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. 2012 Jun 29;287(27):22672-82.
doi: 10.1074/jbc.M112.349225. Epub 2012 May 16.

Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions

Julianne Stack  1 , Andrew G Bowie
Affiliations

Poxviral protein A46 antagonizes Toll-like receptor 4 signaling by targeting BB loop motifs in Toll-IL-1 receptor adaptor proteins to disrupt receptor:adaptor interactions

Julianne Stack et al. J Biol Chem. .

Abstract

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex.

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Figures

FIGURE 1.
FIGURE 1.
A46 impairs ligand-induced TLR4-dependent transcription factor activation. HEK293-TLR4 cells were transfected for 24 h with 150 ng myc-A46 or pCMV-myc (EV) and the NFκB luciferase reporter gene (A), or the pFR luciferase reporter gene along with either IRF3-Gal4 (B) or IRF7-Gal4 (C). Cells were stimulated with 10 ng/ml LPS for 6 h and luciferase reporter gene activity was measured. The data are mean ± S.D. of triplicate samples and are representative of at least five separate experiments. *, p < 0.05; **, p < 0.005; or ***, p < 0.0005 compared with LPS and EV.
FIGURE 2.
FIGURE 2.
The conserved proline of the BB loop is essential for A46 to interact with the TIR adaptors. A, HEK293T cells were transfected with 4 μg of myc-A46. After 48 h, lysates were incubated with GST-TLR TIR domains or GST alone as indicated, and together with input lysate (lane 1), were analyzed by SDS-PAGE and immunoblotting. GST and GST fusion proteins were analyzed by SDS-PAGE and Coomassie staining to demonstrate equal inputs. B–E, HEK293T cells were transfected with A46 and (B) AU1-MyD88 (left) or AU1-MyD88 P200H (right), (C) HA-Mal (upper panel) or HA-Mal P125H (lower panel), (D) Flag-TRAM (upper panel), Flag-TRAM P116H (middle panel) or Flag-TRAM C117H (lower panel) or (E) Flag-TRIF (upper panel) or Flag-TRIF P434H (lower panel) as indicated. After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. F, HEK293T cells were transfected with 8 μg of the indicated TIR adapter molecules. After 48 h, lysates were incubated with GST alone (lane 2) or GST-A46 (lane 3), and together with input lysates (lane 1) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Each immunoblot is representative of three experiments.
FIGURE 3.
FIGURE 3.
A46 inhibits TLR4 signaling by disrupting receptor:adaptor, and not receptor:receptor interactions in the TLR4 complex. A, Coomassie-stained gel showing equal amounts of GST and GST-TLR4 TIR. B–D, HEK293T cells were transfected with the indicated amounts of HA-TLR4 (B), HA-Mal (C), or Flag-TRAM (D), along with increasing amounts of myc-A46 or pCMV-myc and harvested after 48 h. E and F, HEK293T cells were transfected with HA-Mal (E) or Flag-TRAM (F), along with either myc-A46 or pCMV-myc. After 24 h, cells were infected with VACV vWT or vΔA46 (MOI = 1) as indicated. Cells were harvested 24 h after infection. B–F, clarified lysates were incubated with GST alone or GST-TLR4 TIR and, together with input lysates, were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Each immunoblot is representative of at least three experiments.
FIGURE 4.
FIGURE 4.
A46 does not disrupt adaptor:adaptor interactions in the TLR4 complex. A–E, HEK293T cells were transfected with the indicated amounts of myc-MyD88 (A), Flag-TRAM (B) HA-Mal (C), myc-IRAK2 (D) or Flag-TRAF6 (E), along with increasing amounts of myc-A46 or pCMV-myc. After 48 h, lysates were incubated with GST alone or GST fusion protein and, together with input lysates, were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Each immunoblot is representative of at least three experiments.
FIGURE 5.
FIGURE 5.
Receptor:adaptor but not adaptor:adaptor interactions require intact adaptor BB loops. A and B, HEK293T cells were transfected with 4 μg HA-Mal, HA-Mal PH (A) or Flag-TRAM, Flag-TRAM PH and Flag-TRAM CH (B). After 48 h, lysates were incubated with GST-alone or GST-TLR4 TIR, and together with input lysates, were analyzed by SDS-PAGE and immunoblotting. C and D, HEK293T cells were transfected with 4 μg of HA-Mal and AU1-MyD88 (C) or 4 μg of HA-Mal PH and AU1-MyD88 PH (D). After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Each immunoblot is representative of three experiments.
FIGURE 6.
FIGURE 6.
The region of A46 from which the VIPER peptide is derived is required for TLR4 inhibition and TRAM binding. A, HEK293-TLR4 cells were transfected with 2 μg of pCMV-myc, myc-A46, or myc-A46ΔVIPER. After 24 h, clarified lysates were subject to SDS-PAGE and immunoblotting with the anti-myc antibody. B and C, HEK293-TLR4 cells were transfected with pCMV-myc (EV), 50–150 ng myc-A46, or 50–150 ng myc-A46ΔVIPER, and the NFκB (B), or the ISRE (C) luciferase reporter gene. Cells were stimulated with 10 ng/ml LPS for 6 h, and luciferase reporter gene activity was measured. The data are mean ± S.D. of triplicate samples and are representative of at least four separate experiments. D–F, HEK293 cells stably expressing the IL-1 receptor (D), TLR2 (E), or TLR8 (F) were transfected with pCMV-myc (EV), 50–150 ng myc-A46 or 50–150 ng myc-A46ΔVIPER, and the NFκB luciferase reporter gene. Cells were stimulated with either 50 ng/ml IL-1α (D), 20 nm MALP2 (E), 20 ng/ml Pam3Csk4 (E), or 2.5 μg/ml CL075 (F) for 6 h and luciferase reporter gene activity was measured. The data are mean ± S.D. of triplicate samples and are representative of at least four experiments. G, HEK293T cells were transfected with 4 μg of Flag-Mal or Flag-TRAM along with either myc-A46 or myc-A46ΔVIPER. After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. Each immunoblot is representative of three experiments. H, HEK293T cells were transfected with 25 ng of Flag-Mal or Flag-TRAM and 50–150 ng of A46, 50–150 ng of A46ΔVIPER or pCMV-myc (EV), along with the NFκB luciferase reporter gene. The data are presented as percentage inhibition of fold induction and are representative of three experiments. *, p < 0.05 or **, p < 0.005 compared with stimulus only.
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