IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts

Abstract

Virus infection, double-stranded RNA, and lipopolysaccharide each induce the expression of genes encoding IFN-alpha and -beta and chemokines, such as RANTES (regulated on activation, normal T cell expressed and secreted) and IP-10 (IFN-gamma inducible protein 10). This induction requires the coordinate activation of several transcription factors, including IFN-regulatory factor 3 (IRF3). The signaling pathways leading to IRF3 activation are triggered by the binding of pathogen-specific products to Toll-like receptors and culminate in the phosphorylation of specific serine residues in the C terminus of IRF3. Recent studies of human cell lines in culture have implicated two noncanonical IkappaB kinase (IKK)-related kinases, IKK-epsilon and Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1), in the phosphorylation of IRF3. Here, we show that purified recombinant IKK-epsilon and TBK1 directly phosphorylate the critical serine residues in IRF3. We have also examined the expression of IRF3-dependent genes in mouse embryonic fibroblasts (MEFs) derived from Tbk1(-/-) mice, and we show that TBK1 is required for the activation and nuclear translocation of IRF3 in these cells. Moreover, Tbk1(-/-) MEFs show marked defects in IFN-alpha and -beta, IP-10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the Toll-like receptors 3 and 4 by double-stranded RNA and lipopolysaccharide, respectively. Finally, TRIF (TIR domain-containing adapter-inducing IFN-beta), fails to activate IRF3-dependent genes in Tbk1(-/-) MEFs. We conclude that TBK1 is essential for IRF3-dependent antiviral gene expression.

Fig. 6.

Induction of IFN-α and -β, RANTES, and IP-10 is TBK1-dependent. (A) MEFs from Tbk1^+/+ (Left) and Tbk1^-/- (Right) mice were infected with SV for the indicated times. Total RNA (5 μg) was loaded in each lane, and Northern blot analysis was performed by using a mouse IFN-β-specific probe. (B-D) MEFs from Tbk1^+/+ (filled bars) and Tbk1^-/- (empty bars) mice were stimulated with poly(I-C) or LPS, infected with SV or NDV as indicated, or left untreated (none) for ≈16 h. Cell supernatants were harvested, and RANTES, IP-10, or IFN-α levels were measured by ELISA.

Fig. 1.

Purified IKK-ε and TBK1 phosphorylate the C terminus of IRF3. Wild-type (wt) TBK1 (A), IKK-ε (B), or IKK-β (C) was expressed in insect cells by using baculovirus vectors. Kinase activity of the purified proteins was assayed by using various GST-IRF3-(380-427) or GST-IκBα-(5-55) substrates as indicated. A2, S385A, S386A IRF3 mutant; A5, S396A, S398A, S402A, T404A, S405A IRF3 mutant; A7, S385A, S386A, S396A, S398A, S402A, T404A, S405A IRF3 mutant; SS, S32A, S36A IκBα mutant.

Fig. 2.

Reduced IFN-β reporter gene activity in the absence of TBK1. MEFs from Tbk1^+/+(filled bars) and Tbk1^-/- (empty bars) mice were transfected with IFN-β (A), RANTES (B), or IP-10 (C) luciferase reporter genes. Cells were left untreated (none), infected with NDV or SV, or stimulated with poly(I-C) or LPS. Lysates were assayed for luciferase activity.

Fig. 3.

Deficiency of TBK1 leads to impaired induction of IRF3-dependent reporters and normal induction of NF-κB-dependent reporters. MEFs from Tbk1^+/+ (filled bars) and Tbk1^-/- (empty bars) mice were transfected with IFN-β PRDIII-I or ISG-54 ISRE reporter genes (A), IFN-β PRDII (B), or HIV LTR-κB or ELAM luciferase reporter genes (B Lower). Cells were left untreated (none), infected with SV or NDV, or stimulated with poly(I-C) or LPS. Lysates were assayed for luciferase activity.

Fig. 4.

Deficiency of TBK1 leads to defective virus-induced nuclear translocation and phosphorylation of IRF3. (A) MEFs from Tbk1^+/+and Tbk1^-/- mice were transfected with 1.0 μg of an IRF3-GFP fusion protein. Samples were left untreated (none) or infected with 100 HAU of SV and visualized by confocal microscopy. (B) MEFs from Tbk1^+/+and Tbk1^-/- mice were transfected with 500 ng of HA-IRF-3ΔN. Cells were infected 24 h later with SV for the indicated times. Whole-cell lysates were analyzed by Western blotting (WB) with anti-HA antibodies. As a loading control, membranes were stripped and reprobed with anti-calnexin antibodies.

Fig. 5.

TRIF induces IFN-β and RANTES reporter genes by means of TBK1. MEFs from Tbk1^+/+ (filled bars) and Tbk1^-/- (empty bars) mice were transfected with IFN-β (Left), RANTES (Center), or ELAM (Right) luciferase reporter genes and cotransfected with a murine TRIF expression vector or left untreated (none). Lysates were assayed for luciferase activity.