The Jak-STAT signaling pathway is required but notsufficient for the antiviral response of drosophila

Catherine Dostert1, Emmanuelle Jouanguy1,2, Phil Irving1, Laurent Troxler1, Delphine Galiana-Arnoux1,Charles Hetru1, Jules A Hoffmann1 & Jean-Luc Imler1

The response of drosophila to bacterial and fungal infections involves two signaling pathways, Toll and Imd, which both activate

members of the transcription factor NF-jB family. Here we have studied the global transcriptional response of flies to infection

with drosophila C virus. Viral infection induced a set of genes distinct from those regulated by the Toll or Imd pathways and

triggered a signal transducer and activator of transcription (STAT) DNA-binding activity. Genetic experiments showed that the

Jak kinase Hopscotch was involved in the control of the viral load in infected flies and was required but not sufficient for the

induction of some virus-regulated genes. Our results indicate that in addition to Toll and Imd, a third, evolutionary conserved

innate immunity pathway functions in drosophila and counters viral infection.

Innate immunity is an ancient defense that enables multicellularorganisms to detect and fight infectious microbes. The fruit flyDrosophila melanogaster is a powerful model for deciphering themolecular mechanisms of the innate immune system. The fat body,a functional equivalent of the mammalian liver, produces a ‘cocktail’of small cationic antimicrobial peptides in response to infection. Thegenes encoding these peptides are regulated by two evolutionaryconserved signaling pathways1–5. Infections by fungi and mostGram-positive bacteria activate the Toll pathway, which controls theexpression of genes encoding the antifungal peptides Drosomycin andMetchnikowin (as well as many other genes) through Dif, a memberof the transcription factor NF-kB family. In contrast, Gram-negativebacteria mainly activate the immune deficiency (Imd) pathway, whichgoverns expression of the genes encoding many antibacterial peptidessuch as Diptericin and Drosocin via a distinct NF-kB-related protein,Relish. The Toll pathway bears notable similarities to the Toll-likereceptor–interleukin 1 receptor signaling pathway and involves, inaddition to the Toll receptor, homologs of the adaptor moleculeMyD88, the kinase IRAK (encoded by pelle) and the inhibitor IkB(encoded by cactus). In contrast, the Imd pathway is strongly remini-scent of the tumor necrosis factor receptor pathway and includeshomologs of the death domain protein FADD, caspase-8, the kinaseTAK1 and the IKK signalosome. The death domain of Imd itself ismost closely related to that of the essential tumor necrosis factor signal-ing component RIP. In addition to infection with bacteria and fungi,insects are also often exposed to viral infections and in some cases cantransmit viral diseases to vertebrate hosts, including humans6–10.

There is growing interest in viral infections in drosophila8,10–12. Aviral infection model based on the drosophila C virus (DCV; a

member of the dicistroviridae, which share many characteristics withpicornaviridae6,13) has been described. In contrast to bacterial andfungal infection, intrathoracic injection of a suspension of DCV doesnot lead to the inducible secretion of antimicrobial peptides in thehemolymph of infected flies11. These data suggest that flies use adistinct mechanism to counter viral infections. To gather moreinformation about this mechanism, we examined the global hostresponse to DCV infection using high-density DNA microarrays.

Here we show that DCV infection triggers a different transcriptionalresponse in flies than that triggered by bacteria and fungi, suggestingthat drosophila can recognize viral infection and mount a dedicatedresponse. The 5¢ upstream sequences of several DCV-induced genescontained consensus binding sites for the transcription factor STAT14,and DCV infection induced STAT DNA-binding activity. Expressionof some DCV-induced genes required the Jak kinase encoded by hop-scotch (hop)15. These genes were not induced in flies expressing theconstitutively active Tumorous lethal (Tum-l) allele of hop, suggestingthat activation of the Jak-STAT pathway is required but not sufficientfor the triggering of an antiviral response. Flies with hop loss-of-function mutations contained more DCV than control wild-type fliesand succumbed faster to infection. These data indicate that in additionto Toll and Imd, a third, evolutionary conserved innate immunitypathway functions in drosophila and counters viral infection.

RESULTS

DCV triggers a transcriptional response in drosophila

We injected male adult flies with a viral suspension of DCV at a dose(1 � 104 the 50% lethal dose (LD50)) that kills flies in approximately10 d (Supplementary Fig. 1 online) or with buffer only. We extracted

Published online 7 August 2005; doi:10.1038/ni1237

1Centre National de la Recherche Scientifique UPR9022, Institut de Biologie Moleculaire et Cellulaire, 67000 Strasbourg, France. 2Present address: Institut National dela Sante et de la Recherche Medicale U550, Faculte Necker, 75015 Paris, France. Correspondence should be addressed to J.-L.I. (jl.imler@ibmc.u-strasbg.fr).

