Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Antiviral immunity in Drosophila requires systemic RNA interference spread

Nature volume 458, pages 346–350 (2009)Cite this article

Abstract

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations1,2,3,4. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity5,6,7. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response8. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway9 is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 105-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Model for systemic RNAi viral immunity in Drosophila melanogaster.
Figure 2: In vivo dsRNA immunization provides sequence-specific antiviral protection in D. melanogaster.
Figure 3: Increased viral susceptibility of dsRNA-uptake-deficient mutants.
Figure 4: Core RNAi machinery and antibacterial immunity are intact in dsRNA uptake mutants.
Figure 5: Systemic spread of dsRNA follows virus infection and is essential for effective antiviral immunity.

Similar content being viewed by others

References

  1. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004)

    Article  ADS  CAS  Google Scholar 

  2. Dorner, T. & Radbruch, A. Antibodies and B cell memory in viral immunity. Immunity 27, 384–392 (2007)

    Article  Google Scholar 

  3. Roitt, I., Brostoff, J. & Male, D. Immunology (Mosby, 2001)

    Google Scholar 

  4. Voinnet, O. Non-cell autonomous RNA silencing. FEBS Lett. 579, 5858–5871 (2005)

    Article  CAS  Google Scholar 

  5. Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J. A. & Imler, J. L. Essential function in vivo for Dicer-2 in host defense against RNA viruses in Drosophila. Nature Immunol. 7, 590–597 (2006)

    Article  CAS  Google Scholar 

  6. van Rij, R. P. et al. The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. Genes Dev. 20, 2985–2995 (2006)

    Article  CAS  Google Scholar 

  7. Wang, X. H. et al. RNA interference directs innate immunity against viruses in adult Drosophila. Science 312, 452–454 (2006)

    Article  ADS  CAS  Google Scholar 

  8. Roignant, J. Y. et al. Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. RNA 9, 299–308 (2003)

    Article  CAS  Google Scholar 

  9. Saleh, M. C. et al. The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nature Cell Biol. 8, 793–802 (2006)

    Article  CAS  Google Scholar 

  10. Ulvila, J. et al. Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells. J. Biol. Chem. 281, 14370–14375 (2006)

    Article  CAS  Google Scholar 

  11. Vaistij, F. E., Jones, L. & Baulcombe, D. C. Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14, 857–867 (2002)

    Article  CAS  Google Scholar 

  12. Sijen, T. et al. On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107, 465–476 (2001)

    Article  CAS  Google Scholar 

  13. Schubiger, M., Carre, C., Antoniewski, C. & Truman, J. W. Ligand-dependent de-repression via EcR/USP acts as a gate to coordinate the differentiation of sensory neurons in the Drosophila wing. Development 132, 5239–5248 (2005)

    Article  CAS  Google Scholar 

  14. Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004)

    Article  CAS  Google Scholar 

  15. Kennerdell, J. R. & Carthew, R. W. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway. Cell 95, 1017–1026 (1998)

    Article  CAS  Google Scholar 

  16. Hoffmann, J. A. The immune response of Drosophila. Nature 426, 33–38 (2003)

    Article  ADS  CAS  Google Scholar 

  17. Dostert, C. et al. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of Drosophila. Nature Immunol. 6, 946–953 (2005)

    Article  CAS  Google Scholar 

  18. Porter, J. A., Minke, B. & Montell, C. Calmodulin binding to Drosophila NinaC required for termination of phototransduction. EMBO J. 14, 4450–4459 (1995)

    Article  CAS  Google Scholar 

  19. Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B. & Cohen, S. M. bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113, 25–36 (2003)

    Article  CAS  Google Scholar 

  20. Hahn, C. S., Hahn, Y. S., Braciale, T. J. & Rice, C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc. Natl Acad. Sci. USA 89, 2679–2683 (1992)

    Article  ADS  CAS  Google Scholar 

  21. Reed, L. J. & Muench, H. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27, 493–497 (1938)

    Google Scholar 

  22. Okamura, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev. 18, 1655–1666 (2004)

    Article  CAS  Google Scholar 

  23. Cherry, S. & Perrimon, N. Entry is a rate-limiting step for viral infection in a Drosophila melanogaster model of pathogenesis. Nature Immunol. 5, 81–87 (2004)

    Article  CAS  Google Scholar 

  24. Goto, A., Blandin, S., Royet, J., Reichhart, J. M. & Levashina, E. A. Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies. Nucleic Acids Res. 31, 6619–6623 (2003)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to members of the Andino and the O’Farrell laboratories for discussion, technical support and advice on fly work. We thank J. Frydman, P. O’Farrell and M. Vignuzzi for discussions and comments on the manuscript. We also thank T. Cook for advice on the IR EcR eye phenotype, R. Carthew for providing the GMR>IR[white] and Dcr2 fly stocks and M. Siomi for the Ago2 fly stock. B.G. is a Manlio Cantarini fellow. B.B. is a Lebanese CNRSL Fellow. C.J. is a University of Paris VI and Ministère de la Recherche fellow. This work was financially supported by NIH grants AI40085 and AI064738 to R.A., the Institut Pasteur to M.-C.S. and C.A., and CNRS, ANR and ARC grants to C.A.

Author Contributions M.-C.S., M.T. and R.P.v.R. performed dsRNA inoculations and virus infections, normal and reverse northern blotting, western blotting, survival curves, obtained fluorescent images, and prepared and analysed mutant flies. B.G. and V.G. examined systemic spread of dsRNA. The genetic and phenotypic analyses of transgenic flies expressing RNA hairpins were designed and carried out by B.B., C.J. and C.A. M.-C.S. and R.A. designed the experiments, discussed the interpretation of the results and co-wrote the manuscript.

Author information

Author notes

  1. Maria-Carla Saleh & Ronald P. van Rij

    Present address: Present addresses: Institut Pasteur, Viruses and RNA interference, F-75015 Paris, France (M.-C.S.); Department of Medical Microbiology, Nijmegen Center for Molecular Life Sciences, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (R.P.v.R.).,

  2. Michel Tassetto and Ronald P. van Rij: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California, San Francisco 94122-2280, USA,

    Maria-Carla Saleh, Michel Tassetto, Ronald P. van Rij & Raul Andino

  2. Institut Pasteur, Viruses and RNA interference, F-75015 Paris, France,

    Bertsy Goic & Valérie Gausson

  3. Institut Pasteur, Drosophila Genetics and Epigenetics; CNRS, URA 2578, F-75015 Paris, France,

    Bassam Berry, Caroline Jacquier & Christophe Antoniewski

  4. CNRS, URA 2578, F-75015 Paris, France,

    Bassam Berry, Caroline Jacquier & Christophe Antoniewski

Authors
  1. Maria-Carla Saleh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michel Tassetto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ronald P. van Rij
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bertsy Goic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Valérie Gausson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bassam Berry
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Caroline Jacquier
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christophe Antoniewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Raul Andino
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Raul Andino.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures S1-S6 with Legends (PDF 1388 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saleh, MC., Tassetto, M., van Rij, R. et al. Antiviral immunity in Drosophila requires systemic RNA interference spread. Nature 458, 346–350 (2009). https://doi.org/10.1038/nature07712

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07712

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing