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. 2016 May 1;30(9):1086-100.
doi: 10.1101/gad.277533.116. Epub 2016 Apr 28.

Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation

Fansen Meng  1 , Ruyuan Zhou  1 , Shiying Wu  1 , Qian Zhang  1 , Qiuheng Jin  1 , Yao Zhou  2 , Steven W Plouffe  3 , Shengduo Liu  1 , Hai Song  1 , Zongping Xia  1 , Bin Zhao  1 , Sheng Ye  1 , Xin-Hua Feng  4 , Kun-Liang Guan  3 , Jian Zou  2 , Pinglong Xu  1
Affiliations

Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation

Fansen Meng et al. Genes Dev. .

VSports app下载 - Abstract

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies. VSports手机版.

Keywords: IRF3; Mst1; TBK1; host antiviral defense; phosphorylation. V体育安卓版.

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Figures

Figure 1.
Figure 1.
Mst1 attenuates cytosolic RNA/DNA sensing. (A) Transfection of Mst family stress kinases, particularly Mst1, elicited a strong suppression of the IRF3/7-responsive IFNβ promoter in HEK293 cells, which was stimulated by activated RIG-I (caRIG-I). The expression of tagged Mst1–4 and YSK1 were revealed by immunoblotting. n = 3 experiments. (*) P < 0.001, compared with control, by Student's t-test. (B,C) Dose-dependent inhibition of both IFNβ (B) and 5xISRE (C) reporters by low-level Mst1 expression in response to RIG-I or STING-stimulated activation in HEK293 cells. (D) Transfection of TBK1 or IKKε exerted a robust activation of the IRF3-responsive IFNβ reporter, which was suppressed by Mst1 expression, also in a dose-dependent manner. (E,F) Two kinase-dead forms of Mst1, K59R and T183A, did not inhibit the IRF3-responsive IFNβ promoter, which was stimulated by either activated RIG-I (E) or STING (F). n = 3 experiments. (*) P < 0.001, compared with caRIG-I without Mst1; (**) P < 0.001, compared with wild-type Mst1 expression, by Student's t-tests. (G) Mst1 did not inhibit Wnt or Hedgehog signaling, which was, respectively, activated by LiCl treatment or Gli1 cotransfection and assessed by reporter assays from the TCF or Gli1 promoter. (H) siRNA-mediated knockdown of Mst1 in HEK293 cells, as evidenced by anti-Mst1 immunoblotting, boosted IRF3 responsiveness by MAVS, or TBK1 stimulation. (I) Similarly, siRNA-mediated Mst1 depletion effectively enhanced the STING pathway. n = 3 experiments. (*) P < 0.01, compared with control siRNA, by Student's t-test. (J) Treatment of mouse embryonic fibroblasts (MEFs) with H2O2 decreased IRF3 phosphorylation at Ser396 and the formation of IRF3 homodimer in Native-PAGE, which was stimulated by Sendai virus (SeV) infection. (K) SeV infection induced the expression of antiviral genes IFIT1 and ISG15 in control or Mst1−/− MEFs. The effects of H2O2 in ISG inductions were significantly stronger in control MEFs than in Mst1−/− MEFs. (*) P < 0.01, compared with control MEFs; (**) P < 0.01, compared with SeV infection.
Figure 2.
Figure 2.
Mst1 negatively regulates host antiviral defense in primary/cultured cells and mice. (A) Antiviral response of control or Mst1−/− MEFs against SeV infection was measured by mRNA induction at 12 hpi of various ISGs, including IFIT1, ISG15, IRF7, and IFNβ. Mst1−/− MEFs exhibited stronger antiviral responses than control MEFs. (*) P < 0.05, compared with control MEFs, by Student's t-test. (B, bottom panel) Mst1−/− NMuMG epithelial cells were generated by the CRISPR/Cas9 method, and the Mst1 knockout (KO) and rescue were verified by immunoblotting. Cytosolic RNA-sensing signaling in Mst1−/− and control NMuMG was evaluated by IFNβ reporter assay. (Top panel) Stronger IRF3 transactivation was observed by Mst1 ablation but was reversed by the reintroduction of wild-type (WT) Mst1. (C) Antiviral response in Mst1−/− and control NMuMG cells to SeV infection at 6 hpi was measured by quantification of ISGs and IFNβ, and stronger antiviral responses were observed by Mst1 deletion in NMuMG cells. n = 4 