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13thInternationaldsRNAVirusSymposium
ImmunedefensemechanismsofdsRNAviruses
MichelleM.Arnold,Ph.D.LouisianaStateUniversityHealthSciencesCenter–Shreveport
Shreveport,Louisiana,U.S.A.
Whatdefensemechanismsdomicrobesencounterwheninfectingahost?
AnatomicalbarriersInhibitpathogensfromentering
Skin,gastrointestinaltract
InnateimmuneresponseImmediate,non-specificresponsetoinfection
Forviralinfections,largelycontrolledbyinterferon
AdaptiveimmuneresponseAntigen-specific,activatedbyinnateimmune
response,necessaryforviralclearance,servesasimmunologicmemory
TypeIIFNProteins
Genes Numberofeach
Receptor Inducedby
IFN-αIFN-βIFN-εIFN-κIFN-ω
IFNAx*IFNB1IFNE1IFNKIFNW1
131111
IFNAR1andIFNAR2 ViralinfectionOthermicrobialinfections
TypeIIIFNProteins
Genes Numberofeach
Receptor Inducedby
IFN-γ IFNG 1 IFNGR1andIFNGR2 Mitogenic,cytokine,antigenicstimuli
TypeIIIIFNProteins
Genes Numberofeach
Receptor Inducedby
IFN-λ1IFN-λ2IFN-λ3
IL29IL28AIL28B
111
IFNLR1andIL10R2 Viralinfection
Interferons(IFNs)areafamilyofcytokineswithpleiotropicbiologicaleffectsinthehost
*WherexindicatesthenumericaldesignationoftheIFN(e.g.IFNA4,IFNA2,IFNA5,etc.)
Prim
aryfocuso
ntype
IIFNs
Second
aryfocuso
ntype
IIand
IIIIFN
s
Interferons(IFNs)functionasanintegratedsystem
Conceptually,wewillconsidertheIFNresponseintwophases
Ø ProductionofIFNsistransient,andrequiresstimulationbyviruses,microbialproducts,orchemicalinducers
Ø IFNwasdiscoveredinstudiesofviralinterference,usingliveorheat-inactivatedinfluenzavirusesasinducers
Ø Subsequently,othermicrobialproducts(nucleicacids,lipids,polysaccharides,orproteins)wererecognizedtoinduceIFN
Ø Afterbindingtoreceptors,IFNsinitiateasignalingcascadethroughsignalingproteins(thatcanbeactivatedbyothercytokinesaswell)
Ø CellularactionsaremediatedthroughspecificIFN-stimulatedgenes(ISGs),whichunderlietheantiviraleffects
Ø EffectofIFNsimportantforunderstandingmechanismofactionandapplicationsinmedicine
PRODUCTIONofIFNs SIGNALING/EFFECTSofIFNs
SimplifiedIFNinductionandIFNsignalingpathway
Upstreamactivators
Keytranscription
factors
PRRs IRFsNFκB IFNRs ISGs
Effectorsmolecules
Receptormolecules
Keytranscription
factors
PRODUCTIONofIFNs SIGNALING/EFFECTSofIFNs
STATsIFNsCytokines
Somedefinitions:IFN:interferonPRR:pattern-recognitionreceptorIRF:interferonregulatoryfactorNFκB:nuclearfactorkappaB
IFNR:interferonreceptorSTAT:signaltransducersandactivatorsoftranscriptionISG:interferon-stimulatedgene
IFNAR
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
Infectedcellsrapidlyrespondtoviralinfectionsbyactivatingtheinnateimmuneresponse
ViralcomponentsthattriggerRIG-I,MDA5,andTLR3stimulationinclude:
• genomicdsRNAs• dsRNAintermediates(foldedmRNAs)• uncappedmRNAs
IFNAR
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
Interferon(IFN)regulatoryfactorsIRF3andIRF7driveIFN-βexpression
IFNAR
NF-κB
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
IκB UbUbUb
UbUb
SCFβ-TrCP
NF-κBisalsocriticalforIFN-βexpression
IFNAR
IRF7 ISGsNF-κB
IFN-βsignalscellstoproduceIRF7andotherinterferon-stimulatedgenes(ISGs),inhibitingviralspread
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
TheantiviralfunctionsofOASandRNaseLarecoupledtomediateRNAdegradation
✦ OAS(2',5'-oligoadenylatesynthetase)andRNaseLproteinsareconstitutivelyexpressedatlowlevelsasinactivemonomers
