Drosophila Responses in Immunity and Virus-Specific Inducible Mediated Antiviral

27 Pages·2012·1.4 MB·English

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Broad RNA Interference Mediated Antiviral
Immunity and Virus-Specific Inducible
Responses in Drosophila
Cordula Kemp, Stefanie Mueller, Akira Goto, Vincent
This information is current as
Barbier, Simona Paro, François Bonnay, Catherine Dostert,
of April 11, 2019.
Laurent Troxler, Charles Hetru, Carine Meignin, Sébastien
Pfeffer, Jules A. Hoffmann and Jean-Luc Imler
J Immunol 2013; 190:650-658; Prepublished online 19
December 2012;
doi: 10.4049/jimmunol.1102486
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References This article cites 57 articles, 16 of which you can access for free at: ://w://w://w://w://w://w://w://w://w://w://w://w://w://w://w://w://w://w://w
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
The JournalofImmunology
Broad RNA Interference–Mediated Antiviral Immunity and
Virus-Specific Inducible Responses in Drosophila
Cordula Kemp,*,1 Stefanie Mueller,*,1,2 Akira Goto,* Vincent Barbier,* Simona Paro,*
Franc¸ois Bonnay,* Catherine Dostert,* Laurent Troxler,* Charles Hetru,* Carine Meignin,*
Se´bastien Pfeffer,† Jules A. Hoffmann,* and Jean-Luc Imler*,‡
The fruit fly Drosophila melanogaster is a good model to unravel the molecular mechanisms ofinnate immunity and has led to
someimportantdiscoveriesaboutthesensingandsignalingofmicrobialinfections.TheresponseofDrosophilatovirusinfections
remainspoorlycharacterizedandappearstoinvolvetwofacets.Ontheonehand,RNAinterferenceinvolvestherecognitionand
processing of dsRNA into small interfering RNAs by the host RNase Dicer-2 (Dcr-2), whereas, on the other hand, an inducible
responsecontrolledbytheevolutionarilyconservedJAK-STATpathwaycontributestotheantiviralhostdefense.Toclarifythe
contribution of the small interfering RNA and JAK-STAT pathways to the control of viral infections, we have compared the
resistance of flies wild-type and mutant for Dcr-2 or the JAK kinase Hopscotch to infections by seven RNA or DNAviruses D
o
belongingtodifferentfamilies.OurresultsrevealauniquesusceptibilityofhopmutantfliestoinfectionbyDrosophilaCvirusand w
n
cricketparalysisvirus,twomembersoftheDicistroviridaefamily,whichcontrastswiththesusceptibilityofDcr-2mutantfliesto lo
a
manyviruses,includingtheDNAvirusinvertebrateiridescentvirus6.Genome-widemicroarrayanalysisconfirmedthatdifferent de
d
sets of genes were induced following infection by Drosophila C virus or by two unrelated RNAviruses, Flock House virus and fro
Sindbisvirus.Overall,ourdatarevealthatRNAinterferenceisanefficientantiviralmechanism,operating againstalarge m
rangeofviruses,includingaDNAvirus.Bycontrast,theantiviralcontributionoftheJAK-STATpathwayappearstobevirus http
specific. The Journal of Immunology, 2013, 190: 650–658. ://w
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economic losses in crops and animals. Because they like families that sense the viral nucleic acid and trigger the in- u
n
replicate inside cells, and rely for the most part onhost cell mo- duction of IFNs and other cytokines (1). These factors activate o
lecular machineries for their replication, viruses pose specific the production of antiviral molecules, such as protein kinase R l.org
challengestotheimmunesystem.Twomajorstrategiesofantiviral oroligo-29,59-adenylatesynthetase,thatcontaintheinfectionand b/
contributetothe activation of the adaptive immune response (2). y g
In plants, viral nucleic acids are recognized by enzymes of the ue
s
*CNRS-UPR9022,InstitutdeBiologieMole´culaireetCellulaire,67084Strasbourg Dicerfamily,whichproducesmallinterferingRNAs(siRNAs)of t o
C67e0d8e4x,SFtrraasnbcoeu;rg†CCNeRdSex-U,PFRra9n0c0e2;,anIndst‡iFtuatcudlete´BdieosloSgcieienMceosle´dcuelalaireVieet,CUenlilvuelarsiriete´; 21–24nucleotides.ThesesiRNAsarethenloadedontomolecules n A
1dCe.SKt.raasnbdouSr.gM;.6c7o0n8t3ribSutrtaesdbeoquurgalClyedtoexth,iFsrwanocrek. owfitthhecAomrgpolneamuetent(aAryGOse)qfuaemniclyesa;ntdarwgeiltlegduiRdNeAthsemarteowthaerndReiNthAesr pril 1
1
2Currentaddress:Bernhard-Nocht-InstitutforTropicalMedicine,MolecularEnto- sliced by AGO, or their translation is inhibited. This RNA in- , 2
mology,Hamburg,Germany terference(RNAi)mechanismprovidesefficientandsequence- 01
ReceivedforpublicationAugust29,2011.AcceptedforpublicationNovember5, specificprotection against viralinfections(3). 9
2012. RNAi also plays an important role in the control of viral in-
ThisworkwassupportedbytheNationalInstitutesofHealth(PO1AI070167),the fections in insects, as shown by the production of virus-derived
AgenceNationaledelaRecherche(ANR-09-MIEN-006-01),theBalzanFoundation siRNAs in infected flies, and the increased susceptibility to viral
(toJ.A.H.),theEuropeanResearchCouncil(ERCStartingGrantncRNAVIR260767
to S.P.),the Investissement d’AvenirProgram Laboratoire d’Excellence (NetRNA infection of Drosophila mutants for the genes Dcr-2 and AGO2
ANR-10-LABX-36),andtheCentreNationaldelaRechercheScientifique. (3–6). In addition, several reports indicate that an inducible re-
ThesequencespresentedinthisarticlehavebeensubmittedtotheGeneExpression sponse also contributes to the control of viral infections (7–15).
Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE31542 We previously showed that infection with Drosophila C virus
andtotheNationalCenterforBiotechnologyInformationSmallReadArchive
(http://www.ncbi.nlm.nih.gov/sra)underaccessionnumberGSE41007. (DCV),amemberoftheDicistroviridaefamily,leadstoinduction
ofsome130genes(11).Analysisoftheregulationofoneofthese
AddresscorrespondenceandreprintrequeststoProf.Jean-LucImler,IBMC-CNRS/
Universite´ deStrasbourg,RueReneDescartes,Strasbourg,Alsace67084,France. genes, vir-1, revealed the presence of functionally important
E-mailaddress:[email protected] bindingsitesforthetranscriptionfactorSTATinitspromoter.The
Theonlineversionofthisarticlecontainssupplementalmaterial. induction of vir-1, as well as several other DCV-induced genes,
Abbreviationsusedinthisarticle:AGO,Argonaute;CrPV,cricketparalysisvirus; was found to be dependent on the gene hopscotch (hop), which
Dcr-2,Dicer-2;DCV,DrosophilaCvirus;dpi,daypostinfection;DXV,DrosophilaX
encodes the only JAK kinase in Drosophila. Furthermore,hop
virus; FHV, Flock House virus; IIV-6, invertebrate iridescent virus type 6; Imd,
immunedeficiency;MEKK1,MEKkinase1;RNAi,RNAinterference;SINV,Sind- mutant flies succumb more rapidly than do wild-type controls,
bisvirus;siRNA,smallinterferingRNA;TotM,TurandotM;Upd,unpaired;VSV, with a higher viral load, to DCV infection (11). The Toll and
vesicularstomatitisvirus.
immune deficiency (Imd) pathways, initially characterized for
Copyright(cid:1)2013byTheAmericanAssociationofImmunologists,Inc.0022-1767/13/$16.00 their role in the control of bacterial and fungal infections, were
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102486
TheJournal ofImmunology 651
also thought to play a part in the control of viral infections. cells. DCV, CrPV, FHV, and IIV-6 were titrated on Kc167 (DCV, CrPV,
Whereas the Toll pathway was associated with resistance to the and FHV) or S2 (IIV-6) cells by the Reed–Muench method to calculate
50%tissueculture–infectivedoseandconvertedtoPFUwithaconversion
DrosophilaXvirus(DXV)(15),theImdpathwaywasimplicated
factorof0.7.
in the control of Sindbis virus (SINV) (7) and cricket paralysis
virus(CrPV)(9). RNAanalysis
Altogether, the data in the present literature point to the in- Total RNA from infected flies was isolated using TRI Reagent RT bro-
volvement of both RNAi and an inducible expression of effector moanisolesolution(MRC),accordingtothemanufacturer’sinstructions.
molecules tocounter viralinfectionsin insects(5,16).However, TotalRNA,1mg,wasreversetranscribedusingiScriptcDNASynthesis
Kit (Bio-Rad). The reverse transcription was run in the T3000 Thermo-
whereas RNAi was shown to contribute to resistance to several
cycler (Biometra), with the following PCR program: step 1: 65˚C for 5
RNAviruses (with either single-stranded genomes of both polar-
min,step2:4˚Cfor5min,step3:25˚Cfor10min,step4:42˚Cfor60
ities or double-stranded genomes), most studies on the inducible min,andstep5:70˚Cfor15min.Atotalof100ngcDNAwasusedfor
response have so far focused on a single virus. As a result, the quantitativereal-timePCR,usingtheiQCustomSYBRGreenSupermix
global significance of the inducible response for the control of Kit (Bio-Rad). The PCR was performed using the CFX384 Real-Time
System (Bio-Rad) with the following program: step 1: 95˚C for 3 min,
viral infections remains poorly understood. In particular, it is
step2:95˚Cfor10s,step3:55˚Cfor30s,repeated39timesfromstep2.
unclear at present if the JAK-STAT pathway is involved in a PrimersusedforqPCRwereasfollows:RpL32(forward59-GACGCTTC-
generalantiviralresponse,providingbroadantiviralimmunity,or AAGGGACAGTATCTG-39;reverse59-AAACGCGGTTCTGCATGAG-39),
ifitactsspecificallyonacriticalstepinthereplicationcycleofa vir-1(forward59-GATCCCAATTTTCCCATCAA-39;reverse59-GATTAC-
AGCTGGGTGCACAA-39), drosomycin (forward 59-CGTGAGAACCTT-
specificvirusorvirusfamily.Toaddressthisimportantquestion,
TTCCAATATGATG-39;reverse59-TCCCAGGACCACCAGCAT-39),and
we have compared the resistance of a mutant for the JAK-STAT diptericin (forward 59-GCTGCGCAATCGCTTCTACT-39; reverse 59-
pathwaytoinfectionbysevenRNAorDNAviruses.Wefindthat TGGTGGAGTGGGCTTCATG-39). Turandot M (TotM), upd, upd2, and D
hop mutant flies are more susceptible than wild-type controls to upd3 expression levels were quantified using the Brilliant II QRT-PCR o
w
infectionsbytheDicistroviridaeDCVandCrPV,butexhibiteither CoreReagentKit,1-step(Stratagene).Thereactiontookplaceinatotal n
volume of 20 ml, using the Taqman Gene Expression Assay [TotM lo
no or a weak phenotype for other viruses, suggesting that the a
(Dm02362087s1),upd(os)(Dm01843792_g1),upd2(Dm01844134g1), d
e
JAK-STAT pathway–dependent inducible response is virus spe- upd3(custom-designedupd3exon2-ANY),andRpL32(Dm02151827g1), d
cific. Genome-wide transcript profiling shows that infection by all from Applied Biosystems]. We used the 7500 Fast Real-Time PCR fro
two other RNA viruses, Flock House virus (FHV; Nodaviridae) System(AppliedBiosystems)withfollowingPCRprogram:step1:45˚Cfor m
and SINV (Alphaviridae), leads to upregulation of $400 genes, 310mminin,,restpeepat2e:d9359˚Ctimfoers1f0rommins,tesptep3.3I:n9a5l˚lCcafsoers,15gesn,esteexppr4e:ss6i0o˚nCwfoasr http
whichonlypartiallyoverlapwiththoseinducedbyDCV.Overall, normalizedtotheribosomalproteingeneRpL32. ://w
our data indicate that the siRNA pathway exerts broad antiviral ForIIV-6,theexpressionoftheannotatedgenes206R,224L,244L,and w
w
aspceticviifityc ianndducaibffleectrsesbpootnhseRsNcoAntarinbdutiDngNAtovtihreusceos,ntrwoilthofvviriuras-l 2R6e1vRersweaTsraasnssecsrsipedtasbeyssptercainfidc-aslpleyciafidcapRteTd-PfCoRr.geWnee-supseecdifiScupperirmScinrigptaInIdI .jim
followed the manufacturer’s protocol (Invitrogen). Briefly, primer pairs m
infections inDrosophila. were designedto amplify regionsofthe IIV-6genomeexhibitingornot un
