Small RNA Pathways and Antiviral Responses in Insects

20 Pages·2016·2.23 MB·English

VI03CH27-Saleh ARI 10September2016 11:42
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nl Antiviral Responses in Insects
o
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ws.orgperson Vanesa Mongelli and Maria-Carla Saleh
vieFor VirusesandRNAInterferenceUnit,DepartmentofVirology,CNRSUMR3569,Institut
alre16. Pasteur,75724ParisCedex15,France;email:[email protected]
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Annu. Ress provided by I T1CA0lohl.p1irs1yigar4hri6gtt/ihscatlnree(cid:2)n’cssuedrr2evo0vei1:-d6vibroyloAgnyn-u1a1l0R61ev5i-e0w4s2.447 ALtfehicbektyseiotrernevasley.crAtoynroetRmhNearrAkoairbnglteaencrifhseamrreanoccnteerE(iRsatrNitchAo,fii)RnmsNeecActhsiaiasnrtiehsmaintsfiettcoistecbdiortcwhuimbthrvoevanidrtulyvseiarsca,tlianinngd-,
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cc becauseitistriggeredbydouble-strandedRNAmoleculesderivedfromvir-
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tuallyanyvirus,andextremelyspecific,becauseittargetsonlytheparticular
viralsequencethatinitiatedtheprocess.Reviewscoveringthedifferentfacets
oftheRNAiantiviralimmuneresponseininsectshavebeenpublishedelse-
where.Inthisreview,webuildaframeworktoguidefutureinvestigation.
Wefocusontheremainingquestionsandavenuesofresearchthatneedto
beaddressedtomovethefieldforward,includingissuessuchastheactivity
ofviralsuppressorsofRNAi,comparativegenomics,thedevelopmentofde-
tailedmapsofthesubcellularlocalizationofviralreplicationcomplexeswith
theRNAimachinery,andtheregulationoftheantiviralRNAiresponse.
573
VI03CH27-Saleh ARI 10September2016 11:42
INTRODUCTION
InsectswereamongthefirstanimalstocolonizeEarth’secosystemsapproximately500million
Insects: aclassof yearsago(1,2)andnowconstitutethemostdiverseclassoflivingorganismsintermsofdescribed
arthropodswitha species (Figure 1) (3). Insects act as key participants in most terrestrial ecosystems by cycling
three-segmentbody, nutrients, maintaining soil structure and fertility, pollinating plants and dispersing their seeds,
threepairsoflegs,
controllingpopulationsofotherorganisms,andservingasafoodsourceforotherorganisms(4).
compoundeyes,and
Insectshavecapturedhumanattentionsinceearlytimes.Indeed,theearliestobjectsmadeby
onepairofantennae
humanstosymbolizeinsectsdatefromthePaleolithicperiod(10,000yearsago);theseobjectsde-
RNAinterference
pictbeetles(5).Forcenturies,insectssuchasthesilkworm(Bombyxmori)andthehoneybee(Apis
(RNAi): biological
mechanismsguidedby mellifera)havebeenprotectedandcherishedbecauseoftheirsilkandhoneyproduction,respec-
y. smallRNAmolecules tively,butothers,suchasthelocust(Locustaspp.),havebeenbattledbecauseoftheirdevastating
onl (21–30nt)enabling effectsoncrops.Oncetheircapacitytoactasvectorsforhuman,livestock,andplantpathogenswas
se thesequence-specific
u established,insectsbecameaprimaryresearchinterest.Inrecentyears,andparticularlyduringthe
al recognitionofcognate
ws.orgperson nseuqculeeinccaecsidantdartgheetir wZirkitainvgiruosfetsh)iasnrdevinieswec,tmstorsaqnusmitoitetsintgraangsrmiciuttlitnugraalrdbiosevairsuessehsa(vseubchecaosmtheethdeefnogcuues,oWfsetustdNy,illeea,vainndg
vieFor degradation, otherinsectspeciesaside.Today,fewscientistsstudythebehavior,ecology,biology,physiology,
nualre5/16. ttrraannsslcartiipotnioanlaalrrest,or aEnadrthim,rmetuunrintyinogftootbhreoraidnesrecretss.eaCrochnsaibdoeruitnbgatshicatinisnescetctbsiocloongsytiwtuiltleptohseitimveolsytianbfluunednacnetretasxeaarocnh
w.an10/1 regulation onallordersoflife.
