fig. 2a). As previously reported (Wise et al., 1999, 2003), ORFV_NZ2VEGF did not inhibit VEGFR-1 binding to immobilized VEGF-A at any concentration tested (Fig. 2a). Surprisingly, BPSV_V660VEGF significantly inhibited the binding of VEGFR-1 to immobilized VEGF-A from a concentration of 5 µg ml^–1 (P0.05) (Fig. 2a).

View larger version (34K): [in this window] [in a new window]   Fig. 2. BPSVV660VEGF binds the VEGF receptors VEGFR-1 and VEGFR-2. Soluble VEGFR-1–Ig (a) or VEGFR-2–Ig (b) fusion protein (300 ng ml–1) was incubated for 2 h with increasing concentrations of VEGF-A, ORFVNZ2VEGF and BPSVV660VEGF, or with medium alone. The mixture was then added to VEGF-A-coated wells to capture free VEGFR–Ig, which was detected with a biotinylated sheep anti-human Ig, streptavidin–HRP conjugate. The results are presented as the percentage of the maximal absorbance of VEGFR–Ig bound. Values are expressed as mean±SEM (n=2) and are representative of four separate experiments. Soluble VEGFR-3–Ig (c) or NP-1–Ig (d) fusion protein (1 µg ml–1) was incubated for 2 h with increasing concentrations of immobilized VEGF-D or VEGF-A, and ORFVNZ2VEGF or BPSVV660VEGF. Bound Ig fusion protein was detected with a biotinylated sheep anti-human Ig, streptavidin–HRP conjugate. Values are expressed as a binding index, defined as the mean increase in absorbance±SEM at 450 nm over the background (n=2) and are representative of two separate experiments. An asterisk indicates a significant (P0.05) difference from the background level of binding to VEGFR-1, -2 or -3 or NP-1 recorded when no growth factor was added.

To examine the receptor specificity of BPSV_V660VEGF further, we tested its abilities to bind to soluble dimerized Ig fusion proteins containing the extracellular domains of human VEGFR-3 and rat NP-1 using a receptor-binding ELISA.

Whilst VEGF-D showed significant binding to immobilized VEGFR-3 from 200 ng ml^–1 (P0.05), BPSV_V660VEGF and ORFV_NZ2VEGF did not show significant binding to VEGFR-3 at any of the concentrations tested (P0.05) (Fig. 2c).

Consistent with previous reports (Wise et al., 1999, 2003), VEGF-A and ORFV_NZ2VEGF significantly bound NP-1 from a concentration of 200 ng ml^–1 (Fig. 2d). BPSV_V660VEGF, however, did not show significant binding to NP-1 at any of the concentrations examined (P0.05) (Fig. 2d).

BPSV_V660VEGF binds and cross-links VEGFR-1 and VEGFR-2 on the cell surface
The interactions of BPSV_V660VEGF with the receptors VEGFR-1 and VEGFR-2 were tested further in bioassays that detect receptor-binding and cross-linking at the cell surface. These assays made use of BaF3 cell lines expressing chimeric receptors consisting of the extracellular domain of either human VEGFR-1 or murine VEGFR-2 and the transmembrane and cytoplasmic domains of erythropoietin receptor (Makinen et al., 2001; Stacker et al., 1999). Binding and cross-linking of the chimeric receptors induces cell proliferation.

VEGF-A was able to stimulate significant proliferation of cells expressing VEGFR-1 from the lowest concentration tested (4 ng ml^–1, P0.05), whilst ORFV_NZ2VEGF did not induce cellular proliferation (Fig. 3a). BPSV_V660VEGF stimulated proliferation of cells expressing VEGFR-1 from a concentration of 4 ng ml^–1 (P0.05) (Fig. 3a). Surprisingly, BPSV_V660VEGF was as potent as VEGF-A at all concentrations tested (P0.05). The ability of BPSV_V660VEGF to bind and cross-link VEGF-R1 and induce cellular proliferation (Fig. 3a) was greater than its ability to bind VEGFR-1 in the ELISA (Fig. 2a).

