Essential function in vivo for Dicer-2 in host defense against RNA viruses in drosophilaDelphine Galiana-Arnoux1,3, Catherine Dostert1,3, Anette Schneemann2, Jules A Hoffmann1 & Jean-Luc Imler1

The fruit fly Drosophila melanogaster is a model system for studying innate immunity, including antiviral host defense. Infection with drosophila C virus triggers a transcriptional response that is dependent in part on the Jak kinase Hopscotch. Here we show that successful infection and killing of drosophila with the insect nodavirus flock house virus was strictly dependent on expression of the viral protein B2, a potent inhibitor of processing of double-stranded RNA mediated by the essential RNA interference factor Dicer. Conversely, flies with a loss-of-function mutation in the gene encoding Dicer-2 (Dcr-2) showed enhanced susceptibility to infection by flock house virus, drosophila C virus and Sindbis virus, members of three different families of RNA viruses. These data demonstrate the importance of RNA interference for controlling virus replication in vivo and establish Dcr-2 as a host susceptibility locus for virus infections.

Metazoans have developed efficient mechanisms to oppose microbial invaders. Innate immunity is common to all metazoans and serves as a first line of defense. Its hallmarks are the recognition of microorgan-isms by germline-encoded, nonrearranging receptors and rapid induc-tion of effector mechanisms1–3. The fruit fly Drosophila melanogaster is a powerful model for deciphering the genetic mechanisms of innate immunity. Detailed analyses of the immune responses of flies to infec-tion by bacteria or fungi have led to the identification and characteriza-tion of several potent antimicrobial peptides. Inducible expression of the genes encoding these peptides is now known to be regulated by two signaling pathways, Toll and Imd, that also regulate specific activation of DIF and Relish, respectively, two members of the NF-κB family of transcription factors4–6. Additionally, the drosophila Toll pathway is evocative of the mammalian Toll-like receptor–interleukin 1 signaling pathway, whereas components of the Imd pathway have orthologs in mammals that function in the tumor necrosis factor pathway.

In contrast to the wealth of information now available for fungal and bacterial infections, the interaction of drosophila with viruses was not addressed until recently, and possible antiviral responses (and path-ways) in flies were almost completely unknown7–10. Like all organisms, insects are exposed to viral infections, and because some insect viruses threaten insects useful to humans, such as silkworm or honeybees, or can be transmitted by blood-sucking insects to vertebrate hosts (arthro-pod-borne viruses (arboviruses) such as dengue viruses and West Nile virus), these viruses are of increasing importance. Investigation of the response of drosophila to virus infection has shown that drosophila C virus (DCV), a member of the family of dicistroviridae (which are closely related to picornaviridae) and one of the best characterized

drosophila viruses, induces a set of genes distinct from the Toll- and Imd-induced target genes11. A subset of genes induced by DCV, includ-ing the marker vir-1 (virus-induced RNA 1), is regulated by the pathway of the kinase Jak and transcription factor STAT, and flies deficient in the gene hopscotch, which encodes the sole Jak kinase of drosophila12, have increased viral load and sensitivity to DCV infection. Because the Jak-STAT pathway was initially characterized in mammals for its function in interferon signaling, those results indicated the existence of evolutionary ancient and conserved mechanisms of animal host defenses against viral infections. Despite those advances, however, the effector mechanisms that control viral infections in drosophila are not yet understood.

In plants, virus-induced gene silencing, a mechanism mediated by double-stranded RNA (dsRNA) interference, is a potent defense against plant viruses13,14. RNA interference (RNAi) is triggered by long dsRNA processed by the RNase III–like enzyme Dicer into small interfering RNA (siRNA) 20–25 nucleotides in length. These siRNAs then serve as sequence determinants to guide the multisubunit RNA-induced silencing complex (RISC) to target single-stranded RNA molecules for de gradation15. One of the components of the RISC is Dicer, which is thus involved in the two main steps of RNAi.

RNA containing stem-loop structures can also be processed by Dicer to generate ‘micro RNA’ with widespread functions in growth and development16. Evidence that RNAi is an antiviral response in plants includes the identification of virus-derived siRNA in virus-infected plants and of several viral proteins that specifically disable RNAi in infected plants. However, genetic analysis of the contribution ofRNAi to antiviral defenses in plants is complicated by the existence of

1UPR9022 Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire Strasbourg 67000, France. 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. 3These authors contributed equally to this study. Correspondence should be addressed to J.-L.I.(jl.imler@ibmc.u-strasbg.fr).

