Table Of ContentVirology 515 (2018) 165–175
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Virology
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Accessory proteins 8b and 8ab of severe acute respiratory syndrome
T
coronavirus suppress the interferon signaling pathway by mediating
ubiquitin-dependent rapid degradation of interferon regulatory factor 3
HuiHuiWonga,b,1,ToSingFunga,1,ShouguoFangc,d,MeiHuangc,MyTraLeb,DingXiangLiua,c,⁎
aSouthChinaAgriculturalUniversity,GuangdongProvinceKeyLaboratoryMicrobialSignals&DiseaseCo,andIntegrativeMicrobiologyResearchCentre,Guangzhou
510642,Guangdong,People'sRepublicofChina
bInstituteofMolecularandCellBiology,61BiopolisDrive,Proteos,Singapore
cSchoolofBiologicalSciences,NanyangTechnologicalUniversity,60NanyangDrive,637551,Singapore
dAgriculturalSchool,YangtzeUniversity,266Jingmilu,JingzhouCity,HubeiProvince434025,China.
A R T I C L E I N F O A B S T R A C T
Keywords: Severeacuterespiratorysyndromecoronavirus(SARS-CoV)isaninefficientinducerofinterferon(IFN)response.
IFNantagonist It expresses various proteins that effectively circumvent IFN production at different levels via distinct me-
IRF3 chanisms.ThroughtheconstructionofrecombinantIBVexpressingproteins8a,8band8abencodedbySARS-
SARS-CoV CoVORF8,wedemonstratethatexpressionof8band8abenablesthecorrespondingrecombinantvirusesto
ORF8 partiallyovercometheinhibitoryactionsofIFNactivationtoachievehigherreplicationefficienciesincells.We
Ubiquitin-dependentdegradation
alsofoundthatproteins8band8abcouldphysicallyinteractwithIRF3.Overexpressionof8band8abresultedin
the reduction of poly (I:C)-induced IRF3 dimerization and inhibition of the IFN-β signaling pathway. This
counteracting effect was partially mediated by protein 8b/8ab-induced degradation of IRF3 in a ubiquitin-
proteasome-dependentmanner.Takentogether,weproposethatSARS-CoVmayexploittheuniquefunctionsof
proteins8band8abasnovelmechanismstoovercometheeffectofIFNresponseduringvirusinfection.
1. Introduction dimerizeIRF3(Fitzgeraldetal.,2003).TheactivatedIRF3homodimer
thentranslocatestothenucleus,switchingonIFNsynthesis(Linetal.,
Whenchallengedbyaviralinfection,thehostmountsanimmediate 1998;Suhara et al., 2002; Thanos and Maniatis, 1995). Many viruses
innateimmuneresponse,leadingtotheproductionoftypeIinterferons have also evolved strategies to counteract the IFN action, including
(IFN-αandIFN-β)andtheexpressionofhundredsofdownstreamIFN- mechanismsthatallowvirustoevaderecognitionbytheimmunesur-
stimulated genes (ISGs) (Stetson and Medzhitov, 2006; Suhara et al., veillancesystem,soastoinhibitIFNinductionbyhijackingmolecules
2002). Central to the induction of type I IFN is interferon regulatory involvedinIFNactivationpathwaysorbyinhibitingdownstreamsignal
factor3(IRF3)(Hiscott,2007;Taniguchietal.,2001).Theinitialstep transduction (Goodbourn et al., 2000; Versteeg et al., 2007; Weber
of the signaling cascade leading to IRF3 activation is recognition of etal.,2003).
