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INTRODUCTION |
Poxviruses have multiple evasion strategies to counteract the
antiviral host defense. They code for a number of gene products that
are not essential for viral replication. Many of these viral proteins
are directed against the host immune system. They either block the
complement system (1, 2), interfere with cytokine and chemokine
function (3-5), inhibit antigen presentation (6), or influence
inflammatory processes (7, 8). A smart poxviral strategy to evade the
antiviral defense is to encode soluble proteins that prevent the
binding of cytokines to their cognate cellular receptors. Poxviral
cytokine-binding proteins with specificity for mammalian tumor necrosis
factor-
and -
(9-12), interleukin-1
(IL-1
)1 (13-15), IL-2
(16), IL-18 (17-19), interferon (IFN)-
/
(20, 21), IFN-
(22,
23), granulocyte macrophage colony-stimulating factor (16), and various
chemokines (24-26) were identified. The importance of IFN-
in host
defense against poxvirus infections was clearly demonstrated in both
tissue culture and animal model systems (27, 28). The central role of
IFN-
in the host defense against poxviruses probably explains why
this cytokine is one of the main targets of the poxviral immune evasion
strategy. In fact, not only does vaccinia virus code for a soluble
IFN-
-binding protein, but it also targets IFN-
by a virus-encoded
phosphatase that interferes with cytokine signaling (29).
A gene encoding a soluble IFN-
-binding protein was first identified
in the myxoma virus genome (22). Homologous genes were subsequently
identified in the vaccinia virus (23, 30), the variola virus (31, 32),
the swinepox virus (33), the shope fibroma virus (34), and the
ectromelia virus (35). Proteins with IFN-
binding activity
were further detected in supernatants of cowpox virus- and camelpox
virus-infected cells (23). Cells infected with tanapox virus were
reported to secrete a cytokine-binding protein of unknown structure
that can bind IFN-
, IL-2, and IL-5 (36). Soluble IFN-
-binding
proteins of various poxviruses differ in their specificity for IFN-
from different animal species. For example, the myxoma virus M-T7
protein preferentially binds and neutralizes rabbit IFN-
. On the
other hand, the IFN-
-binding proteins of the vaccinia virus, the
cowpox virus, and the camelpox virus (23) have broad specificity. They
recognize human, bovine, and rat as well as rabbit IFN-
. The
IFN-
-binding protein of the vaccinia virus further recognizes and
neutralizes chicken IFN-
(chIFN-
) (37). The mostly broad species
specificity of poxviral IFN-
-binding proteins contrasts with the
situation for cellular IFN-
receptors, which typically show high
affinity for IFN-
from the cognate species only (38). All
poxviral IFN-
-binding proteins described to date show significant
homology to mammalian IFN-
receptors (39). Like their cellular
counterparts, they contain two fibronectin type III domains and exhibit
features of class II cytokine receptor family members. The poxviral
IFN-
-binding proteins lack transmembrane and cytoplasmatic domains.
The fowlpox virus (FPV) belongs to the subfamily chordopoxvirinae. It
is the prototype of the genus avipoxvirus. The complete genome sequence
of a single FPV strain is presently available (40). It contains 260 open reading frames, including that of gene 073, which shows
low homology to viral and cellular IL-18 binding proteins (17-19).
Surprisingly, sequence analysis failed to provide evidence for FPV
genes that might code for IFN-
-binding proteins.
Here we showed that FPV gene 016 codes for a secreted
protein that binds and neutralizes the activity of chIFN-
. This
protein shows no significant homology to known IFN-
-binding proteins of other poxviruses or to any known cellular IFN-
receptors.
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EXPERIMENTAL PROCEDURES |
Cells--
The quail fibroblast cell line CEC-32 and the chicken
macrophage cell line HD-11 were grown in Dulbecco's modified minimal essential medium (DMEM) supplemented with 8% fetal bovine serum and 2% chicken serum. CV-1 cells were grown in DMEM supplemented with
5% fetal bovine serum. Low passage human embryonic fibroblasts and
BALB/c mouse embryo cells were maintained in DMEM supplemented with
10% fetal bovine serum. Primary chicken embryo fibroblasts (CEFs) were
cultured in DMEM supplemented with 7% newborn calf serum.