✦ Activatingligands(suchasviralRNA)induceOAStoformanactivetetramer,whichsynthesizes2',5'-linkedoligomersofadenosinefromATP(pppA(2’p5'A)n)
✦ TheoliogadenylatesspecificallyactivatethelatentmonomericformofRNaseLwhichsubsequentlyleadstodimerization
✦ TheactivedimericRNaseLdegradessingle-strandedRNA,thusinhibitingviralreplicationandtranslation
✦ OASisupregulatedbytypeIIFNs
✦ OASalsofunctionsasaPRR,byvirtueofitsactivationbyviralRNA
NatRevImmunol(2008)8:559-568
PKRinhibitsvirusandhosttranslation
NatRevImmunol(2008)8:559-568
✦ PKR(proteinkinaseR)isamemberofafamilyofproteinkinasesthatregulatesproteinsynthesis
✦ PKRisconstitutivelyexpressedinalltissuesatbasallevel,andismaintainedasaninactivemonomer
✦ Activatingligands(suchasviralRNAorotheractivators)induceaconformationalchangethatallowsformationofanactivedimer
✦ TheactivedimerphosphorylatesEIF2α,whichpreventsrecyclingofGDPandinhibitsproteintranslation
✦ PKRisupregulatedbytypeIandtypeIIIIFNs
✦ PKRalsofunctionsasaPRR,byvirtueofitsactivationbyviralRNA
Autophosphorylationisrequiredfordimerformation
IFNAR
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
WhatarestrategiesusedbyvirusestoevadetheIFNresponse?
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
X
✧ Virusesdonotfullyuncoattorevealthegenome• ThedsRNAgenomeremains
protectedwithintheviralcore/capsidstructure
✧ ViralmRNAsaresynthesizedwith
acapstructurethatmimicshostmRNAcapsb.membrane
penetration
c.transcription
a.attachment,endocytosis
HowdodsRNAviruseshidetheirgenomeandprotectRNAs?
nucleus
b.membranepenetration
c.transcription
a.attachment,endocytosis
HowdodsRNAviruseshidetheirgenomeandprotectRNAs?
factory/viroplasm
nucleus
e.replicationandassembly
d.translation
✧ Virusesdonotfullyuncoattorevealthegenome• ThedsRNAgenomeremains
protectedwithintheviralcore/capsidstructure
✧ mRNAsaresynthesizedwitha
capstructurethatmimicshostmRNAcaps
✧ ViralRNAbindingproteinsmade
duringinfectionmayprotectRNA(factories/viroplasms)
✧ SynthesisofdsRNAgenomeoccursasviralcore/capsidstructureisassembled• ThedsRNAgenomeremains
protectedwithintheviralcore/capsidstructure
IFNAR
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
WhatarestrategiesusedbyvirusestoevadetheIFNresponse?
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
X X
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
SCFβ-TrCP
NSP1NSP1
NSP1 NSP1NSP1
IκB UbUbUb
UbUb
✧ NSP1sharessequencesimilarity,includingaconservedRINGdomain,withviralE3ubiquitinligases
✧ RINGdomainisessentialforIRFandβ-TrCPdegradation
✧ TreatmentwithproteasomeinhibitorspreventsdegradationofIRFsandβ-TrCP
Example:rotavirusANSP1inducesdegradationofIRFsand/or𝛃-TrCPbypolyubiquitinationtotargetproteinstotheproteasome
Example:rotavirusAVP3phosphodiesterasedomaincleavespppA(2'p5'A)ntopreventRNaseLactivation
✦ Activatingligands(suchasviralRNA)induceOAStoformanactivetetramer,whichsynthesizes2',5'-linkedoligomersofadenosinefromATP(pppA(2'p5'A)n)
✦ TheoliogadenylatesspecificallyactivatethelatentmonomericformofRNaseLwhichsubsequentlyleadstodimerization
✦ TheC-terminaldomainofrotavirusAVP3cleaves(pppA(2'p5'A)n)intoATP+2AMP,thuspreventingRNaseLactivationandantagonizingthehostIFNresponse
✦ RotavirusBandGVP3proteinsarepredictedtocontainaC-terminalphosphodiesterasemotif,buttheactivityhasnotyetbeentested
NatRevImmunol(2008)8:559-568
VP3PDE
IFNAR
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
WhatarestrategiesusedbyvirusestoevadetheIFNresponse?