Received 10 March; accepted 20 March; published online 23 March 2006; doi:10.1038/ni1335

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several Dicer-like (DCL) genes (four in the thale cress Arabidopsis thali-ana), and thus so far it has not been possible to assign with certainty virus-induced gene silencing to an individual DCL gene13. Based on the results obtained in plants and because the RNAi machinery is pres-ent in all animals from nematodes to mammals, RNAi has often been postulated to function similarly as an antiviral response in animals. However, that hypothesis remains relatively untested, especially for in vivo attempts. Convincing evidence exists that animal viruses can be targeted by the RNAi-silencing machinery, as treatment of mammalian cell lines, or even mucosal surfaces in vivo, with specific siRNA confers some protection against challenge with the virus against which the siRNA could function17–22. In addition, some animal viruses encode dsRNA-sequestering proteins that have been shown to inhibit RNAi in plant and insect cells23. It is unclear, however, whether those viral proteins function to counter RNAi in animal host cells or whether they have other functions, such as preventing additional responses triggered by dsRNA. Evidence has been presented indicating an important func-tion for RNA silencing for the control of insect flock house virus (FHV) replication in a drosophila cell line24. However, the relevance of that response in vivo was not addressed.

FHV is a member of the nodaviridae, which are small (about 35 nm in diameter) nonenveloped riboviruses with a genome composed of two single-stranded, positive-sense RNAs. This bipartite genome is packaged in an icosahedral capsid assembled from 180 copies of a single type of coat protein25. RNA1 (3,107 nucleotides in FHV) encodes protein A, a 112-kilodalton RNA-dependent RNA poly-merase. A subgenomic RNA3 (387 nucleotides) is produced from

RNA1 in infected cells and encodes the 12-kilodalton B2 protein, which has been shown to inhibit RNAi in a drosophila cell line24. Finally, RNA2 (1,400 nucleotides) encodes the 43-kilodalton capsid protein-α. The nodavirus family is genetically, biochemically and structurally well characterized; these viruses have proven highly trac-table for the study of fundamental issues in virology and represent a powerful model system for exploring virus-host interactions25.

Here we report that FHV infected adult drosophila flies, multiply-ing in many tissues and causing the death of infected flies. Moreover, successful infection of drosophila with FHV was strictly dependent on expression of the B2 inhibitor of Dicer. An absence of B2 led to abrogated virus RNA production and hence virus control in wild-type flies, but did not produce those same phenotypes in flies with a loss-of-function mutation of the gene encoding Dicer-2 (Dcr-2), indicating that RNAi is essential for control of FHV infection. Furthermore, the Dicer-2 mutant flies were also more susceptible to infection with two other RNA viruses, DCV and Sindbis virus (SINV). The data presented here demonstrate the relevance of an RNAi antiviral defense mecha-nism in metazoans in vivo.

RESULTSFHV is a drosophila pathogenFHV caused high lethality after intrathoracic injection of gradi-ent-purified virus into flies. Time to death was dose dependent and occurred between 9 and 16 d after injection. The minimum dose causing 100% lethality was 200 plaque-forming units (PFU; 6 × 104 particles), demonstrating that FHV is a severe pathogen of drosophila

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Figure 1 FHV is a drosophila pathogen. (a) Survival curves after infection with FHV for groups of 20 adult flies injected with various amounts of virus (key). Surviving flies were counted daily. (b) Histology of the fat body of flies 2–5 d after injection of FHV. Semi-thin sections were stained with 1% toluidin blue. Original magnification, ×100. (c,d) RNA hybridization of viral RNA1, RNA2 and RNA3 (c) and immunoblot for capsid protein (d), demonstrating accumulation in infected flies. RNA encoding the ribosomal protein RpL32 serves as a loading control (c, bottom). (e) Immunostaining for FHV coat protein in fat body cells 3 d after infection. (f) Transmission electron microscopy for FHV particles in the cytoplasm of muscle, fat body and trachea cells 5 d after infection. Arrows indicate paracrystalline arrangements of virus particles (enlarged in insets ×4). Scale bars, 50 µm (e) and 0.8 µm (f). NI, not infected. Representative of two (a,b,e,f) or four (b,c) independent experiments.

