Transcriptional activity of interferon regulatory factor (IRF)-3
depends on multiple protein–protein interactions
Hongmei Yang
1
, Charles H. Lin
2
, Gang Ma
1
, Melissa Orr
1
, Michael O. Baffi
1
and Marc G. Wathelet
1
1
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati;
2
Department of
Molecular and Cellular Biology, Harvard University, Cambridge, MA
Virus infection results in the activation of a set of cellular
genes involved in host antiviral defense. IRF-3 has been
identified as a critical transcription factor in this process. The
activation mechanism of IRF-3 is not fully elucidated, yet it
involves a conformational change triggered by the virus-
dependent phosphorylation of its C-terminus. This con-
formational change leads to nuclear accumulation, DNA
binding and transcriptional transactivation. Here we show
that two distinct sets of Ser/Thr residues of IRF-3, on
phosphorylation, synergize functionally to achieve maximal
activation. Remarkably, we find that activated IRF-3 lacks
transcriptional activity, but activates transcription entirely

becomes active only after being exposed to pertinent stimuli,
as is the case for IRF-5 and IRF-7. By contrast, the activity
of IRF-1 and IRF-9 is constitutive. The mechanism by
which these virus-dependent IRFs are activated remains to
be characterized, but it involves the phosphorylation of a
stretch of serine (Ser) and threonine (Thr) residues at their
C-terminal ends. This phosphorylation results in a con-
formational change that allows nuclear accumulation,
DNA-binding and transcriptional activation of target genes
[8–17].
However, the identity of the functionally important
phosphorylation targets remains controversial (Fig. 1A).
Indeed, while Fujita and colleagues propose Ser385/386 to
be the key residues in the activation of human (h)IRF-3
[16,18], Hiscott and colleagues point to the five Ser/Thr
residues between amino acids 396–405 as the responsible
targets [9,19]. Similarly, the identity of phosphorylated
residues is unclear for IRF-7 [10,20]. The candidate residues
in IRF-3 and IRF-7 fall into two groups: the first group
comprises two Ser residues that are conserved among the
human, murine and chicken proteins, while the second
group comprises five or six Ser/Thr residues in a less
conserved region immediately downstream from the first set
(Fig. 1A). In addition, the nature and functional import-
ance of protein–protein interactions in IRF-3-dependent
transcriptional activity remains poorly defined.
Here we first show that IRF-3 activation depends on
synergy between the two sets of Ser/Thr residues. Modifi-
cation of residues within both sets is required to achieve full
DNA binding and transactivation capabilities. Intriguingly,

constructs, the coding sequence of IRF-3 was preceded by a
histidine tag, containing a stretch of six His residues (H6-
IRF-3). Alternatively, the coding sequence of IRF-3 was
fused to the Gal4 DNA-binding domain. Mutants of IRF-3
were generated by PCR and were all verified by sequencing.
Reporter constructs have been described [14].
Cell culture and transfections
HEC-1B (HTB-113, ATCC) cells are derived from a human
endometrial carcinoma and are resistant to IFN; SAN cells
are derived from a human glioblastoma and are lacking type
I IFN genes; 293T cells are a SV40 large T antigen-
expressing highly transfectable derivative of 293 cells, which
are derived from human embryonic kidney cells trans-
formed with human adenovirus type 5. These cell lines were
grown at 37 °C, 5% CO
2
, in Dulbecco’s modified Eagle
medium containing 10% fetal bovine serum, 50 UÆmL
)1
penicillin and 50 lgÆmL
)1
streptomycin.
Sendai virus was obtained from SPAFAS and used at 200
hemaglutinin UÆmL
)1
.
S2 cells were grown at 26 °C, in Schneider’s Drosophila
medium containing 12% fetal bovine serum, 50 UÆmL
)1
penicillin and 50 lgÆmL

