Triggering the Interferon Antiviral Response Through an IKK-Related Pathway

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Science  16 May 2003:
Vol. 300, Issue 5622, pp. 1148-1151
DOI: 10.1126/science.1081315


Rapid induction of type I interferon expression, a central event in establishing the innate antiviral response, requires cooperative activation of numerous transcription factors. Although signaling pathways that activate the transcription factors nuclear factor κB and ATF-2/c-Jun have been well characterized, activation of the interferon regulatory factors IRF-3 and IRF-7 has remained a critical missing link in understanding interferon signaling. We report here that the IκB kinase (IKK)–related kinases IKKϵ and TANK-binding kinase 1 are components of the virus-activated kinase that phosphorylate IRF-3 and IRF-7. These studies illustrate an essential role for an IKK-related kinase pathway in triggering the host antiviral response to viral infection.

The success of host cell defense against viral infection depends on detection of the invading pathogen. Upon recognizing viral antigens, the cell activates a multitude of signaling cascades to produce cytokines that both impede pathogen replication and stimulate immune responses (13). Interferons (IFNs) are well-characterized components of the innate host defense, and rapid induction of IFN expression in response to viral infection requires posttranslational modification of transcription factors, including nuclear factor κB (NF-κB), ATF-2/c-Jun, and interferon regulatory factors (IRFs) IRF-3 and IRF-7 (4, 5). Although activation of NF-κB and ATF-2/c-Jun is well characterized, pathways leading to IRF activation have not been delineated. IRF-3 stimulates the expression of certain IFNs early in infection; IRF-7 then amplifies the expression of other IFN genes not induced during the early stage (68).

IRF-3 and IRF-7 are activated by phosphorylation of their C-terminal domain by a virus-activated kinase (VAK) pathway. This modification permits IRF dimerization, nuclear translocation, and activation of IFN genes (6, 7, 911). A two-hybrid screen identified an interaction between IRF-3 and the C-terminal domain of the IκB kinase α (IKKα) subunit, a component of the multisubunit IKK kinase complex that regulates activation of the NF-κB pathway (12). To determine whether IKK subunits could phosphorylate the C terminus of IRF-3 and IRF-7, IKKα, IKKβ, IKKϵ, and TANK-binding kinase 1 (TBK1) were expressed in HEK293 cells. Only whole-cell extracts from IKKϵ- or TBK1-expressing cells exhibited kinase activity that induced phosphorylation of a fusion protein consisting of the C terminus of IRF-3 and glutathione S–transferase (GST–IRF-3) in an in vitro kinase assay (Fig. 1A and fig. S1A). A GST–IRF-3 substrate mutated in the serinethreonine cluster [GST–IRF-3(5A)] (9) was not phosphorylated when IKKϵ or TBK1 was expressed, and an inactive mutant form of IKKϵ [IKKϵ(K38A)] (13) blocked IKKϵ-induced phosphorylation of GST–IRF-3 (Fig. 1A and fig. S1A). Furthermore, IKKϵ that was transcribed and translated in vitro for use in a kinase assay demonstrated that IKKϵ directly phosphorylates IRF-3 and IRF-7 in vitro (Fig. 1B and fig. S1B), whereas IKKα, IKKβ, and IKKϵ all phosphorylated the IκBα substrate (fig. S1C).

Fig. 1.

IKKϵ phosphorylates the C terminus of IRF-3 and IRF-7 in vitro. (A) Transfected HEK293 cells expressing the indicated IKK isoforms. Whole-cell extract (2.0 μg) was used in an in vitro kinase assay using GST, GST–IRF-3 (amino acids 380 to 427), and GST–IRF-3(5A) (amino acids 380 to 427, with alanine substitutions at positions Ser396, Ser398, Ser402, Thr404, and Ser405) (9). The phosphorylated GST–IRF-3 band is indicated (phosphorylation is indicated by a capital P in a circle). A nonspecific band was phosphorylated in all lanes containing the 5A substrate; this band does not migrate to the position of the 5A substrate. (B) Plasmids encoding Flag epitope–tagged IKKϵ or control plasmid were transcribed and translated in vitro and immunoprecipitated with Ab to Flag, and immunoprecipitates were used in an in vitro kinase assay using GST, GST-IκBα (amino acids 1 to 55), GST–IRF-3, and GST–IRF-7 (amino acids 468 to 503) as substrates. Phosphorylated IKKϵ, IκBα, IRF-3, and IRF-7 bands are indicated.

