Conservation and variation of the parapoxvirus GM-CSF-inhibitory factor (GIF) proteins
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Journal of General Virology (2009), 90, 970–977 DOI 10.1099/vir.0.006692-0
Conservation and variation of the parapoxvirus
GM-CSF-inhibitory factor (GIF) proteins
D. Deane,1 N. Ueda,2 L. M. Wise,2 A. R. Wood,1 A. Percival,1 C. Jepson,1
N. F. Inglis,1 S. B. Fleming,2 A. A. Mercer2 and C. J. McInnes1
Correspondence 1
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ,
C. J. McInnes Scotland, UK
mcinc@mri.sari.ac.uk 2
Virus Research Unit, University of Otago, Dunedin, New Zealand
The GIF protein of orf virus (ORFV) binds and inhibits the ovine cytokines granulocyte–
macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). An equivalent protein
has so far not been found in any of the other poxvirus genera and we therefore investigated
whether it was conserved in the parapoxviruses. The corresponding genes from both the
bovine-specific pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) were
cloned and sequenced. The predicted amino acid sequences of the PCPV and BPSV proteins
shared 88 and 37 % identity, respectively, with the ORFV protein. Both retained the six cysteine
residues and the WSXWS-like motif that are required for biological activity of the ORFV protein.
However, an analysis of the biological activity of the two recombinant proteins revealed that,
whilst the PCPV GIF protein bound to both ovine and bovine GM-CSF and IL-2 with very similar
Received 19 August 2008 binding affinities to the ORFV GIF protein, no GM-CSF- or IL-2-binding activity was found for the
Accepted 10 December 2008 BPSV protein.
INTRODUCTION orthopoxvirus protein (VAC C21L), which inhibits the
activation of the complement cascade (Isaacs et al., 1992).
The parapoxviruses (PPVs), members of the genus
Some, such as the vFLIP protein (MC159) of molluscum
Parapoxvirus of the family Poxviridae, cause a contagious
contagiosum virus, which inhibits apoptosis, may be
pustular dermatitis in ruminants. Orf virus (ORFV) is the
species-specific (Shisler & Moss, 2001; Thurau et al.,
type species of the genus and causes disease in sheep and
2006). ORFV has been shown to encode a protein
goats, whereas bovine papular stomatitis virus (BPSV) and
(GIF) that is capable of binding and inhibiting both of
pseudocowpox virus (PCPV) affect cattle. All three viruses
the ovine cytokines interleukin-2 (IL-2) and granulocyte–
can infect humans. As is common amongst other
macrophage colony-stimulating factor (GM-CSF) (Deane
poxviruses, ORFV has been shown to encode factors that
et al., 2000; McInnes et al., 2005). Such a protein has
either mimic host immunoregulatory proteins (Fleming
not been found so far in any other poxvirus; we there-
et al., 1997; Haig & Fleming, 1999; Seet et al., 2003) or have
fore investigated whether it was conserved in other PPVs.
the potential for interacting with components of the host
immune system (Haig et al., 1998; McInnes et al., 1998,
2005; Deane et al., 2000). In doing so, it is thought that
these virus-encoded factors are capable of subverting the METHODS
host immune response to infection, creating an envir- Viruses. ORFV strain NZ2 (Mercer et al., 2006), PCPV strains BO74
onment suitable for efficient virus replication. Some of the (kindly obtained from Dr Mathias Buettner, Bavarian Health and
poxvirus factors, such as the double-stranded RNA- Food Safety Authority, Oberschleissheim, Germany) and VR634
(Gassmann et al., 1985) and BPSV strains V660 (Gassmann et al.,
binding protein (VAC E3L), which inhibits the antiviral
1985) and A599 (isolated de novo in the UK) were used throughout
effects of interferon (Haig et al., 1998; McInnes et al., this study and were maintained by passage in primary bovine testis or
1998), are conserved across two or more poxvirus genera, fetal lamb muscle cells.
