From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
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ABSTRACT |
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Certain viruses have evolved mechanisms to
counteract innate immunity, a host response in which nuclear factor
B (NF-
B) transcription factors play a central role. African swine
fever virus encodes a protein of 28.2 kDa containing ankyrin repeats similar to those of cellular I
B proteins, which are inhibitors of
NF-
B. Transfection of the African swine fever virus I
B gene inhibited tumor necrosis factor- or phorbol ester-induced activation of
B- but not AP-1-driven reporter genes. Moreover, African swine fever
virus I
B co-immunoprecipitated with p65 NF-
B, and the purified
recombinant protein prevented the binding of p65-p50 NF-
B proteins
to their target sequences in the DNA. NF-
B activation induced by
tumor necrosis factor, as detected by mobility shift assays or by
transfection of
B-driven reporter genes, is impaired in African
swine fever virus-infected cells. These results indicate that the
African swine fever virus I
B gene homologue interferes with NF-
B
activation, likely representing a new mechanism to evade the immune
response during viral infection.
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INTRODUCTION |
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One of the hallmarks of innate immunity consists of the ability of
the infectious agent to generate cytokines that help mount the
inflammatory response and recruit immune cells to the site of
infection. Work carried out in the past several years has identified nuclear factor B
(NF-
B)1 as one of the most
important elements coordinating such responses (1). NF-
B controls
the transcription of many different genes involved in many aspects of
the inflammatory and immunological responses. Of particular importance
for innate immunity are cytokines, cytokine receptors, chemokines,
adhesion molecules, and acute-phase response genes (2-5).
NF-B inducible transcriptional activators are a family of
transcription factors that includes the dorsal gene of
Drosophila and the mammalian genes nfkb-1
(p105-p50), c-rel, relA (p65), nfkb-2
(p100-p52), and relB, all involved in the regulation of gene
transcription (2, 3). NF-
B is composed of homo- or heterodimers,
with the subunit composition of the different complexes defining the
fine specificity of binding to the target sequences and their
transactivating activity (6). All members share a homologous 300-amino
acid Rel region containing the three essential domains for their
activity: the DNA-binding, dimerization, and nuclear localization
domains (5).
In most cells, NF-B factors are present in an inactive form in the
cytoplasm of resting cells, retained through complex formation with a
cytoplasmic inhibitor belonging to another family of proteins termed
I
B, which masks their nuclear localization sequences, therefore
avoiding their binding to DNA (7-9). These inhibitors, which contain
ankyrin repeats as a common structural motif, include I
B-
(10-13), I
B-
(14), Bcl-3 (15, 16), the NF-
B precursor proteins p105 (17) and p100 (18), and the novel I
B-
(referenced in Ref. 19).
In response to a variety of activators, IB-
proteins undergo
phosphorylation of Ser32 and Ser36 (20, 21),
rendering I
B susceptible to proteolysis via the ubiquitin-proteasome
pathway (22, 23). This unmasks the nuclear localization sequence of the
transactivating heterodimers, allowing translocation of active NF-
B
complexes to the nucleus. Furthermore, it has been recently shown that
phosphorylation of tyrosine 42 of human I
B-
leads to activation
of NF-
B without degradation of the I
B protein (24). In addition
to retaining NF-
B in the cytoplasm, I
B-
prevents p65 and c-Rel
binding to DNA in vitro (10, 25).
African swine fever virus (ASFV) is a large DNA virus that infects
different species of suids, causing an acute and frequently fatal
disease (26). ASFV mainly replicates in macrophages and monocytes, and
this may have major effects on the pathogenicity of the disease (27).
Infection by ASFV is characterized by the absence of a neutralizing
immune response, which has prevented the development of a conventional
vaccine. Moreover, it has been speculated that ASFV might have
mechanisms to counteract the host's immune response, as in the case of
other viruses (28, 29). The genome of ASFV is a double-stranded DNA
with a size ranging from 170 to 190 kilobase pairs, depending on the
virus strain. Recently, the entire genome of the ASFV BA71V isolate, a
strain adapted to grow in Vero cells, was completely sequenced, and 151 open reading frames (ORFs) were detected (30). One of these genes,
A238L, contains ankyrin repeats homologous to those found in
the IB family. We show in this report that the protein encoded by
the A238L gene, expressed in Escherichia coli and
purified, behaves as a bona fide I
B-
viral homologue since it
binds p65 NF-
B and prevents the binding of p65-p50 NF-
B dimers to
their target sequence in the DNA. Our work also demonstrates that ASFV I
B specifically inhibits
B-driven reporter genes and that
ASFV-infected cells have an impaired ability to activate NF-
B at the
protein and functional levels.