IKKϵ or TBK1 expressed with either IRF-3 or IRF-7 induced more slowly migrating forms of the transcription factors, as observed by gel electrophoresis (fig. S2, A and B) (9, 14, 15). When IKKϵ was expressed using a Tet-inducible system, more slowly migrating forms of IRF-3 were detected by an antibody (Ab) that recognizes phosphorylation at Ser396, a modification critical for physiological IRF-3 activation (16) (Fig. 2A). To determine whether IKKϵ or TBK1 stimulates IRF-3 and IRF-7 nuclear translocation, subcellular localization experiments were performed by expressing fusion proteins consisting of IRF-3 or IRF-7 fused to green fluorescent protein (GFP) (Fig. 2B and fig. S2C). When expressed alone, each transcription factor localized predominantly to the cytoplasm. However, when expressed with IKKϵ, approximately 35% of IRF-3 and 95% of IRF-7 translocated into the nucleus. Expression of IKKϵ(K38A) resulted in <5% nuclear localization of IRF-3 and IRF-7. Expression of TBK1 also caused nuclear localization of IRF-3 and IRF-7 (fig. S2C). Furthermore, in DNA binding analyses, expression of IKKϵ with IRF-7 resulted in the formation of a protein-DNA complex (Fig. 2C, lane 3) that was supershifted with an Ab to IRF-7 (Fig. 2C, lane 4); expression of IKKϵ and IRF-3 resulted in a protein-DNA complex that supershifted with an Ab to IRF-3 or to Flag (Fig. 2C, lanes 11 and 12). Together these experiments demonstrate that expression of IKKϵ or TBK1 is sufficient to induce phosphorylation, nuclear translocation, and DNA binding of IRF-3 and IRF-7.

Fig. 2.

IKKϵ induces IRF-3 and IRF-7 activation. (A) Vero cells were cotransfected with a Tet-inducible Flag epitope–tagged IKKϵ vector and Myc epitope–tagged IRF-3. Cells were treated with doxycycline (Dox) (1 μg/ml) for 0 to 360 min. Whole-cell extracts (75 μg) were resolved by SD S–polyacrylamide gel electrophoresis and analyzed by Western blot (25) with Ab to IKKϵ, Ab to Myc, and a phosphospecific IRF-3 Ser396 Ab (16). (B) Subcellular localization of GFP–IRF-3 or GFP–IRF-7 was analyzed in transfected COS-7 cells in the presence of control vector (left), IKKϵ (middle), or IKKϵ(K38A) (right). Fluorescence was quantified in living cells at 24 hours after transfection (magnification, ×400). (C) The DNA binding capacity of Flag–IRF-7 (lanes 1 to 6) or Flag–IRF-3 (lanes 7 to 13) was examined in response to IKKϵ expression in bandshift analysis with IRF-7 binding site 1 (IRF-7 BS1) and the IFN-stimulated gene 15 response element (ISG15 ISRE) as probes (8). Specific and nonspecific Ab's (1 μg) were used to identify IRF-3– or IRF-7–specific complexes. Protein-DNA complexes corresponding to IRF-7 or IRF-3 are indicated (*) with supershifted complexes (**).