Materials and Methods ferxohmibiitninfegctaedhiSgh2dceelnlssitwyaosfrsemvearlsleRtNraAnsrceraibdesd.Twoittahl2RNpmA,o1lmofg,eietxhterracftoerd- ol.org
Flystraincultureandinfection wTraarndsc(rFip)taosre.reTvheersreea(cRti)onprwimaserthaenndin2c0u0baUtedoffoSru1pheraStc5ri5p˚tCI.ITIhRenev1ermsel by/
Oregon-R(OR)andywwereusedaswild-typecontrolflies.ThehopM38/msv1, oftheresultingcDNAwasusedtoperform25cyclesofPCR,usingTaq gu
Dcr-2L811fsX,andDcr-2R416Xmutantflieswerepreviouslydescribed(17– DNApolymerase(Invitrogen)andbothFandRprimers.Theprimerpairs es
19).AgenomicrescueoftheDcr-2genewasestablishedwiththeFosmid wereasfollows:206R(forward:59-AAGGAAAGTGGCGAGTACGA-39, t o
FatltyPF4o0s0(21L7)0,74an(dtrtahnesgterannesogmenei.cmcphi-rcobmgo.dsoem) ienswearstedrecaotmthbeinleadndwinitghstihtee rAeTveCrAseC5A9T-ATAGCAACACTATCGC-C3G9,TrTevTeTrsCeT:T5C9-CAATA-3A9)G;C22G4ALA(CfoCrwCaGrdA:A5A9-TCCCAA-C3C9)-; n Ap
edxepfiecriiemnceyntDs,f(D2Rcr)-B2SCm4u5t,anwtshicwheurenccorvoesrssedthewDitchr-t2helocduesfi.cFieonrctyheDref(s2cRue) 2A4C4CLT(CfoCrCwGarGd:A5A9G-TAGTGTAT-A3A9);A2G6A1RGT(GfoGrwTaCrdC:C5A9-TCTATG-3C9,CrCevCeArsTeC:C5G9-ATGATT-- ril 11
BSC45ortheDf(2R)BSC45–Dcr-2rescueline.Flieswerefedonstandard TACTA-39,reverse:59-CTGCAACTGCAGAAATTTGA-39).ThePCRbands , 2
cornmeal–agar medium at 25˚C. All fly lines were tested for Wolbachia weresequencedtoverifytheirviralorigin. 01
9
infectionandcuredwhenevernecessary.Viralstockswerepreparedin10 Statisticalanalysis
mM Tris-HCl, pH 7.5, with the exception of vesicular stomatitis virus
(VSV),whichwasuseddirectlyfromVerocellculturesupernatant[VSV Anunpairedtwo-tailedStudentttestwasusedforstatisticalanalysisof
43109PFU/ml;DCV531010PFU/ml;CrPV13109PFU/ml;FHV datawithGraphPadPrism(GraphPadSoftware).Thepvalues,0.05were
5.53109PFU/ml;DXV4.43107PFU/ml,invertebrateiridescentvirus consideredstatisticallysignificant.Survivalcurveswereplottedandana-
type6(IIV-6)4.431011PFU/ml;andSINV53108PFU/ml].Infections lyzedbylog-rankanalysis(Kaplan–Meiermethod)usingGraphPadPrism
were performed with 4- to 6-d-old adult flies by intrathoracic injection (GraphPadSoftware).
(Nanoject II apparatus; Drummond Scientific) with viral particles, indi-
catedinthefigurelegends.Injectionofthesamevolume(4.6nL)of10 DNAmicroarrayanalysis
mMTris-HCl,pH7.5,wasusedasacontrol.Forbacterialinfection,flies
Foreachsample,Tris-injected,DCV-infected(11),andFHV-andSINV-
were pricked with a thin needle previously dipped in a concentrated
infected, three biologically independent samples comprising 45 male
overnight culture of Escherichia coli and Micrococcus luteus in Luria–
Oregon-Rflieswereused.RNAextraction,biotinylation,andhybridization
Bertanimedium.Infectedflieswerethenincubatedatroomtemperature,
to Affymetrix Drosophila GeneChip microarrays (Affymetrix) were per-
orat29˚CinthecaseofhopM38/msv1andthecorrespondingcontrolflies,
formed as described (20). The Affymetrix Microarray Suite 5.0 (Affy-
and monitored daily for survival, or frozen for RNA isolation and virus
metrix)orExcel(Microsoft)withacombinationofbuilt-infunctionsand
titrationattheindicatedtimepoints.
customformulaewasusedfordataanalysis.Rawdataweresortedwiththe
Cell cultureandvirustitration “absent-marginal-present flags” generated by the Microarray Suite func-
tions.AlthoughanabsentflagmightindicatethatnomRNAofaparticular
VeroRcellsweregrowninDMEM(Invitrogen)supplementedwith10% typewaspresentinasample,marginalflagsandabsentflagsmayindicate
FCS (Biowest), penicillin/streptomycin (Invitrogen), nonessential amino problems with the hybridization; therefore, only data points marked as
acidmix(Invitrogen),10mMpyruvate(LifeTechnologies),and200mML- presentin at least onereplicatewereretained.Theremainingdatamass
glutamine (Invitrogen). Kc167 and S2 cells were grown in Schneider’s foreachmicroarraywasthennormalizedtoitself,making1themedian
medium(Biowest)supplementedwith10%FCS,GlutaMAX(Invitrogen), ofallthemeasurements.Agenewasconsideredinducedifpresentinat
andpenicillin/streptomycin(1003mix,10mg/ml/10,000U;Invitrogen). leastonereplicate,withavirus/Trisratiohigherthan2foratleastoneof
VSVandSINVweretitratedfrominfectedfliesbyplaqueassayonVeroR the time points. Classification of gene functions was analyzed by David
652 VIRUS-SPECIFICINDUCIBLE RESPONSEINDROSOPHILA
BioinformaticsResources6.7(21).ThedatasetforFHVandSINVwas which is characteristic of the accumulation of iridescent viral
submitted to the Gene Expression Omnibus database (http://www.ncbi. particles,thaninwild-typecontrols(Fig.1A).Dcr-22/2flieswere
nlm.nih.gov/geo/)withtheaccessionnumberGSE31542.
significantly more susceptible to IIV-6 infection than were the
Assembly,sequencing,andanalysisofsmallRNAlibraries corresponding wild-type (Fig. 1B). A fraction of Dcr-22/2 flies
injected with buffer also died in the course of the experiment,
The small RNA library of S2 cells and whole flies was constructed as
confirming the increased sensitivity to stress associated with
described (22)and sequenced by the Illumina2GAnalyzer. Reads were
thenalignedtoareferenceconsistingoftheIIV-6genomefromtheNa- mutations of the siRNA pathway (31). The decreased survival
tionalCenterforBiotechnologyInformation(accessioncodeNC_003038) timecorrelatedwitha20-foldincreasedviralloadinDcr-2mutant
usingtheBowtieprogramwithstandardparametersingenomeassembly. flies at 10 d postinfection (dpi) (Fig. 1C). Similar results were
ReadsaligningtotheIIV-6genomewithamaximumofonemismatchwere
obtained when a different null allele of Dcr-2 was used, and the
retainedandanalyzedusingin-housePerlscriptsandExcel.Sequenceswere
submittedtotheNationalCenterforBiotechnologyInformationSmallRead IIV-6 susceptibility phenotype was rescued by a wild-type geno-
Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?) under the acces- mic Dcr-2 transgene (Fig. 1D). The r2d22/2 and AGO22/2 null
sionnumberGSE41007. mutant flies also exhibited increased sensitivity to IIV-6 (Fig.