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EXPERIMENTAL PROCEDURES |
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Cells, Viruses, and Reagents-- Vero (African green monkey) and Jurkat (human) cells were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium containing 10% newborn calf serum. The Vero-adapted ASFV strain BA71V was propagated and titrated as described previously (31). Recombinant TNF (107 units/mg) was a generous gift from Pharmacia Spain. All other reagents were obtained from Sigma, except where indicated.
Plasmids--
pCMV-IB-
was a generous gift of Dr. F. Arenzana-Seisdedos and contains full-length I
B-
under control of
the cytomegalovirus (CMV) immediate-early promoter. The
NF-
B-dependent plasmid p3ConA-Luc vector is driven by
three synthetic copies of the NF-
B consensus sequence of the
immunoglobulin
chain promoter and was a generous gift of Dr. J. Alcamí. The AP-1-dependent plasmid AP-1-Luc vector is driven by three synthetic copies of the AP-1 consensus sequence and
was a generous gift of Dr. J. Moscat.
Expression of the A238L Gene in E. coli-- The A238L ORF lacking the first 8 nucleotides was cloned in the expression vector pRSET-A. Plasmid p2SI' (32), containing the A238L ORF, was digested with AflIII and treated with Klenow fragment. A 1.4-kilobase AflIII restriction fragment was then purified from agarose gels by electroelution. The pRSET-A vector was digested with PvuII, treated with calf intestinal phosphatase, and ligated to the 1.4-kilobase blunt-end AflIII fragment obtained from plasmid p2SI'. E. coli strain BL21 cells were transformed with the recombinant plasmid pRSET-A238L.
Affinity Purification of Recombinant ASFV IB--
A 1-liter
culture of E. coli cells harboring the pRSET-A238L plasmid
was induced with 0.4 mM
isopropyl-
-D-thiogalactopyranoside for 2 h at
37 °C. The fusion protein present in the cell lysate was purified
under denaturing conditions using a 5-ml Ni2+ affinity
chromatography column (QIAGEN Inc.) according to the manufacturer's
instructions. The ASFV I
B protein (eluted from the column with 8 M urea, 0.1 M NaH2PO4,
and 0.01 M Tris-HCl, pH 4.5) was renatured by dialysis
against 25 mM Tris-HCl, pH 8.0, containing 1 mM
EDTA, 1 M NaCl, 5% glycerol, and 0.5% Nonidet P-40.
Preparation of Antibodies--
To prepare anti-peptide
antibodies specific for the ASFV IB protein, a 15-amino acid peptide
(GGSGGCVKKLNKYGK) was synthesized. The last 9 amino acids
of this peptide (underlined) correspond to the carboxyl-terminal region
of ASFV I
B, which is not conserved in cellular I
B-
. The
peptide was conjugated to keyhole limpet hemocyanin and used to
immunize rabbits. The immune serum obtained recognized the recombinant
ASFV I
B protein on Western blots.
Electrophoretic Mobility Shift Assays (EMSA)--
The binding
assays with nuclear extracts from Vero or Jurkat cells were performed
as reported (33), using as 32P-labeled probe either a B
oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') from the immunoglobulin
chain enhancer or an Oct-1 oligonucleotide (5'-TGTCGAATGCAAATCACTAGA-3'). The binding complexes were separated on
a 5% acrylamide gel, and their specificity was determined by competition with a 50-fold molar excess of the same unlabeled oligonucleotide. The protein composition of each DNA-protein complex was identified by supershifting assays using specific polyclonal rabbit
anti-NF-
B antisera. These assays were performed as normal EMSAs,
except that 1 µl of the different anti-NF-
B antisera was added to
the nuclear extract prior to the addition of the probe. The anti-c-Rel,
anti-p50, and anti-p65 antisera were kindly provided by Drs. Nancy Rice
and Alain Israël. In some experiments, purified ASFV I
B was
added to the nuclear extracts.
