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Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates - Nature

  • ️Reis e Sousa, Caetano
  • ️Sun Aug 10 2014

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Acknowledgements

We thank S. Akira and J. Tschopp (deceased) for gifts of mice and cells, as well as N. O’Reilly, the LRI Equipment Park (D. Phillips), and the LRI Protein Analysis and Proteomics Facility (R. George and S. Kjaer) for technical assistance. We also thank P. Maillard and K. Snelgrove for reading the manuscript, P. Tortora, G. Dehò and M. Freire for their insights on the synthesis of poly(I:C) and all members of the CRUK Immunobiology Laboratory for helpful discussions and comments. C.R.S., D.G., S.D. and A.G.V.V. are funded by Cancer Research UK, a prize from Fondation Bettencourt-Schueller, and a grant from the European Research Council (ERC Advanced Researcher Grant AdG-2010-268670). A.J.P. and T.S.D. are supported by Public Health Service award R37 AI038296 and the Elizabeth B. Lamb Center for Pediatric Research. T.F. is supported by the Fundación Ramón Areces. G.H., M.S. and W.B. are supported by the Deutsche Forschungsgemeinschaft (http://www.dfg.de; SFB670 to M.S., W.B. and G.H., DFG SCHL1930/1-1 to M.S., SFB704 to G.H. and W.B., SFB832 and KFO177 to G.H.). G.H. and M.S. are supported by the DFG Excellence Cluster ImmunoSensation. G.H. is supported by the German Center of Infectious Disease (DZIF).

Author information

Author notes

  1. Safia Deddouche & Jan Rehwinkel

    Present address: Present addresses: Drosophila Genetics and Epigenetics, Laboratory of Developmental Biology, CNRS UMR7622, Université Pierre et Marie Curie, Paris, France (S.D.); Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK (J.R.).,

  2. Delphine Goubau and Martin Schlee: These authors contributed equally to this work.

Authors and Affiliations

  1. Immunobiology Laboratory, Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,

    Delphine Goubau, Safia Deddouche, Annemarthe G. Van der Veen, Jan Rehwinkel & Caetano Reis e Sousa

  2. Institut für Klinische Chemie und Klinische Pharmakologie, Universitätsklinikum Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany,

    Martin Schlee, Thomas Zillinger, Marion Goldeck, Christine Schuberth, Winfried Barchet, Janos Ludwig & Gunther Hartmann

  3. Department of Pediatrics, Vanderbilt University School of Medicine, D7235 Medical Center North, 1161 21st Avenue South, Nashville, Tennessee 37232-2581, USA,

    Andrea J. Pruijssers, Jason A. Iskarpatyoti & Terence S. Dermody

  4. Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, D7235 Medical Center North, 1161 21st Avenue South, Nashville, Tennessee 37232-2581, USA,

    Andrea J. Pruijssers, Jason A. Iskarpatyoti & Terence S. Dermody

  5. Instituto de Biología Funcional y Genómica. Consejo Superior de Investigaciones Científicas/Universidad de Salamanca, Zacarías González 2, 37007, Salamanca, Spain,

    Tsutomu Fujimura

  6. Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, D7235 Medical Center North, 1161 21st Avenue South, Nashville, Tennessee 37232-2581, USA,

    Terence S. Dermody

Authors

  1. Delphine Goubau

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  2. Martin Schlee

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  3. Safia Deddouche

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  4. Andrea J. Pruijssers

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  5. Thomas Zillinger

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  6. Marion Goldeck

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  7. Christine Schuberth

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  8. Annemarthe G. Van der Veen

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  9. Tsutomu Fujimura

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  10. Jan Rehwinkel

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  11. Jason A. Iskarpatyoti

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  12. Winfried Barchet

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  13. Janos Ludwig

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  14. Terence S. Dermody

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  15. Gunther Hartmann

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  16. Caetano Reis e Sousa

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Contributions

D.G., M.S., S.D., A.J.P., T.S.D., M.G., W.B., J.L., G.H. and C.R.S. designed experiments and analysed the data. D.G., M.S., S.D., A.J.P., T.F., A.G.V.V., J.R., J.A.I., T.Z., C.S., M.G., J.L. performed experiments. D.G., M.S., A.J.P., T.S.D., G.H. and C.R.S. wrote the manuscript. G.H. and C.R.S. supervised the project.