To determine the ability of IKKϵ and TBK1 to stimulate IFN gene expression, analyses were performed with luciferase expression driven by the IFNA4 and IFNB promoters. Expression of IKKϵ and IRF-7 resulted in 2000-fold stimulation of the IRF-7–responsive IFNA4 promoter, as compared to luciferase expression in the absence of IKKϵ and IRF-7 (Fig. 3A). The IFNB promoter was likewise responsive to IKKϵ expression, resulting in 40-fold stimulation. Sendai virus infection alone or viral infection plus IKKϵ resulted in a 60-fold stimulation of the IFNB promoter, an effect that was blocked by expression of a dominant negative form of IRF-3 (IRF-3ΔN) (Fig. 3B). Activation of the IFNA4 promoter induced by expressing IRF-7 and IKKϵ was blocked by either IKKϵ(K38A) or a C-terminal dominant negative truncation of IKKϵ(1-361) (Fig. 3C). IKKϵ(K38A) also blocked virus-induced activation of the IFNA4 promoter by about fourfold (fig. S3A) but did not interfere with a heterologous NF-κB–dependent promoter (fig. S3C). The capacity of different IKK kinases to stimulate reporter constructs was also investigated. IKKα, IKKβ, IKKϵ, TBK, and NF-κB–inducing kinase (NIK) were each expressed with the IFNA4 promoter, together with IRF-7. Only IKKϵ and TBK1 activated the IFNA4 promoter (Fig. 3D). In contrast, each kinase induced a 10- to 40-fold stimulation of a NF-κB–dependent reporter construct (fig. S3B). These results demonstrate that IRF-7 and IRF-3 stimulate the IFNA4 and IFNB promoters in an IKKϵ- and TBK1-dependent manner.

Fig. 3.

Activation of IRF-regulated IFN promoters by IKKϵ and TBK1. HEK293 cells were transfected with a control plasmid; a luciferase reporter plasmid containing the IFNB or IFNA4 promoters (8); or plasmids encoding IRF-7, IRF-3ΔN, IKKϵ, IKKϵ(K38A), IKKϵ(1-361), IKKα, IKKβ, TBK1, or NIK as indicated. At 8 hours after transfection, cells were infected with Sendai virus at 40 hemagglutination units/ml or left uninfected. Luciferase activity was analyzed 24 hours after transfection as fold activation relative to the basal level of reporter gene in the presence of control vector (after normalization with cotransfected Renilla relative light units) (A and B). (C) Fold activation of IFNA4 in the presence of IKKϵ and IRF-7 is plotted as 100; the relative luciferase activity in the presence of increasing amounts of IKKϵ(K38A) or IKKϵ(1-361) is expressed as a fraction of 100. (D) Luciferase activities were expressed as in (A) and (B), with fold activation relative to the basal level of the IFNA4 promoter in the presence of IRF-7; values represent the average of three experiments, performed in duplicate, with variability shown by error bars.

Specific interfering RNA (RNAi) oligonucleotides were used in lung epithelial A549 cells to down-regulate the expression of TBK1 and IKKϵ (Fig. 4A). RNAi of TBK1 and IKKϵ eliminated IKKϵ expression and blocked TBK1 expression by 70 to 75% (Fig. 4A, lanes 5 to 8). With reduced IKKϵ and TBK1 expression, virus-induced phosphorylation of endogenous IRF-3 (Fig. 4A, lanes 3 and 4) was inhibited (Fig. 4A, lanes 7 and 8). Inhibition of IRF-3 phosphorylation correlated with reduced expression of the IRF-3–responsive IFN-stimulated gene 56 (ISG56) (Fig. 4A, lanes 7 and 8). Activation of the IFNA4 promoter through IRF-7 also decreased by RNAi (Fig. 4B). To assess the effect of IKKϵ expression on viral replication, IKKϵ or IKKϵ(K38A) was expressed with either IRF-3 or IRF-3ΔN. Replication of vesicular stomatitis virus (VSV) in the presence of IKKϵ decreased by 4 logs to 106 plaque-forming units (PFU)/ml (Fig. 4C). Blocking IFN expression by IRF-3ΔN expression restored the VSV titer to 1010 PFU/ml, demonstrating that IKKϵ antiviral activity is IRF-3–dependent (Fig. 4C). Western blot analysis confirmed that IKKϵ expression induced ISG56 expression and inhibited VSV nucleocapsid protein (N) expression (Fig. 4D, lane 3), whereas IRF-3ΔN expression permitted viral replication in the presence of IKKϵ (Fig. 4D, lane 7). An identical experiment with TBK1 yielded similar results (17). Thus, engagement of the antiviral program through IKKϵ or TBK1 was sufficient to induce IFN-stimulated gene expression and inhibit VSV replication.

