Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains

Abstract

The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein which is expressed constitutively in all tissues. In unstimulated cells, IRF-3 is present in an inactive cytoplasmic form; following Sendai virus infection, IRF-3 is posttranslationally modified by protein phosphorylation at multiple serine and threonine residues located in the carboxy terminus. Virus-induced phosphorylation of IRF-3 leads to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the transcriptional coactivator CBP/p300, and stimulation of DNA binding and transcriptional activities of virus-inducible genes. Using yeast and mammalian one-hybrid analysis, we now demonstrate that an extended, atypical transactivation domain is located in the C terminus of IRF-3 between amino acids (aa) 134 and 394. We also show that the C-terminal domain of IRF-3 located between aa 380 and 427 participates in the autoinhibition of IRF-3 activity via an intramolecular association with the N-terminal region between aa 98 and 240. After Sendai virus infection, an intermolecular association between IRF-3 proteins is detected, demonstrating a virus-dependent formation of IRF-3 homodimers; this interaction is also observed in the absence of virus infection with a constitutively activated form of IRF-3. Substitution of the C-terminal Ser-Thr phosphorylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ between amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386 residues, generates a constitutively activated DNA binding form of IRF-3. In contrast, substitution of S385 and S386 with either Ala or Asp inhibits both DNA binding and transactivation activities of the IRF-3(5D) protein. These studies thus define the transactivation domain of IRF-3, two domains that participate in the autoinhibition of IRF-3 activity, and the regulatory phosphorylation sites controlling IRF-3 dimer formation, DNA binding activity, and association with the CBP/p300 coactivator.

FIG. 1

Transactivation of IRF-3 in yeast one-hybrid analysis. (A) Schematic illustration of the IRF-3 protein showing the DBD, NES, proline-rich domain (Pro), and IAD. The wild-type and mutated forms of IRF-3 in the chimeric proteins and their transactivation activities are indicated. ++++, blue colony formation within 30 min; −, white or light blue colonies after 24 h. (B) Representative plate illustrating β-Gal activity. The plasmids encoding chimeric proteins were transformed into yeast strain Y190 carrying GAL1-lacZ reporter; transactivation activity was detected by colony lift filter assay according to the Matchmaker (Clontech) protocol.

FIG. 2

Analysis of intrinsic transactivation activities of various IRF-3 regions fused to the yeast GAL4 DBD. 293 cells were transfected with the (Gal4)₅-TK/CAT reporter plasmid and various GAL4–IRF-3 chimeric expression plasmids as indicated. CAT activity was analyzed at 48 h posttransfection with 10 μg of total protein extract for 1 to 2 h at 37°C. Relative CAT activity was measured as fold activation (relative to the basal level of the reporter gene in the presence of pSG424 vector after normalization to cotransfected β-Gal activity); the values represent the average of three experiments with variability shown by the error bars.

FIG. 3

Removal of the IRF-3 C terminus stimulates intrinsic IRF-3 transactivation potential. 293 cells were transfected with RANTES CAT reporter plasmid and various IRF-3 plasmids expressing truncated forms of IRF-3 as indicated. CAT activity was analyzed at 48 h posttransfection with 5 μg of total protein extract for 1 to 2 h at 37°C. Relative CAT activity was measured as fold activation (relative to the basal level of reporter gene in the presence of CMVBL vector after normalization to cotransfected β-Gal activity); the values represent the average of three experiments with standard deviation shown by the error bars.

FIG. 4

C-terminal IRF-3 truncation induces constitutive DNA binding. (A) EMSA was performed on whole-cell extracts (20 μg) derived from 293 cells transfected with various IRF-3 expression plasmids (5 μg of each) as indicated above the lanes. At 24 h posttransfection, cells were infected with Sendai virus (SV) for 12 h or left uninfected as indicated. The ³²P-labeled probe corresponds to the ISRE of the ISG15 gene (5′-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3′). The position of the IRF-3–CBP complex is indicated by the arrow. Anti-CBP antibody (Ab) A22 (lanes 8 to 10) and anti-IRF-3 antibody F3 (lane 7) were added as indicated to demonstrate the presence of CBP and IRF-3 in the high-molecular-weight protein-DNA complex. (B) Whole-cell extracts (20 μg) from panel A were analyzed by immunoblotting with anti-IRF-3 antibody.