whilst others appear to be genus-specific, such as the
Expression of the PPV GIFs. The entire open reading frames of the
three PPV GIF genes were amplified by PCR and cloned individually
The GenBank/EMBL/DDBJ accession numbers for the sequences of into the pEE14 expression vector (Celltech) (Cockett et al., 1990). The
the PCPV and BPSV GIF genes are EU999744 and EU999745, integrity of the genes was verified by sequencing prior to transfection
respectively. into COS-7 fibroblasts or CHO cells. Stable cell lines expressing each
Supplementary figures showing ELISA and Northern blotting results are of the GIF proteins were established as described previously (Deane
available with the online version of this paper. et al., 2000). Cell lines expressing the recombinant ORFV and PCPV
970 006692 G 2009 SGM Printed in Great BritainParapoxvirus GM-CSF-inhibitory factor proteins
GIF (rGIF) proteins were selected by assaying for GIF activity using a Healthcare Life Sciences), according to the manufacturer’s instruc-
modification of the ovine (ov) GM-CSF-specific ELISA (Entrican et tions, and exposure to Hyperfilm ECL for 0.5–5.0 min before
al., 1996; Deane et al., 2000). The pEE14 vector contains a glutamine development.
synthetase (GS) gene; therefore, cell lines transfected stably with the
plasmid can be selected on the basis of their survival in methionine Ligand blotting. GIF–cytokine binding was detected by ligand
sulphoximine (MSX; Sigma). The cell line expressing the BPSV GIF blotting as described previously (Deane et al., 2000). Briefly, 250–
was selected based on its survival in MSX and upon reactivity with a 400 ng each cytokine was separated by denaturing SDS-PAGE (15 %
rabbit anti-ORFV GIF serum. Verification that the anti-ORFV GIF gel) and then transferred to nitrocellulose membranes. The mem-
serum recognizes the BPSV GIF was obtained by direct ELISA using branes were blocked in blocking buffer, washed in PBS containing
an anti-ORFV GIF mAb (see Supplementary Fig. S1, available in JGV 0.05 % Tween 20 (wash buffer) and then incubated with 125I–GIF
Online). The recombinant proteins used in these studies were (15–25 pM) for 2 h at room temperature. After washing three times
prepared by culturing the cells for 4 days after reaching confluence with wash buffer, bound GIF was detected by autoradiography using
in Glasgow’s modified Eagle’s medium without serum or MSX. The Hyperfilm MP X-ray film (GE Healthcare Life Sciences).
serum-free supernate was harvested, clarified by centrifugation at
1000 g, dispensed in aliquots and stored at 220 uC. Recombinant Scatchard analysis of GIF activity. Purified cytokines were radio-
ORFV and PCPV GIF were purified from CHO cells by affinity iodinated by the chloramine-T method and a soluble ligand-binding
chromatography using purified rovGM-CSF bound to CNBr– assay was performed for each cytokine as described previously (Deane
Sepharose (GE Healthcare Life Sciences). Cell-free supernates from et al., 2000). Briefly, a range of 125I–GM-CSF and 125I–IL-2
the rGIF-transfected cells were applied to the GM-CSF–Sepharose concentrations (2–20 nmol) was incubated with GIF (100 ng)
affinity column. Eluted fractions were tested for GIF activity by using containing 5 % fetal bovine serum for 2 h at room temperature.
the ovine GM-CSF-specific ELISA. Recombinant BPSV GIF was Bound proteins were precipitated by the addition of 20 %
partially purified, from CHO cell-free supernates, by Mono-Q anion- polyethylene glycol (PEG 6000; Sigma) in PBS and incubation on
exchange chromatography followed by gel filtration on Superdex 200 ice for 30 min. The precipitated material was collected under vacuum
(GE Healthcare Life Sciences). For ligand blotting, the purified GIF onto GF/C filter discs (Whatman) and washed four times with ice-
proteins were radio-iodinated by the chloramine-T method cold 10 % PEG 6000 in PBS. The radiolabelled complexes were
(McConahey & Dixon, 1980) and further purified on Superdex 200 detected and quantified in a gamma scintillation counter. The level of
columns in PBS with 0.04 % (v/v) CHAPS buffer, pH 7.2. non-specific binding of 125I-labelled cytokines to the filter discs in the
absence of GIF was measured and the data were adjusted accordingly.