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mRNA 24 h and 48 h after injection and compared the globaltranscriptional profiles of buffer- or DCV-injected flies using Affyme-trix DNA chips. At this stage the flies were still healthy, as indicated bythe finding that the first flies started to die only 3 to 4 d later, and oneof the main target organs for DCV, the fat body10,11, was stillfunctional, as indicated by the finding that both the Toll and theImd pathways could be activated by septic injury (SupplementaryFig. 1 online). Some 140 genes were induced or were upregulated by afactor of two or more in DCV-infected flies, indicating that drosophilaresponded to DCV infection (Fig. 1a and Supplementary Table 1online). Other genes were apparently repressed by DCV infection(data not shown; complete data set available at http://www.ncbi.nlm.nih.gov/geo/, accession number GSE2828). Of the genes that wereupregulated, two thirds were not upregulated by bacterial or fungalchallenges, suggesting a distinct type of response16–18. In addition,most genes encoding antimicrobial peptides regulated by the Tolland Imd pathways were not upregulated or were only weaklyupregulated by DCV infection (Fig. 1b,c), confirming the publishedproteomic analysis11.

The DCV-upregulated genes encoded proteins across a broad rangeof functional categories (Supplementary Fig. 2 and SupplementaryTable 1 online). Notably, one third had unknown functions. Threepatterns of upregulated genes ‘stood out’: some genes (such as Frost)showed similar induction by septic injury and viral infection;some genes (such as Attacin) showed much less induction by DCVinfection than by septic injury with bacteria; and some genes (such asvirus-induced RNA 1 (vir-1), discussed below) showed considerableupregulation by DCV infection but no substantial induction by septicinjury (Fig. 1c). The last group of genes was of particular interest andrepresented a newly identified population of immune regulated genesin drosophila.

Induction of vir-1 by viral infection

We next monitored the expression of vir-1 in response to variousinfectious challenges. Because DCV infection led to tissue damage anddeath of the flies, in contrast to infection with the nonpathogenicEscherichia coli and Micrococcus luteus (Fig. 1c), we used pathogenicGram-negative bacteria (Enterobacter cloacae, Erwinia carotovora andSerratia marcescens), pathogenic Gram-positive bacteria (Staphylococ-cus aureus and Enterococcus faecalis) and pathogenic or opportunisticfungi (Beauveria bassiana and Candida albicans) for comparison inour infection studies. We monitored expression of vir-1 over time,with the last time point in the experiments corresponding to the 50%lethality period. Flies infected with DCV began to die only 6–7 d afterinfection (Supplementary Fig. 1 online). We found that vir-1 was notinduced by pathogenic bacteria or fungi, indicating that it wasspecifically induced as a result of ‘sensing’ either DCV infection or aspecific aspect of DCV-induced pathogenesis (Fig. 2a and data notshown for S. marcescens). Expression of vir-1 was not modified bymany stresses19, such as heat shock, cold shock, mechanical pressure,dehydration or ultraviolet irradiation (Fig. 2b and data not shown).However, infection of flies with another insect virus, flock housevirus (FHV; a member of the nodaviridae family)20, substan-tially induced vir-1 expression (Fig. 2c). We concluded that viralinfection in drosophila led to the induction of a specific set of genes,including vir-1.

A virus-responsive element in the vir-1 promoter

The data reported above raised the issue of the signaling pathwayinvolved in the regulation of the response to viral infection. Genome-wide expression profiling studies have shown that the dynamictranscriptional patterns after microbial challenge can be separatedinto distinct, signaling pathway–specific responses18. A first cluster of

a

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Figure 1 DCV infection triggers a transcriptional response in drosophila. (a) Venn diagram of the genes induced or upregulated (more than twofold) 24 or

48 h after DCV challenge or other microbial infection (with the Gram-negative bacteria E. coli, the Gram-positive M. luteus or the fungus B. bassiana; bottom

circle). Data for bacteria and fungi are from ref. 16 (copyright 2005, National Academy of Sciences, USA) and include the total set of genes induced at the6-, 12- or 48-hour time point. (b) Expression profiles of a set of previously described genes regulated by septic injury (Drs to TotM), along with four DCV-

induced genes (?; CG31764 to CG2081). Dashed horizontal line, twofold induction threshold. Bottom, signaling clusters to which these genes belong (as

defined in ref. 18). Data represent mean of triplicates and the error bars indicate standard deviation. Drs, Drosomycin; IM2, drosophila immune-induced

molecule 2; CecA1, Cecropin A1; Dpt, Diptericin; Dro, Drosocin; Act88F, Actin 88F; fln, flightin; Mlc1, myosin light chain 1; TpnC41C, Troponin C. (c) RNA

blot analysis of the response to septic injury with a mixture of E. coli and M. luteus (E.c.M.l.), injection of Tris buffer or injection of DCV. This experiment

was repeated three times and a representative blot is presented. Fst, Frost; AttA, Attacin-A; RpL32, ribosomal protein L32 (loading control). NI, uninfected.