specificviralpathogen-associatedmolecularpatterns(PAMPs)byhost Severeacuterespiratorysyndromecoronavirus(SARS-CoV)wasthe
pattern recognition receptors (PRRs) in two major pathways. These etiological agent of the SARS epidemic in 2003 (Guan et al., 2003;
includecellsurfacetoll-likereceptors(TLRs),suchasTLR3,TLR7,TLR8 Marra et al., 2003). Apart from four typical structural proteins, nu-
andTLR9,whichsenseviralcomponents(Kumagaietal.,2008;Kumar cleocapsid(N),envelope(E),membrane(M)andspike(S)proteins,and
etal.,2009a,2009b),andcytosolicRNAhelicasessuchasretinoicacid- approximately 16 non-structural proteins (Nsp1-16) involved in viral
inducible gene I (RIG-I) and/or melanoma differentiation-associated replication, SARS-CoV encodes an exceptionally high number of ac-
gene 5 (MDA5), which detect viral RNA (Loo et al., 2008; Onomoto cessoryproteinsthatbearlittleresemblancetoaccessorygenesofother
etal.,2007).Uponbindingtotheirligands,PRRsrecruitadaptorpro- coronaviruses (Liu et al., 2014; Narayanan et al., 2008b). Similar to
teins to set off a series of signaling cascades to phosphorylate and other coronaviruses, SARS-CoV is an inefficient inducer of IFN-β
⁎Correspondingauthorat:SouthChinaAgriculturalUniversity,GuangdongProvinceKeyLaboratoryMicrobialSignals&DiseaseCo,andIntegrativeMicrobiologyResearchCentre,
Guangzhou510642,Guangdong,People'sRepublicofChina.
E-mailaddress:[email protected](D.X.Liu).
1Equalcontribution.
https://doi.org/10.1016/j.virol.2017.12.028
Received10November2017;Receivedinrevisedform22December2017;Accepted25December2017
0042-6822/ © 2017 Elsevier Inc. All rights reserved.
H.H.Wongetal. Virology 515 (2018) 165–175
responseincellculturesystem(Spiegeletal.,2005)andissensitiveto Inthisstudy,weshowproteins8band8abasnovelIFNantagonists.
theantiviralstateinduced byIFNs(Spiegeletal., 2004;Zhengetal., Evidence presented supports thedirect interaction between these two
2004).Itsgenomemaythereforeencodeproteinsthatallowthisvirus proteinsandIRF3.Thetwoproteinswerealsofoundtopartiallysup-
toeffectivelycircumventIFNproductioninordertoovercomelimita- pressIFNinductionbylimitingIRF3activationand/orpromotingthe
tionsimposedbytheIFNaction. proteasome-mediateddegradationofIRF3.
TogetherwithafewstructuralproteinsandNsps,manycoronavirus
accessory proteins could suppress IFN production by targeting different 2. Materialsandmethods
aspects of the IFN signaling cascade (Lim et al., 2016; Liu et al., 2014;
Zhongetal.,2012).Forinstance,SARS-CoVPapain-likeprotease(PLpro) 2.1. Cellculture
attenuatesIFNsynthesisbyabrogatingIRF3phosphorylationandnuclear
translocation by physically interacting with IRF3 (Devaraj et al., 2007). African green monkey kidney Cos-7 and Vero cells, human non-
IFNinductionmediatedbyaconstitutivelyactiveIRF3isalsoinhibitedby small cell lungcarcinoma H1299 cells andhuman hepatocellular car-
thede-ubiquitinationactivityofSARS-CoVPLpro(Matthewsetal.,2014). cinoma Huh7 cells were cultured in Dulbecco's modified Eagle's
Ontheotherhand,Nsp1ofPEDVdoesnotinterfereIRF3phosphorylation medium (DMEM) supplemented with 10% fetal calf serum (Hyclone)
andnucleartranslocation,but interruptstheenhanceosome assemblyof and1%penicillin/streptomycinDMEM(Invitrogen)andmaintainedat
IRF3 and CREB-binding protein (CBP) by promoting proteasomal de- 37°Cwith5%CO .Toinhibittheproteasomeactivity,MG132(Sigma)
2
gradationofCBP(Zhangetal.,2016).Targetingfurtherupstream,SARS- atafinalconcentrationof10µMwasaddedtocells2hpriortoharvest.