wn AntiviralRNAi:
m wale o innateimmune vertLeibkreateevse,riynsoetchtserlaockrgaanniasdmapotinveEiamrtmh,uninesesycststeimntaenradctrewlyitohndlyivoenrsiennpaattehoimgemnus.nIenrecsopnotnraseststtoo
d froCentr rReNspAoinmseebcahsaendisomns copewiththesepathogens.Inaverygeneralmanner,theinnateimmunesystem,whichishighly
dee triggeredbythe conserved in all metazoans, is based on the recognition of conserved pathogen-derived molec-
au
9. DownloBibliotheq pvSirmreasalelonlrciinegtioenfrfdesrRinNgAof urtielvacarotgimonnoittoiioffsnt,hrceeaclTelepodtlol,prIasmt(hPdoR,gaRennsd-)a(Js6as,ko/7cS)i.ataItetndsiingmsneoacllte-stc,ruPalnaAsrMdpuPactttrieoercnnospgan(tPihtAiwoManyPbs,sy)w,PhbRiycRhhsoliensatd-duetncoecsobdtohetehdrhpauapmtitdeorarnca--l
73-58aris - RsmNaAllsR(NsiARNmAolse)c:ules (e.g.,secretionofantimicrobialpeptides,lysozymes,orothermicrobe-targetingsubstances)and
5P cellular(e.g.,programmedcelldeathandautophagy)defenseresponses(6).Thesepathwayswere
v. Virol. 2016.3:nstitut Pasteur - (c(co2teerr1lliallg–--niee2nsnx3podongosteog)nneoonsf)uoosur(svirus) fitasougrbsaftijunedncsettcstovciiofrrintabhleiiindsnrfiaeenncvttisieitvowuinrda(ii1les0sda–oec1tffei2vin)ne.sseienc(t6in,hs8oe,sctt9sd),.etAhfeennoRsetNhaeAgraieinnvtsoetlrubfetairocetnnearcrieail(yaRncNdonAfusie)nrmgvieedacnhddaenfwiesenmrsee,wlmahteeiccrhhsahinsoitwshmne
Rey I Dipteran: amember RNAi-basedresponsesmediaterobustantiviralactivityinplants,nematodes,andinsects(10,
Annu. ovided b ibonyfsDethcitepstpecrrhaea,sreaanncctoeerrodizfeerodnoef 1vi3r,u1s-4d).eArinvetidvidraoluRbNle-AstiriasntdriegdgeRreNdAin(dhsoRstNcAel)lsinfotolloswmianlglitnhteersfeenrsiinnggRanNdAssub(ssieRqNueAnst)ctlheaatvaggueidoef
s pr pairofmembranous thesequence-specificrecognitionofcomplementaryviralRNAmolecules,leadingtotheirdegra-
es wingsandonepairof dationandtheblockadeofviralreplication.Aremarkablecharacteristicofthismechanismrelies
c
Ac hindwings onthefactthatitisbothbroadlyactive,becauseitistriggeredbydsRNAmoleculesderivedfrom

virtuallyanyvirus,andextremelyspecific,becauseittargetsonlytheparticularviralsequencethat
initiatedtheprocess.
Mostofourknowledgeontheinnateimmunesystemininsectscomesfromstudiesofthefruit
fly(Drosophila melanogaster)andthemosquito(6,8).D. melanogasterhasbeentheinsectmodel
ofchoicesincetheearlytwentiethcentury(15),anditisfollowedinpopularitybythemosquito,
the various species of which constitute the main vectors for numerous human viral pathogens
throughout the world (16). Consequently, research on insect immunity is heavily focused on
thesetwodipterans,andtheRNAifieldisnoexception.Reviewscoveringdifferentfacetsofthe
RNAiantiviralimmuneresponseinthefruitflyandthemosquitohavebeenpublishedelsewhere
·
574 Mongelli Saleh
VI03CH27-Saleh ARI 10September2016 11:42
Archaea Protozoa Viruses Bacteria Chromista
0.02% 0.17% 0.2% 0.61% 1.21%
Fungi Plantae
8.14% 21.43%
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Figure1
Insectsrule.TheInsectaclassrepresents86.5%ofallspeciesdescribedintheAnimaliakingdom.Indeed,insectsareamongthemost
extensiveanddiversegroupsoflife.Thesizeofeachcircleisproportionaltothenumberofspeciesineachkingdom,includingviruses.
www.annualreviews.org • SmallRNAsandAntiviralResponsesinInsects 575
VI03CH27-Saleh ARI 10September2016 11:42
(8,10,17).Inthisreview,webuildaframeworkforthediscussionsthatareneededtoadvance
futureinvestigation.Forseveralyears,researchonantiviralRNAiininsectshasbeencentered
onestablishedconceptualframeworks.Weusethisreviewtobrieflydiscusswhatisknownand
MicroRNAs
tofocusontheremainingquestionsandavenuesofresearchthatneedtobeaddressedtomove
(miRNAs):
smallRNAmolecules the field forward, including investigation of the activity of viral suppressors of RNAi, the use
(21–23nt)derived of comparative genomics, the development of detailed maps of subcellular localization of viral
fromhost-encoded replicationcomplexeswiththeRNAimachinery,andtheregulationoftheantiviralRNAiresponse.
