RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions

Abstract

The initial response to viral infection is anticipatory, with host antiviral restriction factors and pathogen sensors constantly surveying the cell to rapidly mount an antiviral response through the synthesis and downstream activity of interferons. After pathogen clearance, the host’s ability to resolve this antiviral response and return to homeostasis is critical. Here, we found that isoforms of the RNA-binding protein ZAP functioned as both a direct antiviral restriction factor and an interferon-resolution factor. The short isoform of ZAP bound to and mediated the degradation of several host interferon messenger RNAs, and thus acted as a negative feedback regulator of the interferon response. In contrast, the long isoform of ZAP had antiviral functions and did not regulate interferon. The two isoforms contained identical RNA-targeting domains, but differences in their intracellular localization modulated specificity for host versus viral RNA, which resulted in disparate effects on viral replication during the innate immune response.

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Fig. 1: ZAP-S interacts with the 3′-UTR of IFN mRNAs.
Fig. 2: CSTF2-mediated alternative polyadenylation generates ZAP-S.
Fig. 3: ZAP-deficient cells have a higher and more prolonged IFN response.
Fig. 4: ZAP-S and ZAP-L localize to different subcellular compartments.
Fig. 5: ZAP-L targets alphavirus RNA at viral replication sites.
Fig. 6: ZAP-S, but not ZAP-L, suppresses IFN.
Fig. 7: Localization of ZAP isoforms determines binding of IFN mRNA.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This project was funded by National Institutes of Health grants (nos. AI108765 and AI135437 to R.S.; no. AI119017 to M.D.D.; and nos. AI104002, AI118916 and AI127463 to M.G.); the Pew Biomedical Scholars program (no. 32011 to M.D.D.); a Research Fellowship from the German Research Foundation (no. SCHW 1881/1-1 to J.S.); T32 training grants (nos. AI106677 and GM007270 to F.W.S.); and T32 training grant (no. GM007240 to A.P.R.). This work was supported in part by the UW Proteomics Resource (UWPR95794). We thank P. von Haller and J. Eng (UW Proteomics Resource) for expert technical assistance with mass spectrometry. We are thankful for the support of W. P. Chang (UW Biology Imaging Facility) for help with super-resolution microscopy. We thank M. A. Davis (UW Immunology) for help with confocal laser scanning microscopy, D. B. Stetson and K. Burleigh (UW Immunology) for providing the IRF3 CRISPR construct, S. N. Sarkar (University of Pittsburgh) for providing STAT1 KO PH5CH8 cells and members of the Savan, Daugherty and Gale laboratories for helpful discussions.

Author information

J.S., F.W.S., M.G., M.D.D. and R.S. designed the study. R.S. directed the study. J.S., F.W.S., A.P.R., K.R.T., L.D.H., S.O., A.F., A.M.K., J.A.R., L.S. and J.L.H. performed experiments and analyzed the data. J.S., M.D.D. and R.S. wrote the manuscript.

Correspondence to Matthew D. Daugherty or Ram Savan.

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The authors declare no competing interests.

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Peer review information Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Integrated supplementary information

Supplementary Figure 1 ZAP-S interacts with the 3′UTR of IFN mRNAs.

(a) ZAP RNA-IP performed in Huh7 ZAP KO cells under UV crosslinking conditions after expression of FLAG-tagged ZAP-S, FLAG-tagged ZAP-L and FLAG-empty vector (EV) control, and stimulation with 0.2 µg/ml RIG-I ligand for 24 h. (b) RNA-IP of endogenous ZAP performed in Huh7 WT and IFNAR1 KO cells after stimulation with 0.5 µg/ml RIG-I ligand for 24 h. Representative experiments of (a) two and (b) three independently performed experiments with similar results are shown.

Supplementary Figure 2 ZAP-deficient cells have a higher and more prolonged IFN response.

(a) Western blot of Huh7 ZAP KO cell pools generated by CRISPR-Cas9 technology using two different guide RNAs. (b) ZAP mRNA expression in ZAP KO cell pools. (c) IFNB and IFNL3 mRNA expression in ZAP KO cell pools compared to WT cells and a single-cell sub-clone after stimulation with RIG-I ligand for 24 h. (a-c) Representative experiments with replicates (n = 3) of two independently performed experiments with similar results are shown. Bars show mean ± SD. (d) Sequencing chromatogram of the CRISPR target region in exon 1 of the human ZAP (ZC3HAV1) gene. Four bacterial clones were sequenced showing the same del C deletion. (e) Alignment of WT and CRISPR targeted ZAP polypeptide sequence (amino acid residue 1-200). The ΔC base pair deletion results in a premature stop codon at amino acid residue 121 (exon 2). (f) Polysome profiling of HPRT and IFNB mRNA after overexpression of ZAP-S (green), ZAP-L (blue), or empty vector (EV, black) control in Huh7 ZAP KO cells and stimulation of the cells with 0.25 µg/ml RIG-I ligand for 18 h. A representative of two independent experiments with similar results is shown. Data were analyzed using one-way ANOVA with Tukey’s post-test. *P < 0.01; **P < 0.001; ns, not significant.

Supplementary Figure 3 ZAP-S and ZAP-L localize to different subcellular compartments.