CoVMproteinpreventsIRF3phosphorylationbyinhibitingtheassembly
of TBK1/IKK complex (Siu et al., 2009), whereas MERS-CoV M protein 2.2. Virusgrowthcurveandplaqueassay
interacts with TRAF3 and disrupts TRAF3-TBK1 association, leading to
reducedIRF3phosphorylation(Luietal.,2016).Likewise,theaccessory To compare the growth kinetics of various recombinant virus
proteinORF4bofMERS-CoVhasbeenshowntospecificallybindtoTBK1 strains, Vero cells were infected with the respective recombinant IBV
andIKKε,therebyinhibitingIRF3phosphorylationandIFN-βproduction strainsatamultiplicityofinfection(MOI)of0.5,andharvestedata4h
(Yangetal.,2015). interval within 20h post-infection for virus titration through plaque
SignalingmoleculesdownstreamofIFNsynthesisarealsotargetsof assay.
SARS-CoV proteins. For instance, SARS-CoV Nsp1 inhibits STAT1- A monolayer of Vero cells seeded on 6-well plates a day prior to
mediated transcription of ISGs by inhibiting its phosphorylation infectionwasinfectedwith200µlof10-foldseriallydilutedvirusstock.
(Wathelet et al., 2007), apart from inducing degradation of a wide After 1h of incubation at 37°C with regular shaking to ensure even
rangeofhostmRNAs(Kamitanietal.,2006;Narayananetal.,2008a). distribution of the virus, cells were washed with PBS and cultured in
SARS-CoV ORF6 blocks STAT1 nuclear translocation by trapping the 3ml of DMEM containing 1% carboxymethyl cellulose (CMC) for 3
nuclear import factors in the endoplasmic reticulum and Golgi appa- days.Thecellswerethenfixedwith4%paraformaldehydeandstained
ratus(Friemanetal.,2007).AmongotherIFNantagonistsidentifiedare with0.1%toluidine.Thenumberofplaqueswascountedinduplicates
nucleocapsid (N) protein of SARS-CoV and PEDV (Kopecky-Bromberg andthevirustiterwascalculatedasplaque-formingunit(Pfu)perml.
etal.,2007;Dingetal.,2014),accessoryprotein4aofMERS-CoV(Siu
et al., 2014), and PLpro domain 2 (PLP2) of MHV-A59 (Wang et al., 2.3. PlasmidConstruction
2011). IFN antagonism mediated by SARS-CoV N protein seemed to
target a very early step of RNA recognition (Lu et al., 2011). Over- The pKTO-Flag-8b and pKTO-Flag-8ab plasmids were previously
expression ofSARS-CoV accessory protein3awasfound to down-reg- described(Leetal.,2007).Thecodingsequencesof8bor8abwerealso
ulate type I IFN receptor by promoting its ubiquitination and sub- subclonedtothepXJ40-Flagvector,whichcontainstheCMVpromoter
sequentdegradationviathelysosomalpathway(Minakshietal.,2009). for expression in mammalian cell lines. For the construction of the
Althoughboth8band8abareencodedbySARS-CoVORF8,theyare pXJ40-Myc-IRF3plasmid,humanIRF3genewasamplifiedfromcDNA
expressedunderdistinctconditions.8abisexpressedasasingleprotein ofH1299cellsusingtheforwardprimer5′-AACGCCTCGACGGAACCC
encoded by the single continuous ORF (ORF8ab) found in SARS-CoV CAAAGCCACGGAT-3′and the reverse primer 5′-GCCGGTACCTTATTG
isolatedfromanimalsandearlystagehumanisolates.Incontrast,asa GTTGAGGTGGTGGGG-3′ prior to ligation into pXJ40-My plasmid at
consequenceofa29-ntdeletionthatresultsintwoseparateoverlapping XhoI and KpnI restriction sites. Truncated mutants were then con-
ORFs(ORF8a⁄ORF8b),mosthumanisolatesobtainedatthemiddleto structed based on the pXJ40-Myc-IRF3 construct using the following
later phase of the epidemic encode instead 8a and 8b as two distinct primers for PCR amplification: for IRF3 (1−133), Forward 5′-CCGCT