Fowlpox Virus--
FPV strain HP1-447 was a gift from Dr.
A. Mayr (University of Munich, Germany). FPV stocks were grown on
primary CEFs. Viral titers were determined by plaque assay on CEFs.
Supernatants of FPV-infected cells containing immunomodulatory proteins
were produced by infecting CEFs at a multiplicity of infection (m.o.i.)
of 0.01 pfu/cell. Cell supernatants were harvested 6 days later and
stored at
20 °C.
Recombinant Vaccinia Viruses--
Vaccinia virus strain
VV
B8R, which lacks the B8R gene, was a gift from Dr.
G. L. Smith (Wright-Fleming Institute, London, Great Britain).
Vaccinia virus VV
B8R-FPV016, which expresses the FPV gene
016 under the control of the vaccinia virus p7.5 promoter,
was constructed by transient dominant selection using VV
B8R and
plasmid pSC11-derived constructs for recombination. Vaccinia virus
stocks were grown on CV1 cells. Titers of virus stocks were determined
by plaque assay on CV1 cells. Cell supernatants containing vaccinia
virus-encoded immunomodulatory proteins were produced by infecting CV1
cells at an m.o.i. of 1 pfu/cell. Supernatants were harvested 24 h
post infection. They were subjected to centrifugation for 25 min at
50,000 rpm in a TLA-120-2.2 fixed angle rotor (k factor, 47).
RNA Analysis--
RNA was isolated using TRIZOLTM reagent
(Invitrogen) according to the manufacturer's protocol. RNA
samples were size fractionated by electrophoresis through agarose gels
containing 4% formaldehyde using standard procedures before blotting
onto nylon membranes and hybridization with the indicated cDNA
probes that were radiolabeled with 32P. cDNAs
containing the complete open reading frames of chicken GBP (41), human
GBP-1 (42), mouse GBP-1 cDNA (42), chicken GAPDH (43), or rat GAPDH
(44) were used as hybridization probes.
Metabolic Labeling of Proteins in the Supernatant of
Poxvirus-infected Cells--
CEFs were infected with FPVs at an m.o.i.
of 10 pfu/cell. At 1 h post infection, the cells were labeled with
71.5 µCi/ml of [35S]Cys/Met Proready mix (Amersham
Biosciences) in a methionine- and cysteine-free labeling medium. Cell
supernatants were harvested 15 h after onset of the labeling
reaction. CV1 cells were infected with vaccinia virus at an m.o.i. of 1 pfu/cell and labeled for 15 h under the same conditions as
described for FPV-infected cells.
Purification of Histidine-tagged Recombinant Proteins from
Escherichia coli--
Purification of histidine-tagged chIFN-
and
MxA by affinity chromatography on nickel chelate agarose was described
previously (45, 46).
Assay for Neutralization of IFN-
-mediated GBP Gene
Induction--
To determine whether culture supernatants contained an
activity that would neutralize the chIFN-
-mediated induction of the GBP gene in CEC-32 cells, 10 units/ml of recombinant
chIFN-
produced by transfected monkey COS cells (46) were
mixed with various dilutions of supernatant from poxvirus-infected
cells and incubated for 1 h at room temperature. CEC-32 cells
(2 × 106 per well) were then treated at 37 °C with
2 ml of the various mixtures for 16 h before RNA isolation. RNA
was subjected to Northern blot analysis and hybridized with
radiolabeled GBP cDNA probes.
