Abstract
RNA viruses are a major threat to animals and plants. RNA interference (RNAi) and the interferon response provide innate antiviral defense against RNA viruses. Here, we performed a large-scale screen using Caenorhabditis elegans and its natural pathogen the Orsay virus (OrV), and we identified cde-1 as important for antiviral defense. CDE-1 is a homolog of the mammalian TUT4 and TUT7 terminal uridylyltransferases (collectively called TUT4(7)); its catalytic activity is required for its antiviral function. CDE-1 uridylates the 3ʹ end of the OrV RNA genome and promotes its degradation in a manner independent of the RNAi pathway. Likewise, TUT4(7) enzymes uridylate influenza A virus (IAV) mRNAs in mammalian cells. Deletion of TUT4(7) leads to increased IAV mRNA and protein levels. Collectively, these data implicate 3ʹ-terminal uridylation of viral RNAs as a conserved antiviral defense mechanism.
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Acknowledgements
We thank M. Tanguy (Gurdon Institute, University of Cambridge) for OrV viral filtrates, L. Frézal for help with OrV RNA fluorescence in situ hybridization, I. Wilkinson for support with the genetic screen, N. J. Lehrbach for help with microinjections and M. Ridyard for lab management. We thank K. Harnish, F. Braukmann and S. Moss for high-throughput-sequencing support. We are grateful to V. N. Kim and H. Chang for sharing information on TAIL-seq and A. C. M. Boon (Washington University School of Medicine) for providing IAV. We thank A. Ashe and P. Sarkies for theoretical input on the screen design. We thank the International C. elegans Gene Knockout Consortium, the TransgeneOme project, J. Ahringer and M.-A. Félix for providing reagents. We thank V. Benes and the EMBL genome core for sequencing support. We thank G. Allen and C. Bradshaw for core bioinformatics support. We thank R. Medhi and D. Zijlmans for help with TUT western blots. This work was supported by the following grants to E.A.M.: Cancer Research UK (C13474/A18583 and C6946/A14492), the Wellcome Trust (104640/Z/14/Z and 092096/Z/10/Z) and The European Research Council (ERC grant 260688). J.L.P. was supported by the Wellcome Trust (093970/Z/10/Z). A.K. is supported by the Wellcome trust (102452/Z/13/Z). D.W. is supported as an Investigator through the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.
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Conceived and designed the experiments: J.L.P., H.J. and E.A.M. Performed the experiments: J.L.P., H.J., E.K., J.K., C.L., M.M. and C.M. Analyzed the data: J.L.P., H.J., T.D.D., K.L.M.R., A.J.E., D.O.C., D.W. and E.A.M. Wrote the manuscript: J.L.P. and E.A.M.
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Supplementary Figure 1 The viral stress sensor (lys-3p::GFP) is constitutively active in some tissues but is induced in the intestine after severe viral infection.
a, Comparison of viral load and the lys-3 and sdz-6 mRNA expression after two days of infection by qRT-PCR, strains as indicated. Dots: independent infection. Samples as in Fig. 3d,e. b, Representative confocal sections (10 × or 20 × magnification, as specified) of the viral stress sensor in wild type and drh-1 mutants without infection. The viral stress sensor exhibited constitutive activity in uninfected individuals, which was restricted to specific tissues. GFP was observed at all developmental stages in the pharynx and the rectum of hermaphrodites. Additionally, hermaphrodites at the L4 larval stage would show a strong GFP signal around the vulva and gravid adults exhibited the GFP in the uterine lumen. In males, GFP was observed in the pharynx and the tail. GFP expression was comparable in wild type and drh-1 mutants and independent of viral infection. Thus, the gene lys-3 is constitutively active in tissues neighboring openings exposed to the environment, the most likely entry points of potential bacterial pathogens. c, Representative confocal sections (20 × magnification) of young adults (strains as indicated) carrying the viral stress sensor. Animals were uninfected (mock) or infected with OrV for four days. The viral stress sensor was strongly induced in the intestine after infection of drh-1 mutants, which is in agreement with the tropism of OrV. Intestinal GFP was most often visible around the collar of the nematodes, in the anterior region of the intestine in young adults. Some infected individuals exhibited a strong GFP signal throughout their entire body (data not shown), suggesting that the induction of the viral stress sensor can spread from cell to cell, like an inflammation process. The viral stress sensor offers an opportunity to easily monitor viral infections in living animals.