proteins (Guanet al.,2003; Oostra etal., 2007). ORF8 ispresumably CGAGCGGATGATGGGAACCCCAAAGCCACG–3′and Reverse 5′–GGGG
acquired from SARS-related coronavirus from greater horseshoe bats TACCCCTCAAGAAGTACTGCCTCCACCAT–3′;forIRF3(399-379),For-
throughrecombination(Lauetal.,2015),andthe29-ntdeletionmay ward5′-CCGCTCGAGCGGATGGATACCCAGGAAGACATTCT-3′andRe-
beanevolutionaryadaptationforenhancingviralpathogenesisinthe verse 5′-GGGGTACCCCTCATCCAGGCAGCGTCCTGTCTC-3′; for IRF3
humanhost.Proteins8a,8band8abmaypossessdifferentbiochemical (241−427), Forward 5′-CCGCTCGAGCGGATGTGGCCAGTCACACTGC
properties and possibly cellular functions (Law et al., 2006; Le et al., CAGA-3′andReverse5′-GGGGTACCCCTCAGCTCTCCCCAGGGCCCT-3′;
2007).Protein8abwasshowntobeaglycosylatedERresidentprotein forIRF3(134−427):Forward5′-CCGCTCGAGCGGATGGATACCCAGG
which can activate ATF6 to modulate the unfolded protein response AAGACATTCT-3′ and Reverse 5′-GGGGTACCCCTCAGCTCTCCCCAGG
(Sungetal.,2009).Protein8awasfoundtoenhanceviralreplication GCCCT-3′. Constitutively active mutant pXJ40-Myc-IRF3-5D was gen-
andinducecelldeath(C.Y.Chenetal.,2007),while8binducesDNA eratedbyperformingsequentialsite-directedmutagenesis PCR(Quik-
synthesis and down-regulates SARS-CoV E protein via a proteasome- Change II site-directed mutagenesis kit; Stratagene) to replace amino
independentpathway(Kengetal.,2006).SARS-CoV8band8abwere acids at positions 396, 398, 402, 404, and 405 with the phosphomi-
alsoshowntobindtobothmono-andpoly-ubiquitinwhenexpressedin meticasparticacid.
cell culture (Keng et al., 2006). Whether these ubiquitin-binding
properties allow them to interact with host cell proteins remains un- 2.4. Westernblotanalysis
known. Interestingly, when an in vivo attenuated recombinant SARS-
CoV lacking the full-length E gene is passaged in mice, the ORF8 se- CellswerelysedinRIPAbufferinthepresenceofproteaseinhibitors
quencemutatestoencodeaPDZ-bindingmotifinprotein8a,andthe (RocheDiagnostics)andphosphataseinhibitors(Pierce).Proteinlysates
virusregainedvirulence(Jimenez-Guardeñoetal.,2015). wereseparatedbyelectrophoresisin8%SDSpolyacrylamidegelsand
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H.H.Wongetal. Virology 515 (2018) 165–175
transferred to nitrocellulose membrane (Amersham Biosciences) via and RANTES mRNAs in treated samples with respect to their mock
wettransfer(Bio-rad).Themembraneswereblockedovernightat4°C treatedcounterpartswasdeterminedbyreal-timequantitativeRT-PCR
with 10% non-fat milk in PBST before probing with specific primary using the SYBR Green method (Roche). Briefly, a 20µl PCR reaction
antibodies,followedbyhorse-radishperoxidase(HRP)-conjugatedanti- containingcDNAtemplate,therespectiveprimersandLightCyclerFast
mouse, anti-rabbit or anti-goat IgG secondary antibodies (DAKO), re- StartSYBRGreenIDNAMastermix(Roche)waspreparedandsubjected
spectively.Thefollowingcommercialprimaryantibodieswereused:β- toaqPCRprogramusingtheLightCycler(Roche).PCRcyclingcondi-
tubulin (Sigma), IRF3 (Santa Cruz Biotechnology), pIRF3(398) (Cell tionscomprisedofaninitialdenaturationstepat94°Cfor10minfol-
Signaling) and β-actin (Santa Cruz Biotechnology). Polyclonal anti- lowedbyanamplificationprogramfor40cyclesof30sat95°C,10sat
bodiesagainstIBVNwereraisedinrabbitsbythislaboratory(Lietal., 55°C,and20sat72°Cwithfluorescenceacquisitionattheendofeach
