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. 2016 Mar;26(3):288-303.
doi: 10.1038/cr.2016.16. Epub 2016 Jan 29.

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Yan Yang  1 , Su-Yun Wang  1 , Zhe-Fu Huang  1 , Hong-Mei Zou  1 , Bing-Ru Yan  2 , Wei-Wei Luo  2 , Yan-Yi Wang  1
Affiliations

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Yan Yang et al. Cell Res. 2016 Mar.

Abstract

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b(-/-) mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b(-/-) mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.

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Figure 1
Figure 1
Mex3B is a positive regulator of TLR3-mediated signaling. (A) Mex3B potentiates TLR3-mediated signaling in 293 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 6 h before luciferase assays. (B) Mex3B potentiates poly(I:C)-induced signaling in 293-TLR3 cells. Reporter assays were similarly performed as in A. (C) Mex3B potentiates poly(I:C)-induced expression of downstream genes in 293-TLR3 cells. The cells were transfected with the indicated plasmids for 24 h, and then treated with poly(I:C) for 3 h before qPCR analysis. (D) Mex3B potentiates poly(I:C)-induced activation of the IFN-β promoter in HeLa and HCT116 cells. Reporter assays were similarly performed as in A. (E) Overexpression of Mex3B has no marked effects on SeV- or cytoplasmic poly(I:C)-induced activation of the IFN-β promoter. The 293 cells were transfected with the indicated plasmids for 24 h, and then infected with SeV or re-transfected with poly(I:C) for 12 h before luciferase assays. (F) Effects of Mex3B-RNAi on expression of Mex3B. The 293 cells were transfected with a Mex3B-RNAi plasmid for 24 h before immunoblot analysis. (G) Effects of Mex3B-RNAi on TLR3-mediated signaling. Reporter assays were similarly performed as in A. (H) Effects of Mex3B-RNAi on poly(I:C)-induced transcription of IFNB1, ISG56 and TNFA genes. The experiments were similarly performed as in C. (I) Effects of Mex3B-RNAi on poly(I:C)- and SeV-induced activation of the IFN-β promoter in HCT116 cells. The cells were transfected with the indicated plasmids for 24 h, and then infected with SeV for 12 h or treated with poly(I:C) for 6 h before luciferase assays. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Student's t-test).
Figure 2
Figure 2
Mex3B is essential for TLR3-mediated signaling in MEFs, BMDMs and DCs. (A) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in MEFs. MEFs (2 × 105) were left untreated, treated with poly(I:C) (20 μg/ml), TNFα (10 ng/ml) or IL-1β (10 ng/ml) for 3 h, or infected with SeV or VSV for 12 h. Total RNA was then extracted for qPCR analysis. (B) Effects of Mex3B deficiency on poly(I:C)-induced phosphorylation of IRF3 and IκBα in MEFs. MEFs (2 × 105) were left untreated, infected with SeV or treated with poly(I:C) for the indicated times. Cells were lysed and immunoblot was performed with the indicated antibodies. (C, D) Mex3B deficiency inhibits poly(I:C)-induced transcription of Ifnb1 gene in BMDMs (C) and DCs (D). The cells (2 × 105) were left untreated, treated with poly(I:C) (20 μg/ml), R837 (20 μg/ml) or PGN (10 μg/ml) for 3 h or infected with SeV, VSV, EMCV or HSV for 12 h before qPCR analysis. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Student's t-test).
Figure 3
Figure 3
Mex3b−/− mice are resistant to poly(I:C)-induced death. (A) Sex- and age-matched Mex3b+/+ and Mex3b−/− mice (n = 8 or 9) were injected intraperitoneally with poly(I:C) for 2 h. Serum concentrations of the indicated cytokines were measured by ELISA. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Student's t-test). (B) Sex- and age-matched Mex3b+/+ and Mex3b−/− mice (n = 12) were injected intraperitoneally with poly(I:C) plus D-galactosamine. The survival of injected mice was monitored every 12 h for 4 days. (C) Mex3b+/+ and Mex3b−/− mice were infected i.p. with HSV-1 at 1 × 107 p.f.u. per mouse (n = 7) for 6 h. Serum concentrations of the indicated cytokines were measured by ELISA.
Figure 4
Figure 4
The RNA-binding activity of Mex3B and proteolytic cleavage of TLR3 are indispensable for Mex3B to potentiate TLR3-mediated signaling. (A) A schematic presentation and expression of full-length Mex3B, TLR3 and their mutants. (B-D) Effects of Mex3B mutants on TLR3-mediated signaling. The 293 cells (1 × 105) were transfected with the IFN-β promoter reporter (0.1 μg) and the indicated plasmids (0.1 μg) for 24 h, and then left untreated or treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays.
Figure 5
Figure 5
