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

B Transcriptional activity in SAN cells of IRF-3 point mutants on the P31·2CAT reporter left graph or as Gal4 fusions on the G5E1bCAT reporter right graph; the sequence of WT and each po

Transcriptional activity of interferon regulatory factor (IRF)-3

depends on multiple protein–protein interactions

Hongmei Yang1, Charles H Lin2, Gang Ma1, Melissa Orr1, Michael O Baffi1and Marc G Wathelet1 1

Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati;2Department 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: marc.wathelet@uc.edu

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)

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

E X P E R I M E N T A L P R O C E D U R E S

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 37C,5% CO2,in Dulbecco’s modified Eagle

medium containing 10% fetal bovine serum,50 UÆmL)1

penicillin and 50 lgÆmL)1streptomycin

Sendaivirus was obtained from SPAFAS and used at 200 hemaglutinin UÆmL)1

S2 cells were grown at 26C,in Schneider’s Drosophila medium containing 12% fetal bovine serum,50 UÆmL)1 penicillin and 50 lgÆmL)1streptomycin

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.35S-Labeled in vitro translated proteins were incubated with GST fusion proteins immobilized on glutathione-sepharose beads in 150 mM KCl,20 mM Tris

pH 8.0,0.5 mM dithiothreitol,50 lgÆmL)1 ethidium bro-mide,0.2% NP-40 and 0.2% BSA (binding buffer) for 1 h

at 4C,followed by two washes with binding buffer and two washes with binding buffer without BSA Extracts from HEC-1B cells labeled in vivo with [32P]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 P31·2CAT 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.

and preclearing on glutathione-sepharose beads,the 32

P-labeled proteins were incubated with immobilized GST

fusions for 2 h at 4C,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

R E S U L T S

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 P31·2CAT

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 transferasegene 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

Ser residues to either Ala (IRF-3A2) or Glu (IRF-3E2)

drastically reduced the ability of IRF-3 to stimulate

P31·2CAT 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·3CAT 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, E5 and E7) led to substantial activation of 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 activityof IRF-3 depends on synergybetween two sets of residues and is entirelydependent 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·3CAT 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).

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

ISG15gene 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 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 vitrotranslation 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).

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,

metabolically32P-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 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 with the data shown in Fig 4C IRF-31)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-31)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 [32P]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)35S-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.

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-31)368and 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-31)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-31)409and IRF-31)388 significantly activated transcription of the ISRE·3CAT 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-31)370and IRF-31)328interacted significantly only with the N-terminus of p300/CBP and displayed no detectable transcriptional activity Thus,even though

IRF-31)328dimerized and bound the ISRE much more efficiently than IRF-31)409 or IRF-31)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-CBP1)1100 and GST-CBP1892)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.

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-CBP1)1100,GST-CBP1892)2441 or their combination

did not result in any activation of the ISRE· 3CAT

reporter (Fig 6B) The inability of GST-CBP1)1100 and

GST-CBP1892)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-CBP1)1100or GST-CBP1892)2441had minimal

effects on the transcriptional activity of Gal4-mCBP1)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·3CAT 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

D I S C U S S I O N

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 vivoboth 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 limitatranscrip-tions 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 lg in

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·3CAT 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].

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-3E7 in mammalian cells, these results led to the

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 P31·2CAT reporter in virus-infected SAN cells (Fig 1B) Therefore,the second set participates minimally in intra- or intermolecular interac-tions when IRF-3 is a dimer as Ala, Glu or phospho-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

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-31)388with 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

C O N C L U S I O N S

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-CBP1)1100or GST-CBP1892)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 CBP1-1100 or CBP1892-2441 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 (as they can do so when fused to the Gal4 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

A C K N O W L E D G E M E N T S

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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