Abstract
Virus-induced expression of interferon (IFN)-A genes is regulated by two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, which are activated by phosphorylation during viral infection by the IKK-related serine/threonine kinases TBK1 and IkappaB kinase epsilon (IKKepsilon). In this study, we demonstrate that three IRF-binding sites located in the virus-responsive element mediate the transcriptional activation of the IFN-A4 promoter by IRF-3. The precise arrangement of these IRF elements is required for synergistic activation of the IFN-A4 promoter following Newcastle disease virus infection or activation by TBK1 or IKKepsilon. The ordered assembly of IRF-3 multimers on the promoter also determines cooperative recruitment of IRF-3 and CREB-binding protein and differential virus-induced expression of IFN-A4 gene promoter compared with IFN-A11. Naturally occurring nucleotide substitutions disrupt two of the IRF elements in the IFN-A11 gene promoter, leading to a dramatic decrease in IRF-3 and CREB-binding protein recruitment and in IRF-3-dependent transcription. Transcription of the IFN-A4 promoter by IRF-7 is mediated by two IRF elements; promoter mutants that carry a reversed IRF element retain the ability to respond to IKKepsilon or TBK1 expression in the presence of IRF-7 but lose the capacity to respond to virus or kinase-induced IRF-3. Interestingly, IKKepsilon or TBK1 stimulates the IRF-7-mediated transcription of IFN-A11, although at a lesser extent compared with IFN-A4. Our data indicate that virus-induced expression of IFN-A genes is dictated by the organization of IRF elements within the IFN-A promoters and that the differential IFN-A gene expression, based on the IRF-3 responsiveness, is partially compensated in the presence of IRF-7 when both factors are activated by IKKepsilon or TBK1.
Highlights
Antiviral defense program [1]
To define the specificity of interferon regulatory factor (IRF)-3 and IRF-7 for the three IRF-elements (B, C, and D) located in the VRE-A4, mutated IFN-A4 promoters were generated by substituting these motifs with the GAAATA motif (Fig. 1), which was previously shown to be unresponsive to both IRF-3(5D) and IRF-7A ectopic expression [46]
Comparison of the data obtained with MB1, MB2, and MB1-2 further indicated that the 5-fold decrease in activation observed with the MB1-2 construct was due to Ϫ94,Ϫ93GT/TA pair substitution and indicated that the sequence GAAAGTGAAAAG (B element) in VRE-A4 defines another binding site for IRF-3
Summary
Cell Culture and Virus Infection—Human embryonic kidney 293 (ATCC CRL 1573), mouse L929 (ATCC CCL 1), and human HeLa S3 cells (ATCC CCL 2.2) were grown in ␣-minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum, glutamine, and antibiotics. Plasmids—Reporter plasmids pIF4T and pIF11T carrying the Ϫ470 to ϩ19 fragment of the mouse IFN-A4 gene promoter and the Ϫ457 to ϩ19 fragment of the mouse IFN-A11 gene promoter, respectively, and the pIF4J and pIF11J constructs containing the Ϫ120 to ϩ19 fragment of both promoters, upstream of the chloramphenicol acetyltransferase (CAT) or luciferase reporter gene have been previously described [45]. HEK293T cells (2 ϫ 106) were plated and transfected 24 h later with 7.5 g of plasmids pIF4T, pIF11T, pIF4J[Brev], or pIF4J[Crev] together with 3 g of pcDNA3-IKK⑀ or pcDNA3-TBK1 in the absence or presence of 1.5 g of pcDNA3-IRF-3. Immunoblotting Assays—HEK293T cells were seeded at 3 ϫ 106 cells/100-mm plate and transfected after 24 h by the calcium phosphate method with 1.5 g of pIF4T-CAT reporter construct, 0.75 g of pIEF-Gal control plasmid, and 6 g of pcDNA3-FLAGIKK⑀ or pcDNA3-FLAGTBK1 either alone or together with 0.75 g of pcDNA3-IRF-3wt or pcDNA3-IRF-7A. Anti-poly(ADP-ribose) polymerase (sc7150), anti-Bcl-xL (sc-8392), and anti-␣-tubulin (sc-5286) were purchased from Santa Cruz Biotechnology
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