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(Fig. 1a). Histological analysis demonstrated morphological defects in the fat body beginning 4–5 d after infection; the most notable changes included a decrease in the number of lipidic granules (Fig. 1b). We detected increasing amounts of the three viral RNAs in infected flies, indicating productive FHV infection (Fig. 1c). Immunoblot analysis indicated that accumulation of the viral capsid protein paralleled an increase in viral RNA (Fig. 1d). Examination of tissues from infected flies by transmission electron microscopy and immunofluorescence showed high concentrations of viral particles in the cytosol of cells from the fat body (Fig. 1e,f). We also detected viral particles in several other tissues, including muscles and tracheae (Fig. 1f). Overall, these data indicate that FHV represents a good model for studying possible antiviral responses in drosophila.

Transgenic model for studying FHV infection in fliesWe established transgenic lines carrying chromosomally integrated con-structs expressing FHV RNA1 or RNA2 using a system consisting of an upstream response element (UAS) activated by the yeast transcriptional activator Gal4 (Fig. 2a). Flies containing a transgene encoding RNA1 had high expression of the RNA even in the absence of a Gal4 ‘driver’ protein (Fig. 2b, lanes 1,2). This strong accumulation of RNA1 was due to production of the RNA by protein A, as shown by hybridization with a sense probe demonstrating high expression of negative-sense RNA1 (Fig. 2c, right) and expression of RNA3, whose synthesis is dependent on rep-lication (by the RNA-dependent RNA polymerase) of RNA1 (ref. 26; Fig. 2b). In contrast, RNA2 was weakly detectable by RNA hybridization even when the UAS-RNA2–transgenic flies were crossed with other strains expressing strong Gal4 ‘drivers’ (Fig. 2b, lanes 3,4). The low amount of RNA2, reflecting its instability, was not unexpected, because the viral cDNAs used to establish the transgenic lines were constructed so that when transcribed, the RNAs produced would have 3′ ends exactly like those produced during virus infection (without polyadenylated tails), which would facilitate their use as templates for the RNA-dependent RNA polymerase protein A but would render them less stable than endog-enous message. Despite its low production, RNA2 produced from the transgene was nev-ertheless sufficiently stable to yield expression of the 43-kilodalton capsid protein-α, which we detected by immunoblot (Fig. 2d).

In contrast, there was high production of FHV RNA2 when individual flies expressing both the RNA1 and RNA2 transgenes were crossed, even in the absence of a Gal4 ‘driver’ (Fig. 2b, lane 7), indicating that the viral RNA–dependent RNA polymerase protein A is suf-ficient for replication of all three FHV RNAs in transgenic flies. Transgenic flies expressing either RNA1 or RNA2 developed normally, had a normal lifespan and were fertile. Likewise, flies carrying both transgenes also developed normally initially; however, they died rapidly after reaching adulthood (Fig. 3a). Death was attributable to viral infection due to infectious virus produced as a result of RNA and protein expression from both transgenes, demonstrated by the fact that transfer of hemolymph from these flies into wild-type flies induced rapid death with associated high viral loads in the recipient flies (Fig. 3b and data not shown).

B2 is required for viral RNA amplification in fliesWe next established transgenic fly lines expressing a variant of RNA1 (RNA1∆B2) containing two point mutations that disrupted the open reading frame of B2 on RNA3 without negatively affecting synthesis of protein A from RNA1, which is translated from a different open reading frame (Fig. 2a). RNA1∆B2 expression was weakly detectable in the transgenic flies, even in progeny produced from crossing the RNA1∆B2-transgenic flies with flies expressing potent Gal4 ‘driver’ lines (Fig. 2b, lanes 5 and 6 versus lanes 1 and 2). Similarly, production of RNA2 by the RNA1-encoded protein A was also weakly detectable in flies containing both the RNA1∆B2 and the RNA2 transgenes (Fig. 2b, lane 7 versus lane 8). Furthermore, as anticipated, flies expressing both transgenes were viable and fertile (Fig. 3a,b). These data indicate that the considerable accumulation of FHV RNAs (and hence produc-tion of infectious virus) in flies expressing both wild-type viral RNAs (Fig. 2b, lane 7) is dependent on B2 and that inactivation of this viral protein substantially reduces replication of all viral RNAs and hence virus production, allowing the transgenic flies to efficiently control amplification of viral RNAs and to survive.