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
X X X
Virusesusedifferentmechanismstopreventnuclearlocalizationoftranscriptionfactors
✧ Someviralproteinspreventtransportacrossthenuclearporecomplex(NPC)• Adenovirus,EBV,HIV-1,HSV-1,
InfluenzaAcanblockNPC–allhavereplicationstepsthatoccurinnucleus
• VSV,cardiovirus,polioviruscanalsopreventNPCtransport–noapparentreplicationstepsinnucleus
✧ ReoviridaeorotherdsRNAviruseshaveyettobeshowntoinhibitNPCtransport
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
IκB UbUbUb
UbUb
SCFβ-TrCP
VP
VPVP
IFNAR
IRF7 ISGsNF-κB
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
WhatarestrategiesusedbyvirusestoevadetheIFNresponse?
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
X XX
IFNAR
NF-κB
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
Example:thereovirusμ2proteinlocalizestonuclearspecklesandalterscellularmRNAsplicing
NS
μ2
NS
μ2 X XXμ2 μ2
✧ μ2localizestothecytoplasmandnucleusofinfectedcells,althoughviralreplicationappearstobeentirelycytoplasmic
✧ μ2interferencewithregulatorsofmRNAsplicingmayalterantiviraland/orproviralprocesses
IFNAR
IRF7 ISGsNF-κB
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
TLR3
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
X
WhatarestrategiesusedbyvirusestoevadetheIFNresponse?
IFNAR
IRF7 ISGsNF-κB
IFN
MAVS
RIG-I
RIG-I
IRF3IRF7
IκB
NF-κB
ISGF3
IRF9STAT1
STAT2
IκB UbUbUb
UbUb
SCFβ-TrCP
Examples:rotavirusNSP3preferentiallyenhancestranslationofviraltranscriptswhilepreventinghostmRNAtranslation
X X
✧ NSP3displacespoly(A)-bindingprotein(PABP)fromeIF4Gtodownregulatehosttranslation
✧ NSP3causesanaccumulationofPABPinthenucleus
✧ NSP3blocksnucleocytoplasmictransportofpoly(A)mRNAs
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
NSP3
PABP
PABPPABPPABP
PABP
PABPPABP PABPPABP
Theseareallwaysinwhichessentialhostproteinlevelsmightbedecreased
Finalthoughts:assessingnewdataforvirusinhibitionoftheinnateimmuneresponse
1.HidethegenomeandprotectRNAs
2.Promotehostproteindegradation
3.Preventtranscriptionfactorsfromlocalizingtothenucleus
4.InhibittranscriptionoralterRNAprocessing
5.Turnofftranslation
Host“ProteinX”,whichisessentialforIFNinduction(orsignaling),isreduceduponviralinfection.Whatdoesthedatashow?
1. Arelevelsofotherhostproteinsequalorvariableduringinfection?2. ArelevelsofhostProteinXrestoredupontreatmentwithproteasomeorlysosome
inhibitors?3. AremRNAtranscriptlevelsofhostProteinXequalorvariableduringinfection?4. IshostProteinXsubcellularlocalizationshowntobealteredduringinfection?5. Arepost-translationalmodificationsofhostProteinX,oritsupstreamactivators,
alteredduringinfection?6. CanoneormoreviralproteinsbeidentifiedtocausethelossofhostProteinX?