Transfection Assays-- Vero or Jurkat cells (1 × 107) resuspended in 0.5 ml of Dulbecco's modified Eagle's medium containing 10 µg of linearized plasmid DNA were subjected to electroporation in a Bio-Rad Gene Pulser (960 microfarads, 250 V). For those experiments with infected cells, 2 h before electroporation, the cells were mock-infected or infected with ASFV or vaccinia virus at a multiplicity of 10 plaque-forming units/cell. After electroporation, the cells were resuspended in 10 ml of fresh Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and cultured overnight at 37 °C before being activated with phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) or TNF (30 ng/ml). Cells were incubated for an additional 4-h period, harvested, and lysed. Luciferase activity was measured in a luminometer and expressed as relative luciferase units, calculated as light emission from the experimental sample divided by light emission from untransfected cells per 106 cells.
Immunoprecipitation--
COS cells (1 × 107), resuspended in Dulbecco's modified Eagle's
medium containing 10 mM Hepes and 10% fetal calf serum,
were transfected by electroporation with 10 µg of pCMV,
pCMV-IB-ASFV, or pCMV-I
B-
linearized plasmids, together with
10 µg of pCMV-p65, as described above. After electroporation, the
cells were resuspended in fresh medium supplemented with 10% fetal
calf serum and cultured overnight at 37 °C. The cells were then
labeled with a mixture of [35S]methionine and
[35S]cysteine (50 µCi/1 × 106 cells;
Amersham Corp.) for 24 h. Lysates were prepared by resuspending the cells at 1 × 107 cells/ml in 1% digitonin lysis
buffer containing protease inhibitors (1% digitonin, 10 mM
triethanolamine, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml
pepstatin) as described (34). The cell extracts obtained from 1 × 107 cells for each immunoprecipitation were precleared
three times with protein A-Sepharose and then immunoprecipitated as
described (34) using 30 µg of ASFV I
B-specific anti-peptide
antiserum or polyclonal anti-I
B-
antiserum (Santa Cruz
Biotechnology, Inc.) and protein A-Sepharose. For re-precipitation
analysis, bound proteins were solubilized by boiling in 400 µl of
SDS-containing buffer (0.4% SDS, 50 mM triethanolamine,
100 mM NaCl, 2 mM EDTA, and 2 mM
2-mercaptoethanol). Subsequently, 100 µl of 10% Triton X-100 and 10 mM iodoacetamide were added to the supernatants. The
supernatants were precleared twice with protein A-Sepharose, diluted
with 1 volume of 1% digitonin lysis buffer, and immunoprecipitated with 20 µg of polyclonal anti-p65 antiserum (Santa Cruz
Biotechnology, Inc.) and protein A-Sepharose. Bound proteins were
eluted by boiling with electrophoresis sample buffer and resolved by
SDS-polyacrylamide gel electrophoresis. For autoradiography, the gel
was exposed on a Fujifilm BAS-MP 20405 imaging plate at room
temperature. The exposed imaging plate was analyzed with a Fuji BAS
1500 analyzer.
Computer Analysis-- Computer analyses of DNA and protein sequences were performed with the software package of the University of Wisconsin Genetics Computer Group. Data base searches were done with the programs FASTA and TFASTA. Protein patterns were searched using the MacPattern program and the PROSITE and BLOCKS data bases. Multiple alignment of protein sequences was performed with the PILEUP program. To identify ankyrin repeats in the ASFV A238L ORF, the method described by Bork (35) was followed.
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RESULTS |
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Homology of A238L to IB-
Proteins--
One of the ORFs of
ASFV, denoted A238L, encodes a protein of 238 amino acids
with a predicted molecular mass of 28.2 kDa, which was previously shown
to have homology to the I
B family of inhibitors of NF-
B (30).
When the A238L deduced protein sequence was compared with I
B-
from different species (human, rat, chicken, and pig) following the
procedure described by Bork (35), it was shown that it contains several
regions that can be aligned with the ankyrin repeats of I
B-
(Fig.