miRNAloci
PIWI-interacting
RNAs(piRNAs): SMALLRNAPATHWAYS,THEIRBIOLOGICALFUNCTIONS,
smallRNAmolecules ANDTHEIRINVOLVEMENTINANTIVIRALDEFENSE
y. (26–30nt)derived
onl fromhost-encoded ThreemainsmallRNA–basedsilencingpathwaysareknownininsects:themicroRNA(miRNA),
se piRNAlocior siRNA,andPIWI-interactingRNA(piRNA)pathways.AlthoughtheyallusesmallRNAs(from
u
al transposableelements 21to30nt)toguidethesequence-specificrecognitionoftargetsequencesbyanARGONAUTE
nualreviews.org5/16. For person A(efArAnRRodGmGGoOrOOtihb)NNeopnArAuoUUctelTTeianEEsse:s (nDsAea.eGtmu1Ore8el–)aa2nnp0ordg)o.aftsaetteienrotfofatmbhreiilieyrfltmyardegemestcbsreairbf,teetrthhreeesceboasgsminsiatolilfoenRa,NcahnAdpsatthdhiefwifrearbyiio(nFloitgghuiecriarelf2ou)rni(gcfoitniro,ant.dhWeetiareilbueisdoegdreeenssceerasiripsc,thitoohnne,
w.an10/1 proteinfamily The miRNA pathway is initiated by the expression of genome-encoded miRNA gene tran-
wn characterizedbyPAZ scripts.TheseprimarymiRNAsarecapableoffoldingbackonthemselvestoformoneormore
m wale o (PIWI-AGO-Zwille) dsRNAstem-loopstructuresthattriggerthepathway.TheprimarymiRNAsareprocessedinthe
ded froe Centr aDnIdCPEIRW(IDdComRa)ins cmeilRl nNuAcl,ewushibcyhaisperxoptoeirntecdotmoptlheexcfoytrompeladsmby(D21R,O22S)H.PAreacnudrsPoArSmHiRANtoAsparoreduthceenthfeurpthreecruprrsoo-r
au proteins:
oq cessedinto21-to23-ntsmalldsRNA(miRNA)duplexesbyanotherenzymaticcomplexformed
9. DownlBibliothe epRnrNodtoaesrieinbsIoIfnIruofacmmletialhsyeeof bmyiRthNeADdIuCpEleRx1p(rDodCuRce1d)ipnrothteisinreaancdtioLnOiQslUoaAdCedIOinUtoSth(LeOAQRGS)O-PNAAoUrTLEO1Q(SA-GPOB1(2)-3c,o2n4t)a.inTinhge
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Acc guidingandcatalyzing harboring 2-nt 3(cid:3) overhangs (22). After being diced, siRNA duplexes are loaded into the AR-
sequence-specific GONAUTE2 (AGO2)-containing RISC. The biogenesis and loading of siRNA duplexes into
recognitionoftarget theRISCrequirestheactivityofLOQSandR2D2asDCR2cofactors.TheLOQS-PDisoform
sequences
andR2D2arerequiredfortheproductionofsiRNAsderivedfromendogenousdsRNAtriggers,
andR2D2isprimarilyrecruitedintheproductionofvirus-derivedsiRNAs(vsiRNAs)(28,29).
Once loaded into the RISC, one strand of the siRNA duplex, termed the passenger strand, is
eliminated from the RISC. The single-stranded siRNA that remains in the RISC, termed the
guidestrand,isthen2(cid:3)-O-methylatedatits3(cid:3)-terminalnucleotidebytheRNAmethyltransferase
DmHEN1(30,31),resultinginamature,activeRISC.Sequence-specificrecognitionmediatedby
theretainedsiRNAguidestrand,whichrequirescompletecomplementarity,theninducestarget
RNAcleavageviatheslicingactivityofAGO2.AlthoughendogenoussiRNAtargetsaremostly
·
576 Mongelli Saleh
VI03CH27-Saleh ARI 10September2016 11:42
a b c
miRNA gene dsRNA Transposon piRNA cluster
DROSHA LOQS ssRNA
Pri-miRNA
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Nucleus DCR2 AUB/PPIWI HEN1
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piRNA
siRNA
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views.orgFor person RISC HEN1 RISC TTrraannssppoossoonn ttrraannssccrriippttAUB/PPIWICH3
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Rey I mRNA.(b)ThesmallinterferingRNA(siRNA)pathwayisinitiatedbydouble-strandedRNA(dsRNA)ofviralorgenomicorigin
Annu. vided b tAbhyRaHtGiEsOrNNec1Ao,UgdniTriezEcet2ds(atAnhGdedOclee2ga)rv-acedodantbtiyaoinDnioInfCgtEhRReIS2taC(rDgbeCytDRR2NC)RAw2ivtaihantdAhGeR2hODe2lp2c.oaTtfahLlyeOtpicQasaSsce.tniTvgihteeyr.rs(etcrs)auTnltdhineisgPedIloiWmubIin-leian-ttseetdrr,aaancntdidendgthsReiRNgNuAiAd(espiasRirReNNdAeA)li,pvmeartehetdwhyatolyatthede
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pr isinitiatedwhenprimarypiRNAsareprocessedfromsingle-strandedRNA(ssRNA)precursorsderivedfromtransposableelement
ess sequences,namedthepiRNAclusters.ZUCCHINIendonuclease(ZUC)cleavestheprimarypiRNAprecursorsandgeneratesthe5(cid:3)
c
Ac endofthematurepiRNAs.TheprecursorsarethenloadedintoPIWIorAUBERGINE(AUB)proteins,trimmedtoreachtheirfinal
lengths,andmethylatedbyHEN1tobecomematurepiRNAs.Cleavageofthecomplementaryactivetransposontranscriptbyprimary
piRNAsloadedintoAUBproteinsinitiatesthesecondbiogenesisroundandleadstotheproductionofsecondarypiRNAsthatareloaded
intoARGONAUTE3(AGO3).Thisamplificationcycleisnamedping-pong.FigureadaptedwithpermissionfromReference103.