2005). extension.Therelativeexpressionofeachgeneiscalculatedusingthe
comparativeΔΔC method,usingthemocktreatedsampleascalibrator
T
2.5. Co-immunoprecipitation andhousekeepinggeneGAPDHasinternalcontrol.
The following primer pairs were used: for GAPDH Forward
Monolayer cells grown overnight in 6-well plates (Nunc) were in- 5′-GACAACTTTGGTATCTTGGAA-3′ and Reverse 5′-CCAGGAAATGAG
fectedwithrecombinantvacciniavirusencodingthebacteriophageT7 CTTGACA-3′; for ISG56 Forward 5′-TCTCAG AGGAGCCTGGCTAAG-3′
RNA polymerase before transfection of plasmids using Effectene re- andReverse5′-CCACACTGTATTTGGTGTCTAGG-3′;forISG15Forward
agent (Qiagen), as previously described (Liu and Inglis, 1991, 1992). 5′-TGGTGGACAAATCGCACGAA-3′ and Reverse 5′-CAGGCGCAGATTC
Briefly, at 20h post-transfection, cells were harvested with 500µl of ATGAAC-3′; for RANTES Forward 5′-GGCACGCCTCGCTGTCATCC
RIPAbufferinthepresenceofprotease(RocheDiagnostics)andphos- TCA-3′ Reverse 5′-CTTGATGTGGGCACGGGGCAGTG-3′; and for IFN-β
phatase inhibitors (Pierce). Lysates were centrifuged at 13,000×g at Forward 5′-CTCTCCTGTTGTGCTTCTCCAC-3′ and Reverse 5′-TAGTCT
4°Cfor15min,andthesupernatantobtainedwasimmunoprecipitated CATTCCAGCCAGTGCT-3′.
directly with antibody-conjugated agarose beads for 2h or with ap-
propriate antibodies followed by incubation with protein A agarose 3. Results
beads (Sigma) for another 2h at room temperature. The im-
munoprecipitatedproteinswereseparatedonSDS-PAGEandanalyzed 3.1. CharacterizationofrecombinantIBVexpressingSARS-CoVproteins
bywesternblotusingappropriateantibodies.TheHRP-conjugatedanti- 8b,8aband8a/b
Mycandanti-FlagantibodieswerepurchasedfromSigma,whileanti-
bodiesagainstIgG,ISG56,β-actinandfull-lengthIRF3werefromSanta In a previous study, we reported the construction of two re-
CruzBiotechnology. combinantIBV(rIBV8bandrIBV8ab)expressingSARS-CoVproteins8b
and8ab,respectively(Leetal.,2007).ToassesstheroleofSARS-CoV
2.6. NativePAGEanalysisofIRF3 8band8abinmodulatingviralreplication,thegrowthpropertiesand
kineticsoftherecombinantviruseswerecharacterizedandcomparedto
Cellswerelysedinbuffer(50mMTris-HCl(pH7.4),150mMNaCl, wildtypeIBV(wtIBV).AnewrecombinantIBVexpressingthe8aand
1mM EDTA, 1% NP-40) containing protease inhibitors and phospha- 8binseparateORFs(rIBV8a/b)wasalsoconstructedasacontrol.Vero
tase inhibitors for 30min at 4°C. Proteins were then separated by cells,knownto lacktheexpression oftypeIIFNs, wereinfectedwith
electrophoresis in 8% non-denaturing polyacrylamide gels, with 1% wild type and recombinant IBV at an MOI of ~0.5 and harvested at
sodium deoxycholate (Sigma) in the cathode buffer. IRF3 monomers every4hoveratimecourseof20hforplaqueassaytodeterminevirus
and dimers were detected by Western blot analysis using polyclonal titers.Consistentwithourpreviousreportontheimpedimentofvirus
antibodiesagainstfull-lengthIRF3(SantaCruzBiotechnology). replication by 8b expression, recombinant viruses expressing 8b, 8ab