added for EMSA involving in vitro translated IRF-3.
Immunoblotting, after SDS/PAGE or native gel electro-
phoresis in the presence of deoxycholate [22], was performed
as described [23], using mouse monoclonal SL-12 [anti-
(IRF-3)] and rabbit polyclonal sc-510 (anti-Gal4, Santa
Cruz) as primary antibodies, and anti-mouse or anti-rabbit
horse radish peroxidase conjugates as secondary antibodies.
The chemiluminescence detection system was from Perkin-
Elmer life sciences.
Pull-down experiments
Glutathione S-transferase (GST)-CBP-N, -M, -C, -p300-N,
-M and -C were described previously [24], GST-CBP-N1,
-N2, -N3, -C1, -C2, -C3 and GST-p300-C1, -C2, -C3 were
generated by subcloning PCR products and verified by
sequencing. GST and GST fusions were expressed in E. coli
BL21 and purified as recommended by the manufacturer
(Pharmacia), and dialyzed against phosphate-buffered
saline-10% glycerol.
35
S-Labeled in vitro translated proteins
were incubated with GST fusion proteins immobilized on
glutathione-sepharose beads in 150 m
M
KCl, 20 m
M
Tris
pH 8.0, 0.5 m
M
dithiothreitol, 50 lgÆmL
)1

analyzed by SDS/PAGE and autoradiography.
RESULTS
Identifying the residues functionally involved in IRF-3
activation is important both for the characterization of the
kinase(s) involved in activation and for our understanding
of the activation mechanism. We undertook a systematic
analysis of the role played by the two sets of Ser/Thr
residues in the virus-dependent activation of IRF-3. To this
end, we examined the phenotypes of a variety of hIRF-3
mutants by performing cotransfections in SAN cells. These
cells are derived from a human glioblastoma and lack all
type I IFN genes. The absence of IFN genes allows us to
avoid the complication of a feed-back loop where virus-
induced IFN in turn activates ISGF-3 (i.e. IFN-stimulated
gene factor 3, a complex of IRF-9, STAT-1 and STAT-2)
and STAT-1 dimers, leading to increased levels of endo-
genous IRF-7 and IRF-1. Increased levels of IRF-1, IRF-7
and ISGF-3 would interfere with the activity of the reporter
plasmid used in these experiments. We used the P31
·2
CAT
reporter, which is driven by the IRF-dependent element of
the IFN-b gene promoter. This reporter is only weakly
inducible by virus alone because of the relatively low affinity
of IRF-3/7 for the P31 sequence (approximately 100-fold
less than for an optimal sequence [14]). However, it can be
strongly stimulated by transfection of wild-type (WT) IRF-
3, thus allowing the phenotype of each mutant to be clearly
assessed (Fig. 1B, middle panel). In addition, we also tested
these mutants as fusion proteins with the Gal4

infection stimulated it further. Simultaneous mutation of
both sets of Ser/Thr residues led to IRF-3 mutants (A7,
A2E5, E2A5 and E7) that were only marginally inducible
upon virus infection by themselves, and not inducible at
all as Gal4 fusions. In conclusion, the results that
mutation of either set of Ser/Thr residues alone is
accompanied by a virus-dependent increase in activity,
and that mutation of both sets is not, strongly suggest that
residues within both sets of Ser/Thr residues are phos-
phorylated in response to virus infection.
IRF-3 activates transcription through CBP recruitment
We next examined the transcriptional activity of IRF-3 in
S2 cells, a Drosophila melanogaster cell line. These cells were
chosen because they do not have any apparent IRF
homolog, and are therefore unlikely to contain a kinase
activity that can specifically phosphorylate the virus-
dependent regulatory domain of IRF-3. In these experi-
ments, we used a reporter driven by the ISRE of the ISG15
gene, which is a high affinity binding site for IRF-3. We
cotransfected pPac plasmids expressing either wild-type or
mutant IRF-3 along with ISRE
·3
CAT and in the presence
or absence of murine (m)CBP (Fig. 2A). In the presence of
mCBP, WT IRF-3 was transcriptionally inactive, but
substitution of either or both sets of Ser/Thr residues with
Glu(IRF-3E2,E5andE7)ledtosubstantialactivationof
the reporter. IRF-3E7 was a much more potent activator
than either IRF-3E2 or IRF-3E5. Immunoblot analysis of
the IRF-3 mutants indicated that IRF-3E5 and E7 levels

