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The RNA helicase DDX46 inhibits innate immunity by entrapping m6A-demethylated antiviral transcripts in the nucleus

Nature Immunology volume 18, pages 1094–1103 (2017)Cite this article

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A Corrigendum to this article was published on 16 November 2017

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Abstract

DEAD-box (DDX) helicases are vital for the recognition of RNA and metabolism and are critical for the initiation of antiviral innate immunity. Modification of RNA is involved in many biological processes; however, its role in antiviral innate immunity has remained unclear. Here we found that nuclear DDX member DDX46 inhibited the production of type I interferons after viral infection. DDX46 bound Mavs, Traf3 and Traf6 transcripts (which encode signaling molecules involved in antiviral responses) via their conserved CCGGUU element. After viral infection, DDX46 recruited ALKBH5, an 'eraser' of the RNA modification N6-methyladenosine (m6A), via DDX46's DEAD helicase domain to demethylate those m6A-modified antiviral transcripts. It consequently enforced their retention in the nucleus and therefore prevented their translation and inhibited interferon production. DDX46 also suppressed antiviral innate immunity in vivo. Thus, DDX46 inhibits antiviral innate responses by entrapping selected antiviral transcripts in the nucleus by erasing their m6A modification, a modification normally required for export from the nucleus and translation.

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Figure 1: DDX46 promotes VSV replication by suppressing VSV-triggered production of type I interferons in macrophages.
Figure 2: iCLIP-seq analysis of DDX46-bound RNA from VSV-infected macrophages.
Figure 3: DDX46 increases the retention of antiviral mRNA in the nucleus and decreases the expression of the protein encoded.
Figure 4: DDX46 recruits ALKBH5 to inhibit VSV-triggered production of type I interferons.
Figure 5: DDX46 affects the m6A modification of transcripts via recruitment of ALKBH5 after viral infection.
Figure 6: In vivo knockdown of DDX46 enhances the innate antiviral response.

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  • 20 September 2017

    In the version of this article initially published, the label 'siDDX46' in the key to Figure 5g is incorrect. That label should read 'siALKBH5'. The error has been corrected in the HTML and PDF versions of this article.

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Acknowledgements

We thank T. Fang, M. Jin and Y. Li for technical assistance; X. Liu, C. Han, S. Xu, Y. Han and T. Chen for discussions; and the SIDANSAI Biotechnology Company (Shanghai, China) for helping to generate Ddx46+/− mice. Supported by the National Key Basic Research Program of China (2013CB530503 and 2012CB518900), the National Natural Science Foundation of China (31390431, 81422037 and 81671564) and the CAMS Innovation Fund for Medical Sciences (2016-12M-1-003).

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  1. Qingliang Zheng and Jin Hou: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology & Center for Immunotherapy, CAMS-Oxford University International Center for Translational Immunology, Institute of Basic Medical Sciences, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China

    Qingliang Zheng & Xuetao Cao

  2. National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China

    Jin Hou, Ye Zhou, Zhenyang Li & Xuetao Cao

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  1. Qingliang Zheng
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Contributions

Q.Z., J.H., Y.Z., and Z.L. performed the experiments; Q.Z., J.H. and X.C. analyzed data and wrote the paper; and X.C. designed and supervised the research.

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Correspondence to Xuetao Cao.

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Integrated supplementary information

Supplementary Figure 1 DDX46 negatively regulates the production of type I interferons after viral infection.

(a) qRT-PCR analysis of Ifnb mRNA in macrophages transfected with the indicated siRNAs after infected with VSV (MOI=10) as indicated time. (b) Confocal microscopy of macrophages stained with DDX46 antibodies. Scale bar, 10 μm. (c,d) qRT-PCR and immunoblot analysis of DDX46 in macrophages transfected with NC, DDX46 siRNA#1 or #2 as indicated for 72 h. (e) qRT-PCR analysis of Ifnb mRNA expression in human THP1 cells transfected with NC or DDX46 siRNA for 72 h and infected with VSV (MOI=10) for indicated time. (f,g) Immunoblot analysis of the indicated protein in RAW264.7 cell clones stably overexpressing DDX46 (f) or transfected with siRNA for 72 h, and infected with VSV (MOI=10) for indicated time (g). (h,i) qRT-PCR analysis of Ifnb, Il6, Tnf and Il1b mRNA in RAW264.7 cells transfected with NC or DDX46 siRNA for 72 h and infected with VSV (MOI=10) (h) or treated with LPS (100ng/mL) (i) as indicated time. (j) Luciferase activity in HEK293T cells after co-transfection with DDX46 and the indicated plasmids. Luciferase activity is presented relative to Renilla luciferase activity. (k-m) Immunoblot analysis of DDX46 in mouse macrophages infected with VSV (k) or HSV (l) or in mouse peripheral blood mononuclear cells (PBMNC), myeloid conventional dendritic cells (cDC), plasmacytoid dendritic cells (pDC) and in human THP1 cells infected with VSV (m) as indicated time. NS: not significant; **p<0.01, *p<0.05 (Student’s t-test). Data are representative of three independent experiments (mean and s.d. of technical triplicates (a,c,e,h -j)) or from three independent experiments with similar results (b,d,f,g,k-m).

Supplementary Figure 2 Gene-ontology (GO) and KEGG analysis of DDX46-bound mRNA with the DAVID tool.