RNAi controls accumulation of FHV RNAs in drosophilaThe 106–amino acid protein B2 has been shown in vitro to bind dsRNA tightly and to block Dicer-mediated cleavage of dsRNA and loading of siRNA into the RISC27,28. The notable consequences of mutations inactivating B2 in FHV suggested that RNAi is critical in controlling virus amplification in vivo. In support of that hypothesis, we detected siRNA corresponding to FHV RNA1 in transgenic fly extracts and in FHV-infected flies (Fig. 3c,d). Detection of siRNA after virus infec-tion has been reported in FHV-infected tissue culture S2 cells and has been correlated with the quantity of dsRNA1 produced in the cells24.

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Figure 2 The FHV protein B2 is required for successful viral RNA amplification in flies. (a) RNA1, RNA2 and RNA1∆B2 constructs used for generating transgenic flies. RNA1∆B2 contains two point mutations that inactivate B2 without affecting RNA1-encoded protein A. (b) RNA hybridization for expression of viral RNA1, RNA2 and RNA3 in transgenic flies (lanes 1–8) versus virus injection (FHV inj; lanes 9–13). *, negative-sense dimeric replicative intermediates26. (c) RNA hybridization to detect positive- and negative-sense viral RNA in transgenic flies. The oligonucleotide probe used to detect negative-sense RNA1 does not cover RNA3. (d) Immunoblot for expression of FHV capsid protein in transgenic flies and FHV-infected flies. Right margin, molecular size in kilodaltons (kDa). yolkG4,yolk-Gal4 driver line. Representative of two (c,d) or three (b) independent experiments.

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That result probably explains the lack of detection of siRNA in FHV RNA1∆B2–transgenic flies, because we detected little dsRNA in those flies. However, normal accumulation of RNA1∆B2 and RNA3 was com-pletely restored when transgenes for the viral RNAs were expressed on a Dicer-2 mutant background, thus establishing in vivo that B2 functions to counter the effects of Dicer-2 (Fig. 3e).

In mammals, treatment of cells with siRNA targeting viral RNA has been shown to confer some protection against virus infection17–22. We thus anticipated that transgenic flies expressing RNA1∆B2 would have activated RNAi machinery, including a ready-made RISC complex for targeting possible single-stranded virus RNA. Indeed, flies expressing RNA1∆B2 were more resistant to infection than were wild-type flies (Fig. 3f). The protection, however, was not 100%, as all the flies eventu-ally succumbed to infection, although with delayed kinetics. However, the transgenic flies showed no increased protection against infection with a different virus, DCV (Supplementary Fig. 1 online), consistent with the interpretation that the partial protection conferred by the expressed FHV RNA1∆B2 was sequence specific to FHV.

Susceptibility of Dicer-2 mutant flies to RNA virus infectionWe next challenged Dicer-2 mutant flies with a low dose of FHV (2 × 102 PFU) and noted an increase of sensitivity to FHV infection, as flies succumbed 1–2 d earlier than did wild-type control flies (Fig. 4a). We noted a two- to threefold increase for both RNA1 and RNA3 in the Dicer-2 mutant flies compared with that in wild-type flies at early time points (Fig. 4b,c). The difference in viral load in wild-type versus Dicer-2 mutant flies was less apparent at later stages of infection (days 5 and 6), possibly reflecting the abrogation of functional Dicer-2 by B2 in wild-type flies. Loss-of-function mutant flies for the Jak Hopscotch are more susceptible to DCV infection than are wild-type control flies, and increased lethality correlates with the absence of induction of

many drosophila genes, including the vir-1 marker gene11. We there-fore monitored vir-1 induction in Dicer-2 mutant flies but did not note any substantial reduction compared with that of wild-type control flies (Fig. 4d). There was an even stronger susceptibility to infection when we challenged flies with a high dose of FHV (2 × 106 PFU), as Dicer-2 mutants died 3 d earlier than did wild-type control flies (Fig. 4e). There was a modest increase in viral RNA at the early stages of the infection in Dicer-2 mutant flies compared with that in control flies (Fig. 4f,g). We noted stronger induction of vir-1 in Dicer-2 mutant flies than in wild-type control flies after infection with a high dose of FHV (Fig. 4h). Dicer-2 mutant flies also succumbed more rapidly than wild-type control flies when they were challenged with a second RNA virus, DCV, and mutant flies contained a higher virus load (Fig. 4i,j). Induction of vir-1 was not reduced in Dicer-2 mutant flies compared with wild-type control flies (Fig. 4k).

