Báo cáo Y học: Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein–protein interactions pdf

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

through the recruitment of the p300/CBP coactivators.
Moreover, we show that two separate domains of IRF-3
interact with several distinct regions of p300/CBP. Interfer-
ence with any of these interactions leads to a complete loss of
transcriptional activity, suggesting that a bivalent interaction
is essential for coactivator recruitment by IRF-3.
Keywords: interferon; IRF-3; coactivator; virus; transcrip-
tion.
Vertebrates respond to infections by first triggering the
innate arm of the immune system upon recognizing generic
microbial products, such as lipopolysaccharides for Gram-
negative bacteria, or dsRNA for viruses [1,2]. A number of
cytokines, including interferons (IFNs), tumor necrosis
factors, and several chemokines and interleukins, are
produced early upon infection. They signal to the organism
the presence of the infection, and alter the behaviour of a
large number of cells in order to expedite pathogen
elimination. The production and action of these cytokines
depends in large part on specific modulations of gene
expression.
IFN regulatory factors (IRFs) have extensive roles in
innate immunity by participating in both the immediate-
early transcriptional response to infection and the secondary
response to cytokines [3–5]. For instance, IFN-b synthesis is
activated directly by virus infection in most cell types, or by
lipopolysaccharide in some specialized cells, processes
requiring IRF-3 and IRF-7 [6,7]. The transcriptional
response as activated by IFN-b in turn depends on IRF-1
and IRF-9 via the JAK-STAT pathway.
IRF-3 is a latent transcriptional activator protein that

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,

we found that IRF-3 lacks intrinsic transcriptional activity,
but activates transcription entirely through the recruitment
of the p300/CREB-binding protein (CBP) coactivators. This
was demonstrated using cells (insect Schneider S2 cells) that
are devoid of endogenous IRF and where the endogenous
Drosophila CBP sequence is functionally unable to
Correspondence to M. G. Wathelet, Department of Molecular and
Cellular Physiology, University of Cincinnati College of Medicine,
231 Albert Sabin Way, Cincinnati, OH45267-0576,
Fax: + 513 558 5738, Tel.: + 513 558 4515,
E-mail:
Abbreviations: CAT, chloramphenicol acetyl transferase; CREB,
cAMP response element binding protein; CBP, CREB-binding pro-
tein; EMSA, electrophoretic mobility shift assay; IFN, interferon;
GST, glutathione-S-transferase; IRF, IFN regulatory factor; ISGF-3,
IFN-stimulated gene factor 3; ISRE, IFN stimulated response
element; STAT, signal transducer and activator of transcription;
WT, wild-type.
(Received 8 August 2002, revised 15 October 2002,
accepted 23 October 2002)
Eur. J. Biochem. 269, 6142–6151 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03330.x
substitute for mammalian p300/CBP. Moreover, we found
that IRF-3 interacts with several distinct regions of p300/
CBP. Finally, we show that these protein–protein interac-
tions are essential for acquiring transcriptional activity.
EXPERIMENTAL PROCEDURES
Plasmid constructs and sequence analysis
Effector constructs for transient transfections of mamma-
lian and insect cells were cloned into pcDbA and pPac
vectors, respectively, using standard methods [21]. In these

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

)1
streptomycin.
Transfections using the calcium phosphate coprecipita-
tion technique were as described [21]. Mammalian cells in
100 mm dishes were transfected with 1 mL of a precipitate
containing 10 lg reporter, 5 lg effector plasmid (except for
Gal4, 1 lg), 5 lg pCMV-lacZ and pSP72 to a total of 25 lg
(5–9 lg) for 18 h, trypsinized, aliquoted for further treat-
ments and harvested 3 days after transfection.
S2 cells were seeded in 6-well plates (3 million cells in
3 mL), transfected the next day with 0.3 mL of a precipitate
containing 250 ng hsp82lacZ, 500 ng reporter plasmid and
effector plasmid mixes as indicated in figure legends (with
pPac added to a total of 5.75 lg), and harvested 2 days after
transfection. Chloramphenicol acetyl transferase (CAT)
activity and b-galactosidase activity were measured in
extracts of transfected cells [21], and CAT activity was
expressed in arbitrary units after normalization to b-
galactosidase activity to control for transfection efficiency.
In vitro
translation, cell extract, EMSA and
Western blot
In vitro translation in rabbit reticulocyte lysates or wheat
germ extracts was performed exactly according to the
manufacturer’s recommendations using the TnT kit
(Promega), linearized pcDbA effector plasmids and T7
RNA polymerase. Whole cell extract preparation, binding
and PAGE conditions for electrophoretic mobility shift
assays (EMSA) were as described [14], except that 5 pmoles
of cold 9–27 IFN-stimulated response element (ISRE) were