1). The similarity between the ASFV and
cellular proteins is 20-24% overall, but is greater in the central
part of the molecules. The cellular I
B proteins contain six ankyrin
repeats (35), each of which is composed of 32-36 amino acids. Only
five repeats of 30-33 amino acids were identified in the viral
protein. The three most carboxyl-terminal repeats co-align with the
corresponding ankyrin repeats of the cellular I
B proteins, while the
second repeat from the amino terminus of the viral protein encompasses
the second and third ankyrin motifs of the cellular proteins. On the
other hand, the first repeat of protein A238L, which has been
identified with a low score with the PROFILESEARCH program, is located
in the multiple alignment between the first and second repeats of the cellular I
B proteins.
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Inhibition of NF-B Activation by the ASFV I
B Gene--
The
above analysis suggested a putative role for the A238L gene
product (hereafter named ASFV I
B) as an inhibitor of NF-
B. To
test this hypothesis, we isolated and inserted the gene into pCMV under
control of the immediate-early CMV promoter, and this plasmid
(pCMV-I
B-ASFV) was transfected by electroporation into Vero or
Jurkat cells, together with a
B-driven reporter gene. When Vero
cells, a cell line susceptible to infection with ASFV, were transfected
with pCMV-I
B-ASFV, an almost complete inhibition of the basal
activity of the
B-driven reporter gene was observed in unstimulated
cells. More interestingly, pCMV-I
B-ASFV completely prevented the
increase in reporter activity observed when the cells were treated with
TNF for 4 h (Fig.
2A).
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Inhibition of Binding of p65-p50 Complexes to B DNA Sequences by
the ASFV I
B Protein--
To study the function of the protein,
recombinant ASFV I
B was purified as described under "Experimental
Procedures." Coomassie Blue staining of the purified protein after
SDS-polyacrylamide gel electrophoresis is shown in Fig.
3. The recombinant ASFV I
B protein was
added to a nuclear extract from Vero cells either untreated or treated
with TNF, and EMSAs were performed. In unstimulated cells, a specific
complex that binds to a
B oligonucleotide was detected (Fig.
4A, lane 1). Upon
treatment with TNF, an additional, slowly migrating complex was
observed (Fig. 4A, lane 2). Those two bands were
specifically competed by an excess of the unlabeled
B
oligonucleotide (Fig. 4A, lane 3), but not by a
mutant
B oligonucleotide (data not shown). Interestingly, when
purified recombinant ASFV I
B protein was added to nuclear extracts
from TNF-stimulated Vero cells, a dose-dependent
displacement of the slowly migrating complex was observed, whereas the
inhibition of the faster migrating band was much less pronounced and
only observed at the higher concentration (Fig. 4A,
lanes 4 and 5). The two complexes detected were
identified by supershifting with specific antibodies against the
different members of the Rel family (Fig. 4B). The fastest migrating complex present in unstimulated Vero cells was supershifted only by anti-p50 antibodies (Fig. 4B, lane 2)
suggesting that it was composed of p50 homodimers. However, the complex
induced after TNF treatment was supershifted by anti-p50 and anti-p65 antibodies, but not by anti-c-Rel antibodies (Fig. 4B,
lanes 2-4). This may indicate that it contains p65-p50
heterodimers, the most commonly induced NF-
B complex in many cell
systems (2-5). ASFV I
B was also able to displace the nuclear
factors from a preformed complex with DNA (Fig. 4C). Again,
it can be seen that the p65-p50 heterodimer was much more efficiently
displaced from the complex than the p50-p50 homodimer (Fig.
4C, lanes 3 and 4).
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Inhibition of NF-B by ASFV Infection--
To confirm the role
of this ASFV I
B protein in infection, Vero cells were infected with
ASFV, and the activity of NF-
B was studied. The ASFV I
B protein
can be detected in infected cells by Western blotting at very early
times after infection and remained up to 16 h post-infection (data
not shown). Moreover, its synthesis was insensitive to cytosine
arabinoside treatment, indicating that the A238L gene is an
ASFV early gene (37). As in the case of many other viral infections (4,
38), a p65-p50 NF-
B complex was detected in the nucleus of
unstimulated ASFV-infected cells (Fig.
7). The amount of this complex, as
determined by densitometric scanning of the films, was always below
20% of that obtained in the presence of TNF in mock-infected cells and
did not further increase with time post-infection (data not shown).
