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YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation

Nature Immunology volume 18, pages 733–743 (2017)Cite this article

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A Corrigendum to this article was published on 18 October 2017

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Abstract

The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKɛ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.

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Figure 1: YAP negatively regulates IFN-β signaling.
Figure 2: YAP deficiency potentiates cellular antiviral responses.
Figure 3: YAP deficiency restricts viral infection in vivo.
Figure 4: Myeloid deficiency in YAP protects mice against viral infection.
Figure 5: YAP associates with IRF3 and represses the dimerization of IRF3.
Figure 6: YAP is targeted for lysosomal degradation by IKKɛ.
Figure 7: IKKɛ phosphorylates YAP at Ser403.
Figure 8: Phosphorylation of YAP at Ser403 by IKKɛ is critical for innate antiviral immunity.

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Change history

  • 20 July 2017

    In the version of this article initially published, the source of the HSV-1 virus stock used in the study was identified incorrectly as a purchase from the Wuhan Institute of Virology, Chinese Academy of Sciences. The correct Acknowledgements section should begin "We thank F. Zhao (Wuhan Institute of Virology, Chinese Academy of Sciences) for HSV-1" and the second sentence of the third subsection of Online Methods ('Cells and reagents') should read "Herpes simplex virus type 1 (HSV-1) was obtained from F. Zhao (Wuhan Institute of Virology, Chinese Academy of Sciences)." The error has been corrected in the HTML and PDF versions of the article.

  • 18 October 2017

    Nat. Immunol. 18, 733–743 (2017); published online 8 May 2017; corrected after print 20 July 2017 In the version of this article initially published, the source of the HSV-1 virus stock used in the study was identified incorrectly as a purchase from the Wuhan Institute of Virology, Chinese Academy of Sciences.

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Acknowledgements

We thank F. Zhao (Wuhan Institute of Virology, Chinese Academy of Sciences) for HSV-1; D. Pan (Johns Hopkins University School of Medicine) for Yap1fl/fl mice; M. Rabelink (Leiden University Medical Center) for shRNA constructs; P. ten Dijke for help with writing; and J. Dai for discussion. Supported by a special program from Ministry of Science and Technology of China (2016YFA0502500 to L.Z.), the Chinese National Natural Science Funds (31571460 to F.Z., 31471315 and 31671457 to L.Z.), PCSIRT (IRT1075 to X.G.) and Jiangsu National Science Foundation (BK20150354 to F.Z.).

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Authors and Affiliations

  1. Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou, Jiangsu, China

    Shuai Wang, Feng Chu, Tong Dai, Liang Gao, Lin Wang, Li Ling & Fangfang Zhou

  2. Life Sciences Institute and Innovation Center for Cell Signaling Network, Hangzhou, Zhejiang, China

    Feng Xie, Zhengkui Zhang, Junling Jia, Jin Jin & Long Zhang

  3. Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California, San Francisco, San Francisco, California, USA

    Bing Yang

  4. Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Leiden, The Netherlands

    Hans van Dam

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Contributions

S.W., L.Z. and F.Z. designed the experiments and analyzed the data; S.W., F.X., F.C., Z.Z., and T.D. performed the experiments; B.Y., L.G., L.W., L.L., J. Jia and J. Jin contributed to writing, discussions and agreement with the conclusions presented; and H.v.D. L.Z. and F.Z. wrote the manuscript.

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Correspondence to Long Zhang or Fangfang Zhou.

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Integrated supplementary information

Supplementary Figure 1 YAP negatively regulates IFN-β signaling.

