Abstract
The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors1,2,3. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses4,5,6,7,8,9,10,11,12,13. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.
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Acknowledgements
We thank the following investigators for contributing viral molecular clones or viral stocks: R. Cattaneo (MV), P. Collins (PIV3, HMPV, RSV), I Frolov (VEEV), M. Heise (SINV), S. Higgs (ONNV), P. Traktman (VV), J. L. Whitton (CVB). We thank E. Jouanguy and J.-L. Casanova for STAT1−/− fibroblasts. We acknowledge the support of C. Zhao, X. Wang and W. Zhang at The Rockefeller University Genomics Resource Center. We thank E. Castillo, B. Flatley, A. Webson, E. Duan and A. McLees for laboratory support, and D. Kraemalmeyer for managing mouse colonies. This work was supported in part by National Institutes of Health grants AI091707 to C.M.R., AI057158 to I. Lipkin (Northeast Biodefense Center, subcontract to C.M.R.), DK095031 to J.W.S., AI057160 to H.W.V., HHSN272200900041CU19 to M.S.D. and H.W.V., AI104972 to M.S.D., AI083025, AI095611 and CEIRS contract HHSN266200700010C to A.G-S., GM076547 and GM103511 to J.D.A. and AI057160 to W.M.Y., an investigator of the Howard Hughes Medical Institute. M.D.G. is supported by T32 AR007279. Additional funding was provided by the Greenberg Medical Research Institute, the Starr Foundation and the Ronald A. Shellow, M.D. Memorial Fund (C.M.R.).
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J.W.S., D.A.M., M.S.D., H.W.V. and C.M.R. designed the project. J.W.S., D.A.M., N.I., M.D.G., B.S., R.D., J.L.E., K.B.M. and R.B.R. performed the experimental work. J.W.S., V.L. and A.V.R. performed clustering analyses. J.W.S., D.A.M., M.S.D., H.W.V. and C.M.R. analysed the results and wrote the manuscript. B.M., A.A., J.D.A., R.M.E., A.G.-S., V.R., M.D.G., W.M.Y., E.J.S. and M.S.D. contributed reagents and technical expertise.
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Extended data figures and tables
Extended Data Figure 1 Antiviral effects of ISGs on virus production of a non-GFP poliovirus.
HeLa cells were transfected with plasmids encoding ISGs and 48 h later infected with P1M (10 m.o.i.) for 16 h. Lysates were collected and viral titres determined by plaque assay on HeLa cell monolayers, as described in Methods. Plaque assays were performed in duplicate. Data represent the average of three independent experiments. Error bars represent s.d.
Extended Data Figure 2 Hierarchical clustering.
a–e, Analyses were performed as described in Methods. In each cluster, one or more ISGs were removed from the analysis in Fig. 3a to determine whether virus clustering is driven by a subset of one or more dominant genes. Blue and green bars underscore +ssRNA and −ssRNA viruses, respectively. f, The top 30 ISG inhibitors from the primary screens were compiled as a gene list and transformed to a binarized vector for clustering using MATLAB Statistics Toolbox (see Methods). A dendrogram was generated from the clustering analysis to show how viruses relate to each other with respect to the ISGs that target them.
Extended Data Figure 3 Co-occurrence of top 20 antiviral ISGs from primary screens.
ISGs were assigned a frequency on the basis of the number of times the gene appeared in a list of the 20 most inhibitory genes from 7 +ssRNA or 5 −ssRNA virus screens. A frequency of 1 indicates an occurrence of 100% across all gene lists. Co-occurrence is reflected by adjacent red and blue bars.
