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Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila

Abstract

Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1–mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

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Figure 1: CrPV antagonizes RNAi in S2 cells.
Figure 2: CrPV-1A is a potent RNAi suppressor and does not interfere in the microRNA pathway.
Figure 3: CrPV-1A inhibits RISC activity downstream of Dicer processing.
Figure 4: CrPV-1A interacts with Argonaute 2 in S2 cells.
Figure 5: CrPV-1A interferes with the function of preprogrammed holo-RISC in vitro and in vivo.
Figure 6: RNAi suppressors determine spread and pathogenesis of Sindbis virus in fruit fly.

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Acknowledgements

We thank J. Frydman and Andino laboratory members L. Gitlin, D. Barnes, M. Flenniken and A. Lauring for useful discussion for preparing the manuscript, R. van Rij and C. Saleh for their help, G. Belsham for critical reading of the manuscript and useful comments, P. Zamore (Univ. Massachusetts Medical School), Mikiko Siomi and Keita Miyoshi (Keio Univ. School of Medicine, Japan) for providing antibody directed against Drosophila Argonaute 2, H. Heidner (Univ. Texas, San Antonio) for providing Sindbis virus plasmid and M. Moritz (Univ. California, San Francisco) for Drosophila embryo extract preparation. This work was financially supported by Pasteur Institute, by the Centre National de la Recherche Scientifique, by grants from the Agence Nationale de la Recherche (AKROSS) and by fellowships from the Lebanese Centre National de la Recherche Scientifique to B.B. as well as by US National Institutes of Health grants AI40085 and AI064738 to R.A.

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Authors

Contributions

A.N. and R.A. designed and interpreted most of the experiments; A.N. performed most of the experiments; B.B. and C.A. designed and performed the IR[white], GFP/bantam sensor and retrotransposon experiments in flies; M.T. and M.K. performed the fly injections; A.A. made some plasmid constructs; C.D. and A.K. carried out the MS analysis; J.G. advised on the project; A.N., R.A., A.A. and C.A. prepared the manuscript.

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Correspondence to Raul Andino.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 501 kb)

Supplementary Video 1

Serial optical section of Drosophila embryos expressing GFP under Bantam miRNA control in the presence or absence of CrPV-1A suppressor. (MOV 6775 kb)

Supplementary Video 2

Serial Optical Sections 2, Bantam expressionCrPV148 (MOV 6835 kb)

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Nayak, A., Berry, B., Tassetto, M. et al. Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila. Nat Struct Mol Biol 17, 547–554 (2010). https://doi.org/10.1038/nsmb.1810

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