(a) Immunofluorescence staining of endogenous ZAP in Huh7 WT and ZAP KO cells. The scale bar represents 20 µm. (b) Subcellular fractionation of ZAP WT and CaaX mutant isoforms after expression in Huh7 ZAP KO cells. For (a, b) representative micrographs and Western blots of three independent experiments with similar results are shown.

Supplementary Figure 4 ZAP-L co-localizes with endolysosomes.

(a, b) Confocal immunofluorescence microscopy of ZAP and (a) Rab5 or (b) Rab7 after co-expression of mCherry-Rab5 or mCherry-Rab7 and tag-less ZAP-S, ZAP-L, or their CaaX mutants in Huh7 ZAP KO cells. The scale bar represents 5 µm. Representative micrographs of three independent experiments with similar results are shown.

Supplementary Figure 5 ZAP-L does not co-localize with the endoplasmic reticulum, peroxisomes or mitochondria.

(a) Confocal immunofluorescence microscopy of ZAP and the endoplasmic reticulum marker Sec61β. Representative micrographs of three independent experiments with similar results are shown. (b) Quantification of co-localization of ZAP-positive and Sec61β-positive pixels. The correlation coefficients (Pearson’s r) of 10 cells were analyzed using the Fiji Coloc 2 plugin. Error bars show mean ± SD. (c) Membrane flotation assay and sedimentation of ZAP-L, ZAP-S, and the endoplasmic reticulum marker calnexin in Huh7 WT cells treated with or without 0.25 µg/ml RIG-I ligand for 24 h. A representative Western blot of two independently performed experiments with similar results is shown. (d, e) Confocal immunofluorescence microscopy of ZAP and (d) the peroxisomal marker PTS1 and (e) the mitochondrial marker COX8 after co-expression of mCherry-tagged PTS1 and COX8 together with tag-less ZAP-L. The scale bar represents 5 µm. (d, e) Representative micrographs of three independent experiments with similar results are shown.

Supplementary Figure 6 ZAP-L targets alphavirus RNA at viral replication sites.

(a) Confocal immunofluorescence microscopy of endogenous ZAP, SINV dsRNA, and E-cadherin in Huh7 WT cells upon infection with SINV Toto (MOI = 1; 6 h post-infection). The scale bar represents 5 µm. (b) Confocal immunofluorescence microscopy of endogenous ZAP, SINV dsRNA, and G3BP1 in Huh7 WT cells upon infection with SINV Toto (MOI = 1; 6 h post-infection). The scale bar represents 5 µm. (a, b) Representative micrographs of three independent experiments with similar results are shown. (c) Replication activity of a Semliki Forest virus (SFV) luciferase replicon in doxycycline-inducible HEK 293 T cells expressing ZAP-S WT, ZAP-L WT, or their respective CaaX motif mutants. A representative experiment with replicates (n = 3) of three independently performed experiments with similar results is shown. Symbols show mean ± SD.

Supplementary Figure 7 ZAP-S, but not ZAP-L, suppresses IFN.

(a) Western blot of ZAP isoform expression and (b) IFNB and IFNL3 mRNA expression in HEK 293 cells after isoform-specific knockdown of ZAP and stimulation with 1 µg/ml RIG-I ligand for 30 h. (a, b) A representative experiment with replicates (n = 3) of three independently performed experiments with similar results is shown. Bars show mean ± SD. (c) IL6 and TNFA mRNA expression in Huh7 cells upon isoform-specific knockdown of ZAP and stimulation with 0.5 µg/ml RIG-I ligand for 42 h. A representative experiment with replicates (n = 3) of two independently performed experiments with similar results is shown. Bars show mean ± SD. (d) ZAP RNA-IP performed in Huh7 ZAP KO cells after expression of FLAG-tagged ZAP-S, FLAG-tagged ZAP-L and FLAG-empty vector (EV) control, and stimulation with 0.2 µg/ml RIG-I ligand for 24 h. A representative experiment of two independently performed experiments with similar results is shown. (e) Expression of IFNB and IFNL3 mRNA and (f) Western blot of IRF1 protein expression in Huh7 WT and ZAP KO cells 48 h after overexpression of IRF1. (e, f) A representative experiment with replicates (n = 3) of three independently performed experiments with similar results is shown. Bars show mean ± SD. Data were analyzed using (b, c) one-way ANOVA with Tukey’s post-test or (e) unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Supplementary Figure 8 Model of ZAP-mediated post-transcriptional regulation during innate antiviral immunity.

Constitutively expressed ZAP-L localizes to endolysosomes and sites of Sindbis virus replication at the plasma membrane to target viral RNA (vRNA) and inhibit viral replication. Escaping vRNA triggers expression and secretion of type I and III IFNs, which engage and signal through the IFN receptors. This induces CSTF2-mediated alternative polyadenylation and alternative last exon usage of ZC3HAV1 and expression of ZAP-S, which then binds to IFN mRNA in the cytoplasm to mediate resolution of the antiviral innate immune response.

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Schwerk, J., Soveg, F.W., Ryan, A.P. et al. RNA-binding protein isoforms ZAP-S and ZAP-L have distinct antiviral and immune resolution functions. Nat Immunol 20, 1610–1620 (2019). https://doi.org/10.1038/s41590-019-0527-6

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