Similar assays were employed to determine whether recombinant FPV016
protein can neutralize the IFN-
of other species. Duck IFN-
produced in COS cells (47) (a kind gift of Dr. U. Schultz, University
of Freiburg, Germany), which is active on quail cells (47), was assayed
on CEC-32 cells. Human IFN-
(Roche Molecular Biochemicals) was
assayed on low passage human fibroblasts, whereas rat and mouse IFN-
(R&D Systems, Wiesbaden, Germany) were assayed on low passage BALB/c
mouse embryo cells.
Assay for Neutralization of chIFN-
-mediated Nitric Oxide
Production in Macrophages--
To measure the neutralization of
chIFN-
-mediated nitric oxide production, 20 units/ml recombinant
chIFN-
from COS cells were incubated with a supernatant from
FPV-infected cells at a 1:4 dilution for 1 h. About 3 × 104 HD-11 cells were then seeded into each well of a
96-well microtiter plate before they were treated at 37 °C with 100 µl of the various mixtures for 24 h. Nitric oxide production was
monitored as a function of nitrite accumulation in the HD-11 culture
medium using the Griess assay (48, 49).
Assay for Neutralization of IFN-
-mediated Antiviral
Activity--
To determine whether culture supernatant of
VV
B8R-FPV016-infected cells contained an activity that would
neutralize chIFN-
, 2-fold dilutions (starting from 200 units/ml) of
recombinant cytokine produced in transfected COS cells were incubated
with culture supernatants at 1:10 dilution for 1 h and then added
to CEFs for 15 h. The cells were then challenged with vesicular
stomatitis virus, and virus-induced damage was assessed 24 h later
as described (50).
Tryptic Digestion of Proteins in the Supernatant of FPV-infected
Cells--
Twenty units of trypsin agarose (Sigma) were incubated with
300 µl of supernatant from FPV-infected cells at 37 °C. Incubation was done under constant rotation on the overhead shaker for 15 h.
The agarose beads were then removed by centrifugation, and the
resulting supernatants were incubated with chIFN-
and then applied
to the indicator cells.
Binding Assay to Demonstrate Direct Interaction of the FPV016
Protein with chIFN-
--
Protein-loaded nickel-agarose beads were
prepared by standard purification procedures for histidine-tagged
proteins in which, however, the final elution step was omitted.
Protein-loaded beads were incubated for 15 h at 4 °C in 1 ml of
buffer I (500 mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 10% glycerol, 0.1% Nonidet P-40,
10 mM 2-mercaptoethanol, 2 mM imidazole, and
proteinase inhibitors) with a 1-ml sample of 35S-labeled
supernatant from poxvirus-infected cells. The beads were washed with 1 ml of buffer I containing 20 mM imidazole and 1 ml of
buffer II (100 mM KCl, 20 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40,
10 mM 2-mercaptoethanol, 20 mM imidazole, and
proteinase inhibitors). Bound proteins were eluted by incubation for 10 min at 95 °C in 50 µl of SDS gel-loading buffer. They were
analyzed by electrophoresis through a SDS-polyacrylamide gel (15%) and
visualized by Coomassie Blue staining followed by autoradiography.
Two-dimensional Gel Electrophoresis--
For isoelectric
focusing, the IPGphor system (Amersham Biosciences) was used.
Separation of proteins by their molecular weights was performed in a
SE600 Hoefer chamber. 100 µg of proteins were dissolved in
isoelectric focusing buffer (8 M urea, 4% CHAPS, 40 mM Tris base), mixed with rehydration solution (Amersham
Biosciences) and applied to an isoelectric focusing strip (pH 3-10).
The strip was subsequently covered with dry strip cover fluid.
Hydration was performed at 30 V for 15 h. After hydration,
isoelectric focusing was started using the following program
parameters: 2 h at 500 V; 2 h at 1,000 V; and 4 h at
8,000 V. Before SDS-PAGE was performed, the isoelectric focusing strip
was incubated for 15 min in SDS equilibration buffer. For second
dimension gel electrophoresis, a 10% SDS-polyacrylamide gel was run at
90 V for 15 min followed by electrophoresis at 275 V for 5 h.
Proteins were visualized by silver or Coomassie Blue staining followed
by autoradiography.