1E).AGO22/2fliescontainedmoreviralDNAthandidwild-type
Results
controls, confirming that this gene participates in the control of
RNAiprovidesbroadantiviralprotectioninDrosophila
infection (Fig. 1F).
Severalindependent studies, includingour own,have established We next sequenced small RNA libraries prepared from IIV-6–
that RNAi, and more precisely the siRNA pathway, serves as an infected S2 cells or adult flies. We observed several hundreds of
efficienthostdefenseagainstRNAviruses.Theseincludeviruses thousandsofreadsmatchingtheIIV-6genomeinbothinfectedS2
with a single-stranded genome of both (+) and (2) polarity and cells and wild-type flies, but not in control noninfected S2 cells D
dsRNA viruses (23–30), and we confirmed that flies mutant for (Supplemental Table I). The large majority of these reads had a o
w
Dcr-2died more rapidly thanwild-type controls when theywere size of 21 nucleotides, which is characteristic for processing by n
lo
infectedwithDCV,CrPV,FHV,SINV,VSV(Rhabdoviridae),and theRNaseDicer-2(Dcr-2).Thispeakwasabsentfromthelibrary a
d
DXV (Birnaviridae) (data not shown). Next, we addressed the prepared from infected Dcr-22/2 mutant flies (Fig. 2A). These ed
question whether the siRNA pathway also participated in the data indicate that Dcr-2 generates 21-nucleotide IIV-6–derived fro
m
controlofaDNAvirusinfection,andinfectedwild-typeandRNAi siRNAsininfectedflies,andraisethequestionofthenatureofthe h
mutant flies with IIV-6 (Iridoviridae). Infection of Dcr-2 mutant substrate used by Dcr-2 in the context of this infection. As pre- ttp
flies led to a more rapid and intense appearance of blue color, viously reported for RNAviruses, the number of reads matching ://w
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FIGURE1. Dcr-2isinvolvedinhostdefenseagainsttheDNAvirusIIV-6.(A)UponinjectionofIIV-6(5000PFU)inwild-type(yw)andDcr-2R416X
mutantflies,typicalblueparacrystallinestructuresappearedearlierintheabdomen(arrowhead)ofthemutantflies.Representativeindividuals10dpiare
shown.(B)Groupsof20wild-type(yw)orDcr-2R416XmutantflieswereinjectedwithIIV-6orTris,andsurvivalwasmonitoreddaily.Thedifference
betweenthewild-typeandDcr-2mutantfliesisstatisticallysignificant.(C)Viraltiteringroupsoffivewild-type(yw)orDcr-2R416Xmutantflieswas
monitored 10 dpi. (D) Rescue of the hemizygous Dcr-2L811fsX for the IIV-6 susceptibility phenotype by a transposon expressing a wild-type Dcr-2
transgene.Dcr-2L811fsXhemizygousflies(Dcr-2L811fsX/Df)aresignificantlymoresusceptiblethanDcr-2L811fsXhemizygousfliescomplementedbyawild-
typeDcr-2transgene(Dcr-2L811fsX/Dfrescue).DfisDf(2R)BSC45,adeficiencythatfullyuncoverstheDcr-2locus.Allcontrolandgenomicrescuedflies
areinCantonSbackground.(E)Survivalrateofwild-type(yw),R2D21,andAGO2414mutantfliesuponIIV-6orTrisinjection.(F)IIV-6DNAloadwas
determinedbyquantitativePCRinfourgroupsofsixfliesoftheindicatedgenotypeat10dpi.Forallpanels,thedatarepresentthemeanandSDofatleast
threeindependentexperiments,andthedifferencebetweencontrolsandmutantfliesisstatisticallysignificant.*p,0.05,***p,0.001.Allexperiments
areperformedat22˚C(A,C,F)or25˚C(B,D,E).
TheJournal ofImmunology 653
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FIGURE2. Virus-derivedsiRNAsinS2cellsandDrosophilaadultfliesinfectedbytheDNAvirusIIV-6.RNAwasextracted5dpifromS2cellsinfected m
u
byIIV-6(MOI0.01)andadultwild-type(yw)ormutant(Dcr-2R416X)fliesinjectedwithIIV-6(5000PFUperfly).(A)SizedistributionofthesmallRNAs n
o
matchingtheviralgenomeinS2cellsandadultfliesoftheindicatedgenotype.(BandC)Distributionofthe21-nucleotidesiRNAsfromtheS2cell(B)and l.o
ywadultfly(C)librariesalongtheIIV-6genome.EachIIV-6–derivedsmallRNAisrepresentedbythepositionofitsfirstnucleotide.TheIIV-6–derived rg
smallRNAsmatchingtheupperandlowerstrandoftheDNAgenomearerespectivelyshownabove(positivereadsnumber)andbelow(negativereads b/
y
number)thehorizontalaxis,whichrepresentsthe212482bpgenome.In(B),thenumberofreadsforfourpeaksgoingoff-scaleisindicatednexttothem,in g
u
italics.(D)Strand-specificRT-PCRwithprimerscorrespondingtotheannotatedviralgenes206R,224L,244L,and261R.Theexperimentwasperformedin e
s
thepresence(+)orabsence(2)ofRT.NI,Noninfected;FandR,forwardandreversestrandprimerusedforreversetranscription. t o
n
A
p
each strand of the viral genome was very similar (Supplemental mutantfliesdiemorerapidlythandowild-typecontrolsfollowing ril 1
TableI).However,unlikeRNAviruses,thevirus-derivedsiRNAs DCV infection, and contain ∼10-fold more virus (Fig. 3A). By 1, 2
were not uniformly distributed along the viral genome. Rather, contrast, we did not observe significant differences in survival 0
1
several hotspots were observed,revealing that specificregionsof between wild-type and hopM38/msv1 mutant flies upon infection 9
theviralgenomegeneratethesiRNAs(Fig.2B,2C).Thesepeaks withthealphavirusSINV(Fig.4A),andtheviraltiters2dpiwere
do not correlate with the intensity of transcription of the viral not significantly different in wild-type and hopM38/msv1 mutant
genome,andsomehighlytranscribedregionsarelocatedinareas flies(datanotshown),indicatingthattheJAK-STATpathwaydoes
not generating significant levels of siRNAs (32). The strong not contribute to resistance to this virus. The hopM38/msv1 mutant
symmetry of the peaks observed in S2 cells and wild-type flies flies, as well as wild-type flies, also resisted infections by the
suggests that these regions are transcribed on both strands and rhabdovirus VSV and by the nodavirus FHV (Fig. 4B, 4C). A
generate dsRNA.Indeed, we coulddetect bidirectional transcrip- slightreductioninsurvivalwasobservedinthecaseofthedsRNA
tion in the areas of the viral genome covered by the peaks (Fig. virusDXV(Birnaviridae)andtheDNAvirusIIV-6(Fig.4D,4E).