View larger version (17K): [in this window] [in a new window]   Fig. 3. BPSVV660VEGF binds and cross-links the VEGF receptors VEGFR-1 and VEGFR-2. The abilities of the viral VEGFs to bind and cross-link VEGFR-1 (a) or VEGFR-2 (b) were tested using specific bioassay cell lines. Bioassay cells were washed and resuspended in dilutions of VEGF-A, ORFVNZ2VEGF, BPSVV660VEGF or medium alone for 48 h at 37 °C. DNA synthesis was quantified by [3H]thymidine incorporation and -counting. Values were expressed as a proliferation index, defined as the mean increase in cell proliferation±SEM over the background (n=2) and are representative of two separate experiments. Proliferation indices that were significantly above that of medium only are indicated by an asterisk (P0.05).

Chemotactic response of THP-1 monocytes to BPSV_V660VEGF
Previous studies have shown that mammalian VEGF family members induce monocyte chemotaxis through their interaction with VEGFR-1 (Clauss et al., 1996; Selvaraj et al., 2003; Shibuya, 2001). Thus, using a transwell assay, we examined the chemotactic response of THP-1 monocytes to treatment with BPSV_V660VEGF. BPSV_V660VEGF was able to induce significant migration of cells from a concentration of 100 ng ml^–1 (P0.05), whilst ORFV_NZ2VEGF did not induce cell migration (Fig. 4a). VEGF-A was more potent than BPSV_V660VEGF, inducing cell migration from the lowest concentration tested (4 ng ml^–1, P0.05) (Fig. 4a). Pre-incubation of THP-1 monocytes with neutralizing antibody against VEGFR-1 significantly inhibited both VEGF-A- and BPSV_V660VEGF-induced migration of cells (Fig. 4b).

View larger version (14K): [in this window] [in a new window]   Fig. 4. BPSVV660VEGF induces VEGFR-1-dependent chemotaxis of THP-1 monocytes. (a) THP-1 monocytes (1x105 cells) were added to the upper chamber of a Transwell insert. The indicated concentrations of VEGF-A, ORFVNZ2VEGF, BPSVV660VEGF or medium alone were added to the lower compartments, the inserts were incubated for 3 h at 37 °C and migrated cells that remained attached to the insert membrane were stained and counted as described in Methods. (b) Where indicated, THP-1 monocytes were pre-incubated with a neutralizing antibody against VEGFR-1 for 16 h and then assayed as described above. Results were expressed as a migration index, defined as the mean increase in cell migration±SEM over the background (n=8) and are representative of two experiments. Migration indices that were significantly above that of medium only are indicated by an asterisk (P0.05). A cross indicates a significant (P0.05) difference between cell migration induced in the presence and absence of antibody.

VEGF-A monomers, consisting of a four-stranded -sheet segment and two -helical segments, dimerize in an anti-parallel, side-by-side fashion, thereby creating at opposite poles two receptor-binding faces, which each contain three variable loop regions that interact directly with the VEGFRs (Keyt et al., 1996; Muller et al., 1997b; Pieren et al., 2006). The dimeric structure predicted for BPSV_V660VEGF was generally very similar to that of both VEGF-A and ORFV_NZ2VEGF, conserving the locations of the anti-parallel -sheets and cysteine residues that form the intra- and inter-chain disulphide bonds responsible for cystine knot formation (Fig. 5). Whilst the loop 2 region was well conserved among the three structures, BPSV_V660VEGF differed significantly from VEGF-A in the loop 1 and loop 3 regions, as seen with ORFV_NZ2VEGF (Figs 1a and 5).