MALDI-TOF MS Peptide Fingerprint Analysis--
MALDI-TOF MS
peptide fingerprint analysis was performed at the Center for Genome
Research, University of Bielefeld, Germany using standard technology.
 |
RESULTS |
Supernatants of FPV-infected Cells Contain Proteins with
chIFN-
-neutralizing Activity--
To identify putative FPV-encoded
proteins with IFN-
-binding activity, we tested whether supernatants
of FPV-infected CEF would neutralize the chIFN-
-mediated induction
of the GBP gene (46) in quail CEC-32 cells. CEFs were
infected for 6 days with FPV strain HP-447 (51) before the culture
medium was harvested and mixed at various ratios with chIFN-
for
1 h before the mixtures were added to the CEC-32 indicator cells.
RNA was isolated at 16 h post onset of cytokine exposure and
subsequently used for Northern blot analysis. As controls, CEC-32 cells
were incubated either with medium alone or chIFN-
alone. Strongly
reduced induction of the GBP gene was observed when
chIFN-
was used in combination with the supernatant from
FPV-infected cells (Fig. 1A).
At a 10
2 dilution of the FPV supernatant, the
GBP signal was almost completely lost. At a
10
3 dilution, the GBP signal was easily
detectable, but it was still less intense than that of the positive
control. A supernatant from vaccinia virus-infected HeLa cells, known
to contain chIFN-
-neutralizing activity (37), neutralized
GBP induction by chIFN-
with comparable efficacy (Fig.
1A).

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Fig. 1.
Supernatants of FPV-infected cells neutralize
the biological activity of chIFN- .
A, inhibition of chIFN- -mediated induction of the
GBP gene. Two different dilutions (10 2 and
10 3) of supernatants from either FPV-infected chicken
embryo fibroblasts (FPV sup) or vaccinia virus-infected HeLa
cells (VV sup) were incubated with 10 units/ml
chIFN- for 1 h, before the samples were added to CEC-32
indicator cells. Untreated cultures ( ) and cultures treated with
chIFN- in the absence of virus supernatant (+) served as
negative and positive controls, respectively. At 16 h post onset
of treatment, RNA was extracted from the CEC-32 cells and subjected to
Northern blot analysis. The membrane was sequentially probed with
32P-labeled chicken GBP and chicken GAPDH cDNAs.
B, inhibition of chIFN- -mediated induction of NO
synthesis. Samples of chIFN- (20 units/ml) were incubated for 1 h with either the untreated or trypsin-treated supernatant of
FPV-infected chicken embryo fibroblasts (final dilution of supernatant
was 1:4), as indicated, before the mixtures were added to chicken HD-11
indicator cells. Untreated cultures (medium) and cultures
treated with chIFN- in the absence of the FPV supernatant
(chIFN- ) served as negative and positive controls,
respectively.
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chIFN-
-mediated induction of the NOS-2 gene in chicken
macrophages (46) was measured to verify the neutralizing activity of
FPV supernatants in an independent second biological test system. For
this purpose, chIFN-
was incubated with supernatants from FPV-infected CEF for 1 h before the samples were added to HD-11 chicken macrophage cells. Nitrite concentration in the culture supernatant was measured 24 h later. As negative and positive controls, the HD-11 cells were incubated with either medium alone or
chIFN-
alone. We found that FPV supernatants potently neutralized the chIFN-
-induced accumulation of nitrite in the culture medium of
HD-11 cells. At a 1:4 dilution, the FPV supernatants blocked this
induction nearly completely (Fig. 1B).
To determine whether the observed chIFN-
-neutralizing activity
resulted from the action of one or more FPV proteins or else from
non-proteinaceous factors, an aliquot of the supernatant was treated
with trypsin that was immobilized on agarose beads. After removal of
the trypsin beads by centrifugation, the treated supernatant was
incubated at a 1:4 dilution with chIFN-
for 1 h as above before
the NO induction assay was performed. The trypsin-treated supernatant
was no longer able to neutralize the activity of chIFN-
(Fig.