Corresponding authors

Correspondence to Delphine Goubau or Caetano Reis e Sousa.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 RNA from reovirus and L-A virus induce a RIG-I-dependent IFN response that requires 5′-diphosphates.

a, Total RNA purified from reoT1L particles (vRNA) was treated or not with calf intestinal phosphatase (± CIP). RNA integrity was verified by gel electrophoresis (left panel) or transfected into HEK293 cells to determine its capacity to stimulate the IFN-β promoter using a reporter assay (right panel). b, L, M, and S reoT1L genome segments were isolated by gel fractionation and treated or not with CIP. An aliquot of the treated samples was electrophoresed in a 0.8% agarose gel to validate RNA integrity (left panel), whereas another was transfected into HEK293 cells to determine its capacity to stimulate the IFN-β promoter using a reporter assay (right panel). c, d, Total L-A RNA as well as gel-purified L-A genomes and transcripts (as in Fig. 1i) were transfected into RIG-I+/− or RIG-I−/− MEFs (c) and MDA5+/+ or MDA5−/− DCs (d). After incubation for 16 h, the relative expression (RE) of ifit1 over gapdh (c) or murine IFN-α levels (d) were determined. Water and ppp-IVT-RNA99nt, poly(dA:dT), or RNA isolated from Vero cells infected with encephalomyocarditis virus (Vero-EMCV-RNA) were included as controls (* = none detected). All experiments were performed at least twice; one representative experiment is shown.

Extended Data Figure 3 Characterization of guanosine sources and IVT-RNA25nt.

a, Representative LC-MS spectra of GMP, GDP, and GTP sources used for the preparation of IVT-RNAs in Fig. 3. Asterisks indicate the expected mass-to-charge ratio (m/z) of the different guanosines. b, IVT-RNA25nt were generated as depicted in Fig. 3a using GMP, GTP or GMP spiked with GTP (GMP + 10% GTP) before being annealed to AS RNA and tested using the IFN-β promoter reporter assay following transfection into HEK293 cells. c, Spectra of 5′pp-RNA24nt and 5′ppp-RNA24nt following MALDI ToF characterization (a.i., absolute intensity). Ions with two charges (m2+) appear exactly at half the expected (m+) mass/charge (m/z) ratio.

Extended Data Figure 4 Phosphatase treatment of poly(I:C) affects RIG-I but not MDA5-dependent IFN-responses.

a, Schematic representation of inosinic acid or cytidylic acid homopolymer synthesis from inosine 5′-diphosphate or cytidine 5′-diphosphate through the action of polynucleotide phosphorylase, which when annealed form the synthetic dsRNA analogue poly(I:C). Whether the synthesized polynucleotides carry a 5′ di- or monophosphate or a mixture of both is unclear. b, IFN-pre-treated MDA5−/− or RIG-I−/− immortalized MEFs were transfected with poly(I:C) ± CIP. IFN induction was quantified 16 h later using an IFN-β promoter reporter assay. c, Poly(I:C) was first cleaved with RNase III for 1 or 5 min before being treated or not with CIP (+/−). Samples were subjected to gel electrophoresis to verify digestion (left panel) or transfected into IFN-pre-treated MDA5−/− MEFs (right panel). Cells were harvested 16 h post-transfection, and IFN-responses were assessed by RT-qPCR for ifit1 expression. Water and ppp-IVT-RNA99nt were included as controls. RE, relative expression. All experiments were performed at least twice; one representative experiment is shown. For PCR data, the mean ( ± s.d.) of triplicate technical replicates is shown.

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Goubau, D., Schlee, M., Deddouche, S. et al. Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates. Nature 514, 372–375 (2014). https://doi.org/10.1038/nature13590

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  • Received: 30 January 2014

  • Accepted: 16 June 2014

  • Published: 10 August 2014

  • Issue Date: 16 October 2014

  • DOI: https://doi.org/10.1038/nature13590