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genes induced is composed of Toll targets; two additional clusters areregulated by the Imd pathway, which bifurcates ‘downstream’ of Imd-Tak1 into Relish-dependent (Imd-Rel group) and Jnk-dependent (cytogroup) branches; and a fourth group is composed of targets of the Jak-STAT pathway18. We selected conventional ‘readouts’ for each of theseclusters and found that none was strongly induced after DCVinfection (Fig. 1b).

We next focused our attention on the promoters of the virus-regulated genes and in particular on vir-1, which was substantiallyinduced by DCV infection. The gene vir-1 is a previously unrecognized

transcript of CG31764, located at cytologicbands 33E3-4 on the ‘left arm’ of the secondchromosome (Fig. 3a and SupplementaryFig. 3 online). Rapid amplification of cDNAends (RACE)–PCR and S1 nuclease mappingshowed that vir-1 transcription was initiatedin a small exon 0 and terminated at the sameposition as the RA and RB transcripts ofCG31764. The vir-1 transcript contained anATG in a favorable Kozak context that enabledsynthesis of a putative 155–amino acid proteinidentical to the C-terminal part of theproducts of CG31764-RA and CG31764-RB(Supplementary Fig. 3 online). The deducedprotein did not contain any structural motifthat might provide a hint as to its possiblefunction. A 2,495–base pair (bp) fragment ofthe vir-1 upstream sequences was able toconfer DCV-inducible expression to a greenfluorescent protein (GFP) or b-galactosidasereporter gene in transgenic flies, indicatingthat the promoter of vir-1 contained a virus-responsive element (Fig. 3b). Experimentsstudying vir-1 promoter truncation further

located the DCV-responsive element to a 190-bp fragment locatedbetween position –516 and position –326 (Fig. 3c). We noted aputative drosophila STAT-binding site14 (5¢-TTTCTAAGAAA-3¢,dyad symmetric sequence underlined; Supplementary Table 2 online)at position –342 in this fragment. Constitutive expression of vir-1occurred in drosophila macrophage-like S2 cells (Supplementary Fig.3 online), and these cells had a constitutive DNA-binding activity to aconsensus STAT site (Supplementary Fig. 4 online). The introductionof point mutations in the consensus drosophila STAT binding site atposition –342 considerably reduced the activity of the vir-1 promoter

vir-1

RpL32

NI 24 48 NI 6 24 120 NI 6 24 96 NI 24 48 168 NI 24 48 96 NI 6 24 NI 6 24 72

DCV E. cloacae E. carotovora C. albicans B. bassiana S. aureus E. faecalisa

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Figure 2 Selective induction of vir-1 after virus infection. (a) RNA blot of vir-1 and RpL32 in flies

infected with DCV, the Gram-negative bacteria E. cloacae or E. carotovora, the fungi C. albicans

or B. bassiana or the Gram-positive bacteria S. aureus or E. faecalis. Above lanes, time of RNA

extraction (after challenge). (b) RNA blot analysis of vir-1 expression in response to infection with

DCV, heat shock (HS; 1 h or 4 h at 37 1C), cold shock (CS; 4 h at 4 1C), mechanical pressure

(MP; squeezing of the thorax with forceps followed by RNA extraction 6 or 16 h later) or dehydration(DH; placement of flies on silica gel for 90 min at 25 1C followed by RNA extraction 6 or 16 h later).

(c) RNA blot analysis of vir-1 expression in flies injected with buffer or a FHV suspension. Above

lanes, time of RNA extraction (after challenge). Each experiment was done at least twice with similar

results. NI, uninfected; UT, untreated.

b

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Figure 3 Characterization of vir-1. (a) The newly identified transcript of CG31764,

vir-1, uses a different promoter to initiate transcription in a small exon 1 that splices

in the last exon of the gene, which is shared by the CG31764-RA and CG31764-RB

transcripts. Top, cytologic bands in the 33E region of the second chromosome;

beside bent arrows, positions of the neighboring genes Pkd2, CG6405 and CG5446.

(b) A 2,495-bp fragment of sequences 5¢ of the vir-1 transcription start site confers

DCV-inducible expression to a GFP transgene. Transgenic flies were injected withTris (uninfected (NI)) or infected with DCV and were examined 72 h later.

(c) Localization of the virus-response element in the vir-1 promoter. Transgenic

flies expressing lacZ under the control of truncated fragments of the vir-1 promoter

were challenged with DCV, and b-galactosidase activity in single flies was measured

72 h later with a fluorimetric assay49. Results are for two independent lines for

each construct (in parentheses: standard deviation; number of flies analyzed).