1B), suggesting that viral proteins were responsible for the
observed neutralization phenomenon.
Proteins in Supernatants of FPV-infected Cells That Bind
Matrix-immobilized chIFN-
--
To identify viral proteins with
chIFN-
binding activity, FPV-infected chicken fibroblasts were
metabolically labeled with radioactive amino acids for 15 h before
the cell supernatant was harvested. Supernatants of uninfected cells
that were metabolically labeled for the same period of time served as a
negative control. The radiolabeled supernatants were then incubated at
4 °C for 15 h with samples of agarose beads that were loaded
with either recombinant histidine-tagged chIFN-
(His-chIFN-
) or a
control protein (His-MxA). After extensive washing, bound proteins were eluted from the beads and analyzed by SDS-PAGE followed by
autoradiography. Beads carrying immobilized chIFN-
specifically
retained two radiolabeled proteins that migrated on the gel as broad
bands at about 32 and 38 kDa (Fig. 2).
These two proteins did not seem to bind to beads carrying the
immobilized control protein. The various faint radioactive signals
present in all lanes probably represent histidine-rich viral
or cellular proteins.

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Fig. 2.
Immobilized chIFN-
is specifically recognized by proteins present in supernatants of
FPV-infected cells. Supernatants of metabolically labeled chicken
embryo cells infected with FPV
(35S-FPV-sup) or uninfected cells
(35S-CEF-sup) were incubated for
15 h with nickel agarose beads loaded with histidine-tagged
chIFN- (His-chIFN- ) or histidine-tagged control
protein (His-MxA). After washing, bound proteins were eluted
from the beads and analyzed by SDS-PAGE and autoradiography. Two
distinct bands (arrowheads) were observed when labeled
supernatant from FPV-infected cells was allowed to react with
immobilized chIFN- . The gel positions of molecular weight markers
are indicated.
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To achieve better separation of FPV proteins with IFN-
binding
activity and obtain sufficient quantities of the critical proteins for
MALDI-TOF MS peptide fingerprint analysis, two-dimensional gel
electrophoresis was performed. For this experiment, infected chicken
embryo cells were maintained for 14 h in serum-free medium before
the culture supernatant was harvested. Proteins were concentrated by
ethanol precipitation, and two-dimensional gel analysis was performed
using 150 µg of protein per gel. To facilitate the detection of
protein spots that might represent the putative soluble IFN-
-binding factor of FPV, we added small amounts of radiolabeled proteins purified
by affinity chromatography using immobilized chIFN-
as described
above. After electrophoresis in the second dimension, a complex pattern
of protein spots was visualized by silver staining (Fig.
3A, left
panel). Autoradiography of the dried gel revealed nine spots
(Fig. 3A, right panel), namely four
prominent spots migrating at about 32-34 kDa and five minor spots
migrating at about 38-42 kDa. The two groups of spots appeared like
pearls on a string, suggesting that they might represent a single
protein with modifications that affect charge and mass. Two of the most prominent spots seen in the autoradiographic picture could be assigned
to protein spots of the stained gel (Fig. 3B,
arrows). These two proteins were also visible on a Coomassie
Blue-stained gel that was run in parallel (data not shown). They were
excised and used for MALDI-TOF MS peptide fingerprint analysis.

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Fig. 3.
Analysis of secreted proteins in supernatants
of FPV-infected cells. A, two-dimensional gel analysis of
supernatant from FPV-infected chicken embryo cells. Isoelectric
focusing (IEF) covered the pH range from 3 to 10. Silver
staining (left panel) was performed to visualize
all of the proteins that were loaded. Autoradiography (right
panel) showed the gel positions of radiolabeled tracer
proteins that were purified from FPV-infected cells by affinity
chromatography using matrix-immobilized chIFN- as described in the
Fig. 2 legend. B, enlargement of the boxed
areas in panel A that contained IFN- -binding
proteins. Two silver-stained protein spots (arrows), which
migrated to the identical gel positions as the radiolabeled
matrix-purified FPV proteins with IFN- -binding activity, were
identified.