IRF-3E5 in S2 cells as the difference in transcriptional
potency between the two mutants was lower when fused to
Gal4.
Taken together, the results in mammalian and insect cells
strongly suggest that residues within both sets must be
modified for maximal activation of IRF-3. However, it is
unclear why the transcriptional activity of IRF-3E5 was
stronger than that of IRF-3E7 in mammalian cells. To
address this question, we investigated the dimerization and
DNA-binding activities of mutant and WT IRF-3 proteins.
Dimerization and DNA-binding activity of IRF-3 mutants
Dimerization was assayed by native PAGE in the presence
of deoxycholate [22], and DNA-binding was assayed by
EMSA using the IFN- and virus-inducible ISRE of the
ISG15 gene. We used 293T cells for the following experi-
ments because the suggestion that the second set but not the
first set of residues was phosphorylated in response to virus
infection was based on work using 293T cells [9,19]. First,
extracts from transfected 293T cells were assayed by EMSA
(Fig. 3A, top panel). Transient expression of WT IRF-3 in
293T cells led to the detection of two new complexes as
compared to extracts from cells transfected with empty
vector (compare lanes 1 and 2 with 3 and 4). The faster
migrating complex corresponds to IRF-3 while the slower
complex, with a mobility very similar to that of virus
activated factor, corresponds to IRF-3 associated with the
p300/CAT coactivators (as determined by supershift
experiments [9,11,13–16], data not shown). Both complexes
became more intense in the presence of virus infection
(compare lanes 3 and 4).

consistent with its stronger transcriptional activity
(Fig. 1B).
Next we investigated the properties of IRF-3 produced by
in vitro translation in wheat germ extracts (Fig. 3C). The
WT and the E2, E5 and E7 mutant proteins were tested for
their ability to dimerize and to bind to the ISRE. Native
PAGE indicated that the in vitro produced mutant and WT
IRF-3 existed predominantly in the monomeric form
(Fig. 3C, top panel) and no specific DNA-binding was
detectable under our standard EMSA conditions (data not
shown). Thus, the ability of IRF-3E5 to dimerize and bind
DNA were significantly different depending on whether it
was produced in vitro or in vivo in mammalian cells. While
IRF-3E5 affinity for the ISRE was approximately an order
of magnitude stronger than that of IRF-3E7 when these
proteins were expressed in mammalian cells, the two
proteins did not display any significant affinity for the
ISRE when produced in vitro. Similarly, native PAGE
analysis revealed that half the IRF-3E5 produced in vivo was
dimeric, while IRF-3E5 produced in vitro and IRF-3E7,
regardless of its source, were predominantly monomeric.
Taken together, these results demonstrate that an additional
modification of IRF-3E5 took place in vivo in mammalian
cells that increased dimerization and DNA-binding activity,
presumably phosphorylation of Ser385 and/or Ser386.
Fig. 3. IRF-3E5 is further modified upon transfection in mammalian
cells. (A) Extracts from 293T cells transfected with empty vector or
vector expressing IRF-3 WT or mutants as indicated (lanes 1–8) were
submitted to EMSA using the ISG15 ISRE (5 lg extract, top panel) or
to immunoblot (IB) analysis using anti-(IRF-3) Ig (SL12) after SDS/

experiments with immobilized GST fusions for a finer
mapping (a representative experiment is shown in Fig. 4C,
and binding values referred to in the text below correspond
to the average of at least three independent experiments).
IRF-3WT bound weakly (1–2% of input) to the N- and
C-terminal regions of CBP, and binding to the correspond-
ing regions of p300 was even weaker ( 0.5–1% of input).
By contrast, binding of either IRF-3E5 or E7 was much
stronger than WT: CBP-N,  20%, CBP-C,  24 and 36%
of input for IRF-3E5 and E7, respectively. Binding to the
corresponding regions of p300 was approximately 2–3 times
weaker.
When binding of IRF-3 to smaller domains of the N- and
C-terminal regions of p300/CBP was examined, most of the
activity was found to reside in the N2 and C2 segments.
Thus, substitution of Ser/Thr residues with Glu in the virus-
regulated domain of IRF-3 led to a strong increase in its
affinity for the N2 and C2 regions of the p300 and CBP
coactivators. These substitutions partially mimicked the
virus-dependent phosphorylation of IRF-3 and allowed us
to recapitulate in vitro the association between IRF-3 and
p300/CBP that takes place when IRF-3 is activated by virus
in vivo (Fig. 4B).
The fact that the interactions between IRF-3 and GST-
CBP-N were not detected using virus-activated proteins
probably reflects the presence of the detergent used to
disrupt the interaction between IRF-3/7 and endogenous
p300/CBP (Material and methods).
Two distinct regions of IRF-3 are required for
interaction with coactivators