(d) Localization of putative STAT92E-binding sites in the proximal upstream

sequences of DCV-induced genes.

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in transfected S2 cells, indicating the importance of this DNA motif inthe regulation of vir-1 expression (Supplementary Fig. 4 online). Wealso noted putative STAT binding sites in the proximal upstreamregions of several other virus-regulated genes (such as CG9080 andCG12780), although there were some exceptions (such as CG2081;Fig. 3d and Supplementary Table 2 online).

Dissection of control and DCV-infected flies transgenic for a lacZreporter gene under control of the vir-1 promoter showed stronginducible staining in the ventral epidermis (Fig. 4a). Double-labelingexperiments with antibody to b-galactosidase (anti-b-galactosidase)and anti-DCV confirmed expression of the reporter gene in the ventralepidermis of DCV-infected flies but failed to detect virus in these cells(Fig. 4b). In contrast, DCV was easily detected in cells of the fat body,as reported before10,11. However, the vir-1 promoter was not induciblein this tissue either in DCV-infected cells or in their neighbors(Fig. 4c,d and data not shown). Similarly, we detected inducible b-galactosidase activity in the oviducts of female flies challenged withDCV but not in epithelial cells forming the periovarian sheath, whichactively supported DCV replication (Fig. 4e,f). These data indicatedthat vir-1 was not induced in the main tissues, which contained DCV.Instead, the data suggested that vir-1 was induced after a signalgenerated by the DCV-infected cells.

Involvement of the Jak-STAT pathway in the antiviral response

The drosophila genome encodes a single STAT factor, STAT92E, whichis regulated by the single drosophila Jak kinase encoded by hop21,22.Electrophoretic mobility-shift assays showed that nuclear extractsfrom whole flies infected with DCV contained increased amounts ofa factor specifically binding to an optimal DNA-binding site for

STAT92E14 (Fig. 5a). This suggested that the Jak-STAT pathway wasactivated during viral infection. To further address the involvement ofthis pathway in the antiviral defense, we studied the response of hopmutant flies15,23 to viral infection and found that induction of vir-1was considerably reduced in loss-of-function hopM38/msvl mutant flies(M38 and msv1 are two alleles of hop that yield viable adults whenused in ‘trans-heterozygous’ combination15; Fig. 5b,c). Althoughinduction of CG12780 (and CG9080; data not shown) was alsoconsiderably reduced or abolished, induction of CG2081 (whichdoes not contain putative STAT-binding sites in its proximal promo-ter; Fig. 3d) was not affected in hop-deficient flies (Fig. 5b). Thesedata indicated that the Jak kinase Hopscotch was required for thevirus-inducible expression of a subset of genes. Neither vir-1 norCG12780 was constitutively expressed in flies expressing the gain-of-function allele Tum-l of hop, which encodes a constitutively activekinase24,25 (Fig. 5b). Notably, Turandot M (TotM)19, which isregulated by the Jak-STAT pathway18,23, was not substantially inducedby DCV infection but it was constitutively expressed in flies express-ing the Tum-l allele of hop. In contrast, infection with FHV ledto substantial induction of TotM (Fig. 5d). These data indicatedthat hop was required but not sufficient for mediating the induc-tion of vir-1 and CG12780. The Jak-STAT pathway exerts impor-tant developmental effects in drosophila and most mutants are notviable. To confirm the finding that induction of vir-1 was mediatedby this pathway, we overexpressed a dominant negative version ofDomeless26, a drosophila homolog of mammalian class I cyto-kine receptors that acts ‘upstream’ of Hopscotch26,27, and noted asignificant and reproducible decrease in vir-1 induction (Fig. 5e).Virus-induced expression of vir-1 was also reduced in flies

*

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a b cX–gal X–gal

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Figure 4 The vir-1 promoter is induced by DCV in the ventral epidermis but not in the fat body. (a) Induction of b-galactosidase activity in the ventral

epidermis of DCV-infected flies (72 h after injection), assessed by X-gal staining. (b) Detection of b-galactosidase and DCV in the ventral epidermis of vir-1–

lacZ flies. Samples from control or DCV-infected flies were double-labeled with anti-b-galactosidase (a–bgal; green; AlexaFluor 488) or anti-VP2 (a–DCV;

red; AlexaFluor 546) and were visualized by confocal microscopy. Scale bar, 50 mm. (c) Expression of b-galactosidase activity in the tracheae (constitutive;

asterisks) and ventral epidermis (inducible; arrowhead) of vir-1–lacZ–transgenic flies, assessed by X-gal staining. Arrows indicate the fat body. (d) Detection

of DCV (green; AlexaFluor 488) in fat body cells of infected flies, visualized by confocal microscopy. Scale bar, 50 mm. (e) Expression of b-galactosidase

activity (X-gal staining) in the female reproductive tract of vir-1–lacZ flies infected with DCV. Arrows indicate the oviduct. (f) Detection of DCV (green;

AlexaFluor 488) in cells of the periovarian sheath, visualized by confocal microscopy with anti-VP2. Scale bar, 100 mm. Original magnifications, �40

(a;e, left), �30 (c) and �25 (e, right). Representative of three independent experiments.