Assay of GIF activity. The presence of biologically active ORFV GIF Scatchard analysis was performed on best-fit plots [bound counts
can be measured indirectly by its ability to interfere with the detection min21/free counts min21 (y-axis) versus bound counts min21 (x-
of ovine/bovine GM-CSF by specific ELISA. Cell-free supernates and axis)], generated by using the Origin software package (OriginLab
cell lysates from GIF cDNA-transfected and virus-infected cells were Corporation).
assayed for GM-CSF-binding activity as described previously (Deane
et al., 2000). Binding of GIF to ovine/bovine IL-2 was similarly Cytometric bead array (CBA) assay. Cell-free supernates were
measured by ELISA. Briefly, recombinant ovine/bovine IL-2 was collected from virus-infected cells and concentrated approximately 10
isolated from CHO cell-free supernates by Mono-Q anion-exchange times by using Centricon YM-10 concentrators (Millipore). These
chromatography followed by gel filtration on a Superdex-200 column and purified rGIF proteins were assayed for chemokine-binding
(GE Healthcare Life Sciences). ELISA plates were coated overnight activity with the human chemokines IL-8, MCP-1, MIP-1a, MIP-1b
with 1 mg IL-2 ml21 in 0.1 M NaHCO3 (pH 9.5) before blocking with and RANTES in a modified CBA assay (BD), carried out as outlined
4 % non-fat milk powder in PBS. Cell-free supernates and cell lysates in the manufacturer’s instructions. Briefly, 50 ml aliquots of infected
from GIF cDNA-transfected and virus-infected cells were assayed for cell supernates, rGIFs (10–20 mg ml21) or control samples were pre-
IL-2-binding activity. Bound GIF was detected with 2 mg ml21 of an incubated with 50 ml individual chemokine standards from
affinity-purified IgG fraction from a rabbit anti-GIF serum, followed Chemokine kit II (1250 pg ml21) for 1 h at 4 uC. The mixture was
by a 1 : 1000 dilution of goat anti-rabbit IgG conjugated with then added to a 50 ml aliquot of mixed capture beads and PE
horseradish peroxidase (HRP; DAKO) in wash buffer [PBS containing detection reagent and incubated for 3 h at room temperature in the
0.02 % Tween 20 (Sigma)]. For colour development, TMB peroxidase dark. After washing, the beads were analysed for bound PE in a
substrate (SureBlue; Kirkegaard & Perry Laboratories) was added; the FACScalibur flow cytometer (BD). Levels of each chemokine present
reaction was stopped after 20–30 min by the addition of 0.1 M HCl in the samples were calculated from the data by using CBA analysis
and A450 was read. The specificity of the interaction between GIF and software (BD).
IL-2 was verified by pre-incubating the IL-2-coated ELISA plate with
an anti-ovine IL-2 mAb, 1E10 (a gift from Dr P. Wood, CSIRO
Animal Health, Parkville, Australia; Pedersen et al., 2002), before
incubating with GIF. RESULTS
The ORFV GIF gene (ORFV117) was found approximately
Western blot analysis. In order to verify protein expression from
20 kbp from the right terminus of the genome (Deane et al.,
the various GIF constructs, the cell-free supernates and/or cell lysates
from the COS-7/CHO cells were electrophoresed in a denaturing 2000; Mercer et al., 2006) and we therefore explored the
SDS–polyacrylamide gel (12 %) and transferred to Hybond ECL corresponding regions of the PCPV, strain VR634, and
nitrocellulose membranes (GE Healthcare Life Sciences) for 3 h at BPSV, strain V660, genomes. Sequencing of the right-hand
2 mA (cm gel)22. The membranes were washed in PBS containing end of the PCPV EcoRI D fragment (Gassmann et al., 1985)
4 % non-fat milk powder for 1 h at room temperature before being revealed a homologue of the ORFV GIF gene. Sequencing
probed with a rabbit anti-GIF IgG fraction (1 mg ml21) in blot wash the ends of the 20.7 kb BPSV BamHI A fragment and
buffer [PBS containing 0.35 M NaCl and 0.5 % (v/v) Tween 80
(Sigma)]. Binding of antibody to immobilized proteins was visualized
comparing the sequence with that of ORFV suggested that
by a further 1 h incubation with a 1 : 1000 dilution of goat anti-rabbit the BPSV GIF gene was likely to be located in this
IgG conjugated with HRP (DAKO) in wash buffer, followed by fragment. We found partial sequences of the BPSV GIF
treatment with the enhanced chemiluminescence reagent ECL (GE gene in SalI subfragments of the BPSV BamHI A fragment,