Interestingly, no further increase in active p65-p50 NF-
B complex
able to bind to the
B probe was observed in the nucleus of
TNF-treated ASFV-infected cells (Fig. 7). This TNF unresponsiveness was
observed at any time post-infection (data not shown).
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DISCUSSION |
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Our results indicate that the ASFV genome encodes a protein with
homology to the ankyrin repeats of IB, which behaves as an effective
inhibitor of NF-
B activity. I
B-
proteins serve many functions.
1) They prevent binding of NF-
B to
B DNA sequences. 2) They
prevent nuclear translocation of active NF-
B. 3) They dissociate
NF-
B from DNA. 4) Some I
B proteins, such as Bcl-3, also serve as
transcriptional activators (7, 39). As deduced from our studies, ASFV
I
B is able at least to prevent NF-
B binding to DNA and to
dissociate already active p65-p50 NF-
B dimers from it.
Comparison of sequences indicates that ASFV IB is more homologous to
the ankyrin repeats present in I
B than to those present in other
proteins. Several residues and motifs have been identified in cellular
I
B-
as essential in the various processes that lead to its
degradation. Amino-terminal serines (20-22) and lysines (40) are the
sites of inducible phosphorylation and ubiquitination, respectively.
The carboxyl-terminal PEST-like sequence also plays a role in
signal-dependent proteolysis (41). Moreover, the
carboxyl-terminal segment between amino acids 269 and 287 of MAD3
renders the protein constitutively unstable, diminishes its interaction
with p65, and is involved in regulating protein half-life (42). In
addition, it has been shown that phosphorylation of Tyr42
leads to activation of NF-
B without proteolytic degradation of the
inhibitory protein (24). All these residues and motifs are absent in
the ASFV I
B protein, suggesting that the viral protein cannot be
regulated and therefore is a natural, constitutive, and potent
suppressor of NF-
B activity.
ASFV IB specifically interacts with p65 NF-
B since it mainly
affects the binding of p65-p50 heterodimers to DNA and
co-immunoprecipitates with the p65 protein. The p65-p50 heterodimer is
the most common transactivating form of the NF-
B/Rel factors and is
induced by proinflammatory cytokines (TNF and interleukin-1) (2-5).
The binding of p50 homodimers to DNA was weakly affected by ASFV I
B, whereas the binding of other nuclear factors was totally unaffected. In
this regard, it is worth mentioning that I
B-
is also able to bind
most of the transcriptionally active heterodimers (p65-p50, c-Rel-p50,
p65-p52, RelB-p50, p65-p65, c-Rel-c-Rel), but not inactive p50-p50 and
p52-p52 (39).
In addition to retaining NF-B in the cytoplasm, I
B-
is able to
prevent in vitro DNA binding of the p65 and c-Rel subunits and displace them from the DNA (10, 25). Binding of p65 to I
B-
or
DNA is mutually exclusive (43). Moreover, I
B-
can be found in the
nucleus, suggesting a role for I
B-
in negatively regulating
transcription (9). ASFV I
B might go to the nucleus, thus sparing the
need for the ankyrin repeat(s) involved in blocking the nuclear
localization system of p65 and providing an explanation for the weak
homology detected in one of the five ankyrin repeats.
On the other hand, NF-B is a key element in coordinating the
inflammatory and immune responses (1). The activation of this factor is
a very effective way of initiating the immune response to viral
infection since it preexists in the cytoplasm and can be rapidly
activated, inducing a large set of genes involved in the immune
response. Thus, there are many examples of NF-
B activation during
viral infection. However, some viruses (human immunodeficiency virus
type 1, human T-cell lymphotropic virus type 1, hepatitis B virus,
herpes simplex virus type 1, CMV, Newcastle disease viruses, SV40,
Sendai virus, Epstein-Barr virus, influenza virus, and adenovirus) take
advantage of the activation of NF-
B for their own benefit to turn on
their own genes (4, 38).