Extended Data Figure 4 Effects of ISGs on ISRE-dependent transcription.
a, 293 cells were transduced with lentiviruses expressing ISGs, followed by transfection with an ISRE reporter plasmid expressing Fluc. Cells were assayed for Fluc activity 24 h after transfection. b, 293 cells were co-transfected with ISG-expressing plasmids and an ISRE reporter plasmid. The cells were then treated overnight with 1,000 U ml−1 interferon-α (IFN-α), followed by Fluc activity assay. Data represent the average of three independent experiments performed in triplicate. Error bars represent s.d. Statistical significance was determined by one-way ANOVA or t-test. *P < 0.05, ***P < 0.001.
Extended Data Figure 5 cGAS mechanistic studies.
a, STAT1−/− fibroblasts were transfected with individual siRNAs targeting STING. Cell lysates were processed 48 h after transfection for western blot with anti-STING- or anti-actin-specific antibodies. From these results, siRNA no. 1 was chosen for additional studies. b, Schematic of cGAS protein sequence and truncation mutants. Red box, α-helix; blue box, β-sheet. Circles denote catalytic residues E225A and D227A. c, STAT1−/− fibroblasts were transduced with lentivirus expressing control or cGAS (wild type and truncation mutants). Cells were infected 48 h after transduction with VEEV-GFP and infectivity was monitored by FACS. Data represent the mean of two independent experiments. Error bars represent s.d. Statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01. d, STAT1−/− fibroblasts were transduced with lentivirus expressing control or cGAS (wild type and mutants). Cells were collected 48 h after transduction and total RNA was analysed for OAS2 mRNA induction relative to RPS11 (top), or protein lysates were analysed for phospho-IRF3 and actin expression by western blot (bottom). e, STAT1−/− fibroblasts were transduced with a puromycin-selectable lentivirus expressing cGAS and placed under selection. At various passages, cells were collected and total RNA was analysed for OAS2 mRNA induction relative to RPS11 (top), or protein lysates were analysed for cGAS and actin expression by western blot (bottom). Western blot and cGAS mRNA data represent one of two independent experiments, each showing similar results. OAS2 mRNA data are presented as the average of two independent experiments, each performed in triplicate. Error bars represent s.d.
Extended Data Figure 6 Gene-targeting strategy to create cGas knockout mice.
a, Mice expressing a cGas exon 2 gene-trap cassette were crossed to FlpE-expressing mice to generate conditional knockouts. These mice were then crossed to Cre-expressing mice to generate the knockout allele with a deletion of exon 2, which contains the cGAS catalytic sites. Mice were backcrossed to remove Cre, and cGas+/− mice were intercrossed to derive cGas−/− mice. b, PCR products from genomic DNA of cGas+/+, cGas+/− and cGas−/− mice using primers outlined in a. c, qRT–PCR of relative cGas expression in lungs (left) or BMMO (right) from wild-type B6 and cGas−/− mice. Data from lung represent means of three mice per group. Data from BMMO were derived from two independent experiments performed in triplicate. Error bars represent s.e.m. Statistical significance was determined by t-test. *P < 0.05, ***P < 0.001.
Extended Data Figure 7 Viral burden in mice infected with WNV.
Wild-type or cGas−/− mice were infected with WNV and viral titres in several regions of the brain were determined by plaque assay. n = 10 mice per group. Statistical significance was determined by t-test.
Extended Data Figure 8 Role for cGAS in BMMO activation.
a, BMMO from wild-type and cGas−/− mice were analysed for baseline expression of chemokines Ccl5 and Cxcl10 by RT–PCR. b, BMMO from wild-type and cGas−/− mice were treated with polyIC (pIC) or transfected with polyIC (Tf-pIC) and Ifnb and Ifit1 levels were determined by qRT–PCR. In both panels, gene expression levels are relative to the housekeeping gene RPS29 and normalized to mock-treated wild-type cells. Data represent two experiments performed in triplicate. Error bars represent s.d. Statistical significance was determined by t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
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Schoggins, J., MacDuff, D., Imanaka, N. et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 505, 691–695 (2014). https://doi.org/10.1038/nature12862
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DOI: https://doi.org/10.1038/nature12862
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