Mst1−/− clones. (*) P < 0.01, compared with control NMuMG cells, by Student's t-test. (D) Mst1−/− and control NMuMG cells were infected with GFP-tagged vesicular stomatitis virus (gVSV). Reduced virus replication, as evidenced by a reduced level of GFP+ cells, was observed in Mst1−/− NMuMG cells by microscopy (top panels) or FACS assay (bottom panels). (E) Survival of ∼8-wk-old Mst1+/+ and Mst1−/− mice given intravenous tail injection of gVSV (2 × 107 plaque-forming units [pfu] per gram). n = 12 mice for each group. P < 0.01, by paired Student t-test. (F) Determination of gVSV loads in mouse organs at 12 hpi in Mst1+/+ and Mst1−/− mice, which were intravenously tail-injected, with gVSV. n = 3 mice for each group. (*) P < 0.01, compared with control Mst1+/+ group, by Student t-test.
Figure 3.
Figure 3.
Mst1 suppresses RNA virus-induced activation of TBK1. (A) Control and Mst1−/− MEFs were subjected to infection by SeV or HSV-1, and virus-induced activation of endogenous TBK1 was visualized by immunoblotting of Ser172 phosphorylation. Stronger TBK1 activation was seen with Mst1 knockout (KO). (B) Similarly, VSV infection-induced activation of endogenous TBK1, also visualized by Ser172 phosphorylation, was stronger in Mst1−/− NMuMG cells. (C) TBK1 activation detected by immunoblotting of phospho-Ser172 (first panel) and IRF3 activation detected by immunoblotting of phospho-Ser396 (third panel) or phospho-Ser386 (fourth panel) were eliminated by cotransfection of wild-type Mst1 (WT) but not by kinase-dead Mst1 mutants (K59R or T183A). (Fifth panel) Also, an obvious mobility shift of IRF3 was seen after coexpression of wild-type Mst1. (D) Similar Mst1-mediated loss of IRF3 phosphorylation and mobility shift were observed when IRF3 was stimulated by activated RIG-I (caRIG-I). (E,F) IKKε activation, which can be recognized by the anti-TBK1 phospho-S172 antibody, was dose-dependently inhibited by Mst1 (E) but not by its kinase-dead mutants (F). (G) Cotransfection of Mst1 with increasing levels of TBK1 showed that Mst1 still suppressed IRF3 transactivation even though Mst1 could not abolish TBK1 activation when it was expressed overwhelmingly. n = 3 experiments. (*) P < 0.001, compared with TBK1 alone control, by Student's t-test.
Figure 4.
Figure 4.
Mst1 associates with and phosphorylates IRF3 to negate its transcriptional activity. (A) Expression of Mst1 wild-type (WT), but not its kinase-dead form, blocked transcriptional activity of constitutively active IRF3 (IRF3 5SD). n = 3 experiments. (*) P < 0.001, compared with IRF3 5SD alone; (**) P < 0.001, compared with wild-type Mst1, by Student's t-test. (B) Phos-Tag SDS-PAGE of transfected IRF3 showed the loss of a faster migrated band in the presence of wild-type Mst1 as another indication for Mst1-induced IRF3 modification. (C) Coexpression of wild-type Mst1 resulted in a clear mobility shift of IRF3 5SD (third lane), which can be negated by cotransfection of phosphatase PPM1B (fourth lane) that we identified as a phosphatase of IRF3 (second lane). (D) Coexpression of PPM1B completely restored Mst1-driven suppression of IRF3 5SD. n = 3 experiments. (*) P < 0.001, compared with wild-type Mst1, by Student's t-test. (E) Lats1/2 double-knockout (dKO) cells were generated by the CRISPR/Cas9 method and verified by immunoblotting of Lats1 expression and TAZ mobility shift and degradation. Cotransfection of Mst1 resulted in the loss of the IRF3 faster migrated band, as in the wild-type or Lats1/2 double-knockout cells. (F) Coimmunoprecipitation assay by differential tags showed the interaction between IRF3 and Mst1, which was enhanced in the presence of Mst1 adaptors such as RASSF proteins and SAV1. (G) Endogenous Mst1 and IRF3 interaction in NMuMG cells was detected by coimmunoprecipitation assay using an anti-Mst1 antibody and was visualized by IRF3 immunoblotting. Note the smaller molecular weight of IRF3 in mouse cells. (H) In