Mex3B regulates poly(I:C)-triggered signaling through TLR3. (A) Mex3B(G83/177D) inhibits poly(I:C)- but not TRIF-induced signaling. The 293 cells (1 × 105) were transfected with the indicated plasmids and increased amounts of a Mex3B plasmid. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for 6 h before luciferase assays were performed. (B) TLR3(C95/122A) inhibits poly(I:C)-induced, TLR3/Mex3B-mediated signaling. The experiments were similarly performed as in A. (C) Knockdown of Mex3B inhibits poly(I:C)- but not TRIF- or TBK1-mediated activation of the IFN-β promoter. The 293-TLR3 cells (1 × 105) were transfected with the IFN-β promoter reporter (0.1 μg) and either a control or Mex3B-RNAi plasmid (0.5 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h followed by either treatment with poly(I:C) (20 μg/ml) or retransfection with TRIF or TBK-1 plasmids (0.1 μg). Luciferase assays were performed 6 h after treatment or 18 h after retransfection. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Student's t-test). (D) Effects of chloroquine on TLR3- and Mex3B-mediated signaling. The 293 cells (1 × 105) were transfected with the indicated plasmids (0.1 μg each) for 24 h. The cells were pretreated with chloroquine for 30 min, and then treated with poly(I:C) (20 μg /ml) for 6 h before luciferase assays.
Figure 6
Figure 6
Mex3B is associated with TLR3 and facilitates the recruitment of TRIF upon poly(I:C) stimulation. (A) Mex3B interacts with TLR3. HCT116(Mex3B-3×Flag) cells (1 × 108), in which a cDNA encoding three copies of Flag tag was inserted immediately before the stop codon of Mex3b gene, or mouse DCs (1 × 108) were treated with poly(I:C) (20 μg /ml) or LPS (20 ng/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. pIC, poly(I:C). (B) Domain mapping of the Mex3B-TLR3 interaction. The 293-TLR3 cells (2 × 106) were transfected with either the full-length or the indicated Mex3B truncation plasmids (5 μg). Coimmunoprecipitation and immunoblot were performed with the indicated antibodies. Ig, mouse IgG; αT3, anti-TLR3. (C) Mex3B interacts with TRIF upon poly(I:C) stimulation. HCT116 (Mex3B-3×Flag) cells (1 × 108) were left untreated or treated with poly(I:C) (20 μg/ml) for the indicated times. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies.
Figure 7
Figure 7
Mex3B colocalizes with TLR3 in the endosome and promotes the processing, stability and ligand binding of TLR3. (A) Mex3B colocalizes with TLR3 in the endosomes. The 293 cells (1 × 105) were cotransfected with Mex3B-HA and TLR3-Flag plus KDEL-GFP (ER marker), EEA1-GFP (early endosome marker) or LAMP-1-GFP (lysosome marker). Twenty hours after transfection, cells were fixed with 4% paraformaldehyde, stained with anti-Flag and anti-HA antibodies, and subjected to confocal microscopy. (B) Mex3B is sorted into the lumen of endosomes. HCT116 (Mex3B-3×Flag) cells (4 × 108) were collected and washed twice with ice-cold PBS. Endosome/lysosome fractions were isolated and either left untreated, treated with proteinase K (5 ng/ml) or treated with proteinase plus 0.1% Triton X-100 for 15 min at room temperature. The samples were then analyzed with the indicated antibodies. (C) Overexpression of Mex3B increases the abundance of processed C-terminal fragment of TLR3. The 293 cells (2 × 105) were transfected with the indicated plasmids. Twenty-four hours after transfection, cells were treated with poly(I:C) (20 μg/ml) for the indicated times before immunoblot. The relative amount of total TLR3 was quantitated by the Bio-Rad Quantity One program. (D) Knockdown of Mex3B affects the stability of TLR3. The 293 cells (2 × 105) were transfected with TLR3 (0.5 μg) and Mex3B-RNAi plasmid (1 μg). Transfected cells were selected with puromycin (1 μg/ml) for 24 h and then treated with poly(I:C) for the indicated times before immunoblot. (E) Mex3B enhances binding of poly(I:C) to TLR3. The 293 cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Cell lysates were mixed at 1:1 ratio as indicated and incubated with biotinylated-poly(I:C) for 1 h before pull-down assays were performed with streptavidin agarose. Precipitates and cell lysates were analyzed by immunoblot with the indicated antibodies.
Figure 8
Figure 8
A working model on the involvement of Mex3B in TLR3-mediated signaling. Upon viral infection, TLR3 transports from the ER to endosomes where it is proteolytically cleaved, resulting in the formation of a very stable “cleaved/associated” TLR3 that represents the primary form of the signaling receptor. The RNA-binding protein Mex3B localizes in the lumen of endosomes, functions as a coreceptor of TLR3 by promoting ligand binding and proteolytic processing of TLR3, resulting in full activation of TLR3.
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References

    1. Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 2011; 34:637–650. - PubMed
    1. Jin MS, Lee JO. Structures of the toll-like receptor family and its ligand complexes. Immunity 2008; 29:182–191. - PubMed
    1. Blasius AL, Beutler B. Intracellular toll-like receptors. Immunity 2010; 32:305–315. - PubMed
    1. McGettrick AF, O'Neill LA. Localisation and trafficking of Toll-like receptors: an important mode of regulation. Curr Opin Immunol 2010; 22:20–27. - PubMed
    1. Matsumoto M, Funami K, Tanabe M, et al. Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 2003; 171:3154–3162. - PubMed

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