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chIFN-
Binding Activity is Encoded by the FPV016
Gene--
Using Matrix Science's MASCOT software, we generated a data
base with all FPV sequence information available in data bases. Virtual
complete and non-complete tryptic digests of every potential FPV gene
product were performed, and the masses of all resulting peptide
fragments were calculated. The MASCOT software was then used to compare
these values to the experimentally derived values. This analysis
yielded single hits for both of the protein samples that we had
retrieved from our two-dimensional gels. Both proteins corresponded to
the predicted product of the FPV016 gene (Mowse Scores of 77 and 71, respectively). The tryptic fragments of the FPV016 protein
identified by MALDI-TOF MS analysis are depicted in Fig.
4. The fragment starting at polypeptide
position 79 was only found in one of the two gel spots, whereas the
other three fragments were present in both samples.

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Fig. 4.
Amino acid sequence of the FPV016
protein. The putative signal peptide is underlined.
Peptide fragments identified by MALDI-TOF MS analysis are shown in
boldface. N- and O-glycosylation sites
are shown in gray boxes. Two cysteine residues
that are believed to form a disulfide bridge in the predicted
immunoglobulin domain are marked by arrowheads.
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The FPV016 gene codes for a polypeptide with 238 residues.
It is located in the left terminal region of the viral genome, which is
a typical position for genes encoding immunomodulatory proteins of
poxviruses (2). Computer-based sequence analysis using program SignalP
(52, 53) identified a potential signal peptide at amino acid positions
1-23, which is followed by a putative signalase cleavage site. Three
potential Asn-X-Ser/Thr N-glycosylation sites are
present. With help of the program NetOGlyc 2.0 (54), a potential
O-glycosylation site was identified that includes Thr at
position 93. The program PFAM (55) identified an immunoglobulin domain
that includes amino acid residues 29-108. Cysteine residues forming a
predicted disulfide bridge are marked by arrowheads in Fig.
4.
Recombinant FPV016 Protein Binds and Neutralizes chIFN-
--
To
confirm that the FPV016 gene product indeed can bind to chIFN-
and
neutralize its biological activity, we constructed several plasmids
designed to direct polymerase II promoter-driven expression of the
FPV016 gene in mammalian or avian host cells. None of these
constructs yielded a biologically active protein (data not shown). To
determine whether poxviral expression systems might work better, we
constructed a recombinant vaccinia virus that lacks the B8R
gene, which encodes a soluble IFN-
receptor, but instead expresses
the FPV016 gene under the control of the vaccinia virus
promoter 7.5 (VV
B8R-FPV016). Supernatants of radiolabeled CV-1 cells
infected with VV
B8R-FPV016 contained a 34-kDa protein that
specifically bound to matrix-immobilized chIFN-
(Fig.
5A). This protein was not
present in supernatants of CV-1 cells infected with the vaccinia virus
strain VV
B8R that carries no FPV genes (Fig.
5A). The 38-kDa IFN-
-binding protein present in
supernatants of FPV-infected cells (Fig. 2) was not observed in
supernatants of VV
B8R-FPV016-infected CV-1 cells. We do not know
whether it was absent or whether its visualization was obscured by a
background band of similar size (Fig. 5A).

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Fig. 5.
Recombinant FPV016 protein binds to
chIFN- and neutralizes its biological
activity. A, FPV016 binds to matrix-immobilized chIFN- .