2D). By contrast, transcription of only one strand of the DNA However,thedifferencebetweenwild-typeandhopM38/msv1mutant
genome was detected for the locus 261R, which is located in flieswasonlystatisticallysignificantinthecaseofDXVinfection.
a region that does not produce significant amounts of siRNAs. Furthermore,wedidnotobservestatisticallysignificantdifferences
Overall, these results indicate that the siRNA pathway in Dro- in the DXVand IIV-6 viral titersin wild-type andhopM38/msv1
sophila can alsoprotectagainsta DNAvirusinfection. mutantfliesin theformat ofour assays(data notshown).
Overall,ourdataindicatethattheJAK-STATpathwayiscriticalfor
The JAKkinase Hopscotchdoesnotconferbroadantiviral
hostdefenseagainstDCV,butplaysaminorroleforDXVandIIV-6
immunity
andisessentiallydispensableinthecaseofFHV,SINV,andVSV.We
To test the contribution of the JAK-STAT pathway in antiviral thereforetestedCrPV,anothermemberoftheDicistroviridaefamily
immunity in Drosophila, we injected loss-of-function mutants of knowntoinfectDrosophila.Weobservedadecreaseinsurvival
the JAK kinase Hopscotch (hopM38/msv1) with different ssRNA, andasignificantincreaseinviraltitersinCrPV-infectedhopM38/msv1
dsRNA, and DNA viruses. As previously described, hopM38/msv1 mutantfliescomparedwithwild-typeflies(Fig.3B).Inconclusion,
654 VIRUS-SPECIFICINDUCIBLE RESPONSEINDROSOPHILA
D
o
w
n
lo
a
FIGURE3. TheJAKkinaseHopscotchisinvolvedinhostdefenseagainstDCVandCrPV.(AandB)Groupsof20wild-type(OR)orhopscotch(hopM38/msv1) de
d
dmautatanretpflrieessenwtetrheeinmjeecatnedanwditShDD.CInVt(h5e00rigPhFtUp)a(nAe)lso,rvCirraPlVtit(e5rPwFaUs)d(eBte)r,manindesduirnvigvraoluwpassomffiovneitoflrieeds2dadilpyi.fTohreDeCxVpe(rAim)eanntdw1adspriepfoeratCedrPtVhre(Be)t.imThees,daantda from
representthemeanandSDofthreeindependentexperiments,andthedifferencebetweenwild-typeandhopmutantfliesisstatisticallysignificant.*p, h
0.05,**p,0.01,***p,0.001.(C)DCVandCrPVinfectiontriggersinductionofthegenesupd2and upd3,whichencodecytokines activatingthe ttp
JAK/STATpathway.FlieswereinfectedwithDCVorCrPV,andexpressionofupd,upd2,andupd3wasmonitoredingroupsof10fliesattheindicated ://w
timepointsbyTaqmanquantitativePCR.Theresultsofatleasttwoindependentexperimentsareshown. w
w
.jim
m
ourdataindicatethattheJAK-STATpathwayinDrosophilaconfers expressioninfliesinfectedbyFHV(2and3dpi)orSINV(4and8 un
o
protectionagainstsomeviruses—inparticular,theDicistroviridae— dpi),andcomparedthedatawiththoseobtainedforDCVinfection l.o
butdoesnotprovidebroadantiviralimmunity. (1and2dpi).Thetimepointsforthisanalysiswerechosentotake rg
intoaccount thedifferentkineticsofreplication andcolonization b/
Induciblegeneexpression inFHV-andSINV-infectedflies ofDrosophilabythedifferentviruses(11,24).Foreachvirus,we y g
u
The above results raised the question of whether an inducible observedalargeoverlapbetweenthegenesinducedatthefirstand es
response contributes to host defense against viruses other than secondtimepoints.Wethenpursuedouranalysis,focusingonthe t o
n
DCVand CrPV. We therefore conducted a genome-wide micro- genes induced either at the first or at the second time point. The A
p
arrayanalysisusingAffymetrixDNAmicroarraystomonitorgene microarraydatarevealedthat487and201geneswereinducedor ril 1
1
, 2
0
1
9
FIGURE4. SusceptibilityoffliesmutantfortheJAK kinaseHopscotchtoinfectionbySINV,VSV,FHV,DXV,andIIV-6.Groupsof20wild-type
(OR)orhopmutantflieswereinjectedwithSINV(A),VSV(B),FHV(C),DXV(D),orIIV-6(E),andsurvivalwasmonitored.ForVSVandSINV,theTris
buffercontrolinjectionisalsoshown,becausehopmutantfliesexhibiteddecreasedsurvivalat29˚Cafterday16uponbothbufferandvirusinjection.
Kaplan–Meieranalysisoftheresultsofatleasttwoindependentexperimentsrevealastatisticallysignificantdifferenceinsurvivalbetweenwild-typeand
hopmutantfliesonlyinthecaseofDXV.*p,0.05.