As shown here and before7,9,11, the FHV and DCV RNA viruses cause lethal infection in drosophila. In contrast, preliminary experi-ments indicated that intrathoracic injection of a third RNA virus, SINV, failed to kill the flies. SINV is an alphavirus (enveloped, positive-stranded genomic RNA) that infects vertebrate hosts and mosquitoes (Aedes spp.), thereby serving as an effective transmis-sion vector. We injected 5 × 102 PFU of SINV intrathoracically into wild-type flies and monitored replication of the viral RNA (Fig. 5a). We confirmed the presence of infecting particles and viral antigens in infected drosophila by electron microscopy and immunofluores-cence (Fig. 5b,c). Although we confirmed that SINV did not kill the infected wild-type flies, infection of Dicer-2 mutant flies led to some 70% mortality (Fig. 5d), which was associated with a considerable increase in viral load (Fig. 5e,f). The susceptibility of Dicer-2 mutant flies to infection was specific to the viruses, as the mutant flies were as susceptible as wild-type flies to infection by pathogenic bacteria (Enterobacter cloacae or Staphylococcus aureus) or fungi (Beauveria bassiana; data not shown).

DISCUSSIONOur data have demonstrated that Dicer-2 is part of a potent effector mechanism in vivo for controlling virus infection in drosophila. Three published studies have indicated involvement of RNAi in antiviral silencing in the nematode Caenorhabditis elegans. Those studies have shown that worms with mutations in rde-1 (which encodes a member of the Argonaute family) or rde-4 (which encodes a dsRNA-binding protein facilitating the loading of siRNA onto the RISC) contain higher

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Figure 3 RNAi-mediated resistance of drosophila to infection with FHV. (a) Survival curves of groups of 20 flies (2 d of age) containing various transgenes (key); flies were monitored daily for survival. (b) Survival curves of flies injected with hemolymph transferred from flies transgenic for RNA1;RNA2 or RNA1∆B2;RNA2 or wild-type flies (WT). Curves for wild-type and RNA1∆B2;RNA2 are superimposed. This experiment was done on two groups of eight flies for each genotype. (c,d) RNA hybridization to detect siRNA of viral origin in FHV RNA1–transgenic flies (c) andFHV-infected wild-type flies 5 d after infection (d). rRNA (c, bottom), negative image of ethidium bromide staining of ribosomal RNA (loadingcontrol). Blots were stripped and rehybridized with a probe detecting the ‘micro RNA’ mir-1 (d, bottom). Right margins, size in nucleotides (nt).(e) RNA hybridization showing accumulation of viral RNA in the absence of B2 in Dicer-2 mutant flies. (f) Survival curves of groups of 12 wild-type or transgenic flies injected intrathoracically with 2 × 106 PFU of FHV. Results are expressed as the percent of surviving flies 9 d after infection (error bars indicate s.d. of quadruplicate samples). P = 0.0002, wild-type versus RNA1∆B2. Representative of two (b–d) or three (a,e) independent experiments.

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viral loads after infection with FHV or the rhabdovirus vesicular sto-matitis virus27,29,30. However, any potential benefit of RNAi for infected worms was not addressed in those studies.

By using FHV and drosophila as a model system, we have shown here that point mutations inactivating the viral protein B2 or the host protein Dicer-2 have substantial effects on viral replication and on the resistance of flies to infection. Although B2 has been shown to bind tightly to dsRNA and to prevent its cleavage by Dicer in vitro27,28, the data presented here have demonstrated critical involvement of B2 in countering the Dicer-2-dependent viral RNA silencing mechanism in vivo. Unfortunately, for technical reasons, an initial attempt to express both the RNA1∆B2 and RNA2 transgenes on a Dicer-2 mutant back-ground, to demonstrate restoration of the accumulation of viral RNAs and a similar effect on virulence, was unsuccessful. Nevertheless, we have formally demonstrated here the importance of Dicer-2 in prevent-ing accumulation of FHV RNA in vivo by showing substantial accu-

mulation of FHV RNA1 and RNA3 in the absence of B2 in Dicer-2 mutant flies.

We have further confirmed the potent antiviral activity of the Dicer-2-dependent RNAi mechanism with two other insect RNA viruses, DCV and SINV, indicating that Dicer-2 has broad antiviral functions in dro-sophila. In particular, our data using SINV have shown that the outcome of the infection (death versus recovery) depended on the presence of a functional Dcr-2 gene. Although we have focused here on drosophila, our findings may be relevant to other insects, including disease vectors that transmit viruses to mammals, including humans31. Indeed, increased viral loads have been reported in Anopheles gambiae mosquitoes with silencing of the gene encoding the Argonaute protein AGO2 (ref. 32), which functions together with Dicer-2 in the RNAi pathway33,34.

