SINTBAD, a novel component of innate antiviral immunity, shares a TBK1-binding domain with NAP1 and TANK

Abstract

The expression of antiviral genes during infection is controlled by inducible transcription factors such as IRF3 (interferon regulatory factor). Activation of IRF3 requires its phosphorylation by TBK1 (TANK-binding kinase) or IKKi (inhibitor of nuclear factor kappaB kinase, inducible). We have identified a new and essential component of this pathway, the adaptor protein SINTBAD (similar to NAP1 TBK1 adaptor). SINTBAD constitutively binds TBK1 and IKKi but not related kinases. Upon infection with Sendai virus, SINTBAD is essential for the efficient induction of IRF-dependent transcription, as are two further TBK1 adaptors, TANK and NAP1. We identified a conserved TBK1/IKKi-binding domain (TBD) in the three adaptors, predicted to form an alpha-helix with residues essential for kinase binding clustering on one side. Isolated TBDs compete with adaptor binding to TBK1 and prevent poly(I:C)-induced IRF-dependent transcription. Our results suggest that efficient signal transduction upon viral infection requires SINTBAD, TANK and NAP1 because they link TBK1 and IKKi to virus-activated signalling cascades.

Figure 1

Identification of SINTBAD. (A) Alignment of N termini of human and murine SINTBAD and NAP1. (B) Northern blot of human tissues probed for SINTBAD (upper panel) and β-actin (lower panel). (C) Domain composition of SINTBAD, NAP1 and TANK. Regions with sequence similarity are highlighted. CC—coiled coils forming domain, TBD—TBK1/IKKi-binding domain as identified in this study; ZnFi—C₂H₂ type zinc finger.

Figure 2

SINTBAD is a TBK1/IKKi adaptor. (A) SINTBAD interacts with TBK1 and IKKi. Lysates from 293ET cells transfected with the indicated combination of luciferase-tagged NEMO, NAP1, SINTBAD, TANK and FLAG-tagged IKKα, IKKβ, TBK1, IKKi were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates. The expression of proteins in lysates was detected by Western blotting. (B) Recombinant SINTBAD binds TBK1. Lysates of 293ET cells were precipitated using MBP-SINTBAD (86–330 aa) immobilized on amylose resin. Uncharged resin was used as a control. Lysates and eluates were blotted for the presence of IKKα and TBK1. (C) SINTBAD binds TBK1 and IKKi at endogenous levels. Lysates of 293ET cells were immunoprecipitated using a polyclonal antiserum against SINTBAD (SIN) or a preimmune serum (PRE). Lysates and precipitates (IP) were blotted for the presence of IKKα, IKKβ, TBK1, IKKi and SINTBAD. (D) Oligomerization of NAP1, SINTBAD and TANK. Lysates from 293ET cells transfected with the indicated combination of luciferase-tagged NAP1, SINTBAD, TANK and FLAG-tagged GFP, NAP1, SINTBAD, TANK were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates. The expression of proteins in lysates was detected by Western blotting.

Figure 3

Importance of TBK1 adaptors for Sendai virus-induced IRF but not NF-κB activation. (A) Lysates from cells transfected with the indicated combinations of shRNAs and FLAG-tagged GFP, NAP1, SINTBAD, TANK, UBC13, TRIF and MAVS were blotted for the presence of FLAG-tagged proteins. Blots for TBK1 and IKKα detected endogenous protein. (B–D) Cells were transfected with the indicated shRNAs and different IRF-dependent luciferase reporters: IFN-β (B), PRD (III-I)₃ (C) or ISRE (D). Two days after transfection, cells were stimulated with or without 150 HAU/ml of Sendai virus. Luciferase activity was measured 16 h later. (E–H) Cells were transfected with the indicated shRNAs and different NF-κB-dependent luciferase reporters: κB (E–G) and PRD(II)₂ (H). Two days after transfection, cells were stimulated with 10 μg/ml of peptidoglycan (E), 50 ng/ml PMA and 1 μM ionomycin (F) or 10 ng/ml of TNF-α (G and H). Luciferase activity was measured 16 h later.

Figure 4

Importance of TBK1 adaptors for the induction of endogenous genes upon infection with Sendai virus. (A, B) Cells were co-transfected with GFP and the indicated shRNAs. Two days after transfection, cells were stimulated with or without 150 HAU/ml of Sendai virus for 16 h. mRNA levels of IP-10 (A) and ISG15 (B) were quantified from GFP⁺ cells isolated by FACS.

Figure 5

Identification of the TBK1-binding domain in SINTBAD and NAP1. (A, B) Schematic representation of deletions in SINTBAD (A) and NAP1 (B). (C–F) Cells were transfected with combinations of FLAG-tagged forms of SINTBAD (C and E), NAP1 (D and F) and luciferase-tagged SINTBAD (C), NAP1 (D) or TBK1 (E and F) as indicated. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates normalized to the binding of GFP. The expression of proteins in lysates was detected by Western blotting.

Figure 6

Characterization of the TBD. (A) Alignment of TBDs from murine and human TANK, NAP1 and SINTBAD. Borders of the alignment correspond to exon boundaries. (B) Cells were transfected with combinations of FLAG-tagged forms of IKKβ, TBK1, IKKi and luciferase-tagged NAP1, SINTBAD, TANK or their respective luciferase-tagged TBDs as indicated. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates. The expression of proteins in lysates was detected by Western blotting. (C) Helical wheel representation of TBD. Human NAP1 (216–250 aa), SINTBAD (291–325 aa) and TANK (135–169 aa) are shown. Bold letters indicate positions conserved in all three adaptors. Positions required and dispensable for TBK1 binding are indicated by black and white arrowheads, respectively. (D) Lysates from cells transfected with luciferase-tagged NAP1 TBD carrying the indicated mutations were subjected to Western blotting. (E, F) Cells were transfected with different forms of luciferase-tagged NAP1 TBD and FLAG-tagged GFP, TBK1 (E) or IKKi (F) as indicated. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates normalized to the binding of GFP. The expression of proteins in lysates was detected by Western blotting.

Figure 7

TBDs compete with binding of full-length adaptors to TBK1. (A–D) Cells were transfected with FLAG-tagged TBK1, YFP-tagged TBDs derived from NAP1, SINTBAD, TANK and luciferase-tagged TANK (A), SINTBAD (B), NAP1 (C), TBK1 (D) as indicated. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates. The expression of proteins in lysates was detected by Western blotting. (E) Cells were transfected with FLAG-tagged TBK1, YFP-tagged NAP1 TBD (wt or L257S) and luciferase-tagged NAP1as indicated. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and lysates. The expression of proteins in lysates was detected by Western blotting.

Figure 8

TBDs prevent poly(I:C)-induced IRF but not NF-κB activation. (A, B) Cells were transfected with IRF (A) or NF-κB (B)-dependent luciferase reporter genes and YFP-tagged TBDs derived from NAP1, TANK and SINTBAD. Two days after transfection, cells were stimulated with 12.5 μg/ml poly(I:C) or 10 ng/ml of TNF-α. Luciferase activity was measured 5 h later. (C, D) Cells were transfected with IRF (C) or NF-κB (D)-dependent luciferase reporter genes and YFP-tagged NAP1 TBD (wt or L257). Two days after transfection, cells were stimulated with 12.5 μg/ml poly(I:C). Luciferase activity was measured 5 h later.