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overexpressing the drosophila homolog of PIAS, a negative regulatorof STAT28 (Fig. 5f,g).

The identification of a mutant strain in which induction of a subsetof genes was substantially impaired allowed us to investigate theprotective effects against virus infection of the transcriptional responsedescribed above. We monitored DCV replication in wild-type andloss-of-function hopM38/msvl flies and reproducibly noted a significantincrease in viral RNA in mutant flies (Fig. 6a). The hop mutant fliesalso contained more capsid proteins than did wild-type flies (Fig. 6b),indicating that the pool of molecules induced by DCV infectionincludes factors that control the viral load in infected flies. We next

monitored the survival of wild-type and hop mutant flies challengedwith DCV and did not note significant differences when we injectedour standard dose of virus (1 � 104 LD50; Supplementary Fig. 5online). This discrepancy in the data regarding DCV accumulationin wild-type and hop mutant flies suggested that the lethality notedfor wild-type flies could be due in part to harmful effects of thestrong response to infection. We therefore injected lower doses ofDCV and found that in these conditions, hopM38/msvl mutant flieswere more sensitive to infection than were wild-type flies. Injectionof 1 � 102 LD50 of DCV induced a weaker response thandid 1 � 104 LD50 in wild-type flies (induction of vir-1 was

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Figure 5 The Jak-STAT pathway is involved in the response to DCV infection. (a) STAT-binding activity of nuclear extracts prepared from control flies (– DCV)

or flies infected with DCV (48 h after infection; + DCV), in the presence (+) or absence (–) of a 50-fold excess of cold competitor (ODBS �50). (b) RNA blot

analysis of the response to DCV infection in wild-type flies (WT) or hop mutant flies (M38/msvl; Tum-l). (c) Real-time quantitative RT-PCR analysis of the

induction of vir-1 in response to infection by DCV in wild-type (WT) and hop mutant (hopM38/msvl) flies. Results were standardized to RpL32, and the value

obtained for infected wild-type flies was considered 100%. Data represent the mean and standard deviation of duplicates. P o 0.01, wild-type versus hop

mutant flies. One representative experiment of three. (d) RNA blot analysis of the expression of the Jak-STAT target gene TotM in response to infection with

DCV or FHV. (e) Quantitative RT-PCR analysis of vir-1 induction in control flies or flies overexpressing a dominant negative version of Dome. DomeDCYT is

expressed under the control of upstream activating sequences (UAS) recognized by the yeast transcription factor Gal4. Gal4 is expressed under the control

of a heat-shock protein promoter. UAS DomeDCYT;hspGal4 flies express DomeDCYT after heat-shock (HS) treatment. Data represent the mean and standard

deviation of duplicates; two independent experiments gave similar results. P o 0.01, DomeDCYT-expressing flies versus controls. (f,g) Induction of vir-1 by

DCV in control flies or flies overexpressing drosophila PIAS (dPIAS), analyzed by RNA blot (f) or quantitative RT-PCR (g), as described in d. RNA blots were

quantified with a BioImager. Data are the results of two independent RNA blots (filled bars) and two independent quantitative RT-PCR analyses (gray bars)

(g). HS, heat-shock treatment.

Figure 6 The Jak-STAT pathway controls the

viral load in DCV-infected flies and resistance to

infection. (a) Quantitative RT-PCR analysis of

the accumulation of DCV RNA in wild-type and

hop mutant flies. Data represent the mean and

standard deviation of duplicates. P o 0.05, wild-

type versus hop mutant flies. One representative

experiment of three. (b) Immunoblot of the

accumulation of VP2 in wild-type or hop mutant

flies. This experiment was done three times withsimilar results. Act b, actin (loading control).

(c) The hopM38/msvl mutant flies succumb faster

than wild-type control flies when injected with a

low dose (1 � 102 LD50) of DCV. Three groups

(15 flies for each genotype) were injected with

DCV and survival was monitored daily. Induction

of vir-1 and accumulation of DCV was assayed by

RNA blot. This experiment was repeated once

with similar results.

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detected only 72 h after injection), and increased viral burden inhopM38/msvl mutant flies correlated with increased susceptibility toinfection (Fig. 6c).