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

ethidium bro-
mide, 0.2% NP-40 and 0.2% BSA (binding buffer) for 1 h
at 4 °C, followed by two washes with binding buffer and
two washes with binding buffer without BSA. Extracts from
HEC-1B cells labeled in vivo with [
32
P]orthophosphate were
prepared and treated with deoxycholate and NP-40 exactly
as described [14]. After 50-fold dilution with binding buffer
Fig. 1. Two sets of residues in IRF-3 are modified in response to virus
infection. (A) Alignment of the C-terminal cluster of Ser/Thr residues
that are potentially phosphorylated in virus-infected cells for human
IRF-7B, murine IRF-7, human IRF-3, murine IRF-3 and chicken
IRF-3. (B) Transcriptional activity in SAN cells of IRF-3 point
mutants on the P31
·2
CAT reporter (left graph) or as Gal4 fusions on
the G5E1bCAT reporter (right graph); the sequence of WT and each
point mutants is indicated on the left of the graphs; C: control,
uninfected cells, V: virus-infected cells.
Ó FEBS 2002 Mechanism of IRF-3 virus-dependent activation (Eur. J. Biochem. 269) 6143
and preclearing on glutathione-sepharose beads, the
32
P-
labeled proteins were incubated with immobilized GST
fusions for 2 h at 4 °C, followed by three washes with
binding buffer. Proteins bound to the beads were eluted
with radioimmunoprecipitation assay buffer and immuno-
precipitated with SL-12 [anti-(IRF-3)] as described [14].
Pulled-down and immunoprecipitated proteins were then

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

DNA-binding domain (amino acids 1–147) by doing
cotransfections with a reporter, G5E1bCAT, where the
chloramphenicol acetyl transferase gene is driven by the E1b
TATA box and five copies of a Gal4 binding site (Fig. 1B,
right panel). This latter approach minimized the interference
due to endogenous IRF proteins associating with the
ectopically expressed IRF-3 mutants.
Phosphorylation of two distinct groups of Ser/Thr
residues is required for virus activation of IRF-3
We mutated the indicated Ser/Thr residues to either
alanine (Ala) or glutamic acid (Glu), as shown in Fig. 1B,
left panel; in many cases Glu can functionally substitute
for phospho-Ser/Thr residues. Mutation of the first set of
SerresiduestoeitherAla(IRF-3A2)orGlu(IRF-3E2)
drastically reduced the ability of IRF-3 to stimulate
P31
·2
CAT activity in virus-infected cells (middle panel).
By contrast, the same constructs behaved differently as
Gal4 fusions (right panel). While Gal4-IRF-3A2 showed
low, uninducible activity, Gal4-IRF-3E2 displayed signifi-
cant activity that was further stimulated by virus infection.
Substitution of the second set of Ser/Thr residues had a
different impact on IRF-3 activity: IRF-3A5 behaved as
WT, except for a slight increase in basal activity; however,
the activity of Gal4-IRF-3A5 was significantly stronger
than that of the WT construct, but importantly, was still
virus-inducible. IRF-3E5 displayed strong basal activity,
which was further stimulated by virus infection. Similarly,
Gal4-IRF-3E5 had very strong basal activity and virus