Fig. 4.

IKKϵ and TBK1 are required for establishing the antiviral state. (A) RNAi-mediated silencing of TBK1 and IKKϵ in A549 cells. Control and TBK1- and IKKϵ-specific RNAi-treated cells were infected with VSV at a multiplicity of infection (MOI) of 100 and were harvested at 0, 2, 4, and 6 hours after infection. Whole-cell extracts (30 μg) were analyzed by Western blot (25) with Ab's to TBK1, IKKϵ, IRF-3, IRF-3 S396, ISG56, VSV N, and β actin. (B) A549 cells treated with control or TBK1- and IKKϵ-specific RNAi oligonucleotides for 48 hours and then transfected with IFNA4-pGL3 and Renilla control plasmid. At 6 hours after transfection, cells were infected with VSV (MOI 10) and harvested 12 hours after infection. Luciferase activity was expressed as relative luciferase activity, relative to the basal level of reporter gene in the presence of control vector (after normalization with cotransfected Renilla relative light units). (C) HEK293 cells were transfected with pEGFP, IKKϵ, or IKKϵ(K38A) and with either IRF-3 or IRF-3ΔN. At 24 hours after transfection, cells were infected with VSV (MOI 100) for 12 hours, and supernatants were analyzed for virus production. Supernatants from transfected cells were analyzed for VSV replication using a standard plaque assay at the time points indicated. Plaques were counted and titers calculated as PFU/ml. Left panel: pEGFP+IRF-3 (solid circles), IKKϵ+IRF-3 (solid triangles), and IKKϵ-(K38A)+IRF-3 (solid squares). Right panel: pEGFP+IRF-3ΔN (open circles), IKKϵ+IRF-3ΔN (open triangles), and IKKϵ(K38A)+IRF-3ΔN (open squares). (D) Whole-cell extracts (80 μg) from the time point 12 hours after infection in (C) were analyzed by Western blot using Ab's to Flag, IRF-3, VSV N, ISG56, and β actin.

IKKϵ and TBK1 were originally shown to phosphorylate Ser36 but not Ser32 of IκBα, which suggests that each possessed a distinct kinase activity as compared to the classical IKKα/IKKβ complex (13, 1820). In response to either tumor necrosis factor α or interleukin 1 expression, TBK1–/– fibroblasts exhibited normal phosphorylation and degradation of IκB, as well as NF-κB binding activity (21, 22). However, NF-κB–directed transcription was reduced, suggesting that TBK1 possessed a direct role in the phosphorylation of transcription factors (21). The present studies demonstrate that IKKϵ and TBK1 are components of the VAK required for IRF-3 and IRF-7 phosphorylation. This pathway appears to confer specificity in establishing the IFN antiviral response, distinct from the broad immunoregulatory functions of the NF-κB/IKK pathway (12). The response may also be cell type–specific, because TBK1 is ubiquitously expressed, whereas IKKϵ activity is inducible in lymphoid and other cell types (13, 1820). The ubiquitous nature of both TBK1 and IRF-3 may reflect their primary role in triggering an early phase of IFN response, whereas IRF-7, which may be the preferred substrate for IKKϵ, mediates amplification of the antiviral response (68). Furthermore, IKKϵ and TBK1 may be functionally linked to IKKα/IKKβ complexes through TANK (TRAF family member–associated NF-κB activator) and IKKγ/NEMO interactions (23, 24).

Supporting Online Material

Materials and Methods

Figs. S1 to S3

  • * These authors contributed equally to this work.

References and Notes

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