From the
Poxviruses encode a large number of proteins that attenuate the
inflammatory and immune responses to infection. In this report we
demonstrate that a number of orthopoxviruses express a type I
interferon (IFN)-binding protein, which is encoded by the B18R open
reading frame in the WR strain of vaccinia virus. The B18R protein has
significant regions of homology with the
The type I interferon (IFNs)
In the
course of using the VV/T7 polymerase system to express the different
subunits of the type I interferon receptor (IFN-R) in mouse SVX.2
cells(9) , we noticed that human
We next designed experiments to determine if
the B18R protein was indeed a type I IFN-binding protein, whether other
poxviruses also encoded an analogous activity, and its cellular
location. Fig. 2A shows that L-929 cells infected with
VV recombinant in which the B18R gene has been inactivated (VV B18RKO),
but not with deletion of the B15R gene (VV B15RKO), have lost the
ability to bind radioiodinated IFN
We next determined if
blocking of binding to the human receptor by the B18R protein was
sufficient to inhibit the IFN
Type I IFNs are the first tier of host defenses against
virus infection in the skin. Poxviruses have been shown to encode at
least three genes that effectively block individual IFN-induced
antiviral mechanisms in L-929 and other
cells(6, 29, 30) ; however, it is not known if
these genes are effective in vivo in keratinocytes or other
cells of the dermis that are productively infected at least in the
ectromelia virus infection of the mouse(31) . Based on in
vitro studies, which documented on abortive ectromelia virus
infection of the peritoneal macrophages pretreated with type I
IFN(32) , it is likely that certain cells in the skin may not
support poxvirus replication if previously exposed to type I IFN. If
this is the case, then the production of the type I IFN-binding protein
early in poxvirus infection and the ability of the protein to bind IFNs
in the extracellular space as well as attached to the cell surface of
infected and uninfected neighboring cells may be critical for the
establishment of the virus in the skin. Interestingly, recent DNA
sequence analysis of variola virus strain Bangladesh-1975 shows an ORF
homologous to B18R (33). Although poxviruses have recently been shown
to encode other proteins that bind cytokines (interleukin-1 (Refs. 18
and 34), tumor necrosis factor (Refs. 35-38), and IFN
Yield of VSV from U-266 cells pretreated with 10 units/ml
IFN
We are especially grateful to Drs. Dennis Hruby
(Oregon State University, Corvallis, OR) and Melanie Spriggs (Immunex,
Seattle, WA) for the VV knockouts. We also thank Drs. M. Brunda
(Hoffman-LaRoche), Paul Trotta (Schering-Plough), G. Adolf (Ernst
Boehringer Institut für Arzneimittelforschung, Vienna, Austria),
L. Ling (Biogen), H. Hochkeppel (Ciba-Geigy, Basel, Switzerland), and
D. Gangemi (Clemson University, Clemson, SC) for providing us with the
different IFNs.
subunits of the mouse,
human, and bovine type I IFN receptors, bound human IFN
2 with high
affinity, and inhibited transmembrane signaling as demonstrated by
inhibition of Fc receptor factor
1/
2 and
interferon-stimulated gene factor-3 formation as well as inhibition of
the IFN
antiviral response. Among viral host response modifiers,
the B18R protein is unique inasmuch as it exists as a soluble
extracellular as well as a cell surface protein and thus should
effectively block both autocrine and paracrine functions of IFN.
