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Molecular and Cellular Biology, April 1999, p. 2465-2474, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Structural and Functional Analysis of Interferon Regulatory Factor 3: Localization of the Transactivation and Autoinhibitory Domains

Rongtuan Lin,1,2,* Yael Mamane,1,3 and John Hiscott1,2,3

Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research,1 and Departments of Microbiology and Immunology3 and Medicine,2 McGill University, Montreal, Canada H3T 1E2

Received 24 August 1998/Returned for modification 28 October 1998/Accepted 4 January 1999

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.

* Corresponding author. Mailing address: Lady Davis Institute for Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec, Canada H3T 1E2. Phone: (514) 340-8222, ext. 4509. Fax: (514) 340-7576. E-mail: mdli{at}musica.mcgill.ca.

Molecular and Cellular Biology, April 1999, p. 2465-2474, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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