transposonsandprotein-codingmRNAs,vsiRNAsrecognizevirus-derivedsequences.Aswiththe
miRNApathway,thesiRNApathwayisubiquitouslyexpressed.
MoleculesinitiatingthepiRNApathwayaresingle-strandedRNA(ssRNA)precursorstran-
scribedfromchromosomallocithatmostlyconsistofremnantsoftransposableelementsequences,
called piRNA clusters (32). Biogenesis of piRNAs involves two steps, primary processing and
secondary amplification. Production of piRNAs is DCR independent and mainly relies on the
www.annualreviews.org • SmallRNAsandAntiviralResponsesinInsects 577
VI03CH27-Saleh ARI 10September2016 11:42
activityofPIWIproteins,asubclassoftheAGOproteinfamily(33).PrimarypiRNAsarepro-
cessedfromssRNAtranscriptsderivedfrompiRNAclusters.ZUCCHINIendonuclease(ZUC)
cleaves primary piRNA precursors and generates the 5(cid:3) end of mature piRNAs (34–36). The
cleavedprecursorisloadedintoPIWIorAUBERGINE(AUB)proteinsandthentrimmedby
an unknown nuclease to reach its final length. After trimming, piRNAs undergo a final 3(cid:3)-end
2(cid:3)-O-methylnucleotidemodificationcatalyzedbyDmHEN1(30,31)toyieldmaturepiRNAs.
PrimarypiRNAsharbora5(cid:3)uridinebias(U1)(37).Cleavageofthecomplementaryactivetrans-
posonRNAbyprimarypiRNAsloadedintoAUBproteinsinitiatesthesecondroundofbiogen-
esis,whichleadstotheproductionofsecondarypiRNAsthatareloadedintoARGONAUTE3
(AGO3).Duringthisping-pong,oramplification,cycle,AUBandAGO3proteinsloadedwith
secondary piRNAs mediate the cleavage of complementary RNA, generating new secondary
y.
nl piRNAsthatareidenticalinsequencetothepiRNAthatinitiatedthecycle.Thecomplementary
o
se secondarypiRNAsusuallyhavea10-ntoverlapandcontainanadenineatposition10(A10)(38).
u
al Most data indicate that the piRNA pathway is mainly active in germ-line tissues, where it acts
nualreviews.org5/16. For person aethsleramoUugengnethntilsot.rhmeeceepngrutoladyr,udcoitaninolynbtyohfecvlsesiaiRRvNiNngAAstp.raaHtnhsowpwoaeysvoewnra,RsvNikrnuAos-wodrnerttirvoaencdsocsnrmitpraitlbilouRnteaNlltAyosstihwleeinthacninptiigvRitNrraalAnrsfepesoaptsouanbreslees
w.an10/1 havebeenfoundbyusingnext-generationsequencinginAedesmosquitoesandmosquitocelllines
wn infectedbyarboviruses(39–44).Thesedescriptivestudieswerefollowedbydirectfunctionalanal-
m wale o yses demonstrating that (a) depletion of PIWI4 protein enhances replication of Semliki Forest
ded froe Centr tviiorunsin(pAoasigt2ivcee-sllesn(s4e5s)saRnNdA(b,)TboogtahvPirIiWdaeI)5waintdhoAuGtiOnt3earfreerrineqguwiriethdvfoirrupsi-RdeNriAvebdiopgiRenNesAispdreordivuecd-
au
oq fromSindbisvirus(positive-sensessRNA,Togaviridae)inthesamecellline(46).Nevertheless,
9. DownlBibliothe fwuhnacttieoxntaelnitnthveivopiRdaNtaArepmatahiwnasycacrocnet,raibnudtemsotroeawntoirvkiranleeddesfetnosebseadsoangeetnoerfaulllmyeucnhdaenrisstman,dnotot
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5P
Rev. Virol. 2016.3:y Institut Pasteur - paminnrefotetiIdvcanutbisricoeoaecllnidtsdsmebcmfay,einRndDaseNel.vteAeminlrsoetltphahamneriseoergoenaigrntsg,utvealdoran,iltfivifsaoeemndnrde(oni4tnfth7ihae)t.othipsoetinRmr,NeiaRgApuNolppaAattisotohstnhwisa,aotyafmntddhaiyuvisneerindtshaoeteeersbpiinomroloomtmgauiopcntapeeleoarperrrsiotpnocoteenbsrsesfeeesrsre,e(q4iwun8iic)rt.lheuVddviiifrrnoaagrll
Annu. vided b iinnftehcetioenstabbyliisnhflmueenntcionfginthfeecteixopnre(4ss9i–o5n3o).fVhiorsatlgimenmoumneesrceasnpoennsceodgeentehseiorrovwirnalmgieRnNesAisn,vwohlviechd
pro functiontoregulateviralgeneexpressionormanipulatethehostimmuneresponse.Amongthe
ss fewinsectviralmiRNAsidentified,mostcomefromviruseswithdouble-strandedDNA(dsDNA)
e
c
c genomes,suchasHeliothisvirescensascovirus(Ascoviridae),Bombyxmorinucleopolyhedrosisvirus,
A
and Autographa californica nucleopolyhedrovirus (51, 52, 54, 55). Nevertheless it has been sug-
gestedthatmiRNA-likemoleculesareencodedbytwoflaviviruses(positive-sensessRNA):West
Nilevirusanddenguevirus(56,57).TheWestNilevirusmiRNA-likemoleculeisthoughtto
facilitateviralreplicationbytargetingcellularmRNA,andthedenguevirusmiRNA-likemolecule
isthoughttoregulateviralreplicationbytargetingaviralnonstructuralproteinsequence(56,57).