http://vir.sgmjournals.org 971D. Deane and others
with the complete sequence being determined by the also used for comparative purposes. No GM-CSF- or IL-2-
primer-walking method. Subsequent publication of the binding activity was found with this strain. To verify that
complete BPSV genome sequence (Delhon et al., 2004) the GIF gene was being expressed by the BPSV strains, a
confirmed this to be the equivalent of the ORFV GIF gene. Northern blot of RNA isolated from infected cells 24 h
The sequences of the PCPV and BPSV GIF genes have been post-infection was probed with a 32P-labelled BPSV GIF
deposited in GenBank under accession numbers EU999744 cDNA (see Supplementary Fig. S2, available in JGV
and EU999745, respectively. An alignment of the predicted Online). As had been reported previously for ORFV GIF
GIF protein sequences from the three viruses is shown in (Deane et al., 2000), no discrete mRNA was detected, but
Fig. 1. rather a smear representative of poxvirus late mRNA
transcripts was present after 24 h. This is also consistent
All three GIF proteins are predicted to be 265 aa in length,
with the T-rich late promoter-like sequences found
with both ORFV and PCPV GIF possessing a 19 aa signal
upstream of the start codons of the GIF genes of all three
peptide, whilst that of BPSV GIF is predicted to be 20 aa in
viruses (data not shown).
length. The ORFV and PCPV predicted proteins share 88 %
identity, whereas both share only 37 % identity with the Supernates from CHO cells transfected with the PPV GIF
predicted BPSV protein. The ORFV and PCPV GIFs share cDNAs were analysed for GIF activity as above. As with the
four conserved potential Asn-linked glycosylation sites, native GIF, the recombinant ORFV and PCPV GIFs bound
only two of which are conserved in the BPSV protein, ovine and bovine GM-CSF and IL-2, but the rGIF from
which has a further three potential sites. The mature ORFV BPSV bound neither (Figs 2 and 3). Again, a Northern blot of
GIF protein is predicted to contain eight Cys residues, the RNA from the BPSV GIF cDNA-transfected cells
whilst both PCPV and BPSV GIFs are predicted to contain indicated that the gene was being expressed (see
seven. Six of these, however, are positionally conserved in Supplementary Fig. S3, available in JGV Online). In addition,
all three proteins. The WDPWV motif, important for the the cell-free supernate from the BPSV GIF cDNA-transfected
biological activity of ORFV GIF (McInnes et al., 2005), is cells was analysed by liquid chromatography electrospray
perfectly conserved in the PCPV GIF sequence, but ionization tandem mass spectrometry to verify that the BPSV
although present in the BPSV GIF sequence, it shows two GIF protein was being produced (data not shown).
amino acid changes, WSPWT.
Western and ligand-blot analysis
Native and recombinant GIF activity
In order to verify that each of the three PPV GIF species
Native GIF activity was assessed in the supernates from was being expressed, partially purified proteins from the
fetal lamb muscle cell cultures 72 h post-infection with the transfected CHO cells were blotted onto nitrocellulose and
three species of PPV at an m.o.i. of 10. Whilst both ORFV probed with an IgG fraction of a rabbit antiserum raised
and PCPV native GIFs were found to bind both ovine and against ORFV GIF. Under reducing conditions, protein
bovine GM-CSF and IL-2, GIF from BPSV did not bind bands were detected at approximately 43 and 55 kDa
either cytokine (Figs 2 and 3). As the BPSV strain used (Fig. 4). The 43 kDa protein represents the native and
(V660) is a well-used laboratory strain that has been highly glycosylated form of the predicted 28 kDa
passaged in culture numerous times, a second BPSV strain monomer, whereas the 55 kDa form is thought to be a
(A599) that has not been repeatedly passaged in vitro was GIF dimer (McInnes et al., 2005).
Fig. 1. Alignment of the predicted mature
amino acid sequences of the three PPV GIF
proteins. The predicted ORFV GIF sequence
is given as the consensus and identical amino
acids in the other proteins are indicated by an
asterisk (*). Missing amino acids are indicated
by a dash (–). The predicted signal peptides
are not shown. The six Cys residues and
potential glycosylation sites are shaded and
the WDPWV motif known to be important for
biological activity of ORFV GIF is boxed.