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

were similar, and significantly lower than that of IRF-3 WT
or IRF-3E2. Remarkably, we found that none of the IRF-3
constructs activated the reporter in the absence of mCBP,
suggesting that IRF-3 by itself is devoid of intrinsic
transcriptional activity.
Fig. 2. The transcriptional activity of IRF-3 depends on synergy between
two sets of residues and is entirely dependent on mammalian CBP. (A)
Transcriptional activity in S2 cells of transfected IRF-3 point mutants
(0.5 lg) in the presence or absence of cotransfected CBP (1.5 lg) on
the ISRE
·3
CAT reporter (top panel) and the expressed proteins were
detected by Western blotting (bottom panel; n-sp. stands for nonspe-
cific). (B) Transcriptional activity in S2 cells of transfected Gal4-IRF-3
point mutants (1 lg) in the presence or absence of cotransfected CBP
(1.5 lg) on the G5E1bCAT reporter (top panel) and the expressed
proteins were detected by Western blotting (bottom panel, the top
band in each lane corresponds to full-length fusion protein and the
same pattern was observed using anti-Gal4 Ig instead of SL12).
6144 H. Yang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
To exclude the influence of possible differences in DNA-
binding activity on transcriptional potency, we also tested
the same mutants as Gal4 fusions, using G5E1bCAT as
reporter plasmid (Fig. 2B). We observed again that none of
the Gal4-IRF-3 constructs activated the reporter in the
absence of mCBP. Gal4-IRF-3E2 was approximately as
strong an activator as Gal4-IRF-3E5 when differences in
expression levels were taken into account. IRF-3E7 was the
most potent activator of all, either by itself or as a Gal4
fusion, and probably bound the ISRE more effectively than

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).

The presence of these complexes correlated with the
amount of dimeric IRF-3 detected by native PAGE
(Fig. 3B, lanes 3 and 4), where untransfected HEC-1B cells
extracts were used as a reference to show the change of IRF-
3 mobility that corresponds to a monomer (lane 9,
uninfected) to dimer transition (lane 10, virus-infected).
Thus, in uninfected but transfected cells, IRF-3 dimerizes
and forms the transcriptionally competent complex with
p300/CBP (lane 3). By contrast, when cells are not
transfected, virus activated factor is only detected in virus-
infected cells [14]. Given that DNA transfection alone is
sufficient to activate some endogenous IFN-stimulated
genes, including ISG15 [25], these results suggest that the
formation of the activated IRF-3/coactivator complex in
uninfected cells (lane 3) is an artefact of transfection.
When a plasmid directing the expression of IRF-3E5
was transfected in 293T cells, both IRF-3 complexes could
be detected in control or virus-infected cells by EMSA and
virus infection had little effect on the abundance of either
complex (compare lanes 5 and 6 in Fig. 3A). However,
when IRF-3E7 was expressed in 293T cells, only the IRF-
3/coactivator complex could be detected, and at a level
much lower than what was observed for IRF-3E5, despite
their similar expression levels (compare lanes 5 and 6 with
7 and 8, bottom panel). In these extracts, approximately
half IRF-3E5 was dimeric whether cells were infected or
not, while IRF-3E7 was predominantly monomeric
(Fig. 3B). Thus, IRF-3E5 expressed by transient transfec-
tion dimerized much more efficiently and had a much
greater affinity for the ISRE than IRF-3E7 (Fig. 3),

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/

PAGE (10 lg extract, bottom panel); C: control, uninfected cells, V:
virus-infected cells. (B) The same extracts as in A (lanes 1–8) and
extracts (10 lg) from uninfected (lane 9) or virus-infected (lane 10)
HEC-1B cells, were analyzed by native-PAGE and immunoblot ana-
lysis using SL12. (C) Proteins were produced by in vitro transcription/
translation using wheat germ extracts and cDNA encoding wild-type
and the indicated IRF-3 mutants; control protein (ctrl) is luciferase;
translated proteins were detected by immunoblotting using SL12 after
native-PAGE (top panel) and SDS/PAGE (bottom panel).
Ó FEBS 2002 Mechanism of IRF-3 virus-dependent activation (Eur. J. Biochem. 269) 6145
On the basis of these data, we conclude: (a) maximal
dimerization, DNA-binding and transcriptional activity of
IRF-3 required modifications within both sets of Ser/Thr
residues in the C-terminal virus regulatory domain and (b)
IRF-3 has no intrinsic transcriptional activity and depends
on its ability to associate with CBP to activate transcription.
IRF-3 interacts with multiple domains of the p300/CBP
coactivators
As shown above, IRF-3 is functionally entirely dependent
on p300/CBP for transcriptional activation. We examined
the interaction between the coactivators and IRF-3 pro-
duced in vivo or in vitro by pull-down/immunoprecipitation
assays (Materials and methods). As shown in Fig. 4B,
metabolically
32
P-labeled IRF-3 associates specifically with
the C-terminal 550 amino acids of mCBP in a virus-
dependent manner. Thus, endogenous virus-activated IRF-
3 can interact with the C-terminal domain of CBP. We used
proteins produced by in vitro translation in pull-down