Our results, in conjunction with the information now available on RNAi, particularly in plants13,14, lead us to propose that in flies Dicer-2 detects and cleaves newly synthesized viral dsRNA, generating siRNA

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Figure 4 Dicer-2 mutant flies are more sensitive than wild-type flies to virus infection. (a–h) Survival and viral RNA production of groups of 20 Dcr-2L811fsX mutant flies (Dcr-2–/–) or wild-type flies injected with 2 × 102 PFU (a–d) or 2 × 106 PFU (e–h) of FHV. (a,e) Survival curves. Data represent the mean and s.d. of triplicates. P = 0.018, 0.023, 0.016 and 0.025, wild-type versus Dicer-2 mutant for days 9–12, respectively. (b,c,f,g) RNA hybridization and quantification of viral RNA, analyzed with FHV RNA1 (b,f) or RNA3 (c,g) probes and normalized to signals for RpL32. RNA1 expression at day 6 in wild-type flies was arbitrarily considered 100%. (d,h) RNA hybridization and quantification of induction of vir-1 expression. Expression of vir-1 was normalized to signals for RpL32 and maximum expression in wild-type flies was arbitrarily considered 100%. (i) Survival curves of groups of 20 Dicer-2 mutant or wild-type flies challenged by intrathoracic injection of DCV (1 × 104 of a 50% lethal dose). Data represent the mean and s.d. of triplicates. One representative experiment of three. (j,k) RNA hybridization and quantification of accumulation of DCV RNA (j) and induction of vir-1 (k) in groups of 20 Dicer-2 mutant and wild-type flies. Results represent the mean and s.d. of three experiments. P = 0.049 and 0.028, viral load in wild-type versus mutant flies at days 2 and 3, respectively. Induction of vir-1 induction is enhanced in Dicer-2 mutant flies at days 1–3 (P = 0.034, 0.0002 and 0.028, respectively).

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that then specifically recognizes viral RNA and ‘guides’ the RISC to degrade the viral RNA. That proposed mechanism is supported more strongly by data obtained using plants, in which a nucleic acid–based antiviral defense was first described35. In particular, A. thaliana plants deficient in the Dicer factor DCL2 have increased susceptibility to infec-tion by the RNA virus turnip crinkle virus, with higher viral titers and a more deleterious disease phenotype than that of wild-type plants. However, DCL2-mutant A. thaliana are as susceptible as wild-type plants to infection by two other RNA viruses, turnip mosaic virus and cucumber mosaic virus, indicating that DCL2 does not have a general function in antiviral defenses against RNA viruses36.

We have demonstrated an opposite result for drosophila, for which all cases tested so far have indicated that Dicer is a global antivirus defense mechanism. The difference in results obtained with plants and drosophila probably reflects the fact that A. thaliana expresses four DCL factors, which may have partially overlapping functions37. In contrast, D. melanogaster has two Dicer genes: Dcr-1, which controls the pro-duction of ‘micro RNA’ and accomplishes important developmental functions; and Dcr-2, which controls the production of siRNA34 and participates in the control of viral infection. In contrast, there are no indications thus far that the sole Dicer protein found in mammals, which is essential for development38, participates in the control of viral infections. Instead, mammals seem to have a diverse set of cytosolic receptors (RIG-I and MDA5) and transmembrane receptors (Toll-like receptors 3, 7, 8 and 9) that recognize viral RNA or DNA and trigger antiviral responses39,40.

Despite its importance, as demonstrated here, RNAi is certainly not the sole effector mechanism controlling virus infection in flies. The modest increase in viral RNA in FHV-infected Dicer-2 mutant flies, in contrast to the considerable effect on survival, was unexpected. Further experiments will be needed to determine whether the small differ-

ences in viral RNA concentrations in whole flies reflect tissue-specific requirements for Dcr-2 and can explain the enhanced death of infected flies or if they indicate that Dicer-2 exerts an additional function beneficial to the host other than ‘dicing’ viral RNA.

In addition to RNAi, other antiviral effec-tor mechanisms in plants and metazoans have been characterized, including programmed cell death, which has been reported to partici-pate in the control of viral infections and can be blocked by specific viral inhibitors such as the baculovirus caspase inhibitor p35 (ref. 41). Furthermore, as discussed above, infection of drosophila with DCV triggers induction of some 150 genes by a factor of two or more. At least some of those genes encode proteins that participate in controlling the infection, an hypothesis supported by the fact that the genes are not induced after virus infection in Jak-deficient flies, mutant flies that have higher viral loads than wild-type control flies and are generally more susceptible to infection11.

