immune defense mechanisms of dsrna viruses · protein (pabp) from eif4g to downregulate host...

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


✦  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


✧  mRNAsaresynthesizedwitha

capstructurethatmimicshostmRNAcaps

✧  ViralRNAbindingproteinsmade

duringinfectionmayprotectRNA(factories/viroplasms)

✧  SynthesisofdsRNAgenomeoccursasviralcore/capsidstructureisassembled•  ThedsRNAgenomeremains


IFNAR

MAVS

RIG-I

RIG-I

IRF3IRF7

TLR3

IκB

NF-κB

ISGF3

IRF9STAT1

STAT2

IκB UbUbUb

UbUb

SCFβ-TrCP







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


VP3PDE

IFNAR

MAVS

RIG-I

RIG-I

IRF3IRF7

TLR3

IκB

NF-κB

ISGF3

IRF9STAT1

STAT2

IκB UbUbUb

UbUb

SCFβ-TrCP







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







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






X


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






Host“ProteinX”,whichisessentialforIFNinduction(orsignaling),isreduceduponviralinfection.Whatdoesthedatashow?

1.  Arelevelsofotherhostproteinsequalorvariableduringinfection?2.  ArelevelsofhostProteinXrestoredupontreatmentwithproteasomeorlysosome

inhibitors?3.  AremRNAtranscriptlevelsofhostProteinXequalorvariableduringinfection?4.  IshostProteinXsubcellularlocalizationshowntobealteredduringinfection?5.  Arepost-translationalmodificationsofhostProteinX,oritsupstreamactivators,

alteredduringinfection?6.  CanoneormoreviralproteinsbeidentifiedtocausethelossofhostProteinX?

immune defense mechanisms of dsrna viruses · protein (pabp) from eif4g to downregulate host...

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