GST-CBP fusions. IRF-3 truncated to amino acid 388, i.e.
between the first and second set of Ser/Thr residues, bound
effectively to GST-CBP fusions at a level very close to that
observed for IRF-3
1)409
. However, IRF-3 further truncated
to amino acids 370 bound poorly to GST-p300C, -CBP-C
or -CBP-C2, while binding to GST-p300N, -CBP-N or
-CBP-N2 was very similar to that of other IRF-3
Fig. 4. IRF-3 interacts with multiple domains of p300/CBP. (A) Pri-
mary structure of mCBP: the position of functional domains is indi-
cated, and regions fused to GST protein and used in this study are
mapped below. (B) Extracts from control [C] or virus-infected [V]
HEC-1B cells labeled in vivo with [
32
P]orthophosphate were treated
with deoxycholate/NP-40 to dissociate IRF proteins from p300/CBP,
the detergent concentration was decreased by dilution, and the diluted
proteins were incubated with the indicated GST fusions immobilized
on glutathione sepharose. Proteins retained on the GST fusions were
eluted and immunoprecipitated with anti-(IRF-3) Ig (SL12). Immu-
noprecipitated proteins were analyzed by SDS/PAGE and autoradi-
ography. (C)
35
S-labeled IRF-3 WT, E5 and E7 were produced by
in vitro transcription/translation using rabbit reticulocyte lysates and
incubated with the indicated GST fusions of murine CBP and human
p300 immobilized on glutathione sepharose for pull-down experi-
ments. Proteins retained on the GST fusions were analyzed by SDS/
PAGE and autoradiography. 20% of IRF-3 protein input is shown on

the target residues are absent from IRF-3
1)368
and shorter
truncations. Rather, the slower migrating forms most likely
corresponded to dimers and higher order oligomers. The
same truncations were assayed for their ability to bind the
ISRE by EMSA, and the amount of DNA-binding,
normalized to the amount of protein, is charted in Fig. 5D,
along with the ratio of dimeric to monomeric forms.
Truncation to amino acids 409 or 388 resulted in a small
increase in the proportion of the dimeric form and in low
levels of detectable DNA-binding activity, as compared to
full-length IRF-3. Further truncation resulted in a much
higher proportion of the dimeric form and in higher levels of
binding to the ISRE up to amino acids 328–308. IRF-3
1)288
(Fig. 5D) and shorter forms (Fig. 5A and data not shown)
displayed reduced DNA-binding and dimerization. Thus,
there was a strong correlation between the ability of IRF-3
to dimerize and its ability to bind DNA. These data,
together with previous results [14,19], suggest that progres-
sive truncations from the C-terminus of IRF-3 removed a
domain that prevented dimerization, and the ability to bind
DNA that accompanied it. Further truncations eventually
affected the dimerization domain whose C-terminal end-
point is located between amino acids 308 and 288 (Fig. 5).
Each of IRF)3¢s multiple interactions with coactivators
is essential for activity
We have shown above that IRF-3 transcriptional activity
was entirely dependent on its ability to associate with

, it failed to activate tran-
scription in the absence of contact with the C-terminal part
of CBP.
Next, we investigated the ability of N- and C-terminal
fragments of CBP to interfere with the ability of IRF-3E7
and full-length CBP to activate transcription (Fig. 6B).
Expression of GST-CBP
1)1100
and GST-CBP
1892)2441
Fig. 5. Two domains of IRF-3 are involved in interactions with coacti-
vators. (A) Proteins were produced by in vitro transcription/translation
using rabbit reticulocyte lysates and cDNAs encoding WT and the
indicated IRF-3 mutants; control protein (ctrl) is luciferase; translated
proteins were analyzed in the presence of GST, GST-CBP-N and
GST-CBP-C2 by EMSA using the ISG15 ISRE as a probe (left panel);
translated proteins were detected by immunoblotting (right panel) after
SDS/PAGE. (B)
35
S-labeled IRF-3 WT, E7, 1–409, 1–388 and 1–370
were produced by in vitro transcription/translation and incubated with
the indicated GST fusions immobilized on glutathione sepharose for
pull-down experiments. Proteins retained on the GST fusions were
analyzed by SDS/PAGE and autoradiography. Twenty-five per cent of
IRF-3 proteins input is shown on the right. (C) Proteins were produced
by in vitro transcription/translation using rabbit reticulocyte lysates
and cDNAs encoding WT and the indicated IRF-3 truncations; con-
trol protein (ctrl) is luciferase; translated proteins were analyzed by
deoxycholate-PAGE (top panel) or SDS/PAGE (bottom panel) and
immunoblotting with SL12; the wavy pattern of migration for the