The role of viral and cellular miRNAs during the antiviral response remains an enigma, but it
seemstoconstituteakeyfeatureofanintricatehost-pathogenstruggle,inwhichbothvirusand
hostmiRNAsandproteinsaimtocontrolvirusandhostgeneexpression(58–60).Theelucida-
tionofsuchinteractionsrequirescomplexandrefinedresearchthatfullyaddressesthebiological
implicationsandconsequencesbeyondprovidingmeredescriptionsandmiRNAannotations.
·
578 Mongelli Saleh
VI03CH27-Saleh ARI 10September2016 11:42
VIRALSUPPRESSORSOFRNAiANDVIRALCOUNTER-DEFENSE
MECHANISMS
TheexistenceofviralproteinswiththecapacitytointerferewithdifferentcomponentsoftheRNAi
machinery,theviralsuppressorsofRNAi(VSRs),highlightstherelevanceoftheRNAiantiviral
defenseasthecenterofthehost-pathogenencounter.Severalviralsuppressorshavebeenidenti-
fiedininsectviruseswithpositive-sensessRNAgenomes(Nodaviridae,Dicistroviridae,Flaviviridae,
Nora viruses with unassigned family), dsRNA genomes (Birnaviridae), and DNA genomes (As-
coviridae,Iridoviridae)(61).Curiously,viralsuppressorsfornegative-sensessRNAviruseshavenot
beenreported.TheseVSRsexerttheiractionbyfourmainmechanisms:(a)bindinglongdsRNA
topreventDCR2cleavage,(b)bindingsiRNAstopreventtheirloadingintotheRISC,(c)directly
y. interactingwithDCR2orAGO2proteinstopreventtheiraction,and(d)degradingsiRNAs.In
nl
o somecases,oneVSRproteinmaycombinemorethanoneofthesemodesofaction(forreviews
e
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u ofinsectVSRs,see10,61).
al
ws.orgperson repMorotesrtVsySstResmhsaovrerbeeceonmcbhianraancttevriirzuesdesus(i6n2g,e6x3)viwvoithsoysuttemcosnasnidderinatitoisnsuoefctuhletunraetuursainlgcoRnNteAxti
vieFor ofaninfectionandtheroleoftheVSRinpathogenesisandtransmission.Despitetheutilityof
alre16. these RNAi and VSR reporter systems, they have important limitations. Viral proteins usually
nu5/ displaymorethanonefunction,andtheirectopicoverexpressioninasystemdesignedtodetect
n1
w.a10/ suppressionmightproducefalse-positiveresults.Inthesetypesofexperiments,itisdifficultto
wn
m wale o pecrtoovpeicwahlleytehxeprreasvseirdadlspRroNteAi-nbiwnidthingapdrsoRtNeinAs-abcintadsinVgSdRosminarinepiosroterriasssnaoytsainVpSlaRn.tsF(o6r4)e.xIanmdepelde,,
d froCentr Escherichia coli RNase III and mammalian orthoreovirus (MRV) σ3 protein expressed in plants
dee both display VSR activity. Strikingly, E. coli RNase III is a global gene expression regulator
au
oq
9. DownlBibliothe ieannffidbciamecnteecdryiiaa(t,6e5isn).flseMuveeRnrVcailnσagc3tpiivosistatitemrsauntlhstcriforuiupngtcihtoionouantlaltchopenrotvrtioerilanlotlfihfmaetRcsyeNcrlvAees(s6taa6sb)a.iliWvtiyrhiooernthmoeurRtMNerARcaVtprsaσind3slpacrtaoinotenbianel
73-58aris - considered to be a VSR in its natural biological context remains unknown. Another limitation
5P ofstudyingVSRsisthehostspecificityofVSRactivity.Forexample,VP1proteinsencodedby
v. Virol. 2016.3:nstitut Pasteur - DVaDcS..tmiRamssemlabaniyVgorigSnaaRntsetseriAnracGNatiOonDrg2.awm(v6iier7tluha).snAO(oDGgfamOastleel2rlkNaSnn2oVdw)cbnealnlolidncrkseDeipncrotogrsVottepaSrhrRgisleyas,tsittmcehlmemea.BivgDa2rgaipenmr.somHNteNooinwrVaeo-vVlfiekFPre,l1oDvccirkiamunHsmi(onDNuteisVmreamvcVitrNPou1Vnslc()yFaanHwcntiVtoahst)
Rey I has been most extensively studied. Its absence during infection in adult flies causes a defect in
Annu. vided b FwHasVcornedpulicctaetdionusainngdDth.emaeclcaunmogualsatetrio,nwhoifcFhHisVn-odterthiveednastiuRrNalAhso(s6t8o)f.FTHheVs.tHudoywtehvaetrf,otuhnisdktihnids
o
pr ofdirectexperimentalevidenceabouttheroleofVSRsinthevirallifecycleandduringinfection
s
es ofthehostislackingforVSRsfromotherinsectviruses.