(a) Immunoblot analysis of Yap knockdown efficiency with sh-Yap (#1 to #4 independent constructs) in Raw264.7 cells. (b) IFN-β-Luc and PRDs I-III-Luc activity in Raw264.7 cells depleted for Yap and stimulated with SeV for 12 h. Results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 (two-tailed Student's t-test.) (c) qPCR analysis of sh-YAP #1 efficiency (left panel) and IFNB1 mRNA level (right panel) in control and YAP depleted THP1 cells followed by SeV infection at the indicated time points. (d) IFN-β-Luc and PRDs I-III-Luc activity in HEK293T cells transfected with respective reporters and YAP2 or YAP4 expression plasmids followed by stimulation with SeV for 12 h. (e) qPCR analysis of HEK293T cells transfected with empty vector (Co.vec) and YAP2 or YAP4 expression plasmids for 48 h followed by Sev infection (left) or Poly (I:C) transfection at the indicated time points. All qPCR results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.) (f) Fluorescence microscopy of VSV–GFP levels in HEK293T cells transfected with YAP2 or YAP4 expression plasmids followed by infection for 12 h with VSV–GFP (MOI 0.1) (bright-field, upper; fluorescence, bottom). Scale bars, 100 μm. Representative results are shown of least two independent experiments.

Supplementary Figure 2 YAP deficiency potentiates IFN-β signaling.

(a, b) qPCR analysis of Ifnb1, Ccl5 and Cxcl10 mRNA levels in wild type and Yap1+/- mouse bone marrow derived macrophages (BMDMs) infected with SeV (a) or stimulated with 5'-pppRNA for the indicated time points. (c, d) qPCR analysis of Ifnb1 levels in wild type and Yap1+/- BMDMs transfected with Poly (I:C) (c) or infected with HSV-1 (MOI, 10) (d) for the indicated time points.(e, f) qPCR analysis of Ifnb1, ccl5 and Cxcl10 mRNA levels in wild type and Yap1+/- MEFs infected with SeV (e) or stimulated with 5'-ppp RNA (f) for the indicated time points. (g, h) qPCR analysis of Ifnb1 levels in wild type and Yap1+/- MEFs transfected with Poly (I:C) (g) or infected with HSV-1 (MOI, 10) (h) for the indicated time points. All qPCR results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.) (i) Survival of Yap1+/+ and Yap1+/− mice (n=10 for each group) infected intraperitoneally with VSV (5×108 PFU per mouse). *P < 0.05 (two-way analysis of variance (ANOVA)). (j) Representative hematoxylin-and-eosin-stained images of lung sections from mice as in a. Scale bars, 100 μm. (k) HSV-1 viral titers in brain of Yap WT and heterogeneous mice (n = 6 mice per group) infected with HSV-1 for 72 h. (l) Survival of Yap1+/- and WT mice (n = 10 mice per group) after intravenous injection of HSV-1 (2×107 PFU per mouse). *P < 0.05, *** P < 0.01 (two-tailed Student's t-test in k or two-way analysis of variance (ANOVA) in l).

Supplementary Figure 3 IFN-β signaling is upregulated in YAP-deficient macrophages.

qPCR of Ifnb1, Cxcl10 and Ccl5 mRNA levels in Yap1fl/fl Lyz2-Cre+ and Yap1fl/fl Lyz2-Cre- peritoneal macrophages infected or stimulated with SeV, 5'-pppRNA, VSV(MOI=1) or HSV-1 (MOI=0.1). All qPCR results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 and **P < 0.01 (two-tailed Student's t-test.)

Supplementary Figure 4 YAP retains IRF3 in the cytoplasm.