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

We next examined which regions of IRF-3 are required for
interaction with p300 and CBP. IRF-3WT and mutant
derivatives were produced by in vitro translation in rabbit
reticulocyte lysates and assayed for their ability to bind to
the ISRE in the presence or absence of GST-CBP-N or
GST-CBP-C2 by EMSA (Fig. 5A). In the absence of GST-
CBP fusions, binding of IRF-3 to the ISRE was undetect-
able for the WT protein and very weak for IRF-3E7 and the
C-terminal truncations 1–409, 1–388 and 1–370 (lanes 4, 7,
10, 13 and 16). Truncations of IRF-3 to amino acids 328,
264 or 241 resulted in much stronger binding to the ISRE
(lanes 19, 22 and 25). In the presence of GST-CBP-N a
doublet of supershifted bands migrating slowly were
detected for all IRF-3 constructs tested. By contrast, in
the presence of GST-CBP-C2, the doublet of supershifted
bands was detected for only some of the constructs: IRF-
3WT, IRF-3E7 and the truncations to amino acids 409 and
388 (lanes 6, 9, 12 and 15). Truncations to amino acid 370
and shorter did not produce a supershift in the presence of
GST-CBP-C2 (lanes 18, 21, 24 and 27).
These interactions were also probed in the absence of
DNA using pull-down assays (Fig. 5B). Binding of IRF-3
WT to the N- and C-regions of p300 and CBP was weak,
while substitution of both sets of Ser/Thr residues with Glu
led to much stronger binding for IRF-3E7, in agreement
withthedatashowninFig.4C.IRF-3
1)409
, a construct that
displays constitutive transcriptional activity in mammalian
cells (data not shown), also led to stronger binding to the

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 right. Binding to p300-C2 was reproducibly stronger than binding
to p300-C. This could be due to poor accessibility to the C2 domain in
p300-C, or imperfect folding of p300-C.
6146 H. Yang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
truncations. Taken together, these results indicated that the
C-terminal region of IRF-3 including the second set of Ser/
Thr residues is dispensable for binding to the C2 region of
the p300 and CBP coactivators, and that the C-terminal
end-point of this interaction domain is located between
amino acids 370 and 388. By contrast, binding to the N2
region of CBP could be achieved with only the first 241
amino acids of the protein.
Mapping IRF-3 dimerization domain
IRF-3 truncations’ ability to bind DNA varied considerably
depending on the end-point of each truncation. Thus, full-
length protein produced by in vitro translation does not bind
DNA, while truncation at amino acids 328 resulted in
strong binding (Fig. 5A [14]). We generated a series of
truncations at  20 amino acid intervals to map the
domains of IRF-3 required for DNA binding and dimeri-
zation more precisely (Fig. 5C,D). When analyzed by
deoxycholate-PAGE and immunoblotting, IRF-3 WT pro-
duced in vitro was predominantly in a faster migrating form,
as observed in Fig. 3C. Progressive truncations from the
C-terminus led to the detection of slower migrating forms
(Fig. 5C, top panel). In the case of virus-activated IRF-3
(Fig. 3B), the slower migration on deoxycholate-PAGE
could be due to dimerization of the protein, or simply to the
phosphorylation of its C-terminus. However, the latter is
unlikely as the proteins are produced in vitro and because

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

mammalian p300/CBP (Fig. 2), and that IRF-3 physically
interacted with distinct regions of these coactivators (Figs 4
and 5). The importance of each of these contacts for IRF-3¢s
ability to activate transcription was tested in S2 cells. We
first investigated which IRF-3 truncations would be active
in insect cells (Fig. 6A). Interestingly, both IRF-3
1)409
and
IRF-3
1)388
significantly activated transcription of the
ISRE
·3
CAT reporter in the presence of mammalian coac-
tivators, and these truncations interacted with both the
N- and C-terminal regions of p300/CBP (Fig. 5A,B). By
contrast, IRF-3
1)370
and IRF-3
1)328
interacted significantly
only with the N-terminus of p300/CBP and displayed no
detectable transcriptional activity. Thus, even though IRF-
3
1)328
dimerized and bound the ISRE much more efficiently
than IRF-3
1)409
or IRF-3
1)388