(
)are the
cornerstone of the early antiviral host response in the skin, which is
the site of replication of the majority of poxviruses. The
keratinocytes of the epidermis have been shown to be potent producers
of both IFN
and IFN
(1) , which in turn induce the
transcription of a large number of genes, some of which encode proteins
with potent antiviral activity such as the RNA-dependent protein kinase
and the 2`,5`-oligo nucleotide A systems (for review see Refs. 2 and
3). Accordingly, poxviruses have evolved effective counter measures in
the form of proteins that block the activation of the RNA-dependent
protein kinase (ORF E3L; Refs. 4 and 5), the phosphorylation of
initiation factor eIF2
(ORF K3L; Ref. 6), and possibly the action
of 2`,5`-oligo nucleotide A system (ORF D11L; Ref. 7). Despite the
effectiveness of these virus genes at blocking the antiviral activity
induced by the pretreatment of certain cell lineages with type I IFNs,
they appear to be ineffectual in other cell types such as macrophages,
which are known to support the replication of the poxvirus ectromelia in vivo(8) . This suggests that type I IFNs can block
poxvirus replication in other ways and could imply existence of
additional virus gene(s) for blocking type I IFN action.
I-IFN
2 was
cross-linked to a cell surface protein with an approximate mass of
62-68 kDa (data not shown). This binding protein was initially
believed to correspond to the
subunit of the type I IFN-R;
however, a more detailed characterization revealed that the IFNaR3
(anti-
subunit) and the IFNaR
1 (anti-
subunit) mAbs
failed to immunoprecipitate
I-IFN
2 cross-linked to
this IFN-binding protein and to block
I-IFN
2
binding, respectively (data not shown). These results raised the
possibility that the 62-68-kDa protein either corresponded to an
IFN
2-binding protein encoded in the VV genome, or that VV
infection of L-929 cells induced the expression of an IFN
2-binding
protein. This second possibility was highly unlikely as mouse L-929
cells do not bind human IFN
2(2, 10) . Fig. 1A shows that SVX.2 cells infected with VV/T7
polymerase, but not mock-infected cells, expressed the 62-68-kDa
(82-88 including 20 kDa corresponding to radioiodinated
IFN
2) IFN
-binding protein on their cell surface. Similar
results were obtained with parental L-929 cells (data not shown) and
confirmed the viral origin of this protein. Fig. 1A also
shows that binding of radioiodinated IFN
2 to the VV protein(s) was
blocked by different human type I IFNs including IFN
1 (data not
shown), IFN
2, IFN
, IFN
, IFN
7, IFN
8, but not
by huIFN
. Natural murine IFN
at a concentration of 1
10
units/ml was less effective in blocking binding
than human type I IFNs. Furthermore, the anti-
subunit antibody
IFNaR
1 failed to block binding of radiolabeled IFN
2
supporting the viral origin for this type I IFN binding activity.
Figure 1:
Vaccinia virus encodes a type I
IFN-binding protein. A, a culture of mouse SVX.2 cells
expressing constitutively the human type I IFN-R subunit was
infected with 0.1 plaque-forming units/cell of VV/T7 polymerase, for 16
h at 37 °C. Radioiodination of IFN
2 and affinity cross-linking
methods were performed as described previously (39-41). The
specific activity of the radioiodinated IFN
2 was 47
µCi/µg. Affinity cross-linking was performed in the presence or
absence of a 500-fold excess of unlabeled IFN
2, IFN
,
IFN
, IFN
7, and IFN
8. Natural murine IFN
and
human IFN
were used at 1
10
and 6.5
10
units/ml, respectively. The IFNaR
1 mAb was used at
a final concentration of 100 µg/ml. Similar results were obtained
in parental L-929 cells infected with VV/T7 polymerase (data not
shown). B, the B18R protein has homology with the
subunits of the type I IFN-R. Comparison of the protein sequences
obtained from GenBank was performed using the multiple alignment
program MACAW (42). The different blocks within the binding domains (BDI and BDII, corresponding to
the NH
- and COOH-terminal binding domains, respectively)
are indicated with a B, followed by the number of the block as
originally described by Bazan (15). The presence of a block in the
first or second fibronectin module of the binding domain is denoted by
absence or presence of a primesymbol, respectively (i.e.BD II-B6 or BD II-B6`). Those residues
that are common to the B18R protein and the one or more
subunits
are boxed. The shaded areas correspond to regions of
homology found with computer program MACAW (42). Asterisks indicate those highly conserved residues previously described in
cytokine receptors (15).
