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Antiviral immunity in Drosophila requires systemic RNA interference spread

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.

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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.

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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.

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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

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