By contrast, activation of NF-B may be deleterious for viruses that
need to establish latency or that have a long infecting cycle, as would
be the case for a large virus, such as ASFV. Moreover, cells of the
macrophage/monocyte lineage play a central role in the immune defense,
producing cytokines, NO synthase, etc., genes whose expression is
controlled by NF-
B (3, 4). NF-
B is induced in macrophages by a
variety of cytokines, TNF being one of the most important. As
macrophages are the principal host cells for ASFV, it would be
evolutionarily advantageous for this virus to inhibit the activity of
NF-
B in the infected cells.
The number of virus-encoded gene products that influence host
mechanisms and the viral strategies to evade immune responses continue
to expand (28, 29). Cytokines regulate the inflammatory (i.e. interleukin-1 and TNF) and immune responses and may be
directly antiviral (i.e. interferon) or destroy
virus-infected cells (i.e. TNF). Therefore, cytokines
constitute good targets for virus evasion strategies. During evolution,
some large viruses, such as poxviruses, have acquired from their
mammalian hosts a variety of genes that encode proteins that appear to
specifically target cytokines to presumably avoid their antiviral and
proinflammatory activity. Thus, they encode homologues of cytokine
receptors (interleukin-1 receptor type 2, TNF receptor, interferon-
receptor, and interferon-
/
receptor) that block cytokine activity
(44, 45). The functional significance of these genes in vivo
is poorly understood, but there is evidence that they influence
pathogenicity.
ASFV is a large virus that has also acquired genes that may
affect the cytokine response (30). However, its strategy seems to be
different from that used by other viruses. Instead of targeting proinflammatory cytokines individually, it expresses an IB-like protein that blocks NF-
B, a key common effector downstream
from many of them. The results described in this work may explain
previous data from our laboratory showing that TNF has no antiviral
activity in ASFV-infected macrophages (46).
Recently, a report showing that ASFV-infected macrophages have an
impaired ability to secrete inflammatory cytokines has been published
(47). The authors proposed that ASFV IB might be responsible for
this effect on the basis that transfection of the ASFV I
B gene
inhibited luciferase expression under control of a fragment of the
interleukin-8 promoter that contains, in addition to the
B site,
other sequences likely involved in promoter activity. Our work provides
a characterization of the molecular basis of this effect, showing for
the first time the following. 1) ASFV I
B specifically inhibits
B-driven reporter genes since the reporter gene used in our
experiments is driven by a promoter consisting exclusively of NF-
B
consensus sequences. 2) The ASFV I
B protein specifically interacts
with p65 NF-
B, preventing the binding of p65-p50 NF-
B dimers to
their target sequence in the DNA. It is important to note that these
experiments have been performed with a purified recombinant ASFV I
B
protein, which provides direct evidence that the ASFV gene encodes a
protein with I
B-like activity. 3) In addition, we show that
ASFV-infected cells have an impaired ability to activate NF-
B at the
protein and functional levels.
In summary, our results provide evidence of a possible mechanism of
host evasion by viruses, the inhibition of NF-B activity by an
I
B-like protein encoded by ASFV. This may have important biological
implications. ASFV and other viruses have survived under adverse host
conditions and have adapted to block the action of a key mediator of
the inflammatory and immune responses, NF-
B. Studies with ASFV I
B
may help to clarify the role of NF-
B, the structure-function
relationships of I
B, and eventually the mechanism of TNF signal
transduction.
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ACKNOWLEDGEMENTS |
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We thank Margarita Salas for critical reading
of the manuscript; Juan Pablo Albar for preparation of the ASFV IB
peptide; and M. Chorro, Pedro Bonay, and A. Villarraso for excellent
technical assistance.
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FOOTNOTES |
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* This work was supported by Dirección General de Investigación Científica y Técnica Grants PB93-0160-C02-01 and BIO95-0115, European Community Grant AIR-CT93-1332, Fondo de Investigaciones Sanitarias Grant 95/089, the Comunidad Autónoma de Madrid, and an institutional grant from the Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 34-1-3978436;
Fax: 34-1-3978490; E-mail: Evinuela{at}Trasto.cbm.uam.es.
1
The abbreviations used are: NF-B, nuclear
factor
B; ASFV, African swine fever virus; ORF, open reading frame;
TNF, tumor necrosis factor; CMV, cytomegalovirus; EMSA, electrophoretic
mobility shift assay; PMA, phorbol 12-myristate 13-acetate.
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REFERENCES |
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