, 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

various IRF-3 truncations on deoxycholate-PAGE was highly repro-
ducible. (D) Proteins used in C were analyzed by EMSA as in A, and
the amount of protein bound to the ISRE, expressed in arbitrary units
(open triangles), was plotted along the ratio of dimeric to monomeric
IRF-3 (filled squares) determined from quantification of C.
Ó FEBS 2002 Mechanism of IRF-3 virus-dependent activation (Eur. J. Biochem. 269) 6147
reduced transcriptional activation by up to 95 and 70%,
respectively, suggesting that these fusion proteins effectively
interacted with IRF-3E7 in transfected insect cells. In the
absence of full-length mCBP, coexpression of IRF-3E7 with
GST-CBP
1)1100
, GST-CBP
1892)2441
or their combination
did not result in any activation of the ISRE · 3CAT
reporter (Fig. 6B). The inability of GST-CBP
1)1100
and
GST-CBP
1892)2441
, alone or in combination, to activate
transcription together with IRF-3E7 was not due to a lack
of transcription potential of these CBP fragments. Indeed,
expression of various Gal4-mCBP fusion constructs in
insect cells revealed that, as observed in mammalian cells
[26], both the N- and C-termini of mCBP displayed intrinsic
transcriptional activity (Fig. 6C). Furthermore, coexpres-
sion of GST-CBP
1)1100

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-

3. The failure of p300DNR and IRF-3 to activate
transcription might similarly reflect a decreased affinity of
p300 for IRF-3 upon removal of part of its N–terminal
interaction domain. The basis for p300DE1a lack of activity
in this assay remains to be determined. However, this result
suggests that the E1a region of p300, which is known to
interact with general transcription factors such as TFIIB, p/
CAF and RNA polymerase II [28–31], also plays an
essential role in IRF-3 transcriptional activity.
DISCUSSION
Previous studies have identified IRF-3 and IRF-7 as
essential mediators of the transcriptional response in virus-
infected vertebrate cells [9–14,16,32–34]. Indeed, each
protein becomes hyperphosphorylated following virus
infection, dimerizes, accumulates in the nucleus and
activates transcription of a specific set of genes. Moreover,
in vivo both proteins are found to be physically associated
with the promoter of the IFI-56K and IFN-b genes, in a
virus-dependent manner [14]. Finally, gene targeting
experiments further demonstrated that IRF-3 and IRF-7
play essential yet distinct roles in the response to virus
infection [33,35]. However, detailed investigations of the
mechanism by which IRF-3 and IRF-7 activate transcrip-
tion have been hampered by a number of limitations
inherent to the experimental systems used. These include:
(a) the presence of endogenous IRFs, which can associate
with transfected IRFs; (b) the presence of the endogenous
p300/CBP coactivators and the unavailability of animals
or cell lines null for them due to embryonic and cellular
lethality; (c) the existence of a feedback loop involving

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-

tants, schematically represented in (F), DNR (D3–173), DBromo
(D1071–1241), DE1a (D1739–1871), DSRC (D2042–2157) [27].
6148 H. Yang et al. (Eur. J. Biochem. 269) Ó FEBS 2002
SV40 large T in COS cells). In this paper, we have
examined the molecular events by which IRF-3 activates
transcription in response to virus infection using a
combination of approaches that circumvent the limitations
discussed above, allowing us to reconcile previously
conflicting interpretations and to further define the
mechanism by which IRF-3 activates transcription.
Modification of both sets of Ser/Thr residues
is essential for full activation of IRF-3
In SAN cells, the transcriptional activity of essentially all
Gal4-IRF-3 constructs where only one set was mutated was
still virus-inducible, while the activity of all constructs where
both sets were mutated was not (Fig. 1B), suggesting that
some residues, within each set, are phosphorylated upon
infection. This conclusion is strongly supported by the
observation that IRF-3E7 is much more active than either
IRF-3E2 or IRF-3E5 in insect cells, where the absence of
additional post-translational modifications of IRF-3E5
uncovers the synergy between the two sets of Ser/Thr
residues in the activation of IRF-3 (Fig. 2).
In contrast to SAN cells, constructs like Gal4-IRF-3E5
are constitutively active in 293T cells, with no further
stimulation by virus ([19], data not shown). Together with
the observation that IRF-3E5 is a stronger activator than
IRF-3E7inmammaliancells,theseresultsledtothe
conclusion that the first set of residues is not phosphory-
lated upon virus infection, but might play a regulatory

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.