vitro kinase assay with separately purified IRF3 and Mst1 displayed the signal of IRF3 phosphorylation in the presence of Mst1 adaptor protein SAV1. Mst1 autophosphorylation was also seen and was boosted by SAV1. (I) Amino acid sequences around the Thr75 and Thr253 residues of IRF3 were similar to a reported consensus motif for Mst1 (Miller et al. 2008). (J) Nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) assay, which analyzed the modifications on purified IRF3 that was coexpressing with kinase-dead or wild-type Mst1, showed Mst1-induced phosphorylation of IRF3 at the Thr75 and Thr 253 residues as well as reduced phosphorylation at the Ser396 residue. (K) Transfection of phosphomimetic IRF3 mutants revealed that the transcriptional activity of IRF3 5SD was nearly lost when either Thr75 or Thr253 was phosphorylated to an extent comparable with Mst1 cotransfection. n = 3 experiments. (* and **) P < 0.001, compared with control of IRF3 5SD expression, by Student's t-tests. (L) Phosphatase PPM1B, which restored Mst1-driven suppression on IRF3 5SD, failed to restore the transcriptional activity of IRF3 5SD with mimetic phosphorylation on Thr75 or Thr253. (*) P < 0.001, compared with control of IRF3 5SD expression, by Student's t-tests.
Figure 5.
Figure 5.
Mst1-mediated IRF3 phosphorylation disrupts IRF3 homodimerization and DNA binding. (A,B) Phosphomimics of Thr253 did not prevent TBK1-mediated IRF3 phosphorylation on its C terminus, as measured by phospho-S396 immunoblotting of samples from the in vitro kinase assay (A) or from cell lysates (B). (C) Electrophoresis on a Native-PAGE gel exhibited a severely impaired formation of IRF3 dimerization when Thr253 was phosphomimicked (fourth lane). Note that there was a comparable level of IRF3 phospho-Ser396 modification (first panel) but with a drastically reduced IRF3 homodimer level on phosphomimetic T253D (second panel). (D,E) A SeV-induced (D) or VSV-induced (E) homodimer of endogenous IRF3 was observed and enhanced in Mst1−/− NMuMG cells, revealing a robust IRF3 homodimerization with Mst1 ablation. Note that E and Figure 3B were from the same experiment. (F) Ribbon representation of the C-terminal IAD of IRF-3 showing that Thr253, Arg211, Arg213, Lys360, and Arg361 are located at the same dimer interface. Thr253 phosphorylation by Mst1 would likely impair IRF3 dimer formation. (G) An immunofluorescence assay showed that IRF3 nuclear import, which was stimulated by MAVS cotransfection, was largely similar between the wild type and the T75D mutation. (H, second lane) In a biotin-labeled DNA pull-down assay, activated IRF3 (IRF3 5SD) was shown to bind the ISRE sequence. Mst1 coexpression weakens DNA binding of activated IRF3, while a phosphomimetic of the Thr75 residue (T75D) also displayed severely impaired binding to the ISRE sequence. As a control, Mst1 did not block binding of Smad3 to its SBE DNA sequence. (I) Ribbon representation of the IRF3:DNA complex (left panel) or the modeled Thr75 phosphorylated IRF-3:DNA complex (right panel). Side chain conformational changes of Thr75 were potentially disruptive for the IRF3 DNA binding by disturbing hydrogen bonds between Arg78 and nucleotides.
Figure 6.
Figure 6.
Model for Mst1-driven suppression of cytosolic RNA/DNA sensing and antiviral defense. Mst1 impedes cytosolic RNA/DNA sensing by a dual mechanism. Mst1 directly modified IRF3 at the Thr75 and Thr253 residues, which severely disrupted the activated IRF3 for homodimerization and binding to ISRE elements. Meanwhile, Mst1 also prevented the RNA virus-induced activation of TBK1 kinase by an unexplored manner, thus further keeping IRF3 at rest. In accordance with this, expression of Mst1 dampened antiviral host defense at both the cellular and whole-animal levels, while knockout or knockdown of Mst1 boosted the antiviral sensing and response.
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