TheJournal ofImmunology 655
D
o
w
n
lo
a
d
e
d
FIGURE5. MicroarrayanalysisofgeneinductionfollowinginfectionbyDCV,FHV,orSINV.(A)Venndiagramshowingthenumberofupregulated fro
genes(byafactorofatleast2)followinginfectionbythethreeviruses.Thetotalnumberofgenesregulatedbyeachvirusisindicatedinparentheses.(B) hm
FHVandSINVinducemembersofthesamegenefamilies,butFHVtriggersastrongerresponse.Thenumbersofgenesbelongingtosevengeneontology ttp
functionalcategoriesinducedbybothFHVandSINVorbyFHVonlyareshown.(C)Expressionofvir-1andTotMbyquantitativePCRnormalizedforthe ://w
expressionofthehousekeepinggeneRpL32.Groupsof10wild-type(OR)flieswereinjectedwithTrisbufferorthevirusesDCV,CrPV,FHV,SINV,VSV,
w
DXV,orIIV-6orprickedwithaneedledippedinaconcentratedpelletoftheGram-positivebacteriumM.luteusandtheGram-negativebacteriumE.coli. w
RNAwasextractedat6h,1d,2d,3d,and4dafterchallenge.ThedatarepresentthemeanandSEsofatleasttwoindependentexperiments.Thepvalues .jim
werecalculatedforeachtimepointindividuallyversustheTris-injectedcontrol.*p,0.05,**p,0.01,***p,0.001. m
u
n
o
upregulated by a factor of at least 2 upon infection by FHVand FHVarealsoinducedbySINV(Fig.5A).Itisintriguing,though, l.org
SINV, respectively. When analyzed with the same criteria, 166 thatmanyofthegenesinducedsolelybyFHV,butnotbySINV, b/
genes wereinducedbyDCV (Fig. 5A,Supplemental Table II). aremembersofthesamegenefamiliesasthegenescoinducedby y g
Thedataofthistranscriptomicanalysiscallfortwocomments. both FHVand SINV. This peculiarity underlines the basic simi- ue
s
First, we note that 42 genes were induced by all three viruses laritiesbetweenthetranscriptionalresponsetothetwoviruses.In t o
(Fig.5A).Wecomparedthissetofgeneswithmicroarraystudies addition, several genes associated with cell death are induced by n A
performedonfliesinfectedbyfungiandbacteria(bothextra-and FHV, but not SINV, which may reflect the higher virulence of p
intracellular) to identify a potential signature specific for viral FHV(Fig.5B,SupplementalTablesII,III).Only22%and16%of ril 1
1
infections(SupplementalTableIII).Weobservedthatanumberof the genes induced by SINVand FHV, respectively, are also in- , 2
genes, such as Frost, are upregulated similarly by all types of duced by DCV, indicating that DCV, on one hand, and FHVand 0
1
infections, suggesting that they are induced by the stress of the SINV, on the other hand, trigger different inducible responses 9
infection, rather than by recognition of specific characteristics (Fig.5A).Wedidnotdetectinourmicroarraysexpressionofthe
of the infecting microorganism. Of interest, other genes, such as genes encoding the unpaired (Upd) cytokines, which activate the
Vago, Obp99b, Mal-B1, Nmda1, CG8147, CG1572, l(2)gd1, JAK-STATpathwayinDrosophila.However,quantitativeRT-PCR
CG14906, CG10911, and Tsp42EI, appear to be induced only in analysisrevealedthatupd2andupd3,butnotupd,areinducedor
response to viral infections, and may represent the core of an upregulated followingDCVand CrPVinfection (Fig. 3C).
inducibleantiviralgeneexpressionprogram.ThecaseofObp99b
Virus-specificpattern ofgeneinduction
is particularly striking, as this gene is strongly upregulated by
FHV, SINV, and DCV, but inhibited following other types of in- To further characterize the transcriptional response triggered by
fection. Clearly, the regulation and function of this molecule different viruses, wild-type flies were injected with DCV, CrPV,
deserves further investigation. The genes CG4680, Eip75B, Sp7, FHV, SINV, VSV, DXV, and IIV-6, and gene induction was
and CG10916 are induced both by the viruses and by the intra- measured at 6 h postinfection and 1, 2, 3, and 4 dpi. Gene ex-
cellular bacterium Listeria (33), suggesting that theymay partic- pression was monitored by quantitative RT-PCR, which provides
ipate in the defense against intracellular intruders (Supplemental a more accurate quantification of gene expression than does hy-
Table III). bridizationtoshortoligonucleotideprobesonmicroarrays(34).We
Asecondcommentisthatthemajorityofupregulatedgenesare monitoredexpressionoftheDCV-inducedgenevir-1(11)andof
inducedbyonlyoneortwooftheviruses,revealingvirus-specific TotM,which,accordingtothemicroarrays,isinducedbyFHVand
responses. Of interest, 84% of the genes upregulated by SINV SINVinfection.Weconfirmedtheinductionofvir-1byDCVand
arealsoinducedbyFHV,pointingtoastrongsimilaritybetween FHV (11) and detected a milder but significant induction of this
theresponsestothetwoviruses.FHVinducedahighernumberof gene by CrPV infection. By contrast, no induction of vir-1 by
genesthandidSindbisvirus,andonly34%ofthegenesinducedby SINV, VSV, DXV, and IIV-6 was observed (Fig. 5C). For TotM,
656 VIRUS-SPECIFICINDUCIBLE RESPONSEINDROSOPHILA
we confirmed the induction by FHVat different time points. In cells, as demonstrated by the critical role played by the dsRNA
addition, we observed that TotM expression was significantly in- receptorTLR3inthesensingofherpesvirusinfectioninmammals
ducedbyDCVatlatetimepointsofinfection(4dpi).Wenotethat (39, 40). Our data are consistent with a model whereby dsRNA
induction of TotM by SINV, VSV, and DXV was 10–20 times generated from convergent transcription of the IIV-6 genome is
strongerthantheinductionbyFHV(Fig.5C).TheDNAvirusIIV- processed by Dcr-2 and triggers RNAi. Thus, we conclude that
6 did not induce TotM at any measured time point. Interestingly, RNAi provides an efficient and highly specific RNA-based de-
we observed different profiles for vir-1 and TotM induction after fense against many types of viruses in Drosophila and probably
viralchallenge.Overall,thevirusesthatkillwild-typefliesrapidly other insects. This conclusion parallels the situation described
(within10d),suchasDCV,CrPV,andFHV,werepotentinducers in plants. Thevertebrates, which largelyrely on the induction of
ofvir-1,whereaslesspathogenicviruses,suchasSINV,VSV,and IFNstocounterviralinfections,appeartobetheexceptionamong
DXV, did not induce vir-1. The opposite trend was observed for multicellular organisms (1).Ofinterest,however,theDExD/H
TotM,whichwasmostpotentlyinducedbySINV,VSV,andDXV. box helicase domains found in Dcr enzymes and RIG-I–like
Thedifferentpatternofinductionofvir-1andTotMsuggeststhat receptors,whichsensethepresenceofviralRNAsincellsinfected
thetwogenesmayberegulateddifferently,eventhoughbothwere byRNAandDNAviruses,arephylogeneticallyrelated(10).This
previouslyshowntoberegulatedbytheJAK-STATpathway(11, findingsuggeststhatanessentialdomainofacoremoleculefrom
17). Indeed, the MAP3K MEK kinase 1 (MEKK1) and the Imd theancestralantiviralresponse,RNAsilencing,wasatsomepoint
pathways are also known to contribute to the induction of TotM recruited to sense viral RNAs invertebrates and to subsequently
induction insomecontexts(17,35). activate a signalingpathwayleading toproduction ofIFNs.