972 Journal of General Virology 90Parapoxvirus GM-CSF-inhibitory factor proteins
Fig. 2. Cell-free supernates from virus-
infected cells or from CHO cells transfected
with expression plasmid constructs of the
three PPV species of GIF were incubated with
recombinant ovine GM-CSF (filled bars) or
bovine GM-CSF (empty bars) for 1 h at 37 6C.
The mixtures were then subjected to the GM-
CSF-specific ELISA. A high ELISA reading
indicates no interference in the detection of
GM-CSF (”ve GIF activity), whilst a low ELISA
reading indicates interference with detection
(+ve GIF activity), as indicated by the bar on
the right. Values are means±SEM of quad-
ruplicate samples. *P,0.001 compared with
the medium control.
The partially purified proteins were then radio-iodinated above that of the non-specific control samples was
and tested for GM-CSF- and IL-2-binding activity by detected.
ligand blotting (Fig. 5). Under normal blotting conditions,
125
I-labelled PCPV GIF bound both sets of cytokines
Investigation of chemokine binding by PPV GIFs
strongly. In contrast, 125I-labelled ORFV GIF bound all
four cytokines only under less stringent washing conditions It had previously been suggested that the ORFV GIF
and, in addition, a much longer exposure time to X-ray protein showed significant similarity to a family of
film was required to detect binding. No binding was poxvirus chemokine-binding proteins; more specifically,
observed with 125I-labelled BPSV GIF, even with mild those found in members of the genera Orthopoxvirus and
washing conditions and extended exposure times. Leporipoxvirus that bound a variety of CC chemokines
(Seet et al., 2003). In addition, significant similarity was
also found between it and another ORFV protein, encoded
Determination of PPV GIF binding constants
by a gene found upstream of the GIF gene in the ORFV
The affinity of the binding of PCPV and ORFV GIFs to genome. This protein also displayed chemokine-binding
both ovine and bovine GM-CSF and IL-2 was determined activity, but in addition to binding CC chemokines, was
by Scatchard analysis (Table 1). Although no binding also shown to bind the C chemokine lymphotactin. As a
activity had been detected with the BPSV GIF in either the consequence of the similarity between the sequences of
ELISA or ligand-blot assays, it was still included in this these proteins, it had been suggested that the GIF protein
analysis. However, again no binding activity significantly may also exhibit chemokine-binding activity. As no GM-
Fig. 3. Cell-free supernates from virus-
infected cells or from CHO cells transfected
with expression plasmid constructs of the
three PPV species of GIF were incubated with
ovine IL-2 (filled bars) or bovine IL-2 (empty
bars) pre-bound to 96-well plates. After
washing to remove non-specifically bound
protein, the PPV GIFs were detected by using
an affinity-purified IgG fraction from a rabbit
anti-GIF serum, followed by a 1 : 1000 dilution
of goat anti-rabbit IgG conjugated with HRP.
Values are means±SEM of quadruplicate
samples. *P,0.01 compared with the anti-IL-
2+GIF control.
http://vir.sgmjournals.org 973D. Deane and others
Fig. 4. Western blot of PPV GIF proteins with a rabbit anti-rGIF-
ORFV GIF polyclonal antiserum. Partially purified recombinant PPV
GIF proteins were separated on SDS-PAGE gels (12 %) run under
reducing conditions. Proteins were electrotransferred to nitrocel-
lulose sheets and probed with an IgG fraction of a rabbit antiserum
raised against rGIF-ORFV. Bound antibody was visualized by
treating the blots with an HRP-conjugated goat anti-rabbit IgG
fraction and ECL reagent. The positions of molecular mass markers
are shown on the left. The 43 kDa protein isolated from rGIF-
ORFV represents the native and highly glycosylated form (of the
predicted 28 kDa monomer). The 55 kDa form is thought to be a
GIF dimer.
CSF- or IL-2-binding activity had been detected for the
BPSV GIF, we explored the possibility that it could bind CC
chemokines. This was done by using a modified CBA assay.