Supernatants of radiolabeled CV-1 cells infected with either vaccinia
virus strain VV B8R-FPV016 or VV B8R were allowed to interact with
matrix-immobilized chIFN- as described in the Fig. 2 legend. Bound
proteins were eluted from the matrix and analyzed by SDS-PAGE and
autoradiography. Molecular weight markers are indicated. The extra
band, which appeared to represent the FPV016 protein, is marked by an
arrowhead. B, FPV016 blocks the GBP-inducing
activity of chIFN- . Three different dilutions (1:5, 1:10, and 1:20)
of the supernatants of CV-1 cells infected with either vaccinia virus
strain VV B8R-FPV016 or VV B8R were incubated with 10 units/ml of
chIFN- for 1 h before the mixtures were added to CEC-32
indicator cells. Untreated CEC-32 cultures ( ) and cultures treated
with chIFN- alone served as negative and positive controls,
respectively. At 16 h post onset of cytokine treatment, RNA was
extracted from the various cell cultures and subjected to Northern blot
analysis. The membrane was sequentially hybridized with radiolabeled
chicken GBP and chicken GAPDH cDNA probes.
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To show that the recombinant FPV016 protein can neutralize the activity
of chIFN-
, we compared the activities of the supernatant of CV-1
cells infected with either VV
B8R-FPV016 or VV
B8R. Three different
dilutions of supernatant (1:5, 1:10, and 1:20) were incubated with 10 units/ml chIFN-
for 1 h before the mixtures were added to
CEC-32 indicator cells. At 16 h post cytokine treatment, RNA was
isolated and subjected to Northern blot analysis using a radioactive
GBP cDNA probe. The supernatant of VV
B8R-FPV016-infected cells
clearly neutralized the activity of chIFN-
(Fig. 5B). It blocked the GBP-inducing activity of chIFN-
almost completely when
used at a 1:5 dilution, and it was partially effective at 1:10 and
1:20. Control supernatant of VV
B8R-infected cells did not block the
GBP-inducing activity under these experimental conditions (Fig.
5B). At a 1:10 dilution, the supernatant of
VV
B8R-FPV016-infected cells had no detectable neutralizing effect on
the antiviral activity of chIFN-
at any concentration that we tested
(data not shown).
We next determined whether the FPV016 gene product might bind and
neutralize IFN-
from other species. The amino acid sequences of duck
and chicken IFN-
are 67% identical, and both proteins are active on
quail cells (47). The supernatant of VV
B8R-FPV016-infected cells
neutralized the activity of duck IFN-
with remarkably good efficacy,
whereas the control supernatant of VV
B8R-infected cells did not
(Fig. 6A). Interestingly, the
supernatant of VV
B8R-FPV016-infected cells also neutralized the
activity of human IFN-
(Fig. 6B), but it did not
neutralize mouse (Fig. 6C) and rat (Fig. 6D)
IFN-
.

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Fig. 6.
Recombinant FPV016 protein neutralizes the
biological activity of IFN- from ducks
(A) and humans (B) but fails to
neutralize IFN- from mice (C)
and rats (D). Different dilutions of supernatants of
CV-1 cells infected with either vaccinia virus strain VV B8R-FPV016
or VV B8R were incubated with IFN- (10 units/ml) of the various
species for 1 h before the mixtures were added to either CEC-32
cells (duIFN- ), human embryonic fibroblasts
(huIFN- ), or BALB/c mouse embryo cells
(muIFN- and ratIFN- ). Untreated cultures
( ) and cultures treated with the various IFN- alone served as
negative and positive controls, respectively. At 16 h post onset
of cytokine treatment, RNA was extracted from the various cell cultures
and subjected to Northern blot analysis. The membrane was sequentially
hybridized with radiolabeled avian or mammalian GBP and GAPDH cDNA
probes, respectively.
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|
 |
DISCUSSION |
Sequence analysis of the complete FPV genome (40) yielded no
evidence for the existence of a gene for a soluble IFN-
-binding protein in this virus. Nevertheless, our experiments with the supernatants of FPV-infected chicken embryo cells clearly showed the
presence of an activity that neutralized chIFN-
(Fig.