Since the IFN-binding protein associated with VV infection has the
ability to bind both human and mouse type I IFN (although with
different affinities), we searched for homology between the VV encoded
surface and secreted proteins and the known type I IFN-R subunits.
Protein alignment analysis revealed that the product of the B18R gene
had significant homology with the mouse(11) , human(12) ,
and bovine (13, 14) subunits of the type I IFN-R (Fig. 1B). Bazan (15) has proposed that the
subunit of the human type I IFN-R (12) has duplicated
200-residue binding domains (Fig. 1B, BD I and BD II, for the amino- and carboxyl-terminal binding domains,
respectively). Each binding domain is formed by approximately two
100-residue modules with similarity to fibronectin type III repeats and
can be further subdivided in seven blocks (i.e.BDII-B4; Ref. 15). The alignment in Fig. 1B shows that the homology between B18R and the
subunits begins in block 7 of the second module of the binding
domain I (BD I-B7`) and extends without gaps to the third
block of the first fibronectin module of the binding domain II (BD
II-B3). Homology was also observed between B18R and parts of
blocks 4 and 5 (BD II-B4 and BD II-B5) of the first
fibronectin module of binding domain II, and blocks 2, 3, and part of 6
of the second fibronectin module (BD II-B2`/3`/6`). Some
distinctive common features conserved in the B18R protein and found in
binding domain II that deserve mentioning were (i) the
prolines in block 1, (ii) the tryptophan residues in
blocks 2 and 3 of the first module and block 3 of the second module, (iii) the characteristic pattern of aromatic residues
separated by 3 residues in block 3, and (iv) the conserved
tyrosine in block 6. These data suggested that these regions should
play a major role in the interaction between the receptor and IFN
.
It is also worth mentioning that only one cysteine is conserved in the
viral protein, considering that cysteine pairs are postulated to be
necessary for folding of receptor proteins. The reason for this
difference is unclear.
2. These results confirmed that
the B18R gene product was a type I IFN-binding protein and that
orthopoxviruses cowpox and ectromelia also encoded a similar binding
activity in their genomes (Fig. 2A). The Lister strain,
which has a naturally occurring deletion of the B18R gene(16) ,
also failed to show type I IFN binding activity. The B18R protein was
originally identified as an early surface antigen (S antigen) on the
surface of poxvirus-infected cells(17) , and recently shown to
be a secreted protein(18) . The deduced amino acid sequence of
the B18R protein has an amino-terminal hydrophobic region consistent
with a signal sequence, but lacked an obvious membrane anchor, and thus
a means to localize to the plasma membrane. We sought to determine the
relative amounts of the B18R IFN
binding activity in the culture
supernatant and on the surface of virus-infected cells. Fig. 2B shows that very high levels of IFN
2 binding
activity were present in conditioned medium of cells infected with the
VV WR strain, cowpox virus, ectromelia virus, VV B15RKO, but not in the
Lister strain or VV B18RKO. For all viruses, the binding activity
observed in conditioned medium in this experiment corresponded to
approximately 12 times as much as that associated with the cell surface
of L-929 cells (data not shown). Binding studies with L-929 cells
revealed the B18R protein to have a single binding site with a K
of 440 pM and to be present in
an excess of 16,800 binding sites/cell (data not shown). This compares
with 1,200 high (36 pM) and 8,200 low (700 pM)
affinity receptors/cell on the surface of U-266 cells and a few hundred
receptors/cell observed in most normal tissues(2) . Thus, the
high affinity of the B18R protein for type I IFNs and its abundance
both on the cell surface and in the extracellular milieu indicated that
the B18R protein could be an extremely powerful blocker of type I IFN
autocrine and paracrine functions.