or GST-CBP
1892)2441
had minimal
effects on the transcriptional activity of Gal4-mCBP
1)2441
(Fig. 6D), demonstrating that the inhibitory effect on the
activity of the IRF-3/CBP complex was not due to
interference with CBP transcriptional activity but to inter-
ference with the interaction between IRF-3 and CBP. Taken
together, these results demonstrate that the transcriptional
activity of IRF-3 is dependent on simultaneous contact with
both the N- and C-termini of CBP, and on the physical
integrity of CBP.
We also tested the ability of IRF-3E7 and human p300 or
mutant derivatives [27] to activate transcription from the
ISRE
·3
CAT reporter in S2 cells (Fig. 6E,F). The level of
activation achieved by IRF-3 and p300 WT was approxi-
mately twofold lower than that reached by the IRF-3/CBP
combination. Deletion of the p300 Bromo domain
(DBromo, D amino acids 1071–1241) resulted in a significant
reduction of p300s ability to activate transcription in
combination with IRF-3. The other deletions tested,
p300DNR (D amino acids 3–173), p300DE1a (D amino
acids 1739–1871) and p300DSRC (D amino acids 2042–
2157) all completely failed to activate transcription in the
presence of IRF-3. The inability of p300DSRC to activate
transcription with IRF-3 was expected, as this deletion
removes one of the two major interaction regions with IRF-

virus-induced IFN that leads to the formation of ISGF3
and the induction of both IRF-1 and IRF-7, all
transcription factors that can activate the reporters used
to monitor the transcriptional activity of IRF-3; (d) the
ability of DNA transfection per se to undesirably stimu-
late the virus-activated signal transduction pathway to
some extent and (e) the presence of viral gene products in
certain cell lines that can potentially interfere with p300/
CBP function (e.g. E1a and SV40 large T in 293T cells;
Fig. 6. All interactions between IRF-3 and coactivators are essential for
transcriptional activity. (A) Transcriptional activity in S2 cells of
transfected IRF-3 deletion mutants (0.5 lg) on the ISRE
·3
CAT re-
porter in the presence of cotransfected p300/CBP (1.5 lg). (B) S2 cells
were transfected with IRF-3E7 (0.5 lg), mCBP (0.5 lg) and the in-
dicated GST-CBP fusions (0.5 and 2 lg in the presence of CBP, 2 lgin
its absence) together with the ISRE
·3
CAT reporter. (C) Transcrip-
tional activity in S2 cells of the indicated Gal4-mCBP fusions (2 lg) on
the G5E1bCAT reporter. (D) S2 cells were transfected with Gal4-
mCBP
1)2441
(0.5 lg), the indicated GST-CBP fusions (0.5 and 2 lg)
together with the G5E1bCAT reporter. (E) Transcriptional activity in
S2 cells of transfected IRF-3E7 (0.5 lg) on the ISRE
·3
CAT reporter in
the presence of cotransfected p300 (1.5 lg), WT or the indicated mu-

role in the phosphorylation events at the C-terminal end
of IRF-3, e.g. they could be part of the surface of the
protein recognized by the virus-activated kinase that
would phosphorylate the downstream Ser/Thr residues
[19]. However, IRF-3E5 has no constitutive activity in
L929 cells [18]. Thus, the transcriptional potential of IRF-
3E5 and its virus-dependence are cell type specific. What
accounts for these differences? One possibility is that IRF-
3E5 could be additionally modified when transfected in
mammalian cells. 293T cells can be transfected with very
high efficiencies, and a high transfection efficiency in turn
would result in high levels of dsRNA production by
symmetric transcription of the transfected plasmids (limi-
tation #4). Accordingly, subsequent virus infection would
not result in any further increase in transcriptional
activity. Alternatively, it is possible that substitution to
E5 primes IRF-3 for its phosphorylation either by the
genuine virus-activated IRF-3 kinase or by another
endogenous kinase. The possibility that IRF-3E5 is further
modified upon transfection into mammalian cells has
considerable support from the DNA binding and dime-
rization experiments (Figs 3 and 5). That is, IRF-3E5
dimerized and bound the ISRE much more effectively
than IRF-3E7 when these proteins were produced in
transfected 293T cells, a difference that was absent in
wheat germ extracts (or in insect cells). Thus, IRF-3E5
can be additionally modified in some transfected mam-
malian cells, and this modification is presumably phos-
phorylation of Ser385/386 as mutation of these residues to
either Ala or Glu led to much weaker DNA binding.