(a) IFN-β-Luc activity (left) and IFNB1 mRNA in HEK293T cells depleted with YAP and transfected with cGAS+STING, RIG-I N-terminal 2CARD (RIG-IN), MAVS, TBK1, IKKɛ or IRF3-5D expression plasmids as indicated. Results are shown as mean + sd of triplicates of at least two independent experiments. *P < 0.05 (two-tailed Student's t-test.) (b) Immunoblot (IB) of total cell lysate (TCL) and immunoprecipitate derived from HEK293T cells transfected with Myc-IRF3 and Flag-YAP1 or 2 expression plasmids as indicated. (c) IB of IRF3 dimerization (Native gel), p-IRF3, p-TBK1, p-IKKɛ, total IRF3, TBK1 or IKKɛ in HEK293T cells transfected with Flag-YAP2 expression plasmid and treated with SeV for the indicated time points. (d) IB of nuclear and cytoplasm fractions derived from HEK293T cells transfected with IRF3-Flag, IRF3-5D-Flag and YAP4-HA as indicated. (e) IB of TCL and immunoprecipitates derived from HEK293T cells transfected with Flag-Importin α5 or Flag-Importin β1 along with IRF3-5D-Myc. Data are representative of three independent experiments with similar results (b-f).

Supplementary Figure 5 YAP is promoted for lysosomal degradation by IKKɛ.

(a) Immunoblot of immunoprecipitates derived from HEK293T cells transfected with Flag-YAP2 plasmid and treated with SeV or poly (I:C) for 12 h. Cells were treated with NH4Cl (10 mM) for 4 h before harvest. (b) IB analysis for endogenous YAPs of control or IKKɛ depleted MCF10A cells treated with SeV for 12 h. (c) IB of HEK293T cells transfected with Flag-YAP4 and Myc-IKKɛ and treated with NH4Cl (10 mM), Chlq (100 μM), MG132 (10 μM) and control DMSO for 6 h. Data are representative of three independent experiments with similar results (a-c).

Supplementary Figure 6 YAP is phosphorylated by IKKɛ.

(a) Immunoblot of HEK293T cells transfected with expression vectors for Flag-YAP2 wt or Flag-YAP2 5SA (in which all the previously reported by Lats1 or 2 and CK1 phosphorylated Serines at positions 61, 109, 127, 128, 131, 163, 164, and 381 are changed to Alanine), with or without Myc-IKKɛ expression vectors as indicated. (b) Mass spectrometry identification of 4 phosphorylation sites in Flag-YAP2 5SA targeted by IKKɛ. (c) IB of HEK293T cells transfected with Myc-IKKɛ and Flag-YAP2 5SA derivatives carrying the indicated additional phosphorylation site mutations. (d) Sequence alignment of IKKɛ-mediated phosphorylation sites in YAP orthologues of different species. (e) IB of cell lysates of HEK293T cells transfected with Flag-YAP4 WT, 4SA or S403A mutant with or without Myc-IKKɛ expression plasmid as indicated. (f) Validation of the antibody against phosphor-Serine 403 of YAP. Parental, YAP1-deleted (KO) and YAP S403A-mutated (KI) HEK293T cells were treated with SeV for 8 h. Chlq (100 μM) were added to the cells 6 h before harvest. Cells were then harvested for immunoprecipitation with anti-YAP antibody followed by anti-p-S403 YAP immunoblot analysis. Data are representative of three independent experiments with similar results (a,c,e,f).

Supplementary Figure 7 IKKɛ-mediated phosphorylation of YAP at Ser403 is critical for the innate antiviral response.

(a) Immunoblot of IRF3 dimeration, and p-IRF3, p-TBK1, p-IKKɛ, total IRF3, TBK1 and IKKɛ in parental and YAP S403A knock-in (KI) A549 cells. Data are representative of three independent experiments with similar results. (b) qPCR analysis of IFNB1 mRNA levels (left panel) and ELISA analysis of IFN-β secretion in parental and YAP S403A-mutated A549 cells stimulated with SeV, VSV (MOI, 0.1) or transfected with cGAS and STING or poly (dA:dT) (right panel) for 12 h. qPCR and ELISA results are shown as mean ± sd of triplicates of at least two independent experiments.

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Wang, S., Xie, F., Chu, F. et al. YAP antagonizes innate antiviral immunity and is targeted for lysosomal degradation through IKKɛ-mediated phosphorylation. Nat Immunol 18, 733–743 (2017). https://doi.org/10.1038/ni.3744

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