Figure 2:
The B18R gene encodes a type I IFN-binding
protein as do genomes of other poxviruses. Cultures of L-929 cells (2
10
cells/lane) were infected for 16 h with 0.1
plaque-forming units/cell of the indicated viruses. Cells were
harvested, and affinity cross-linking was performed as described in
Fig. 1A. The B15RKO, B18RKO, and B15/18RKO correspond to WR
recombinant viruses in which the B15R, B18R, and both B15R and B18R
genes have been knockout by insertional inactivation (34). The
82-88-kDa band corresponds to the B18R protein cross-linked to
one molecule of
I-IFN
2 (62-68 kDa
corresponding to the B18R protein (18) and 20 kDa of
I-IFN
2). Two additional bands also detected by
affinity cross-linking (51 and 100 kDa) are indicated by arrows. B, the B18R protein is present in the
conditioned medium of cells infected with various poxviruses. Five
hundred microliters of conditioned media from various infection
conditions were incubated with 5 nM
I-IFN
2,
cross-linked, and immunoprecipitated with an anti-IFN
serum.
Immunoprecipitates were analyzed as in Fig. 1A. Quantitation
of the B18R protein was carried out using a PhosphorImager (Molecular
Dynamics, Sunnyville, CA). C, VV B18R protein blocks binding
to the normal human type I IFN-R. The indicated amounts of conditioned
medium from HeLa cells infected with VV B15RKO or VV B18RKO was
preincubated in a final volume of 200 µl with 5 nM radioiodinated IFN
2 for 30 min at 4 °C. Then, the mixture
was added to human U-266 cells and the cross-linking procedure
performed as described above. The IFNaR
1 mAb was used as
specificity control to block binding of
I-IFN
2 to
the human receptor (no conditioned medium was added). Arrows indicate the position of the different subunits of the type I
IFN-R and the B18R cross-linked to IFN
2. The high molecular mass
complex contains the
and
subunits, and probably other
receptor-associated proteins.
To test whether the B18R protein
could ``sequester'' IFN from the type I IFN-R,
I-IFN
2 was preincubated with increasing
concentrations of conditioned medium obtained from cells infected with
VV B15RKO or VV B18RKO prior to the addition to uninfected U-266 cells
and cross-linking analysis. Fig. 2C shows that there was
a progressive decrease in
I-IFN
2 binding to the
human receptor (arrow,
and
subunits) expressed in
human U-266 cells in the presence of increasing concentrations of
conditioned medium containing the B18R protein (VV B15RKO). The
decrease in IFN
2 binding to the human receptor was paralleled by
an increase in binding to the B18R protein. One hundred microliters of
conditioned medium (50% of the final reaction volume) blocked binding
to the human receptor in a way comparable as the anti-
subunit
antibody IFNaR
1 (data not shown). No effect on binding was
observed when U-266 cells were incubated with conditioned medium
obtained from cells infected with the VV in which the B18R gene has
been deleted (data not shown). These data demonstrated that the B18R
protein was highly effective in blocking binding of type I IFN to the
normal cell receptor, even when present at levels higher than observed
under physiological conditions. Furthermore, this experiment also shows
that the B18R protein was detected on the surface of uninfected U-266
cells, indicating that this IFN-binding protein is first secreted into
the supernatant and then binds back to the cell membrane. Similarly,
experiments with uninfected L-929 cells and supernatant containing the
B18R protein demonstrated that B18R binds to a cell surface component
with saturable kinetics (data not shown).