Some antimicrobial peptide genes were also upregulated ac-
Virus-specificinducedgeneexpression inDrosophila
cordingtothemicroarrays,suggestinganoverlapbetweenantiviral D
immunity and antibacterial–antifungal defenses. We observed an Microarraysarepowerfultoolstomonitortheglobaltranscriptome o
w
enrichment for genes regulated by the Toll pathway [e.g., the ofinfectedcellsandcomparetheresponsetodifferentinfections. n
lo
cytokineSpaetzle(Spz)andtheantifungalpeptidesDrosomycine Despiteitslimitationsforaccuratemeasurementsofthemagnitude a
d
e
(Drs)andMetchnikowine(Mtk)]intheDCV-specificsetofgenes ofexpressionchanges,thistechnologyprovidesusefulinformation d
(Supplemental Table II). We also noted an enrichment of Imd on changes in gene expression (34). In this article, using whole- fro
m
pathway–regulatedgenes,suchastheantibacterialpeptidesAttacin- genome Affymetrix microarrays to analyze the transcriptome of
h
Aand -C, Diptericin-B, and the transcription factor Relish, in fliesinfectedbyDCV,FHV,orSINV,wereporttheexistenceofvirus- ttp
thegenesupregulatedbybothDCVandFHV.However,whenex- specific responses to infection. These results are in keeping with ://w
pressionofdiptericinanddrosomycin—twomarkersofactivation a previous study pointing to autophagy as an antiviral defense w
w
oqfuatnhteitaItmivdeaRnTd-PTCoRll,pnaothnweaoyfs,threesvpieructsievseltyri—ggweraesdmaonniintodruecdtiobny mvierucsheasniwsme uasgeadinbsetloVnSgVt,obduitffneoretnDtCfaVm,iilniefsecatniodnp(r1e4se).ntTdhiefftehrerenet .jim
m
comparabletothatofbacterialandfungalinfections,althoughthe characteristics that make them valuable for the current study. For u
n
wounding associated with the injection procedure clearly led to example, 1) DCVand FHV replicate rapidly and kill Drosophila o
someexpressionof thegenes (SupplementalFig.1). uponinjection,whereasSINVdoesnotatthedoseused(11,24);2) l.org
DCVisanaturalpathogenofDrosophila,whereasFHVandSINV b/
Discussion havenotbeenfoundinwildDrosophilapopulations(41);3)FHV y g
We have investigated the involvement of RNAi and the evolu- and DCV possess, respectively, a strong and moderate viral sup- ue
s
tionarilyconservedJAK-STATsignalingpathwayintheresistance pressorofRNAi,whereasSINVpresumablydoesnot(28,42,43). t o
toapanelofsevenvirusesrepresentingseveralimportantfamilies, The three viruses also have different tissue tropism and may be n A
icnocnlturdasintigngthpeicaturbreo:voirnustehseSoInNeVhaanndd, aVbSrVo.adOaunrtidvairtaalpimromviudneitya athsesoicnifaetcetdedwhitohstt.isFsuoer-esxpeacmifipcle,mFoHdiVficwataisonrseciennttlhyesphhoywsinoltoogbyeoaf pril 1
1
basedonRNAicontributingtothedefenseagainstbothRNAand cardiotropicvirus,affectedbypotassiumchannelsregulatingheart , 2
DNAviruses,andontheotherhand,avirus-specifictranscriptional function(44),whereasDCVinfectioncausesintestinalobstruction 01
response involving the JAK-STAT pathway but playing a critical (S.ChtarbanovaandJ.-L.Imler,manuscriptinpreparation). 9
role onlyin thecase ofDicistroviridaeinfection. Comparison of the transcriptomes of the flies infected by the
three viruses revealed more similarities between FHVand SINV
RNAiprotectsagainstaDNAvirus infection
than between each of these and DCV. This may reflect the co-
Thepresentstudyextendsworkfromseveralgroups,includingour evolutionofDCVwithitshost,andthefactthatthisvirusmayhave
own, showing that flies mutant for the siRNA pathway are more learnedtowardofftheantiviralarsenalofitshost. Indeed,DCV
sensitivethanwild-typefliestoalargepanelofRNAviruses,and inducesfewergenesthandoesFHV,eventhoughthetwoviruses
revealsthatDcr-2isalsorequiredforthecontroloftheDNAvirus replicate with similar kinetics and lead to the rapid death of the
IIV-6.Wenote,however,thattheincreaseofviraltiterinsiRNA flies. The genes induced by FHVand SINVencode chaperonins
pathway–mutantfliesisnotasstrongasinthecaseofsomeRNA (Tcp or Hsp), glutathione transferases, cytochrome P450s, stress
viruses [e.g., VSV (25)]. This finding could reflect either the ex- markers (Tot family), thioester-containing proteins, and cyto-
pression of a viral suppressor of RNAi by IIV-6 or the fact that skeletal regulators, suggesting an involvement of oxidative stress
onlyaportionoftheviralgenomeistargetedbysiRNAs.Indeed, andphagocytosisintheresponsetotheseviruses.Thetwoviruses
this virus encodes an RNaseIII enzyme, which could cleave alsoupregulatethegeneegghead(egh),whichencodesamolecule
siRNA duplexes, as previously reported in plants infected by the involved in the uptake of dsRNA and antiviral immunity (27).
sweet potato chlorotic stunt virus (36). The involvement of Dcr- DespitethelargeoverlapbetweenthegenesupregulatedbyFHV
mediated immune responses against DNA virus infections was and SINV, the former induce a more intense transcriptional re-
previously noted in plants, in which secondary structures in the sponse than the latter. This observation may reflect the more ag-
transcribed viral RNAs, or dsRNAs formed from overlapping bi- gressive replication of FHV in Drosophila. Indeed, the genes
directional transcripts, can be processed into siRNAs (37, 38). specificallyinducedbyFHVincludenotonlyadditionalmembers
Production of dsRNA from DNA viruses also occurs in animal ofthefamiliesmentionedabove(Hsp,Tcp,Gst,cytP450,thioester-
TheJournal ofImmunology 657
containingproteins),supportingtheideaofamoreintenseresponse, as flies mutant for either RNAi or the inducible JAK-STAT
but also genes associated with cell death. In addition, FHVupre- pathway succumb toinfection 2–3 d before the controls, with an
gulatesseveralmoleculespreviouslyconnectedtoinnateimmunity ∼10-foldincrease inviral titer.
inDrosophila,suchasHel89B(45),POSH(46),orMEKK1(35), Interestingly, even though hop mutant flies appear to be spe-
ormoleculesthatmay downmodulatethestrong responsetovirus cifically sensitive to Dicistroviridae, other viruses activate the
infection (e.g., the genes CG9311 and Pez, encoding tyrosine JAK-STATpathway.Indeed,weobservedaslightincreaseinthe
phosphatases). Finally, we note that FHVinduced eight genes lethality of hop mutant flies postinfection with DXVand IIV-6.