View larger version (33K): [in this window] [in a new window]   Fig. 5. Structural conservation among VEGF-A, ORFVNZ2VEGF and BPSVV660VEGF. Ribbon representation of the VEGF-A (PDB identifier 2VPF; Muller et al., 1997a) and ORFVNZ2VEGF (PDB identifier 2GNN; Pieren et al., 2006) dimers and the predicted structure of the BPSVV660VEGF dimer, which was derived as described in Methods. One monomer of each dimer is shown in a darker shade. The conserved intramolecular and intermolecular disulphide bonds are shown in yellow and green, respectively. The variable loop regions have been numbered. The locations of amino acids of VEGF-A predicted to be involved in receptor binding are marked. These residues are coloured to indicate that they have been implicated in mediating the binding of VEGF-A to VEGFR-1 (red), VEGFR-2 (blue) or to both receptors (green). Where these residues are conserved in ORFVNZ2VEGF and BPSVV660VEGF, they are coloured in a similar manner. Residues of VEGF-A that form the groove implicated in VEGFR-1 binding are boxed (grey) with lettering in white or in a similar manner to that described above. In ORFVNZ2VEGF and BPSVV660VEGF, the equivalent residues are boxed (grey) and with dark grey lettering, or if conserved with VEGF-A, in a similar manner to that described above. The locations of additional amino acids of BPSVV660VEGF that are conserved with VEGF-A but not ORFVNZ2VEGF are shown in black or in a similar manner to that described above. Residues Ser-94–Asn-99 within loop 3 of one ORFVNZ2VEGF monomer are missing, as they were disordered due to their intrinsic flexibility in the determined structure (Pieren et al., 2006).

Structural determinations have revealed a groove between loop 1 and loop 2 at each end of the VEGF-A dimer that is believed to interact with the region linking domains 2 and 3 of VEGFR-1 (Wiesmann et al., 1997). Residues forming this groove in VEGF-A are indicated in Fig. 5. It has been postulated that in ORFV_NZ2VEGF a salt bridge across this groove between Arg-46 and Glu-64 may prevent binding to VEGFR-1 (Mercer et al., 2002; Pieren et al., 2006). However BPSV_V660VEGF conserves most of the groove-forming residues with ORFV_NZ2VEGF, including Arg-46 and Glu-64, and the composition of the groove does not provide a ready explanation for the ability of BPSV_V660VEGF to bind VEGFR-1 (Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Determination of DNA sequence from the genome termini of BPSV strain V660 revealed the presence of a VEGF-like gene not near the right terminus, as has been observed in the other parapoxviruses, but near the left genome terminus. This is similar to the location of a VEGF-like gene recently identified in BPSV strain AR02 (Delhon et al., 2004), although the two genes show only 71 % amino acid sequence identity within the VHD. Purified BPSV_V660VEGF was found to be a glycosylated disulphide-linked homodimer with monomeric subunits of 28 kDa on SDS-PAGE. Receptor-binding analysis revealed that BPSV_V660VEGF was functionally more similar to mammalian VEGF-A than the other viral VEGFs in that it showed significant binding to VEGFR-1 and induced VEGFR-1-dependent chemotaxis of monocytes.

This is the first description of a viral VEGF, BPSV_V660VEGF, showing significant recognition of VEGFR-1. Despite these functional differences, sequence and structural analyses revealed that BPSV_V660VEGF is predicted to be very similar to ORFV_NZ2VEGF, and differs from VEGF-A in the loop 1 and 3 regions, the receptor-linker groove and in the specific residues implicated in VEGF receptor interactions. BPSV_V660VEGF does, however, share twelve residues with VEGF-A that are not found in ORFV_NZ2VEGF or in any other viral VEGF (Fig. 1a). Of these residues, only Tyr-21 and Gln-79 have been implicated in mediating the binding of VEGF-A to VEGFR-1 and VEGFR-2, or VEGFR-2, respectively (Keyt et al., 1996; Li et al., 2000; Muller et al., 1997b). The remaining residues (Val-14, Ser-74, His-86, Glu-93, Met-94, Leu-97, Gln-98, Asn-100, Glu-103 and Lys-107) are positioned outside the known receptor-binding face and would seem unlikely to interact directly with either receptor (Fig. 5). However, they may influence the orientation of loop 3 and the width of the receptor-linker groove, and hence the interaction of BPSV_V660VEGF with the VEGFRs. Consistent with this concept, three of these residues, Val-14, Glu-93 and Glu-103, are also shared by the VEGFR-1-specific ligands VEGF-B and PlGF.