signaling pathway. One of the
earliest events in IFN
signaling is tyrosine phosphorylation of
the Jak kinases (19, 20, 21, 22) and the
activation of the transcription factors Stat1
, Stat1
, and
Stat2(23) . To determine the effect of the B18R protein on IFN
signaling, whole cell extracts were prepared from U-266 cells treated
with IFN
2, and the IFN
-dependent activation of the Stat1 and
Stat2 transcriptional regulators was assessed by electrophoretic
mobility shift assay (EMSA) with probes encoding IFN
response
region (GRR) present in the Fc
1 receptor gene (Fc receptor for
IgG) (24, 25) and interferon-stimulated response element
(ISRE)(23, 26, 27) . Fig. 3A shows that low levels of basal FcRF
2 were observed in cells
treated with conditioned medium from cells infected with VV B15RKO and
VV B18RKO, but not from mock-infected cells (lanes1, 7, and 13). Preincubation of 1,000 units/ml IFN
2
with conditioned medium obtained from B15RKO-infected cells (lane10), but not from mock-infected (lane4) or B18RKO-infected cells (lane16),
completely blocked the activation of FcRF
1, FcRF
2, and ISGF3 (Fig. 3B) in U-266 cells. FcRF
1/
2 DNA binding
activity was blocked by an excess of unlabeled GRR oligonucleotide and
was supershifted by an anti-Stat1 serum indicating that Stat1 was
present in these complexes (Fig. 3A). Interestingly, the
basal level of activation of FcRF
1/
2 detected after treatment
with conditioned medium obtained from either VV B15RKO- or VV
B18RKO-infected cells (in the absence of IFN) was specific as it was
blocked by an excess of unlabeled GRR probe and was supershifted by the
anti-Stat1 antibody. The nature of this basal FcRF
1/
2
activation is under investigation. Fig. 3C shows that
IFN
-dependent activation of FcRF
2 is completely blocked by
conditioned medium obtained from either VV B18RKO- or VV
B15RKO-infected cells, but not by medium from mock-infected cells.
Presumably it was the B8R protein(28) , and not the B15R or B18R
proteins, that was responsible for inhibition of the IFN
activation of the Jak/Stat pathway.
Figure 3:
The B18R protein inhibits activation of
Stat1 and Stat2. A, whole cell extracts were obtained
from U-266 cells treated with 1,000 units/ml IFN
2 previously
incubated for 30 min in the presence of conditioned medium from VV
B15RKO-, VV B18RKO-, or mock-infected cells. EMSA was performed using a
GRR probe (24) in the presence of a 100-fold excess of unlabeled GRR or
0.5 µl of anti-Stat1 serum for supershifts. The positions of
FcRF
1, FcRF
2, and free GRR are indicated. B, the
same whole cell extracts from A were used for EMSA with and
ISRE probe. The position of ISGF3 and free ISRE are indicated. C, whole cell extracts were prepared in the same form as in A except that cells were treated with 10 ng/ml IFN
. A GRR
probe was used for EMSA.
Finally, we tested the ability
of the B18R protein to reverse the IFN2 mediated inhibition of
vesicular stomatitis virus (VSV) replication in U-266 cells. As shown
in , as little as 1 µl of conditioned culture
supernatant from VV B15RKO infection completely reversed the inhibition
of VSV replication induced by 10 units/ml IFN
2, whereas identical
and greater amounts (50 µl, data not shown) of mock or VV B18RKO
culture supernatant had no effect. In the absence of IFN
2, VSV
replicated equally well in cultures treated with supernatants from VV
B15RKO and VV B18RKO infections, and to a level similar to that
observed in cultures treated with IFN
2 and VV B15RKO culture
supernatant.
(Ref.
28)), only the type I IFN-binding protein appears to be able to exist
in both a soluble and surface form. The importance of the poxvirus type
I IFN-binding protein in the pathogenesis of the natural infection is
not known. We have determined that inactivation of the B18R gene
increases the mouse intracranial LD
value only by 7-fold
over VV wild type.
(
)This value was lower than
expected, but may be due to the lower affinity that the VV B18R protein
has for mouse IFN
(Fig. 1) or to the fact that VV is
not a natural pathogen of the mouse. A more precise understanding of
the role of the B18R gene in poxvirus pathogenesis will come from the
study of a B18R
poxvirus in its natural host. We are
currently initiating such studies in ectromelia virus, the causative
agent of mousepox.
Table: B18R protein blocks the anti-viral effects of
IFN2
2 mixed with 1 µl of the indicated culture supernatant for
24 h prior to challenge with VSV. Data are presented as the mean of
triplicate values ± standard error of the mean.
response region; ISRE,
interferon-stimulated response element.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.