encoding factors with RNA binding domains, including four In Aedes mosquitoes, the JAK/STAT pathway was also shown to
DExD/Hboxhelicases,whichmayparticipateinthesensingand activate a defense against Dengue, a member of the Flaviviridae
neutralization of viral nucleic acids. This specificity may reflect family (54). Wealso notethat theJAK-STATpathway–regulated
aresponseofthehosttocountertheeffectofthestrongsuppressor genevir-1(11)isinducedbyDCVandCrPV,butalsoFHV,even
of RNAi B2,a dsRNA-bindingprotein (47). thoughhopmutantfliesresistFHVinfectionmuchasdowild-type
Anintriguingaspectofthetranscriptomeofvirus-infectedflies flies.Onehypothesistoexplainthisapparentparadoxisthatsome
istheupregulationofgenesregulatedbytheTollandImdpathways. genesmaybeinducedinaJAK-STAT–independentmannerinthe
WeobservedanenrichmentofTollpathwaytargetgenesinduced contextofviralinfections.Forexample,thegeneTotM,whichis
infliesinfectedbyDCV,butnotFHVorSINV,suggestingthat inducedbyseveralvirusesnormallyresistedbyhopmutantflies,
DCVinfectiontriggersthispathway.Amongthegenesinducedby can be induced by theMEKK1 pathway, in addition to the JAK-
DCV,butnotbythetwootherviruses,wealsonotethepresenceof STATpathway(35).Indeed,weobservedthatTotMremainsfully
Ect4,whichencodesaTIRdomaincytoplasmicmolecule.The induced by FHVand SINV in hop mutant flies (C. Dostert and
mammalian ortholog of this gene, SARM, was proposed to par- J.-L. Imler, unpublished observations). However, this hypothesis D
ticipateasanegativeregulatorofTLRsignalinginsomeantiviral cannot account for the induction of vir-1 by FHV, because it is o
w
defenses(48).TwoothergenesregulatedbyDCVandpossibly strongly reduced in hop mutant flies (C. Dostert and J.-L. Imler, n
lo
establishingaconnectionbetweenRNAsilencingandtheinducible unpublishedobservations).Thisfindingsuggeststhatsomeaspects a
d
e
responseareworthmentioning:headcasewasidentifiedinascreen of the JAK-STAT–induced response may be redundant of other d
as a regulator of the siRNA pathway (49), whereas CG9925 en- defenses for FHV, but not for DCV. The fact that FHV triggers fro
m
codesaproteinwithaTudordomain,acharacteristicofseveral a stronger transcriptional response than does DCV (Fig. 5) is
h
componentsofthePiwi-interactingRNApathway(50). consistent withthis hypothesis. ttp
UnliketheToll-regulatedgenes,severalgenesregulatedbyImd A keyquestion pertains to the nature of thereceptor detecting ://w
were induced in flies infected by DCVor FHV, although not by DicistroviridaeinfectionandtriggeringtheJAK-STAT–dependent w
w
SinINthVe. TrehgeulTaotilolnanodfIbmacdtepraiathlwaanydsfpulnagyaal iwnefellc-tcihoanrsa,cttherroizuegdhrtohlee isnudbusectibolefrgeesnpeosn,sein.cOluudrindgatathpeoiJnAtKto-StThAeTi–nrdeugcutiloatnedofgaensepevciirfi-1c .jim
m
regulationofgenesencodingantimicrobialpeptides.Thesegenes (11),byfast-killingvirusessuchasDCVandCrPV,butalsoFHV, u
n
arealsoupregulatedbyviralinfection,althoughnotsignificantly, which replicate rapidly to high titers upon injection in flies. Of o
compared withbuffer injection. Thislowlevelofinductionmost note,vir-1inductionisnotaffectedinfliesexpressingthedsRNA- l.org
likely explains our inability to detect antimicrobial peptides in binding protein B2, or in Dcr-2 mutant flies, indicating that this b/
thehemolymphofDCV-infectedflies(51).Althoughnotformally geneisnotinducedfollowingsensingofdsRNA(10).Thisfinding y g
establishing that the Toll and Imd pathways participate in the suggests that sensing tissue damage and/or cell death could con- ue
s
antiviral response, these results certainly do not rule out such a tributetothisinducibleresponse,ahypothesiscorroboratedbythe t o
role(7,9,15).Alternatively,inductionoftheantimicrobialgenes association of the JAK-STAT pathway with the cellular response n A
mayinvolvethetranscriptionfactorFOXO,aknownregulatorof toavariety of stresses (17,55–57). p
stressresistance,andmayoccurindependentlyoftheTollandImd Inconclusion,ourdataconfirmthat,beyondRNAi,aninducible ril 1
1
pathways (52). Whatever the mechanism of induction, the bio- response contributes to the control of some viral infections in , 2
logicalsignificanceofthisweakinductionofmoleculesnormally Drosophila. However, this response is complex, and great care 0
1
active in the micromolar range is unclear. One possibility is that should be exercised beforegeneralizing the resultsobtained with 9
theDrosophilaantimicrobialpeptidescarryadditionalfunctions one single virus species. This unexpected complexity probably
that do not require high-level expression. For example, some reflectstheintricateassociationofviruseswiththeirhostcellsin
mammalianb-defensinsplayadualroleininnateimmunityand, differenttissues,theirdifferentstrategiesofreplicationorprotein
in addition to their antibacterial properties, interact with chemo- expression, ortheiracquisition ofsuppressors of hostdefense.
kine receptors with affinities in the nanomolar range, thus medi-
ating chemoattraction of phagocyticcells (53).
Acknowledgments
Dicistroviridae-specificcontributionoftheJAK-STATpathway
WethankEstelleSantiagoandMiriamYambaforexcellenttechnicalassis-
toantiviralimmunity tance, Phil Irving for help with the microarray experiments, Anette
An unexpected finding reported in this article is that hop mutant Schneeman(TheScrippsResearchInstitute,LaJolla,CA)forproviding
DXVandCrPV,TrevorWilliams(Veracruz,Mexico)forprovidingIIV-6,
flies have a clear phenotype for DCVand CrPV, but not for the
and Ste´phanie Blandin and Dominique Ferrandon for critical reading of
othervirusestested.ThisobservationindicatesthattheJAK-STAT
themanuscriptandhelpfulsuggestions.Themicroarrayanalysisandthe
pathway,inadditiontoRNAi,participatesinhostdefenseagainst
deep sequencing wereperformed at the PlateformeBiopuces et Se´quen-
members of the Dicistroviridae family. DCV infection leads to c¸age,InstitutdeGe´ne´tiqueetdeBiologieMole´culaireetCellulaire,Stras-
induction of the genes encoding the cytokines Upd2 and Upd3, bourg,France.
whichmaysubsequentlyactivatetheJAK-STATpathwayinnon-
infectedcells,triggeringanantiviralprogramofgeneexpression.
Altogether, our results highlight that the contribution of the in- Disclosures
ducibleresponsetothecontrolofDCVissimilartothatofRNAi, Theauthorshavenofinancialconflictsofinterest.
658 VIRUS-SPECIFICINDUCIBLE RESPONSEINDROSOPHILA
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