1A). The simplest explanation for these discrepant results
was that an FPV-encoded protein with no significant homology to
previously described IFN-
receptors exhibited IFN-
-neutralizing
activity. Our biochemical approach described in this paper demonstrated that this assumption was correct and that the FPV016 gene is
encoding the critical viral factor.
The calculated molecular mass of the secreted form of the FPV016 gene
product is ~24,000. However, our SDS gel analysis of viral proteins
with high affinity for matrix-immobilized chIFN-
showed that the
factor in question presented itself as a pair of diffuse bands with
apparent molecular masses of about 32-34 and 38-42 kDa (Fig.
2). This difference in molecular mass probably resulted from
glycosylation of the mature protein. In fact, sequence analysis showed
that the FPV016 protein has three potential N-glycosylation sites and one potential O-glycosylation site. Strong
glycosylation of soluble IFN-
-binding proteins of other poxviruses
has been described (22, 23). It is of interest to note that our
MALDI-TOF MS analysis of two gel spots with slightly different
migration properties clearly showed that they both contained proteins
encoded by the FPV016 gene and that one peptide, which
presumably contains a glycosylated threonine residue, was missing in
one of these proteins. Because the extent of glycosylation cannot be
predicted, MALDI-TOF MS analysis most likely failed to detect the
glycosylated peptide. These results suggested that
O-glycosylation at threonine 93 does occur but that it is
probably incomplete. Differences in glycosylation at this and other
putative glycosylation sites of the FPV016 gene product might further
explain its appearance in multiple forms on two-dimensional gels (Fig.
3).
The FPV016 gene is localized in the terminal region of the
viral genome. This localization is characteristic for genes that code
for immunomodulatory proteins in other poxviruses (2). By expressing
the FPV016 gene with the help of a vaccinia virus that lacks
its own soluble IFN-
-binding protein, we verified that the FPV016
gene product is indeed active. Supernatants of host cells infected with
the FPV016-expressing vaccina virus contained chIFN-
binding and
neutralizing activity, whereas supernatants of control cells infected
with the parental strain did not. A computer search for characteristic
protein motifs in the FPV016 protein revealed the presence of an
immunoglobulin domain. This was unexpected, because all known viral and
cellular IFN-
receptors contain fibronectin type III domains rather
than immunoglobulin domains (39). We thus identified a new type of
viral IFN-
-binding protein. This finding readily explains why our
previous experiments failed to reveal the identity of the FPV016 gene
product. Those attempts were all based on the assumption that
IFN-
-binding proteins of FPV and other poxviruses have similar structures.
Most poxviral genes with immunomodulatory functions seem to represent
pirated cellular genes (3). Exceptions are the poxvirus-encoded type I
IFN-binding proteins that contain Ig domains, whereas all known
cellular type I IFN receptors are composed of fibronectin type III
domains (20, 21). Similarly, the FPV016 gene might have
evolved independently of cellular genes with analogous functions. However, because IFN-
receptors of birds have not yet been
characterized at the molecular level, the alternative possibility
remains that FPV016 indeed represents a hijacked chicken
gene and that avian and mammalian IFN-
receptors have fundamentally
different primary structures. It is further possible that chickens
possess an as yet unidentified gene that codes for a soluble
IFN-
-binding protein that is used to regulate the IFN-
response
and that FPV has pirated this gene.
FPV and other avian poxviruses are presently being evaluated as vaccine
vectors for use in humans. These viral vectors are considered to be
safe because they fail to replicate productively in mammalian cells.
Our finding that FPV016 is capable of binding and neutralizing human
IFN-
(Fig. 6B) is relevant in this context. Because early
IFN-
synthesis is known to drive TH1-type immune responses, it is
conceivable that FPV vaccine vectors that lack the 016 gene
and, as a consequence, permit the accumulation of higher local
concentrations of active IFN-
might exhibit enhanced performance.