c
c
A An interesting facet of VSRs is their potential capacity to interfere with the normal activ-

ity of small RNA pathways other than the vsiRNA pathway, due to their mode of action. Al-
thoughtheectopicexpressionofVSRsinplantswasfoundtointerferewiththemiRNApathway
andleadtopleiotropicdevelopmentaldefects(69,70),analogousexperimentsperformedusing
D.melanogasterhavedemonstratedthatVSRsdonotaffectglobalmiRNAbiogenesisandfunction
(71).TheseresultscanbereconciledbythefactthatplantsiRNAandmiRNApathwaysshare
biogenesis factors, and both vsiRNAs and miRNAs can be loaded into AGO1 (72), whereas in
flies,themainfactorsofthesepathwaysarenotcommonlyshared.Additionally,ectopicexpression
ofsomeVSRssuppressesRNAiinducedbyexogenousdsRNAinembryosandbyendogenous
siRNAsinadultflies.Theseresultssuggestthat,althoughVSRsmightnothaveaconspicuous
www.annualreviews.org • SmallRNAsandAntiviralResponsesinInsects 579
VI03CH27-Saleh ARI 10September2016 11:42
effectonthemiRNApathway,theyappeartoblocktheendogenoussiRNApathway,aresultthat
isfurthersupportedbyotherworkfromthesamegroup(73).DeterminingwhetherVSRsinter-
ferewithsmallRNApathwaysinageneralmannerrequiresmoreresearchonD.melanogaster,as
wellasonotherinsectspecies.
Finally,becausethepiRNApathwayisinvolvedintheantiviralresponse,shouldweexpectto
identifyVSRsofthepiRNApathway?Ifso,whatwouldbethecounter-defensemechanismof
virusesagainstantiviralpiRNAs?
COMPARATIVEGENOMICS:NATURALVARIATIONSOFRNAi
COMPONENTSAMONGINSECTS
y.
nl Asmentionedabove,mostofwhatweknowabouttheantiviralroleofsmallRNA–basedpathways
o
se comesfromstudiesofD.melanogasterandmosquitoes.Nevertheless,otherinsectsofeconomicin-
u
al terest,suchashoneybees(A.mellifera),silkworms(B.mori),redflourbeetles(Triboliumcastaneum),
nualreviews.org5/16. For person ptasotnruoeddsleifpeonldarcneu(t7no4dvf–iser7mrus7sat)-al.tlnrTRdanhNinsegAmetsixhtpiteseitnrceoginelceshedoeemfotefeinpcRdtteNeordgoAebiindyiosnnu(easdxpgtif-hefgenidereness,neowrtrathtfyoiioptreneaflsfsifeeoeqscf,utiiaennnnsgedctcinhtthsgerhiainpnasssde)cb,(btea)leritenfheebscheuyiocsnelwegonafesxbRatyNemn(Aaes)itivhateohsldyea
w.an10/1 ofpestcontrol.However,theincreasingnumberofgenomesequencingprojects,with138insect
wn genomesequencesnowavailable(78),allowsfordetailedcomparativegenomicandphylogenetic
m wale o analysesoftheRNAipathwaycomponentspresentindifferentinsectspecies.Table1showsthe
ded froe Centr npruomtebienrfoafmgielynemceompbieesrsfo,rinthseomkeeyicnosemcptoonrdenertssoanfdthsepsemciaelsl.RTNhiAslpisatthiswnayost,etxhheaDusCtiRveabnudtAilGluOs-
au
oq tratesthatsmallRNA–encodinggenesdifferincopynumberamonginsectspecies.Phylogenetic
9. DownlBibliothe aenxpalaynsdeesdoofrsmcoanltlrRacNteAddpuatrhinwgatyhceocmouprosneeonftesvhoaluvteioinnd(i7c9a–te8d1)t.hFaotrloexcaimenpcleo,dminigRNthAesgeemneoelexcpualne-s
73-58aris - asinodnsthaerehofouusnefldyin(Mthuescpaedaomapehstiidca()A.Gcyretnheoseipxhpoannspiiosunmso),ftshiReNRuAssairaenfowuhnedatinapthhiedlo(Dcuiustra(Lphociussntoaxmiai)-,
5P