Chemokine-binding activity was not detected with any of
the recombinant GIF proteins or supernates from the
BPSV-infected cells. However, chemokine binding was
detected in both ORFV- and PCPV-infected cells (Fig. 6).
This, however, differed between the two viruses, with
MCP-1, MIP-1a, MIP-1b and RANTES binding being
found with supernates from ORFV-infected cells, whilst
only RANTES binding was detected with the supernates Fig. 5. Partially purified ovine and bovine GM-CSF and IL-2 were
from PCPV-infected cells. This suggests that a factor(s), separated by SDS-PAGE and transferred to nitrocellulose. These
were then probed with radio-iodinated rGIF-ORFV (a), rGIF-PCPV
other than GIF, produced by ORFV and PCPV is
(b) or rGIF-BPSV (c); the blots were washed with 0.02 % Tween
responsible for binding these chemokines.
20 in PBS and then subjected to autoradiography. The gel in (b)
was exposed for 48 h; those in (a) and (c) were exposed for
7 days.
DISCUSSION
Orthologues of the ORFV GIF protein were found in the
two bovine-specific PPVs BPSV and PCPV. In addition to
amino acid similarity with them. In addition, some of the
sharing 88 % overall amino acid identity with the ORFV
features that are key to the biological activity of GIF, such
GIF protein, the PCPV protein was found to contain the
as the six Cys residues, two of the putative Asn-linked
six Cys residues, the Asn-linked glycosylation sites and the
glycosylation sites and the WSXWS-like motif, are
WDPWV (WSXWS-like) motif, all of which were shown to
conserved in the BPSV protein. However, we did not
be necessary for the biological activity of ORFV GIF
detect any interaction between it and any of the cytokines
(McInnes et al., 2005). As might have been predicted from
tested. This may be due to a variety of factors. BPSV V660
the presence of these motifs, we were able to show that the
is a laboratory strain of the virus that has undergone serial
PCPV protein was able to bind GM-CSF and IL-2.
passage in culture and could therefore potentially have lost
Although it is only 37 % identical to the ORFV and PCPV the ability either to express the GIF gene or, as a result of
proteins, the BPSV GIF protein shares approximately 60 % cumulative mutations to the gene sequence, to produce
974 Journal of General Virology 90Parapoxvirus GM-CSF-inhibitory factor proteins
Table 1. Binding affinities (in pM) of PPV GIF proteins with ovine and bovine GM-CSF and IL-2
GIF Ovine GM-CSF Ovine IL-2 Bovine GM-CSF Bovine IL-2
rGIF-ORFV 405–500 1110–1390 400–468 496–556
rGIF-PCPV 248–278 536–714 237–263 250–338
rGIF-BPSV NA NA NA NA
NA, Not applicable.
biologically active GIF. We demonstrated, however, that the sequence reported here is representative of the true
the GIF mRNA from V660 and A599 was expressed BPSV GIF sequence and it is considered unlikely that the
similarly to that of PCPV GIF. We also tested cell-free few changes in amino acids noted would have resulted in
supernates from cells infected with A599, a different strain an inactive protein. It is worth noting, however, that the
of BPSV that has been passaged fewer than 10 times since WSXWS-like motif that is found in the BPSV GIF protein
its isolation. However, again no GM-CSF- or IL-2-binding is different from those found in the ORFV and PCPV
activity was detected with this virus. The V660 sequence proteins. This motif is found in a number of cytokine
reported here is 95.5 % identical to that of the published receptors and is thought to be linked to the tertiary
BPSV sequence (Delhon et al., 2004) and 98 % identical to structure of the receptor molecules (Bazan, 1990; Quelle
another isolate (B177; kindly provided by H.-J. Rziha, et al., 1992; Baumgartner et al., 1994; Ronco et al., 1995).
