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

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your

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FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS INNATE IMMUNE PATHWAYS ACT SYNERGISTICALLY TO CONSTRAIN RNA VIRUS EVOLUTION IN _DROSOPHILA MELANOGASTER_ Article 10

March 2022 RETROTRANSPOSON ACTIVATION DURING _DROSOPHILA_ METAMORPHOSIS CONDITIONS ADULT ANTIVIRAL RESPONSES Article 17 November 2022 TWO CGAS-LIKE RECEPTORS INDUCE ANTIVIRAL IMMUNITY IN

_DROSOPHILA_ Article 14 July 2021 REFERENCES * Baulcombe, D. RNA silencing in plants. _Nature_ 431, 356–363 (2004) Article  ADS  CAS  Google Scholar  * Dorner, T. & Radbruch, A.

Antibodies and B cell memory in viral immunity. _Immunity_ 27, 384–392 (2007) Article  Google Scholar  * Roitt, I., Brostoff, J. & Male, D. _Immunology_ (Mosby, 2001) Google Scholar  *

Voinnet, O. Non-cell autonomous RNA silencing. _FEBS Lett._ 579, 5858–5871 (2005) Article  CAS  Google Scholar  * 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  * 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  * 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  * 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  * 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  * 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  * 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  * Sijen, T. et al. On the role of RNA amplification in dsRNA-triggered gene silencing. _Cell_ 107, 465–476 (2001) Article  CAS  Google Scholar  * 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  * 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  * 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  * Hoffmann, J. A. The immune response of _Drosophila_. _Nature_ 426, 33–38 (2003) Article  ADS  CAS  Google

Scholar  * 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  * 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  * 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  * 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  * Reed, L. J. &

Muench, H. A simple method of estimating fifty percent endpoints. _Am. J. Hyg._ 27, 493–497 (1938) Google Scholar  * 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  * 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  * 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 * 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.)., * Michel Tassetto and

Ronald P. van Rij: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Microbiology and Immunology, University of California, San Francisco 94122-2280,

USA, Maria-Carla Saleh, Michel Tassetto, Ronald P. van Rij & Raul Andino * Institut Pasteur, Viruses and RNA interference, F-75015 Paris, France, Bertsy Goic & Valérie Gausson *

Institut Pasteur, Drosophila Genetics and Epigenetics; CNRS, URA 2578, F-75015 Paris, France, Bassam Berry, Caroline Jacquier & Christophe Antoniewski * CNRS, URA 2578, F-75015 Paris,

France, Bassam Berry, Caroline Jacquier & Christophe Antoniewski Authors * Maria-Carla Saleh View author publications You can also search for this author inPubMed Google Scholar * Michel

Tassetto View author publications You can also search for this author inPubMed Google Scholar * Ronald P. van Rij View author publications You can also search for this author inPubMed 

Google Scholar * Bertsy Goic View author publications You can also search for this author inPubMed Google Scholar * Valérie Gausson View author publications You can also search for this

author inPubMed Google Scholar * Bassam Berry View author publications You can also search for this author inPubMed Google Scholar * Caroline Jacquier View author publications You can also

search for this author inPubMed Google Scholar * Christophe Antoniewski View author publications You can also search for this author inPubMed Google Scholar * Raul Andino View author

publications You can also search for this author inPubMed 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 POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR

FIG. 4 POWERPOINT SLIDE FOR FIG. 5 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: 04 October 2008 * Accepted: 03

December 2008 * Published: 08 February 2009 * Issue Date: 19 March 2009 * DOI: https://doi.org/10.1038/nature07712 SHARE THIS ARTICLE Anyone you share the following link with will be able to

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