The biological significance of the interaction of BPSV_V660VEGF with VEGFR-1 in the context of viral infection remains unknown. VEGFR-1 mediates VEGF-induced dendritic cell activation, monocyte migration and pro-inflammatory gene expression (Autiero et al., 2003; Gabrilovich et al., 1998; Selvaraj et al., 2003; Shibuya, 2001), which are important antiviral responses. BPSV lesions, however, appear histologically similar to other parapoxvirus lesions (Groves et al., 1991; Horner et al., 1987; Nagington et al., 1967). Differences in the inflammatory response to BPSV infection that might arise as a consequence of VEGFR-1 activation by BPSV_V660VEGF may not be evident due to a number of viral or host factors. For example, unlike the other parapoxviruses, which characteristically infect the muzzle or teats, BPSV preferentially infects the mucosal lining of the tongue or oral cavity (Griesemer & Cole, 1960; Jolly & Daniel, 1966; Snider et al., 1982), which is a tolerogenic microenvironment characterized by the production of the anti-inflammatory cytokines, IL-10 and transforming growth factor- (Novak et al., 2004; Szpaderska et al., 2003; Tlaskalova-Hogenova et al., 2004). In the mucosal environment, a selective pressure against VEGFR-1 recognition may therefore be less pronounced than in a cutaneous infection. The potential pro-inflammatory activities of the BPSV VEGFs, induced via VEGFR-1, could also be counteracted by anti-inflammatory virulence proteins, which have been described in a number of parapoxviruses, including an IL-10 homologue and a chemokine-binding protein (Delhon et al., 2004; Fleming et al., 2000; Haig et al., 2002; Imlach et al., 2002; Seet et al., 2003).

Our results showed that BPSV_V660VEGF, like other parapoxviral VEGFs, is a biologically active member of the VEGF family that interacts with the main mitogenic receptor, VEGFR-2, with high affinity. VEGFR-2 mediates VEGF-induced vascular dilation, dermal oedema, proliferation of endothelial cells and epidermal hyperplasia (Ferrara, 2004; Savory et al., 2000; Wilgus et al., 2005; Wise et al., 2003). This interaction with VEGFR-2 is likely to contribute to the proliferative and highly vascularized nature of BPSV lesions (Nagington et al., 1967).

Numerous VEGF family members, including VEGF-A, PlGF, VEGF-B and the VEGFs from ORFV strains NZ2, NZ10 and D1701, have been shown to interact with the VEGFR-2 co-receptor NP-1 (Fuh et al., 2000; Wise et al., 1999, 2003). BPSV_V660VEGF, however, resembled the VEGFs from ORFV NZ7 and PCPV in that it failed to bind NP-1 (Tokunaga et al., 2006; Wise et al., 2003). Recent findings have implicated a motif, TRPPRR, found at the C terminus of the ORFV NZ2-like VEGFs, in this interaction with NP-1 and in heparin binding (Tokunaga et al., 2006; von Wronski et al., 2006). This motif is related to that of an immunostimulatory peptide, Tuftsin (TKPR), and a higher affinity antagonist (TKPPR), that were shown to selectively bind NP-1 and block binding of VEGF-A to that receptor (von Wronski et al., 2006). This motif is not found at the C terminus of the ORFV NZ7, PCPV or BPSV VEGFs and its absence may contribute to the failure of these ligands to bind NP-1.

The evolutionary significance of the sequence and functional divergence between BPSV_V660VEGF and the other major variants of the parapoxviral VEGFs remains unknown. The BPSV VEGFs, however, clustered genetically between the VEGF-A and the parapoxviral VEGFs, and this is reflected functionally. The BPSV VEGFs may therefore represent a more ancestral mammalian-like VEGF that has been under less selection pressure to lose VEGFR-1 binding, or may have resulted from a more recent and independent recombination event between a mammalian VEGF and BPSV.

	ACKNOWLEDGEMENTS

 
This work was partially supported by the Health Research Council of New Zealand. L. M. W. was supported in part by the University of Otago Health Sciences Career Development Programme Postdoctoral Fellowship Award. We thank Ellena Whelan and Nicola Real for expert technical assistance and Wessel de Graaf for construction of the BPSV expression vector.

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Received 21 September 2006; accepted 14 November 2006.

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