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Mitochondria-localised ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS

Nature Cell Biology volume 21, pages 1346–1356 (2019)Cite this article

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Abstract

In the past two decades, emerging studies have suggested that DExD/H box helicases belonging to helicase superfamily 2 (SF2) play essential roles in antiviral innate immunity. However, the antiviral functions of helicase SF1, which shares a conserved helicase core with SF2, are little understood. Here we demonstrate that zinc finger NFX1-type containing 1 (ZNFX1), a helicase SF1, is an interferon (IFN)-stimulated, mitochondrial-localised dsRNA sensor that specifically restricts the replication of RNA viruses. Upon virus infection, ZNFX1 immediately recognizes viral RNA through its Armadillo-type fold and P-loop domain and then interacts with mitochondrial antiviral signalling protein to initiate the type I IFN response without depending on retinoic acid-inducible gene I-like receptors (RLRs). In short, as is the case with interferon-stimulated genes (ISGs) alone, ZNFX1 can induce IFN and ISG expression at an early stage of RNA virus infection to form a positively regulated loop of the well-known RLR signalling. This provides another layer of understanding of the complexity of antiviral immunity.

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Fig. 1: Viral infection induces Znfx1 expression, an IFN-stimulated gene.
Fig. 2: ZNFX1 deficiency impairs cellular antiviral RNA response.
Fig. 3: ZNFX1 is essential for host defence against RNA virus in mice.
Fig. 4: ZNFX1 initiates IFN-based antiviral responses.
Fig. 5: ZNFX1 binds to viral RNA.
Fig. 6: ZNFX1 localises to mitochondria and interacts with MAVS.
Fig. 7: ZNFX1 induces IFN signalling in an RLR-independent manner.

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Data availability

RNA sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE132979. Source data for Figs. 1–7 and Extended Data Figs. 1–7 have been provided as Statistics Source Data. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the National Natural Science Foundation (NNSF) of China under grants 31770943, 81430099 and 31900661 and by the Natural Science Foundation of Guangdong Province of China under grants 2015A030306043, 2018A030313924 and 2018A030313051.

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Authors and Affiliations

  1. State Key Laboratory of Biocontrol, Guangdong Province Key Laboratory of Pharmaceutical Functional Genes, School of Life Science, Sun Yat-sen University, Guangzhou, China

    Yao Wang, Shaochun Yuan, Xin Jia, Yong Ge, Tao Ling, Meng Nie, Xihong Lan, Shangwu Chen & Anlong Xu

  2. School of Life Science, Beijing University of Chinese Medicine, Beijing, China

    Anlong Xu

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Contributions

Y.W., S.Y. and A.X. conceived the ideas and designed the experiments. Y.W., X.J., Y.G., T.L., M.N. and X.L. performed the experiments. Y.W., S.Y. and X.J. analysed the data. S.Y., Y.W., X.J. and S.C. contributed to editing the manuscript. S.Y. and A.X. supervised the research and wrote the paper. S.Y. and X.J. are joint co first authors.

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Correspondence to Anlong Xu.

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Extended data

Extended Data Fig. 1 Bioinformatics analysis of in vitro transcription-sequencing APA sites (IVT-SAPAS) data of VSV-infected macrophages and previously collected viral infection microarray data.

a, IVT-SAPAS revealed the genes with transcriptional changes in MDMs infected with VSV for 0, 2, 4, 8, 16, 24 hrs. b-c, The percentage of GFP+ cells of FACS analysis of A549 cells transfected with control siRNA or the indicated siRNAs followed by VSV-eGFP infection for another 6 hrs (n = 3 independent experiments). d, qRT-PCR revealed the mRNA expression of target gene in A549 cells transfected with indicated siRNAs for 48 hrs (n = 3 independent experiments). e-f, RNA levels of Znfx1 and Rig-I are significantly increased after different virial infection in different cell types. g, Schematic representation of ZNFX1 promoter containing core region bound by STAT1, STAT2, IRF1 and IRF9. For e, n = 3 independent experiments. Data in f, n = 4 wells for SeV infected epithelial cells and n = 6 wells for uninfected cells; n = 5 samples for IVA infected pDCs; n = 4 independent experiments for SeV infected monocytoid cells; n = 2 independent experiments for SeV or HIV infected Macrophages or mDCs. All data are shown as the mean ± s.d. Statistical differences were detected using two-tailed unpaired Student’s t-tests.