and 8a/b replicated at a slightly slower rate, compared to wild type
2.7. IFN-βreporterassays virus during the first 12h of infection (Fig. 1a & b). At 16h post-in-
fection,however,thethreerecombinantIBVswereabletoattaintiters
Huh7cellsseededona12-wellplateweretransfectedwithatotalof comparabletothatofwildtypevirus(Fig.1b).Expressionofprotein8b
4μgoftheappropriateplasmidsusingLipofectamine2000(Invitrogen) or 8ab could not be detected in cells infected with rIBV8a/b using
accordingtomanufacturer'sinstructions.pIFN-β-LucandpRL-TKwere Westernblotanalysis(datanotshown),furthersupportingourprevious
purchased from Promega. 10µg of poly (I:C) complexed with 10µl observation that 8b is not expressed from this construct (Le et al.,
Lipofectamine2000wasthenintroducedintothecells20hlater.Cells 2007).Takentogether,theseresultsdemonstratethattheinclusionof
werelysed20hposttreatmentinpassivelysisbuffer(Promega)andan 8band8abdoesnotrenderdetectableenhancementeffectsonthere-
aliquot of the lysates was measured for firefly and Renilla luciferase plicationandgrowthofIBVinculturecells.Theslightlyslowergrowth
activitiesaccordingtothemanufacturer'sinstruction(Promega). ratesobservedfortherecombinantvirusesatearlytimepointsofthe
infectioncyclemaybeduetotheintroductionofextrasequencesinto
2.8. Invitroassemblyandtranscriptionoffull-lengthcDNAclones theIBVgenome.Thisisconsistentwithourpreviousobservationsthat
suchgeneticmanipulationsmayalterthereplicationofIBVincells(Le
Construction of recombinant IBVs (rIBVs) was carried out essen- etal.,2007;Shenetal.,2009).Asexpressionofproteins8a,8band8ab
tially as previously described (Fang et al., 2007; Le et al., 2007; Tan didnotrendergrowthadvantagestoIBVinnormalculturedcells,these
et al., 2006). To generate the 8b mutant (8bm) containing lysine to proteinsmaynothavedirectfunctionsinviralreplication,especiallyin
arginine mutations at all three positions, standard PCR site-directed aheterogeneousgenomecontext.
mutagenesis was performed using the construct containing ORF8 in-
sertion as a template. The genotypes of rIBVs were validated by se- 3.2. Expressionof8band8abconfersgrowthandreplicationadvantagesto
quencing. rIBV8bandrIBV8aboverwtIBVandrIBV8a/binthepresenceofpoly(I:C)
2.9. RNAIsolationandQuantitativeRT-PCR Theabilitytosubvertthehostinnateimmuneresponseisacritical
factorforestablishingeffectivevirusreplication.Todetermineif8band
Total RNA was isolated using the Trizol reagent (Invitrogen) as 8abmayhavearoleincounteractingtheactionofIFN,oneofthemost
describedbymanufacturer'sprotocol.ThreeµgtotalRNAwasreversed commonandpotenthostanti-viraldefensemechanisms,weexamined
transcribed (Roche). The relative abundance of IFN-β, ISG15, ISG56 therelativereplicationefficacyofrIBV8b,rIBV8abandrIBV8a/binthe
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H.H.Wongetal. Virology 515 (2018) 165–175
Fig. 1.Growth kinetics for recombinant viruses. a. Titration by plaque assay.