Rev. Virol. 2016.3:y Institut Pasteur - gv2wArieaitandrstipeo7psr;,e9itna)Aw.n)n,Toisto)hhtp,yheedpeipRelveiseuRs,rosNssafeinaaAsndnpptweCsach(tuiCheelewsaaxtmoaafygppGeoghnneliooendstrseu,aisstn.heflaDexo(prtursiaepdendlatidsncfleeuaodfltsuiiaiorennnsbdb)e,aoHetnththdaleerpdfp(reeTiuvag.ienatcranaflstsdihytfiao,Rcnsaauenstausidslomtitanah)tn,oeotrwhfh);heosameupnasaadterlaflalmpsyRihot(Nois8dqi2Adsu;;iawttphlosaaeeostpshpsfwae(rNreoaayfiamssisgtouotanhrriidaeee
Annu. vided b watidtheelyspperecsieesntleavmelo.nginsects,indicatingthatthesechangesarenotrareandhaveoccurredeven
pro Although gene duplications are a frequent source of novelty in host genome defense reper-
ss toires,theevolutionaryfateofspecificduplicatescanvary.Inmostcases,oneoftheduplicated
e
c
c copieswillbeeliminated;alternatively,bothcopiesmayberetainedbutmayacquiredifferentiated
A
profilesofexpressionordifferentfunctions(83,84).ThefrequentexpansionofgenesintheRNAi
pathwayisoftenaccompaniedbychangesintheirevolutionaryrates.Someofthesechangesmight
be explained by the involvement of these genes in host-pathogen interactions. For example, in
D.melanogaster,genesinvolvedinpathogendefenseevolvemuchmorequicklythantherestofthe
genome.AGO2isaclearexampleofthisobservation,andPIWIandAUBalsoevolveextremely
quickly(85),whichisthoughttobeattributabletoadaptationtotheever-changinglandscapeof
transposition activity in the fly. Another interesting example arises when studying the involve-
mentofthepiRNApathwayinantiviralresponsesintwodipterans,thefruitflyandthemosquito.
Althoughvirus-derivedpiRNAsarepresentinmosquitoes,recentworkfromourlab(47)hasindi-
catedthattheyarenotproducedduringviralinfectionsinD.melanogaster.Althoughfliesencode
·
580 Mongelli Saleh
VI03CH27-Saleh ARI 10September2016 11:42
3
O
G — 2 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 1 1
A A
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cting NA) AUB — 0 0 1 0 1 3 2 2 1 — 1 1 1 1 1 0 0 0
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PIWI-inte (pi PIWI, WI/AUB — 8 5 1 1 1 0 1 1 1 1 1 1 1 1 1 7 2 6
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y.
m www.annualreviews.orgale on 10/15/16. For personal use onl meinsectordersandspecies Small interfering RNA(siRNA)miRNA) CR1AGO1DCR2AGO2 1122 2211 1212 1111 1112 1111 1112 1111 1111 1111 1111 1111 1111 1111 21—2 —1—3 1111 1111 1112
Annu. Rev. Virol. 2016.3:573-589. Downloaded fro Access provided by Institut Pasteur - Paris - Bibliotheque Centr ofgenecopiesforkeycomponentsofthesmallRNApathwaysinso MicroRNA( SpeciesCommonnameDROSHAD LocustamigratoriaLocust1 AcyrthosiphonpisumPeaaphid1 DiuraphisnoxiaRussianwheataphid1 NilaparvatalugensBrownplanthopper1 TriboliumcastaneumRedflourbeetle1 ApismelliferaHoneybee1 NasoniavitripennisParasitoidwasp1 CamponotusfloridanusBullant1 HarpegnathossaltatorIndianjumpingant1 BombyxmoriSilkworm1 ManducasextaTobaccohornworm1 DanausplexippusMonarchbutterfly1 HeliconiusmelpomenePostmanbutterfly1 DrosophilamelanogasterFruitfly1 MuscadomesticaHousefly1 GlossinamorsitansTsetsefly— AedesaegyptiYellowfevermosquito1 AnophelesgambiaeMalariamosquito1 CulexpipiensHousemosquito1 numberisnotknown;0indicatesthatcopieswerenotfound.
