Abstract
Zika virus (ZIKV) is a flavivirus linked to multiple birth defects including microcephaly, known as congenital ZIKV syndrome. The identification of host factors involved in ZIKV replication may guide efficacious therapeutic interventions. In genome-wide transcriptional studies, we found that ZIKV infection triggers aryl hydrocarbon receptor (AHR) activation. Specifically, ZIKV infection induces kynurenine (Kyn) production, which activates AHR, limiting the production of type I interferons (IFN-I) involved in antiviral immunity. Moreover, ZIKV-triggered AHR activation suppresses intrinsic immunity driven by the promyelocytic leukemia (PML) protein, which limits ZIKV replication. AHR inhibition suppressed the replication of multiple ZIKV strains in vitro and also suppressed replication of the related flavivirus dengue. Finally, AHR inhibition with a nanoparticle-delivered AHR antagonist or an inhibitor developed for human use limited ZIKV replication and ameliorated newborn microcephaly in a murine model. In summary, we identified AHR as a host factor for ZIKV replication and PML protein as a driver of anti-ZIKV intrinsic immunity.
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Data availability
The authors declare that data supporting the findings of this study are available as Supplementary Tables 1–4. Sequencing data have been deposited into the Gene Expression Omnibus (GEO) under the SuperSeries accession nos. GSE147093 and GSE147094. Data from ZIKV-infected patients were accessed at accession no. GSE139181. Data from ZIKV-infected brain organoids were accessed at accession no. GSE129180. Source data are provided with this paper.
Change history
10 August 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41593-020-0700-0
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Acknowledgements
This work was supported by grant nos. NS102807, NS087867, ES02530, AI126880, AI093903 and AI100190 from the National Institutes of Health, RSG-14-198-01-LIB from the American Cancer Society, and RG4111A1 and JF2161-A-5 from the National Multiple Sclerosis Society to F.J.Q., who also received support from the International Progressive MS Alliance. C.C.G. was supported by Universidad de Buenos Aires (grant no. 20020160100091BA), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET, grant no. PIP11220170100171CO) and Agencia de Promoción Científica y Tecnológica (ANPCyT) (grant no. BID-PICT 3080). C.C.G. is member of the Research Career CONICET. F.G. was supported by a Du Pre grant from the International MS Foundation and a postdoctoral fellowship from CONICET. J.P.S.P. was supported by FAPESP (grant nos. 2017/26170-0 and 2017/22504-1). C.M.P. and N.G.Z. received an FAPESP fellowship (grant nos. 2017/11828-0 and 2016/07371-2). We thank all members of the Garcia and Quintana laboratories for helpful advice and discussions.
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F.G., I.B., C.M.P., M.F.T., L.R., M.S.R.V., V.R., A.B., L.M.S., M.C.T., S.M., C.G.V., M.A.W., N.G.Z., N.B.S., C.D.M., Z.W. and J.P.S.P. performed in vitro and in vivo experiments. F.G., Z.L. and E.C.T. performed bioinformatics analysis. I.B, L.G., E.B.D., D.S., J.P.S.P., C.C.G. and F.J.Q. provided unique reagents and discussed and/or interpreted findings. F.G., C.C.G. and F.J.Q. wrote the manuscript. J.P.S.P, C.C.G. and F.J.Q. designed and supervised the study and edited the manuscript.
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F.J.Q. is a member of the Scientific Advisory Board of Kyn Therapeutics. D.S. is a co-founder of and holds equity in Hercules Pharmaceuticals, The Netherlands. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 AHR signaling is activated by ZIKV infection in human brain organoids and in patients.
a, IPA analysis of RNA-seq data (GEO accession GSE129180) of ZIKV-infected human brain organoids. Pathways enriched in ZIKV-infected NPCs compared to control cells are shown (n = 11 per condition). Dashed red line indicates P = 0.05. P values were determined using Fisher’s exact test b, IPA analysis identified AHR as an upstream regulator. P value was determined using Fisher’s exact test c, IPA analysis of RNA-seq data (GEO accession GSE139181) of placental tissue from ZIKV-infected pregnant women compared to healthy controls (n = 3 patients per condition). Pathways enriched in ZIKV-infection are shown. Dashed red line indicates P = 0.05. P values were determined using Fisher’s exact test.
Extended Data Fig. 2 ZIKV infects NPCs in vitro.
a, NPCs were infected with ZIKV (MOI = 1) and 48 h postinfection, cells were fixed for immunofluorescence against ZIKV NS5 (green). Nuclei were counterstained with DAPI (blue). Images were acquired using an Olympus FV1000 confocal laser scanning microscope. Scale bar = 50 µm. Images are representative of 3 independent experiments. b, AHR modulation in NPCs. NPCs were treated with I3S or CH223191. 24 h after treatment, NPCs were harvested for qPCR analysis of AHR, CYP1A1, IDO1 and TDO2. Data represent the mean ± SD (n = 3 independent experiments). P values were determined by a two-sided Student’s t-test.