FIG. 5

Intramolecular association between the N- and C-terminal domains of IRF-3. 293 cells were transfected with Myc-tagged IRF-3(328-427) and expression plasmids encoding IRF-3 deletions (5 μg of each plasmid) as indicated above the lanes. At 24 h posttransfection, cells were infected with Sendai virus (SV) (+) for 12 h or left uninfected (−). (A) Whole-cell extracts (200 μg) were immunoprecipitated (IP) with anti-Myc antibody 9E10 (αmyc); immunoprecipitated complexes were run on an SDS–10% polyacrylamide gel and subsequently probed with anti-IRF-3 antibody (lanes 1 to 9) or anti-Flag antibody M2 (lanes 10 and 11). (B) The same membrane was also reprobed with anti-Myc antibody 9E10. (C) Whole-cell extracts (WCE; 20 μg) were run on an SDS–10% polyacrylamide gel and probed with anti-IRF-3 antibody (lanes 1 to 9) or anti-Flag antibody (lanes 10 and 11) to assess the level of transgene expression.

FIG. 6

IRF-3 homodimer formation after virus infection. 293 cells were transfected with Flag-tagged and Myc-tagged IRF-3 expression plasmids (5 μg) as indicated above the lanes. At 24 h posttransfection, cells were infected with Sendai virus (SV) for 12 h (+) or left uninfected (−). (A) Whole-cell extracts (WCE; 200 μg) were immunoprecipitated with anti-Myc antibody 9E10; immunoprecipitated complexes were run on an SDS–10% polyacrylamide gel and subsequently probed with anti-Flag antibody M2. (B) The membrane in panel A, reprobed with anti-Myc antibody 9E10 to assess the level of expression of transfected protein.

FIG. 7

Analysis of IRF-3 point mutations for transactivation potential. 293 cells were transfected with RANTES CAT reporter plasmid and various IRF-3 plasmids expressing C-terminal point mutations as indicated in on the left. At 24 h posttransfection, cells were infected with Sendai virus for 16 h (dark bars) or left uninfected (light bars). The point mutations are indicated by open circles (mutated to Ala) and closed circles (mutated to Asp): 2A, S396A/S398A; 3A, S402A/T404A/S405A; 5A, S396A/S398A/S402A/T404A/S405A; 5D, S396D/S398D/S402D/T404D/S405D; J2A, S385A/S386A; J2D, S385D/S386D; and NES, L145A/L146A. CAT activity was analyzed at 48 h posttransfection with 5 μg of total protein extract for 1 to 2 h at 37°C. Relative CAT activity was measured as fold activation (relative to the basal level of reporter gene in the presence of CMVBL vector after normalization to cotransfected β-Gal activity); the values represent the average of three experiments with variability shown in the error bars.

FIG. 8

Effects of IRF-3 point mutations on DNA binding activity. (A) EMSA was performed on whole-cell extracts (20 μg) derived from 293 cells transfected with various IRF-3 expression plasmids as indicated. At 24 h posttransfection, cells were infected with Sendai virus for 12 h or left uninfected. The ³²P-labeled probe corresponds to the ISRE of the ISG15 gene: 5′-GATCGGGAAAGGGAAACCGAAACTGAAGCC-3′. (B) Whole-cell extracts (20 μg) from panel A were analyzed by immunoblotting with anti-IRF-3 antibody. IRF-3(5D), IRF-3(5D-J2A), and IRF-3(7D) proteins migrated more slowly than wtIRF-3, at a position consistent with phosphorylated IRF-3; arrows indicate positions of the different forms of IRF-3.

FIG. 9

Effects of specific mutations in the C-terminal inducible phosphorylation sites on IRF-3–CBP complex formation. 293 cells were transfected with Myc-tagged IRF-3 expression plasmids (5 μg) as indicated above the lanes. At 24 h posttransfection, cells were infected with Sendai virus (SV) for 12 h (+) or left uninfected (−). Whole-cell extracts (300 μg) were immunoprecipitated with anti-CBP antibody A22 (αCBP). Immunoprecipitated complexes (A and B) or 20 μg of whole-cell extracts (WCE) (C) were run on an SDS–8% polyacrylamide gel and subsequently probed with anti-Myc antibody (A and C). The membrane in panel A was reprobed with anti-CBP antibody 9E10 (B) to verify expression of the transfected genes.

FIG. 10

Schematic representation of IRF-3 activation and dimerization by virus-induced phosphorylation. IRF-3 exists in the cytoplasm of uninfected cells. Intramolecular association between the C terminus and the DBD maintains IRF-3 in a latent state by masking both DBD and IAD regions of the protein. Virus-induced phosphorylation (P) of the Ser/Thr residues in the aa 396 to 405 cluster leads to a conformational change in IRF-3 that relieves C-terminal autoinhibition and exposes both DBD and IAD regions. The opened conformation of IRF-3 is now able to homodimerize; translocation to the nucleus leads to DNA binding at ISRE- and PRDI/PRDIII-containing promoters. The presence of a NES element ultimately shuttles IRF-3 from the nucleus and terminates the initial activation of virus-responsive promoters. Pol, polymerase.