Supplementary Figure 2 A cde-1-deletion allele does not complement the screen isolate ovid-9.
a, Workflow of cde-1/ovid-9 (mj414) × cde-1 (tm1021) F8 recombinant family generation. A similar strategy was used to construct the cde-1 (mj414) × drh-1 (ok3495) F8 recombinant families. All animals were homozygous for the viral stress sensor (mjIs228). b-c, Number of families that activated the viral stress sensor in more than 20% of individuals after four days of infection with OrV. Approximately 50 individuals scored per family. Bars: number of families meeting criteria as indicated.
Supplementary Figure 3 cde-1 mutants show horizontal transmission of OrV infection.
Workflow and data monitoring the inter-individual transmission of OrV infection (in strains as indicated) using the viral stress sensor.
Supplementary Figure 4 Intestinal expression of cde-1 confers antiviral immunity.
a, Representative confocal sections (20 × magnification) of OrV in vivo RNA FISH. b, Representative confocal section (10 × magnification) of a C. elegans L4 larva expressing cde-1::GFP. As two previous reports disagreed about the expression pattern of CDE-1 (Olsen, A. et al. Science 312, 1381–1385, 2006; van Wolfswinkel, J. C. et al. Cell 139, 135–148, 2009), we used fosmid-recombineering to generate transgenic animals driving GFP expression from an endogenous genomic context. c, Diagram of the cde-1 rescue transgene, using the intestine-specific promoter of the gene vha-6. This transgene was injected in cde-1 null mutants. d, Viral load as measured by qRT-PCR of OrV RNA1 genome in adults two days after infection. Bars: average value; error: SEM; five independent infections. One-tailed student’s t-test: *** p < 0.001, **p < 0.01. e, Incidence of male in the progeny of hermaphrodites left to self-fertilize at 25 °C, in strains as indicated.
Supplementary Figure 5 CDE-1 is not required for general miRNA homeostasis.
a, Non-templated nucleotides at the 3ʹ end of the different classes of endogenous and antiviral small RNAs as indicated. RNA was isolated from young adults after two days of infection with OrV. b, miRNA expression in cde-1 (tm1021) mutants as compared to wild type, samples as in a. Dots: individual miRNA; reads per million averaging two independent C. elegans culture plates. c, piRNAs and endogenous 22G-RNAs abundance in cde-1 (tm1021) mutants as compared to wild type, normalised to library size. Dots: independent C. elegans culture plate. Samples as in a.
Supplementary Figure 6 CDE-1-depleted animals show high expression of stress-response genes during OrV infection.
a, Fold change in the length of poly(A) tails (measured by TAIL-seq) in cde-1 mutants compared to wild type. RNA was isolated from young adults after two days of OrV infection. Turkey boxplot. b, Differential mRNA expression in cde-1 (tm1021) compared to wild type, two days of OrV infection (mRNA-seq). Turkey boxplot; dots: outliers.
Supplementary Figure 7 The 3′ end of the OrV genome contains CDE-1-dependent nontemplated U tails.
Supplementary Figure 8 The terminal uridylyltransferases TUT4(7) restrict IAV infection.
a, Protein level of the IAV NP measured by immunofluorescence (FACS). Error: SEM in three independent infections. b, Level of expression of the IAV NP mRNA normalized to Gapdh in MEF cells of different genotypes as indicated. Bars: average value; error: SEM; three independent infections.
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Le Pen, J., Jiang, H., Di Domenico, T. et al. Terminal uridylyltransferases target RNA viruses as part of the innate immune system. Nat Struct Mol Biol 25, 778–786 (2018). https://doi.org/10.1038/s41594-018-0106-9
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DOI: https://doi.org/10.1038/s41594-018-0106-9
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