Strictly speaking, there is at present evidence for two types of responses to virus infection in drosophila: degradation of viral RNA by the RNAi machinery and cytokine-medi-ated induction of many genes (via hopscotch-encoded Jak activated by the gp130-related cytokine receptor Domeless), some of which

may counter viral infection. The coexistence of those two types of response may reflect an important difference in RNAi in plants versus drosophila: whereas RNAi is cell autonomous in drosophila42, in plants the RNAi response triggered in infected cells spreads systemically to the plant to induce protective RNAi at distant sites43,44. That cell-to-cell transfer of the silencing signal is essential for the host to counter viral infection, as the presence of dsRNA is in most cases detected after viral replication at a stage at which the cells may not succeed in blocking or destroying the virus. We propose that in drosophila, RNAi functions to limit viral replication in infected cells and is coupled to other defense mechanisms triggered by cytokine signaling in uninfected cells. A prin-cipal challenge for future work will be to elucidate how the integration of these responses allows drosophila to resist viruses.

METHODSFly strains and infections. OregonR and yw flies were used as wild-type controls. Dcr-2L811fsX mutant flies (Dcr-2–/–) have been described34. The ‘yolk-Gal4’ driver line has been described45. Flies were fed standard cornmeal-agar medium at25 °C. Adult flies 4–6 d of age were used in infection experiments. Viral stocks were prepared in 10 mM Tris-HCl, pH 7.5. Infections were done as described9,11 by injection (Nanoject II apparatus; Drummond Scientific) of 4.6 nl of a viral suspension (DCV, 2 × 1011 of a 50% lethal dose/ml; FHV, 4 × 1011 PFU/ml; SINV, 1 × 108 PFU/ml) into the thoraces of adult flies. Injection of the same volume of 10 mM Tris-HCl, pH 7.5, was used as a control. For hemolymph transfer experi-ments, hemolymph was collected from donor flies using the same apparatus and was immediately injected into host flies. Infected flies were then incubated at22 °C. All survival experiments were done at 22 °C.

Construction of FHV RNA–transgenic lines. The pUAS-T vector46 was first modified to shorten the length of hsp70 sequences separating the transcriptional start site from the polylinker. A PCR fragment was amplified using pUAS-T as a template and the primers 5′-GCTTGGCTGCATCCAACGC-3′ (sense) and5′-AAAGAATTCTTTCGCTTAGCGACGTGTTC-3′ (antisense); the product

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Figure 5 Dicer-2 confers protection of drosophila against infection with SINV. (a) RNA hybridization of SINV RNA accumulation in drosophila tissues after intrathoracic injection of 5 × 102 PFU of SINV.(b) Immunofluorescence for SINV E1 glycoprotein (green) in fat body cells. Scale bar, 40 µm.(c) Transmission electron microscopy of SINV particles detected in muscle cells surrounding the midgut (arrows). Scale bar, 500 nm. Representative of three (a) or two (b,c) experiments. (d) Survival curves of flies challenged by intrathoracic injection of 5 × 102 PFU of SINV and monitored daily. Data represent the mean ± s.d. of three groups of 20 flies. This experiment was repeated twice with similar results. (e) RNA blot for SINV genomic RNA obtained from surviving flies on day 15 of the experiment in d. RpL32 serves as a loading control. (f) RNA hybridization and quantification of accumulating SINV RNA in Dicer-2 mutant and wild-type flies (20 flies for each sample). SINV RNA at day 8 inwild-type flies was arbitrarily considered 100%.