mber atthe
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u t
Table1N Order Orthoptera Hemiptera Coleoptera Hymenoptera Lepidoptera Diptera Dashindicates
www.annualreviews.org • SmallRNAsandAntiviralResponsesinInsects 581
VI03CH27-Saleh ARI 10September2016 11:42
threePIWIproteins(PIWI,AUB,andAGO3),thePIWIfamilyisexpandedtoeightmembers
(PIWI1–7andAGO3)inthemosquitoAedesaegyptiandtoseveninCulexpipiens(86).Itispossible
thatduringspeciationanddiversificationofpiRNApathwayproteins,thepiRNApathwaygained
functionsbeyondtherepressionoftransposonactivityinmosquitoeswhileremainingexclusively
focusedonthecontroloftransposonsinthefly.Alternatively,thepiRNApathwaymayhavelost
anancestralantiviralfunctionsincethelastcommonancestoroffliesandmosquitoes.Withthe
advent of new sequencing technology and increasing access to metagenomic data, the field of
comparativegenomicsandevolutionshouldcontinuetoexpandknowledgeabouttheorigin,role,
anddiversityofRNAigenesininsects.Thesestudieswillestablishafoundationforcomprehen-
sivedesignsofwet-labapproachestodifferentinsect-viruscombinationstounlockthesecretsof
antiviralRNAiininsects.
y.
nl
o
e
s
al u WHEREANDWHEN?SUBCELLULARLOCALIZATION
nualreviews.org5/16. For person OStrheepeFvlepircTraaeltHsieionnEndcieeRnptoNeefnrmvAdierien-adlVtiadsItsteRRusdUN(iseeASsesIhtiaNanbvlTteeh1EieniRcdneiA1clla0.Ct)Ie.nTdAmdItOhdoiastNttiotcnhaaeslelasy,n,tRthivNeirAsaolvuiRrrcuNeseAosifctmahnaebcseheivrneiertarrylotdirssaRntNrsicgArgisbeeriseddvinirbtaoyl
w.an10/1 aviralDNAformbycellularreversetranscriptases.ThetranscriptionoftheseviralDNAformsis
wn involvedinthedsRNAbiogenesisthattriggerstheRNAiresponse(seefigure1in87).However,a
m wale o questionsurroundsthisevidence:WhenandwhereinthecelldoestheRNAimachineryencounter
ded froe Centr theVviirraallrreepplliiccaattiioonnaisppaacraotmusp?lex and regulated process that may occur in thenucleus (dsDNA
au
oq virusesandsomenegative-sensessRNAviruses)orthecytoplasm(positive-sensessRNAviruses,
9. DownlBibliothe drespRlNicaAtivoinruosfesm,aonsdtpsoomsiteivnee-gsaetnisvee-ssseRnNseAssvRirNusAesviirsuassesso)c(iFaitgedurwei3th).,Danedsploitceabliizoelodgtioc,ahldoisftf-edreernicveesd,
73-58aris - mvireomnbmraennetst.oPsouspitpiovert-sveinraslersespRliNcaAtivoinruasnedsfaossremmvbelsyi.cTulhareivnivraalgdinsaRtNionAsitnoteursmeeasdiaanteoipstmimaiizneldyedne--
5P
Rev. Virol. 2016.3:y Institut Pasteur - tt(ft8rehooc8ettm)heG,deaeorinclnegydsitoidodfseopeorrlmitavhBseeemudssnefytfomrharovreTmoimruoigdgbmaahrveiat,ipnoraooicndrhudaeose-f,snlrtifdokrrurmeoicmastatudrutruhuteocerstipen(uh8ngr8aedg)sio,nopw(fs8eloha9cmsi)tcm.iheoTsinachfrorweeerscitetPiohcnimucnmoleruemenmcmatbevabrdinaretindrdosoaGoueot.osfhVlsegtthirirreumfuocsferetamusFmrflbeairlrsovya,imnvoNierroosistddahpaanehvedr,ierofrfiraupdomlaemeen-s,
Annu. vided b iclaiellse,dsuvcirhopalsaIsrmidsotvhiraitdhaeo,uPseoxvviirraildaree,palincdatiRoeno.vIirnidtahee,cfoasremoefldescRtrNonA-dreenosveirucysetos,prlaespmlicicatiinocnluosfiothnes
pro genometakesplaceinsideviralparticles.ViraldsRNAproducedduringviralreplicationismainly
ss localizedinsideviralmembranousorproteinstructures,whichmaybeamechanismtoprotector
e
c
c delayrecognitionofPAMPsinvertebrates(90,91).Thesamerationalecanbeappliedtoinsect
A
virusesandantiviralRNAi.
ThesubcellularlocalizationofRNAiisnotpreciselyunderstood,althoughithasbeenproposed
tooccurinthecytosol.Inplantsandmammals,RNAiproteinsareassociatedwithendomembrane
systemsinthecell(92).Forinsects,informationisscarce.R2D2andDCR2havebeenproposedto
localizeindiscretecytoplasmicbodiesinDrosophilacells,termedD2bodies(93),andtheAGO1-
containing RISC localizes to mRNA decay centers, suggesting that this is the final step of the
RNAipathway,whentargetRNAisdegraded(94).
InDrosophilasomaticcellsfromgermtissue,theprimarypiRNAbiogenesismachinery(PIWI
proteinandothers)localizesinperinuclearcytoplasmicstructurescalledYbbodies(95).Ingerm-
linecells,bothprimaryandsecondarypiRNAbiogenesismachinerieslocalizeinotherperinuclear,
·
582 Mongelli Saleh