(a) Gel electrophoresis analysis of cDNA for removing free RT primers and size selection. (b) Gel image of amplified cDNA libraries and extraction of the indicated size ranges. (c,d) Overlap of two biological replicates for iCLIP in uninfected or infected RAW264.7 cells. Numbers show the sum of genes identified in each sample. (e) Overlap of transcripts identified by iCLIP of DDX46 in uninfected or infected RAW264.7 cells. Numbers in c,d,e show the sum of genes identified in each sample. (f,g) GO and KEGG analysis of the 1000 most enriched DDX46-bound mRNAs in uninfected cells but not in that of the infected cells. (h,i) GO and KEGG analysis of the 1000 most enriched DDX46-bound mRNAs in infected cell but not in that of uninfected cell. * indicates the enriched antiviral signaling pathway upon viral infection. Data are representative of three independent experiments with similar results (a,b).

Supplementary Figure 3 DDX46 associates with ALKBH5 to inhibit the expression of antiviral transcripts after viral infection.

(a) Fold enrichment of Gapdh and Ticam1 transcripts in DDX46-RNA co-immunoprecipitation versus RNA-protein input control in RAW264.7 infected with VSV (MOI=10). (b) The relative changes in PSI values observed in 1449 transcripts between wildtype and DDX46+/− macrophages by RNA-seq. (c) RAW264.7 cells were transfected with plasmids for 24 h, and then the cells were fixed, permeabilized, and stained with FISH probes for Tbk1 transcript. Scale bar, 10 μm. (d) RAW264.7 cells were fixed, permeabilized, and stained with FISH probes for Mavs transcript, and then stained with DDX46 and SC-35 antibody. Scale bar, 10 μm. (e) Immunoblot analysis of the indicated protein in macrophages transfected with NC or siRNA for 72 h, and infected with VSV (MOI=10) for indicated time. (f) Immunoblot analysis of the indicated protein in RAW264.7 cells transfected with NC or siRNA for 72 h, and infected with VSV (MOI=10) for indicated time. NS: not significant. (Student’s t-test). Data are representative of three independent experiments (mean and s.d. of technical triplicates (a)) or from three independent experiments with similar results (c-f).

Supplementary Figure 4 DDX46 associates with ALKBH5 to inhibit MAVS expression after viral infection.

(a) PAGE gel resolution of immunoprecipitated DDX46 and its associated proteins from peritoneal macrophages infected by VSV for the indicated time. Different bands were analyzed by MS. Arrow indicates the band of the protein detected by MS. (b) IP and immunoblot analysis of HEK293T cells after co-transfection of DDX46 with ALKBH5 upon various stimulation as indicated. (c) Luciferase activity (left) and immunoblot (right) analysis in HEK293T cells after co-transfection with MAVS, SRPK2, and ALKBH5 as indicated. (d) IP and immunoblot analysis of HEK293T cells after co-transfection of DDX46 mutants with ALKBH5 as indicated upon VSV (MOI=10) infection for 6 h. (e,f) Luciferase activity (left) and immunoblot (right) analysis in HEK293T cells after co-transfection with MAVS and DDX46 mutants or truncates as indicated. NS: not significant; *p<0.01 (Student’s t-test). Data are representative of three independent experiments (mean and s.d. of technical triplicates (c(left),e(left), f(left))) or from three independent experiments with similar results (a,b,c(right), d,e(right), f(right)).

Supplementary Figure 5 DDX46-inhibited production od type I interferons is dependent on the recruitment of ALKBH5.

(a,b) qRT-PCR analysis of Ifnb mRNA in RAW264.7 cells transfected with plasmids plus siRNAs as indicated, and infected with VSV (MOI=10) for 12 h. (c) Immunoblot analysis of ALKBH5 in Alkbh5−/− RAW264.7 cells. (d) Fold enrichment of the indicated mRNA transcripts in m6A in vitro IP versus mRNA input control. (e) RAW264.7 cells were fixed, permeabilized, and stained with FISH probes for MAVS transcript, and then stained with DDX46 and ALKBH5 antibody. Scale bar, 10 μm. NS: not significant; *p<0.01 (Student’s t-test). Data are representative of three independent experiments (mean and s.d. of technical triplicates (a,b,d)) or from three independent experiments with similar results (c, e).

Supplementary Figure 6 DDX46 negatively regulates the production of inflammatory cytokines and Ifnb in macrophage.

(a) qRT-PCR analysis of Il6, Tnf and Il1b mRNA in peritoneal macrophage cells from Ddx46+/+ or Ddx46+/− mice infected with VSV (MOI=10) as indicated time. (b) qRT-PCR analysis of Ifnb, Il6, Tnf and Il1b mRNA in peritoneal macrophage cells from Ddx46+/+ or Ddx46+/− mice and treated with LPS (100ng/mL) for indicated time. (c) Working model for the mechanism of RNA helicase DDX46 which recruits ALKBH5 to inhibit antiviral innate immunity by nuclear entrapping m6A demethylated antiviral transcripts via m6A modification. Ac: Acetylation. *p<0.05 (Student’s t-test). Data are representative of three independent experiments (mean and s.d. of technical triplicates (a,b)).

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Zheng, Q., Hou, J., Zhou, Y. et al. The RNA helicase DDX46 inhibits innate immunity by entrapping m6A-demethylated antiviral transcripts in the nucleus. Nat Immunol 18, 1094–1103 (2017). https://doi.org/10.1038/ni.3830

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