Friedrich-Loeffler-Institute, Federal Research Institute for The sequence of the motif varies according to the cytokine
Animal Health, Tübingen, Germany), which we have being bound and, therefore, it may be that the amino acid
sequenced (results not shown). Therefore, it is likely that changes within the BPSV GIF molecule could alter its
Fig. 6. An adaptation of the CBA assay (BD) was used to assess chemokine-binding activity in cell-free supernates of PPV-
infected cells or PPV GIF-transfected cells. Supernates were pre-mixed with a known amount of the standard chemokines
MCP-1 (diagonally hatched bars), MIP-1a (filled bars), MIP-1b (horizontally hatched bars), RANTES (shaded bars) or IL-8
(empty bars) before mixing with the capture beads and detection reagent. This was incubated for 3 h at room temperature
before binding was measured using a FACScalibur flow cytometer (BD). A high reading indicates no binding activity in the
supernates, whereas a low reading indicates interference by the cell-free supernates of binding between the chemokines and
the capture beads. Data represent mean±SEM binding of quadruplicate samples. *P,0.001 compared with medium taken from
untransfected CHO cells.
http://vir.sgmjournals.org 975D. Deane and others
binding specificity in comparison to the ORFV and PCPV (corresponding to VACV F13L) shares approximately 95 %
proteins. identity with the equivalent ORFV protein, but only 84 %
identity with that of BPSV (Inoshima et al., 2001).
The GIF proteins of the PPVs remain unique, with no
Similarly, there is approximately 68 % identity between
corresponding proteins yet found in any of the other
ORFV and PCPV VEGF proteins, but only 55 % between
genera of the subfamily Chordopoxvirinae. This is despite
the PCPV and BPSV VEGFs (Ueda et al., 2003). This
many computer-based structural-analysis programs sug-
difference is also reflected in the biological activities of the
gesting a close relationship with the family of type II virus-encoded VEGF proteins. In addition to binding
chemokine-binding proteins (vCCI) encoded by members VEGFR-2, the BPSV VEGF also has the distinct capability,
of the genera Orthopoxvirus and Leporipoxvirus (Carfi et al., amongst the poxviral VEGFs, of binding VEGFR-1 and
1999; Seet & McFadden, 2002). This relationship is inducing monocyte migration (Inder et al., 2007). It had
primarily due to sequence similarity found between the originally been thought that the two cattle viruses would be
GIF protein and a chemokine-binding protein also more similar to each other than either would be to ORFV.
encoded by the PPVs (ORF 112). This protein has been However, the data published here for the three GIF
shown to be related to the vaccinia virus (VACV) proteins would appear to reinforce the suggestion that
chemokine-binding protein C23L and to possess both C- PCPV is related more closely to ORFV than to BPSV. As it
and CC- chemokine-binding activity (Seet et al., 2003). As would appear that PCPV does not cause disease in sheep or
we did not find any GM-CSF- or IL-2-binding activity with goats and ORFV does not cause disease in cattle, a more
the BPSV GIF protein, we investigated whether the GIF detailed comparison of the ORFV and PCPV genomes may
proteins exhibited CC chemokine-binding activity. No reveal critical elements that help to dictate host range
such activity was detected with any of the rGIF proteins, within the PPVs.
despite the fact that the supernates from both ORFV- and
PCPV-infected cells appeared to interact with human CC
chemokines. This activity was presumably due to the PPV ACKNOWLEDGEMENTS
chemokine-binding protein characterized previously.
This work was funded by the Scottish Government and the Health
Interestingly, no activity was detected from BPSV-infected
Research Council of New Zealand, and made extensive use of reagents
cells. The putative BPSV chemokine-binding protein developed under the BBSRC/RERAD Immunological Toolbox (grant
(Delhon et al., 2004) is predicted to be only 41 % identical nos BBS/B/00255 and MRI/094/04).
to the ORFV protein (the PCPV protein is yet to be
characterized), with even lower similarity to the vCCI
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hamster ovary cells using glutamine synthetase gene amplification.
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Deane, D., McInnes, C. J., Percival, A., Wood, A., Thomson, J., Lear, A.,
has a role to play in the proliferation of ORFV and PCPV
Gilray, J., Fleming, S., Mercer, A. & Haig, D. (2000). Orf virus encodes a
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The full genomic sequences of ORFV and BPSV have been Bermelillo, A., Lehmkuhl, H. D., Piccone, M. E., Kutish, G. F. & Rock,
D. L. (2004). Genomes of the parapoxviruses ORF virus and bovine
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is not known, but the small amount of sequence that has Fleming, S. B., McCaughan, C. A., Andrews, A. E., Nash, A. D. &
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