DISCUSSION

Our data suggest that flies respond to DCV infection by inducing adedicated transcriptional response that is mediated in part by the Jak-STAT pathway. In particular, we have shown that DCV triggered adrosophila STAT DNA binding activity in infected flies and thatmutation of this binding site at position –342 in the vir-1 promotersuppressed its activity in S2 cells. Induction of vir-1 was substantiallyreduced in Hopscotch-deficient flies. The physiological relevance ofour findings was demonstrated by the finding that infection with adifferent insect virus, FHV, also induced vir-1 and the established Jak-STAT target gene TotM. These genes are probably involved incontrolling the infection, as shown by the increased viral burdenand accelerated death of hop loss-of-function mutant flies afterinjection of DCV.

These experiments raise the issue of the nature and mode of actionof the induced antiviral molecules. Ectopic overexpression of vir-1 intransgenic flies (or ‘knockdown’ of vir-1 by RNA interference) did notaffect resistance to DCV infection (data not shown). Thus, althoughwe focused our attention on the regulation of vir-1 expression becauseof its considerable inducibility by DCV, we do not have formal proofthat this gene participates in the immune control of viral infection.However, because of its specific induction in response to virusinfections (DCV and FHV), the function of vir-1 warrants furtherinvestigation. Future experiments should test the potential inductionof vir-1 by other viruses, such as DNA viruses, and by otherintracellular pathogens29. Other potential effector mechanisms includethe production of antimicrobial peptides30,31, apoptosis32 or RNAinterference7,33,34. Although our microarray analysis indicated thatsome genes encoding antimicrobial peptides were induced, quantita-tive analysis by RNA blot showed that these genes were only weaklyupregulated; in agreement with those findings, we did not detectantimicrobial peptides in the hemolymph of DCV-infected flies byMALDI-TOF mass spectrometry11. However, CG12780 encodes adeduced protein that shares an N-terminal domain with membersof the Gram-negative binding protein–b-glucan recognition proteinfamily. Two members of this family are essential in the response tobacterial and fungal infections35,36 (M. Gottar and D. Ferrandon,personal communication); future experiments should test whether theGram-negative binding protein–like molecule encoded by CG12780 isinvolved in the detection or neutralization of viral particles. Also, thelist of genes induced by DCV includes Damm, which encodes one ofthe seven drosophila caspases37, suggesting a potential connectionbetween DCV infection and apoptosis. In contrast, none of the genesencoding known components of the RNA interference machinerywere notably upregulated by DCV. These considerations raise thepossibility that functional characterization of the hop-dependent genesselectively induced by DCV infection may demonstrate previouslyunknown antiviral strategies.

Our results have identified a previously unknown function for theJak-STAT pathway in drosophila. This pathway was initially character-ized for its involvement in embryonic segmentation15 and was latershown to control multiple other aspects of development, such as eyeformation, sex determination, spermatogenesis and oogenesis, trachealand gut morphogenesis21,22. Activation of the kinase Hopscotch iscontrolled by the receptor Domeless26,27, which is a member of thefamily of type I cytokine receptors and most closely resembles gp130and the related subunits that participate in the receptors for the

cytokines interleukin 6, LIF, OSM and interleukin 31 (ref. 38).Domeless is activated during development by the secreted factorUnpaired (Upd)39. Activation of the Jak-STAT pathway in responseto bacterial infection has also been reported in insects18,23,40–42. Inbacteria-challenged drosophila, the Jak-STAT pathway regulatesexpression of thiol-ester protein 1 (ref. 41) and the genes TotA andTotM18,23. Bacterial infection induces the hemocyte-specific expressionof the Upd family cytokine Upd3 that is necessary for the Dome-Hop-STAT92E–dependent expression of TotA in the fat body23. However,the biological relevance of these findings remains unclear, becauseTotA is also induced by many stressors, such as heat shock, dehydra-tion, mechanical pressure or injection of detergents19,23, and hopmutant flies control bacterial infections much like wild-type controlflies. Our results have now demonstrated that the Jak-STAT pathway isinvolved in the control of viral infection in drosophila. This pathwaywas initially characterized in mammals for its involvement in inter-feron signal transduction43–45. The importance of this pathway in theinnate control of viral infections in mammals is best demonstrated bygenetic studies linking STAT1 deficiencies in mice and humans withincreased susceptibility to infection46,47. Our data suggest that thefunction of the Jak-STAT pathway in the control of viral infections hasbeen conserved through evolution. Thus, the fruit fly seems to rely onat least three evolutionary conserved signaling pathways, Toll, Imd andJak-STAT, to counter infections by fungi, bacteria and viruses, con-firming the unexpected complexity of the innate immune system inthis model organism.

