Rt. Lin et al., Structural and functional analysis of interferon regulatory factor 3: Localization of the transactivation and autoinhibitory domains, MOL CELL B, 19(4), 1999, pp. 2465-2474
The interferon regulatory factor 3 (IRF-3) gene encodes a 55-kDa protein wh
ich is expressed constitutively in all tissues. In unstimulated cells, IRF-
3 is present in an inactive cytoplasmic form; following Sendai virus infect
ion, IRF3 is posttranslationally modified by protein phosphorylation at mul
tiple serine and threonine residues located in the carboxy terminus. Virus-
induced phosphorylation of IRF3 leads to cytoplasmic to nuclear translocati
on of phosphorylated IRF3, 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 n
ow demonstrate that an extended, atypical transactivation domain is located
in the C terminus of IRF3 between amino acids (aa) 134 and 394, We also sh
ow that the C-terminal domain of IRF-3 located between aa 380 and 427 parti
cipates in the autoinhibition of IRF3 activity via an intramolecular associ
ation 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 IRF3 homodimers; this intera
ction is also observed in the absence of virus infection with a constitutiv
ely activated form of IRF3. Substitution of the C-terminal Ser-Thr phosphor
ylation sites with the phosphomimetic Asp in the region ISNSHPLSLTSDQ betwe
en amino acids 395 and 407 [IRF-3(5D)], but not the adjacent S385 and S386
residues, generates a constitutively activated DNA binding form of IRF3. In
contrast, substitution of S385 and S386 with either Ala or Asp inhibits bo
th DNA binding and transactivation activities of the IRF-3(5D) protein. The
se studies thus define the transactivation domain of IRF-3, two domains tha
t participate in the autoinhibition of IRF3 activity, and the regulatory ph
osphorylation sites controlling IRF-3 dimer formation, DNA binding activity
, and association with the CBP/p300 coactivator.