dimerization, ISRE–binding and interaction with coactiva-
tors (Fig. 5), resulting in transcriptional activity in insect
(Fig. 6) or mammalian cells (data not shown).
In vivo, this intramolecular interaction is naturally
disrupted when IRF-3 becomes phosphorylated in virus-
infected cells. This conformational change involves the loss
and gain of molecular interactions, and the two sets of
phosphorylated residues could play distinct roles in this
process. IRF-3 dimerizes and binds to DNA much more
efficiently when the first set is phosphorylated than when it
is not or when it is substituted with Ala or Glu residues
(Figs 1 and 3). These results thus suggest the first set of Ser
residues are involved in a new intra- or intermolecular
interaction where phosphorylated Ser385/386 interact with
another domain of IRF-3 either on the same molecule or on
the dimerization partner, and this gain of interaction can
only be inefficiently mimicked by glutamic (or aspartic) acid
substitution. By contrast, phosphorylation of the second set
of Ser/Thr residues seems to be primarily involved in the loss
of the intramolecular interaction that keeps IRF-3 in the
inactive form. Indeed, ectopic expression of IRF-3 WT,
IRF-3A5 and IRF-3E5 lead to very similar levels of
transcriptional activation from the P31
·2
CAT reporter in
virus-infected SAN cells (Fig. 1B). Therefore, the second set
participates minimally in intra- or intermolecular interac-
tionswhenIRF-3isadimerasAla,Gluorphospho-Ser/
Thr residues within this set all displayed the same phenotype
in these assays. However, unphosphorylated Ser/Thr resi-

in CBP [37] (Figs 4 and 5). Because Gal4 binds DNA as a
dimer, the dimeric conformation of IRF-3 is presumably
favored in these experiments, thus exposing a region of the
protein that is not accessible under physiological conditions,
either because IRF-3 is in its monomeric conformation or
because it interacts with p300/CBP. It is possible that the
transcription potential of IRF-3 in yeast cells is due to a
spurious interaction between this region of IRF-3 and a
component of the yeast transcription machinery.
Virus-dependent phosphorylation of IRF-3 leads to
strong association with p300 and CBP [12,14–16,18,38],
and this association involves multiple interactions (Figs 4–
6). There is an interaction between the N-terminal half of
IRF-3 (amino acids 1–241) and N-terminal fragments of
p300 and CBP. This was further mapped to CBP-N2
(amino acids 267–462 of CBP), which contains the CH1/
TAZ1 domain. This region of the coactivator is known to
interact with a large number of transcription factors,
including RelA, STAT-2 and p53. There is another
interaction between a central region of IRF-3 (amino acids
139–386) and the C-terminal part of p300 and CBP, more
specifically the C2 region that contains the recently
described IBiD domain, which is known to interact with
TIF-2, Ets2 and E1a ([37] and references therein). The first
set of Ser residues may not be directly involved in binding of
CBP/p300, as changing them both to Ala or Glu had little
effect on the association of IRF-3
1)388
with GST-CBP-C2
in vitro (data not shown). However, a peptide extending

coactivators to stimulate transcription. Simply recruiting the
coactivators’ intrinsic transcription potential to IRF-3 is not
sufficient, as shown by the failure of GST-CBP
1)1100
or
GST-CBP
1892)2441
alone or in combination to activate
transcription together with IRF-3E7 in insect cells. By
contrast, such fragments are sufficient to stimulate the
activity of other transcription factors [39]. This failure is not
due to an inability of CBP
1-1100
or CBP
1892-2441
to (a)
interact with IRF-3E7 in the transfected cells (as they can
interfere with transcriptional activation mediated by IRF-
3E7 and full-length mCBP), or to (b) independently activate
transcription(astheycandosowhenfusedtotheGal4
DNA-binding domain, Fig. 6). Rather, these results and the
effect of p300 deletions underscore how multiple interac-
tions between IRF-3 and a mammalian coactivator are
indispensable to activate transcription.
ACKNOWLEDGEMENTS
We would like to thank T. Collins, R. Goodman, W. Lee Krauss and
D. Livingston for kindly providing reagents, and Maria Czyzyk-
Krzeska and Nelson Horseman for critical reading of the manuscript.
This work was supported by a Dean Research Award to M. G. W.,
and by grant from the National Institutes of Health (AI20642) to Tom

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