Our genetic data suggest that detection of viral infection triggersproduction of a cytokine of the Upd family that leads to activation ofDome and Jak-STAT–dependent induction of a set of genes that helpcontrol the viral load in infected flies. Indeed, the vir-1 promoter wasnot induced in DCV-infected cells of the fat body and periovariansheath but was substantially induced in epithelial cells of the ventralepidermis or in the oviduct, in which we failed to detect DCV. Theseobservations are consistent with a model in which vir-1 expression iscontrolled by a cytokine that relays information regarding the infec-tion. This is reminiscent of the response to fungi, in which detection ofinfection by secreted proteins in the hemolymph activates the cytokineSpaetzle, which in turn binds to and activates the Toll receptor, therebytriggering the expression of antifungal factors1–5. Our data have alsoshown that Hopscotch activation (as ‘mimicked’ by expression of thegain-of-function allele Tum-l) was not sufficient to trigger vir-1expression, indicating that the vir-1 promoter may integrate signalsfrom different pathways. This situation is reminiscent of the regulationof the cecropin A, attacin A and defensin genes, which depend on boththe Toll and Imd pathways and show reduced inducibility in Toll-deficient flies but are not constitutively expressed in flies carrying thegain-of-function Tl8 allele48. The existence of at least one otherpathway involved in the response to DCV infection is furthercorroborated by the fact that CG2081 remains fully inducible in hoploss-of-function mutant flies. Ultimately, systematic mutagenesis ofdrosophila and screening based on monitoring the markers vir-1 andCG2081 reported here should lead to identification of the genenetworks involved in the response to DCV infection. Such studieswill presumably demonstrate previously unknown aspects of theinnate antiviral response in mammals.

METHODSFly strains and infections. OregonR and ywDD1;cnbw flies were used as wild-

type controls49. The hop alleles used (M38, msv1 and Tum-l) as well as the UAS-

dpias– and UAS-domeDCYT–transgenic lines have been described23–25. Stocks

were raised on standard cornmeal–agar medium at 25 1C. Adult flies 4–6 d of

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age were used in infection experiments. For heat-shock induction of transgene

expression, flies were placed at 37 1C for 45 min and were allowed to recover for

6 h at 25 1C before immune challenge. Control experiments indicated that

heat-shock treatment did not interfere with the induction of vir-1 (data not

shown). Infections by bacteria and fungi were done as described50. Viral stocks

were prepared in 10 mM Tris-HCl, pH 7.5. Infections were done by injection of

4.6 nl of a viral suspension (DCV, 2 � 1011 LD50/ml; FHV, 4 � 1011 plaque-

forming units/ml) into the thoraces of adult flies11. Injection of the same

volume of 10 mM Tris-HCl, pH 7.5, was used as a control. Infected flies were

then incubated at 22 1C.

DNA microarray analysis. For each experimental challenge, three biologically

independent samples comprising 45 male OregonR flies were used. RNA

extraction, biotinylation and hybridization to Affymetrix drosophila GeneChip

microarrays (Affymetrix) were done as described16. The Affymetrix Microarray

Suite 5.0 (Affymetrix) or Excel (Microsoft) with a combination of built-in

functions and custom formulae were used for data analysis. Raw data were

sorted with the ‘absent-marginal-present flags’ generated by the Microarray

Suite functions. Although an ‘absent flag’ might indicate that there was no

mRNA of particular type present in a sample, ‘marginal flags’ and ‘absent flags’

may indicate problems with the hybridization; therefore, only data points

marked as ‘present’ were retained. The remaining data mass for each micro-

array was then normalized to itself, making the median of all the measurements

one. Subsequent analysis of the results was done on the genes present in all

three replicates of a sample type.

RNA analysis. RNA extraction and analysis by RNA blot or quantitative

RT-PCR were done as described50. The qPCR kit from Eurogentec and an

I-cycler iQ (Biorad) were used for real-time PCR analyses, done in duplicate.

The expression of the gene of interest was normalized to expression of the

RNA encoding ribosomal protein L32 (RpL32) for each sample. Statistical

analysis of the data was done with Student’s t-test. The primers used were

as follows: RpL32 forward, 5¢-GACGCTTCAAGGGACAGTATCTG-3¢, and

reverse, 5¢-AAACGCGGTTCTGCATGAG-3¢; vir-1 forward, 5¢-GATCCCAATT

TTCCCATCAA-3¢, and reverse, 5¢-GATTACAGCTGGGTGCACAA-3¢; and

DCV forward, 5¢-TCATCGGTATGCACATTGCT-3¢, and reverse 5¢-CGCATAA

CCATGCTCTTCTG-3¢.