Extended Data Fig. 3 Effect of I3S and CH223191 on HepG2 cells viability and proliferation.
a, HepG2 cells viability after treatment for 24 h with different concentrations of I3S and CH223191 was evaluated by an MTT assay. Drug concentrations used for experiments are highlighted in color. Data represent the mean ± SD (n = 3 independent experiments). b, HepG2 cells were incubated for 24 h or 48 h with the indicated concentrations of CH223191 and the number of cells was quantified using a hemocytometer. Data represent the mean ± SD (n = 3 independent experiments). P values were determined by a one-way ANOVA followed by Dunnet’s post-hoc test.
Extended Data Fig. 4 Vero cells show functional AHR signaling.
Vero cells were treated with I3S or CH223191. 48 h after treatment, cells were harvested for qPCR analysis of AHR, CYP1A1, and CYP1B1. Data represent the mean ± SD (n = 3 independent experiments for AHR, CYP1A1; n = 4 for CYP1B1). P values were determined by a two-sided Student’s t-test.
Extended Data Fig. 5 NaF and TSA inhibit the IFN-I pathway.
Schematic representation of the IFN-I pathway. TSA inhibits the assembly of STAT1/STAT2/IRF9 complex and NaF impairs its translocation to the nucleus.
Extended Data Fig. 6 HP163 is a second generation AHR antagonist.
a, SUM149 cells were transfected with the AHR-driven pGudLuc reporter construct and treated with DMSO (vehicle) or 20 μM HP163. 24 h later, cells were assayed for pGudLuc (luciferase) activity and normalized to the CMV-green fluorescence signal. Data represent the mean ± SD (n = 3 independent experiments). P values were determined by a one-way ANOVA followed by Tukey’s post-hoc test b, Murine MOC1 oral cancer cells were treated with DMSO (vehicle) or 20 μM HP163 for 30 min and then treated with DMSO, 100 μM Kyn or 1 nM TCDD. Nuclear and cytoplasmic extracts were prepared and AHR protein quantified by Western immunoblotting. White spaces between gel pieces indicates cropping of the original image. Data are representative of three independent experiments. c, Quantification of Western immunoblotting bands from the three experiments described in (b). Data are presented as mean ± SD (n = 3 independent experiments). P values were determined by a two-sided Student’s t-test.
Extended Data Fig. 7 AHR activation boosts DENV replication in vitro.
a, A549 cells were treated with I3S (50 μM) or CH223191 (2 μM) for 24 h. Then, cells were harvested for qPCR analysis of CYP1A1. Data represent the mean ± SD (n = 3 independent experiments). P values were determined by a two-sided Student’s t-test. b, AHR activation boosts DENV replication. A549 cells were pretreated with the indicated concentrations of I3S for 24 h and infected with DENV-2 (MOI = 0.1). 48 h postinfection supernatants were harvested for plaque assay. Data represent the mean ± SD (n = 3 independent experiments). P values were determined by a one-way ANOVA followed by Dunnet’s post-hoc test.
Extended Data Fig. 8 ZIKV-driven AHR activation suppresses IFN-I dependent and IFN-I independent mechanisms that limit viral replication.
IDO1/TDO2 upregulation induced by ZIKV infection boosts the generation of Kyn, which activates AHR to inhibit IFN-I dependent and IFN-I independent mechanisms that limit ZIKV replication as follows: 1) AHR activation limits IFN-I. 2) AHR also limits NF-kB activation, suppressing PML expression. AHR signaling may also operate in a similar manner to promote DENV replication.
Supplementary information
Supplementary Tables
Supplementary Table 1: List of differentially expressed genes in ZIKV-infected HepG2 cells compared with mock-infected HepG2 cells. Supplementary Table 2: IPA analysis of ZIKV-infected HepG2 cells. Supplementary Table 3: IPA analysis of RNA-seq data from fetal mouse CNS samples. Supplementary Table 4: IPA analysis of PCR array data from fetal mouse CNS samples.
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Giovannoni, F., Bosch, I., Polonio, C.M. et al. AHR is a Zika virus host factor and a candidate target for antiviral therapy. Nat Neurosci 23, 939–951 (2020). https://doi.org/10.1038/s41593-020-0664-0
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DOI: https://doi.org/10.1038/s41593-020-0664-0
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