Source data

Extended Data Fig. 2 ZNFX1 deficiency impairs antiviral immune response in vitro and in vivo.

a, Quantitative RT-PCR (qRT-PCR) analysis of Znfx1 mRNA expression in A549 and L929 cells transfected with control siRNA or ZNFX1 siRNA 1#, 2# or 3# for 48 hrs (n = 3 independent experiments). b, Western blot analysis of ZNFX1 protein expression in A549 cells transfected with control siRNA or human ZNFX1 siRNA 1# for 48 hrs. c, ELISA of IFN-α or IFN-β production in cell supernatants from A549 cells with target gene knockdown for 48 hrs followed by VSV infection or poly I:C stimulation for another 12 hrs (n = 4 independent experiments). d, qRT-PCR analysis of VSV mRNA expression (left panel) and plaque assay analysis of VSV titer (right panel) of A549 cells transfected with RIG-I, ZNFX1 expressing plasmids or empty vector plasmid for 24 hrs and then infected with VSV at an MOI of 2 for 16 hrs (n = 3 independent experiments). e, FACS analysis of Znfx1+/+ and Znfx1-/- 293T cells followed by VSV-eGFP infection at 0.5 MOI for the indicated time points (n = 3 independent experiments). f, Znfx1-/- 293T and A549 clones were generated by the CRISPR-Cas9 method. Deficiency of target genes in the KO clones were confirmed by immunoblotting analysis. g, qRT-PCR analysis of viral mRNA transcripts in VSV, EMCV, H1N1 and HSV-1 infected A549 cells with control siRNA (si Control) or Znfx1-specific siRNA (si ZNFX1) (n = 3 independent experiments). h, ELISA of IFN-α in supernatants of BMDMs from WT and Znfx1-/- mice infected with VSV or HSV-1 for 16 hrs (n = 5 independent experiments). All data are shown as the mean ± s.d. P values were calculated using two-tailed unpaired Student’s t-test. For b, f, the experiments were repeated three times, independently, with similar results obtained.

Source data

Source data

Extended Data Fig. 3 ZNFX1 positively regulates IFN-β signaling.

a, Illustration of the CRISPR-Cas9 strategy to generate Znfx1-deficient mice and primer design used in (b). b, Genotyping of the ZNFX1 mutant pups. c, Immunoblot analysis of ZNFX1 protein levels in Znfx1+/+ and Znfx1-/- MEFs cells. d, qRT-PCR analysis of Ifnb1 and ISGs mRNA levels in A549 cells transfected with siControl (si Ctrl) or si ZNFX1 for 48 hrs and then infected with VSV for the indicated time points (n = 3 independent experiments). e, qRT-PCR analysis of Ifnb1 and ISGs mRNA expression in Znfx1+/+ and Znfx1-/- 293T cells followed by VSV infected with increasing MOI (0.5 and 1) for 0, 8 and 16 hrs (n = 3 independent experiments). For b, c, the experiments were repeated three times, independently, with similar results obtained. Data in d, e are the mean ± s.d. P values were calculated using two-tailed unpaired Student’s t-test.

Source data

Source data

Extended Data Fig. 4 ZNFX1 localizes to mitochondria and interacts with MAVS.

a, FACS analysis of Znfx1-/- A549 cells transfected with ZNFX1 and its mutants expressing plasmids or empty vector (EV) for 24 hrs followed by VSV-eGFP infection at an MOI of 2 for 6 hrs. b, Endogenous level of ZNFX1 protein in mitochondrial fractions in WT and Mavs-/- 293T with or without VSV infection at 1 MOI for 6 hrs. COX-IV was used as the loading control. Data are representative of at least three independent experiments.

Source data

Extended Data Fig. 5 The expression of Znfx1 in different tissues and cell types, and the phylogenetic tree of Znfx1.

a-c, The expression of Znfx1, Rig-I and Mda5 in different tissues and cell types as per BioGPS. d, Phylogenetic tree of ZNFX1 and RIG-I using an amino acid sequence alignment among different species.

Extended Data Fig. 6 Alignment of ZNFX1 amino acid sequences in human, mouse, rat and zebrafish.

Shading indicates sequence conservation, with darker gray indicating a higher degree of conservation.

Extended Data Fig. 7 Work model of mitochondria-localized ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS.

Upon RNA virus infection, ZNFX1 induces type I interferon response by interacting with MAVS in the early stage, thus primes the expression of a number of ISGs, including RIG-I and MDA5. The induced sensors further enhance the antiviral immune response by amplifying ISGs expression.

Supplementary information

Reporting Summary

Supplementary Tables 1

Conservative analysis of ZNFX1 and DDX58 in different species.

Supplementary Tables 2

Information about the primers used in the study.

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Wang, Y., Yuan, S., Jia, X. et al. Mitochondria-localised ZNFX1 functions as a dsRNA sensor to initiate antiviral responses through MAVS. Nat Cell Biol 21, 1346–1356 (2019). https://doi.org/10.1038/s41556-019-0416-0

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