Characterization of the vir-1 transcript. The vir-1 cDNA was cloned by 5¢-and 3¢-RACE with the Marathon kit from Clontech and the following primers:

forward primer (3¢-RACE), 5¢-CGATTCCTCAGACGATGAAGACGGCGAGG-

3¢; reverse primer (5¢-RACE), 5¢-CGATCCTGATCCTGATCGAATTCGG-3¢.The transcription start site of vir-1 was determined with S1 nuclease mapping

and the 5¢ 32P-labeled oligonucleotide 5¢-AATGATGGCACCTCTTTACG

CGTACTGATCACTTTTTTGACTGAACTGCCGTCAAGTCGAGCTTAGG-3¢.Labeled oligonucleotide probes (0.3 ng; 5 � 104 c.p.m.) were hybridized

overnight at 55 1C with 50 mg of RNA in hybridization buffer (1 M NaCl,

0.17 M HEPES, pH 7.5, and 0.33 mM EDTA). The samples were then

treated for 30 min at 37 1C with 300 U of nuclease S1 in S1 digestion buffer

(0.28 M NaCl, 0.05 M sodium acetate, pH 4.5, and 4.5 mM ZnSO4), were

precipitated with ethanol, were resuspended in formamide loading dye and

were separated by denaturing PAGE. MatInspector software from Genomatix

(http://www.genomatix.de) was used to search 1,000 base pairs of upstream

sequences for vir-1, CG12780, CG9080 and CG2081 for putative transcription

factor binding sites.

Construction of reporter strains. The phk-2 promoter in the pCasper

transformation vector pJL265 (ref. 11) was replaced with a NotI–NheI PCR

fragment containing 2.6 kilobases of vir-1 upstream sequences (GenBank

accession number AE003636, nucleotides 6,472–9,399). This fragment includes

exon 0 of vir-1, the first intron and the first exon to the first ATG, which is used

to initiate translation of green fluorescent protein (replacement of Ser at

position 65 by Thr). The same PCR fragment, as well as shorter versions

(GenBank accession number AE003636, nucleotides 6,472–7,801, 6,472–7,421

and 6,472–7,231), were cloned between the NotI and NheI sites upstream of the

sequences encoding b-galactosidase in the pCasper transformation vector

pJL300. The resulting constructs were injected into embryos of a w– strain

(w1118) to obtain transgenic lines. At least three independent lines were

analyzed for each construct. The expression of the reporter transgenes was

monitored as described49.

Electrophoretic mobility-shift assay. Nuclear extracts from whole flies were

prepared in the presence of 100 mM vanadate as described40. An aliquot of the

extract (20 mg protein) was incubated for 1 h at 25 1C with 6 � 104 c.p.m.

(10 fmol) of a [g32P]ATP–end-labeled optimum drosophila STAT binding

site14 (top strand, 5¢-ACTGGGATTTTTCCCGGAAATGGTC-3¢) in 20 ml of

binding buffer (0.2 M HEPES, pH 7.9, 0.25 M NaCl, 1 mM EDTA and 1 mM

vanadate) containing 2 mg poly(dI:dC). The samples were then analyzed by

standard gel-shift assay.

Antibodies and immunostaining. Mouse monoclonal antibodies to b-

galactosidase were purchased from Promega. Polyclonal antibodies to DCV

were prepared in rabbits against a peptide corresponding to residues 16–47 of

the DCV coat protein VP2 (ref. 13). Immunoblots with this antiserum showed

a band with the expected molecular weight (30 kDa; ref. 6) for protein extracts

from DCV-infected flies but not those from uninfected control flies (Fig. 6b).

Immunohistochemistry experiments confirmed that this antiserum specifically

recognized DCV (Fig. 4b,d,f, uninfected controls). AlexaFluor 488– or

AlexaFluor 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

X-100 and blocked with 1% BSA for 30 min, samples were incubated overnight

at 4 1C with anti-VP2 or anti-b-galactosidase (1:500 dilution). Labeling with

secondary antibodies was done at 25 1C for 4 h. Slides were mounted in

Vectashield medium (Vector Laboratories) and were examined by confocal

microscopy (Zeiss LSM510).

Accession codes. GEO: full dataset of genes regulated by DCV infection,

GSE2828. Genbank: vir-1 cDNA, DQ143902. BIND (http://bind.ca): 312131.

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

ACKNOWLEDGMENTSWe thank S. Ozkan for help with transgenesis; M.-C. Lacombe for assistancewith real-time quantitative RT-PCR analysis; J. Mutterer and D. Zachary forhelp with the confocal microscopy; E. Santiago for technical support; D.Ferrandon and J. Royet for discussions and critical reading of the manuscript;N. Perrimon, J. Castelli-Gair Hombria and J. Darnell for providing fly stocks;and A. Schneemann for the FHV stock and discussions. Supported by CentreNational de la Recherche Scientifique and Ministere de la Recherche et dela Technologie (Actions Concertees Incitatives Physiologie Integrative andMicrobiologie, and Programme puces Affymetrix); Institut Universitairede France; Ligue contre le Cancer (E.J.); Centre National de la RechercheScientifique (D.G.A.); and Ministere de la Recherche du Grand-Duche deLuxembourg (C.D.).

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

Received 20 April; accepted 30 June 2005

Published online at http://www.nature.com/natureimmunology/

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