Additional evidence that the first set of Ser residues is
phosphorylated in response to virus infection comes from
studies where IRF-3A5 can be in vitro phosphorylated,
but only using extracts from virus-infected cells [22].
Mechanism of IRF-3 activation
IRF-3 exists in primarily two forms, one of which is
monomeric, exhibits weak affinity for DNA or p300/CBP,
and shuttles between the cytoplasm and the nucleus, with a
dominance of export over import. Another form of IRF-3 is
dimeric, binds efficiently to DNA, interacts strongly with
the p300/CBP coactivators and resides in the nucleus. The
equilibrium between the monomeric and dimeric forms of
IRF-3 is affected by phosphorylation of the Ser/Thr
residues in its C-terminal regulatory domain. When these
residues are unphosphorylated, the monomeric form of
IRF-3 is predominant, while phosphorylation shifts the
equilibrium towards the dimeric form.
When IRF-3 is in its monomeric form, there is an
intramolecular interaction involving the C-terminal domain
and a region C-terminal of the DNA-binding domain
(amino acids 98–240). Lin et al. [19] mapped the minimal
C-terminal domain to amino acids 380–427, and our results
suggest that maximal interaction involves amino acids 328–
427). The region (amino acids 98–240) with which the
C-terminal domain interacts is part of IRF-3 dimerization
domain, whose C-terminal end point we mapped between
amino acids 288 and 308.
Interference with this intramolecular interaction leads to
the activation of IRF-3. In vitro, removal of only the
C-terminal 17 residues is sufficient to lead to low levels of

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-

dues within the second set must participate in direct contacts
with the amino acids 98–240 domain in the monomeric
form, as this intramolecular interaction can be disrupted by
substitution to either Ala or Glu. It is important to note that
we do not claim that all residues become phosphorylated in
either set. Rather, our experiments only suggest that residues
within each set are modified upon virus infection and that
there is a functional synergy between phosphorylated
Ó FEBS 2002 Mechanism of IRF-3 virus-dependent activation (Eur. J. Biochem. 269) 6149
residues present in these sets. Additional experiments are
required to determine exactly which residues within each set
become phosphorylated in infected cells.
Interaction with a coactivator is essential for IRF-3
transcriptional activity
Unprecedently, the transcriptional activity of IRF-3 is
entirely dependent on its interaction with a mammalian
coactivator as demonstrated by transfection experiments in
insect cells (Figs 2 and 6), and is consistent with the
observation that E1a can strongly interfere with IRF-3
activity in mammalian cells [36]. Intriguingly, while the yeast
genome contains no CBP homolog, a Gal4-IRF-3 fusion is
transcriptionally active in yeast cells ([9] and our unpub-
lished results). Even full-length IRF-3 fused to Gal4 is active
in those cells, unlike what we observed in insect cells in the
absence of mCBP (Fig. 3B). Mapping of the IRF-3
activation domain in yeast identified residues 134–394 as
the minimum region required for transcriptional activity, an
unusually large activation domain as noted by the authors
[19]. This domain contains amino acids 139–386, which is
the minimal domain required for interaction with the IBiD

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

from amino acids 375–427 could compete with the interac-
tion between GSTDp300 (amino acids 1752–2221) and
virus-activated IRF-3 only when the peptide was phosphor-
ylated at position 385 and 386. Based on this latter result, it
was proposed that when Ser385/386 are phosphorylated,
these residues make direct contact with p300 [18]. It is
possible though it appears not very likely that phospho-
Ser385/386 could make simultaneous contacts with another
region of IRF-3 and with p300/CBP. As phosphorylation of
Ser385/386 seems to be required for a new interaction that
promotes both dimerization and DNA binding, even in the
absence of p300 or CBP, an alternative or complementary
explanation for the effect of the phosphorylated peptide on
the interaction between IRF-3 and p300 would be that this
latter interaction is dependent on IRF-3 adopting the
dimeric conformation. In this scenario, the peptide would be
competing for the intra- or intermolecular interaction
involving phospho-Ser385/386 and another domain of
IRF-3, preventing dimerization and hence interaction with
p300/CBP.
CONCLUSIONS
In summary, IRF-3 is phosphorylated on two sets of Ser/
Thr residues within its C-terminus upon virus infection.
Phosphorylation of both sets is functionally important for
full dimerization, DNA-binding, p300/CBP interaction and
transcriptional activity, and each set might play distinct
roles in the conformational switch that accounts for IRF-3
activation.
Activated IRF-3, in turn, entirely depends on simulta-
neous interactions with multiple domains of the p300/CBP

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

Maniatis, Harvard University, during its initial phase.
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