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was digested with SphI and EcoRI and was used to replace the corresponding frag-ment in pUAS-T. In the resulting vector (pJL309), the EcoRI site of the polylinker is located 42 base pairs downstream of the hsp70 transcription start site. FHV RNA1 or RNA2 was subcloned in this vector as an EcoRI or BamH1 (respectively) fragment from the vector pBacPAK9-R1δ or pBacPAK9-R2δ, which contains the cDNA of FHV RNA1 or RNA2 followed by the hepatitis delta virus (HδV) ribozyme cleavage site47. For the construction of the B2 mutations (RNA1∆B2), the plasmid pBacPAK9-R1δ was used to introduce two point mutations at posi-tions 2739 (T to C) and 2910 (C to A) of RNA1. These mutations closed the open reading frame of B2 on the subgenomic RNA3 by changing the initiating Met at position 1 to Thr and the Ser at position 58 to a stop codon and did not affect replication of RNA1 and RNA3 (ref. 48). The mutations were introduced sequen-tially by overlap extension PCR with Pfu polymerase. Primers were designed such that the final PCR product contained nucleotides 1,998–3,107 of RNA1, the entire HδV ribozyme sequence and nucleotides 1,261–1,373 of pBacPAK9. The PCR product was purified, was digested with ApaI and NotI and was ligated into pBacPAK9-R1δ digested with the same enzymes. After transformation ofE. coli DH5-α, plasmid DNA was purified and sequenced to confirm the presence of the desired nucleotide changes. The resulting three constructs were injected into embryos of a w– strain (w1118) to obtain transgenic lines. The percentage of transgenic lines obtained was as expected for the UAS-RNA2 construct but was much lower for UAS-RNA1 (three lines for more than 200 embryos injected) and UAS-RNA1∆B2 (two lines for 200 embryos injected).

RNA analysis. RNA was isolated with TRIzol reagent (Gibco-BRL) according to the manufacturer’s instructions and was analyzed by RNA blot using standard procedures. The primers used to generate the cDNA probes were as follows: RpL32 forward, 5′-GACGCTTCAAGGGACAGTATCTG-3′, and reverse, 5′-AAACGCGGTTCTGCATGAG-3′; FHV RNA1 and RNA3 forward, 5′-GGACC-GAAGTGCGGTGATG-3′, and reverse, 5′-CAGTTTTGCGGGTGGGGGG-3′; FHV RNA2 forward, 5′-CGGAACAACTTCAACATCTAGG-3′, and reverse, 5′-AACCAAATTGGGCTTATTGTGG-3′; SINV forward, 5′-AAGAGCGAC-CAAACGAAGTGG-3′, and reverse, 5′-CACATCTAGGAAACTGGTAGTG-3′. The oligonucleotides used to detect positive- and negative-sense FHV RNA1 were 5′-TCTGCCCTTTCGGGCTAGAAC-3′ and 5′-CAAGACGCCCATTT-GATGAAGC-3′, respectively. RNA blots were quantified with a Bioimager (FujixBAS 2000). Signals for specific RNA signals were normalized to signals for the ‘housekeeping gene’ RpL32, used as a loading control. For siRNA detection, 15 µg of total RNA was separated by electrophoresis through 17.5% polyacrylamide-urea gels. Ethidium bromide staining was used to ensure equal loading. The primers described above were used to prepare the FHV RNA1 probe to detect siRNA. A DNA oligonucleotide complementary to the ‘micro RNA’ mir-1 was end-labeled with [γ-32P]ATP using T4 kinase and was used as a size marker for the position of siRNA.

Antibodies and immunostaining. Polyclonal antibodies to FHV capsid have been described47. Polyclonal antibodies to SINV glycoprotein E1 were a gift from J. Strauss (California Institute of Technology, Pasadena, California). Alexa Fluor 488– or Alexa Fluor 546–labeled secondary antibodies were purchased from Molecular Probes and were used at a dilution of 1:500. Standard procedures were used for immunostaining. Flies were dissected in PBS containing 4% formaldehyde and were fixed for 20 min. After being washed with PBS containing 0.1% Triton-X100 and blocked with 1% BSA for 30 min, samples were incubated overnight at4 °C with antibody to FHV or to SINV (1:500 dilution). Labeling with second-ary antibodies was done at 25 °C for 4 h. Slides were mounted in Vectashield medium (Vector Laboratories) and were examined by confocal microscopy (Zeiss LSM510).

Statistical analysis. Student’s t-test was used for statistical analysis.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank E. Santiago for technical assistance; S. Ozkan and R. Walther for help with transgenesis; D. Zachary for help with electron microscopy; R. Carthew for Dicer-2 mutant lines; J. Strauss for anti-SINV; J. McCauley for titered stock of SINV; and O. Voinnet for discussions and comments. Supported by Centre National de la Recherche Scientifique, Ministère de la Technologie et de l’Enseignement Supérieur (ACI Microbiologie to J.-L.I.), the National Institutes of

Health (GM053491 to A.S.), a Centre National de la Recherche Scientifique post-doctoral fellowship (D.G.A.) and the Ministère de la Recherche du Grand-Duché du Luxembourg (C.D.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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