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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 Interferons (IFNs) are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation, and immune activation (32). Virus infection induces the transcription and synthesis of multiple IFN genes (11,23,32); newly synthesized IFN interacts with neighboring cells through cell surface receptors and the Jak-STAT signalling pathway, resulting in the induction of over 30 new cellular proteins that mediate the diverse functions of the IFNs (6,13,15,28). Among the many virus-and IFN-inducible proteins are the growing family of interferon regulatory transcription factors (IRFs), including IRF-1, IRF-2, IRF-3, IRF-4/Pip/ICSAT, IRF-5, IRF-6, IRF-7, ISGF3␥/p48, and ICSBP (21). All of the family members share a high degree of homology in the Nterminal DNA binding domain (DBD) with the five characteristic tryptophan repeats (21). Structurally, the Myb oncoproteins share homology with the IRF family, although their relationship to the IFN system is unclear (31). Recent evidence also demonstrates the presence of a virally encoded analogue of cellular IRFs in the genome of human herpesvirus 8 (25).IRF-3 was originally identified as a member of IRF family on the basis of (i) homology with other IRF family members and (ii) binding to the IFN-stimulated regulatory element (ISRE) of the ISG15 promoter (1). This protein is distinct from cIRF-3, an avian protein which demonstrates homology to the IRF family members (10). IRF-3 is expressed constitutively in a variety of tissues, and the relative levels of IRF-3 mRNA do not change in virus-infected or IFN-treated cells. IRF-3 demonstrates a unique response to viral infection. Recent studies with IRF-3 demonstrate that virus-and doublestranded RNA (dsRNA)-inducible phosphorylation represents an important posttranslational modification, leading to cytoplasmic to nuclear translocation of phosphorylated IRF-3, association with the CBP/p300 coactivator, and stimulation of DNA binding and transcriptional activities (18,20,26,(33)(34)(35). Overexpression of IRF-3 significantly enhances virus-mediated expression of type I (alpha/beta) IFN and results in the induction of an antiviral state (14). Virus-induced phosphory...

Recent studies implicate the interferon regulatory factors (IRF), IRF-3 and IRF-7, as key activators of Type 1 interferon genes, as well as the RANTES (regulated on activation normal T cell expressed) chemokine gene. Both IRF-3 and IRF-7 are regulated in part by virusinduced C-terminal phosphorylation, leading to nuclear translocation, stimulation of DNA binding, and transcriptional activities. Structure-function studies with IRF-7 suggested a complex organization of the C-terminal region, with a constitutive activation domain located between amino acids 150 -246, an accessory inducibility region at the very end of IRF-7 between amino acids 467 and 503, and an inhibitory region (amino acids 341-467) adjacent to the C-terminal end that interferes with transactivation. Furthermore, an element that increases basal and virus-inducible activity is located between amino acids 278 and 305. A transcriptionally active form of IRF-7 was also generated by substitution of Ser-477 and Ser-479 residues with the phosphomimetic Asp. IRF-7, particularly IRF-7(S477D/S479D), was a strong transactivator of type I interferon and RANTES chemokine gene expression. Unlike wild type IRF-3, IRF-7 overexpression was able to stimulate inteferon gene expression in the absence of virus infection. Using tagged versions of IRF-7 and IRF-3, the formation of homo-and heterodimers was detected by co-immunoprecipitation. These results demonstrate that IRF-3 and IRF-7 transcription factors possess distinct structural characteristics that impart complementary rather than redundant functional roles in cytokine gene activation.