Representativeimagesshowedthatcomparablenumbersofplaqueswereformedbythe
respectiverecombinantvirusesat16hpost-infection.b.Growthcurveofwildtypeand
recombinantvirusesoveratimecourse20h.Plaqueassayswereperformedwithcell
lysatesinfectedwiththerespectiverecombinantvirusesatanMOIof0.5andharvested
every4hpost-infectionoveratimecourseof20h.10-foldseriallydilutedvirusstockwas
thenusedforplaqueassaytodeterminevirustiter.Errorbarshowsstandarddeviation
from3independentexperiments.
presenceofpoly(I:C).Forthispurpose,H1299cellswereinfectedwith
wildtypeortherecombinantIBVsfor8hpriortomockorpoly(I:C)
transfection. Cells werethenfurtherincubated for10hbeforelysates
were harvested for analysis of viral protein expression (Fig. 2a). In Fig.2.RecombinantIBVsexpressing8band8abreplicatemoreefficientlyinthe
agreement with our growth kinetics studies earlier, the recombinant presenceofIFNinductionbypoly(I:C)treatment.a.WesternblotanalysisofIBVN
proteinexpressionincellsinfectedwithwildtypeandrecombinantIBVsinthepresence
virusesreplicatedto similarlevelsby18h(Fig.2a)intheabsenceof
andabsenceofIFNinduction.CellswereinfectedwithwtIBV,rIBV8b,rIBv8aborrIBV8a/
poly (I:C) treatment although the wildtype virus wasobserved to re-
bfor8hbeforetransfectionwithpoly(I:C).Wholecelllysatecollectedat10hpost-poly
plicate slightly faster as indicated by a higher abundance of IBV N (I:C)treatmentwasanalyzedforIBV-N.Actinwasprobedasaloadingcontrol.TheN
protein(Fig.2a). proteinexpressionefficiencywascalculatedasthebandintensityoftheproteinafter
Notsurprisingly,replicationofwildtypeandthethreerecombinant beingnormalizedtothebandintensityofactin,withtheNproteinexpressionfromcells
viruses were severely suppressed in cells stimulated by poly (I:C) infectedwithwtIBVafterpoly(I:C)treatmentas1.b.Relativetitersofwildtypeand
(Fig. 2a), reflecting the sensitivity of coronavirus to interferon inter- recombinantIBVreleasedfrominfectedcellsinthepresenceorabsenceofIFNinduction.
The number of infectious particles released in the supernatants was quantified using
vention. Compared to wild type and rIBV8a/b, however, rIBV8b and
plaqueassayintriplicates,andtheaveragenumberofPfuforeachtreatmentwascal-
rIBV8ab were observed to replicate significantly better and express culated.Therelativeamountofvirusproducedafterpoly(I:C)treatmentwasexpressedas
higher levels of N protein in cells stimulated by poly (I:C) (Fig. 2a). a percentage of their respective control cells not treated with poly (I:C). Error bars
Usingplaqueassay,viraltitersattainedininfectedcellsexposedtopoly showedstandarddeviationfrom3independentexperiments.Errorbarsshowedstandard
(I:C)treatmentwerecomparedtotheirrespectivemock-treatedcoun- deviationfrom3independentexperiments.
terparts. While poly (I:C)-treatment reduced the virus titers of wtIBV
(from 1.1×106 to 2.75×105) and rIBV8a/b (from 1×106 to 4×104) Western blot analysis with antibodies against the Flag epitope. As
drastically by 87.5% and 96%, respectively, titers of rIBV8b (from shown inFig. 3b,proteins8b and8ab wereconsistently pulled down
8.2×105to4.1×105)andrIBV8ab(from7.9×105to1.98×105)were with IRF3 in samples where they were co-expressed with IRF3. Simi-
reducedbyamoremodest50%and75%,respectively(Fig.2b).These larly,whentheexperimentwasrepeatedusinganti-Flagcoatedagarose
resultssuggestthatproteins8band8abmayhaveafunctionalrolein beads, IRF3 co-precipitation was detected with both proteins. These
modulatingtheIFNpathway.Asexpressionofprotein8a(fromrIBV8a/ resultsdemonstratethatIRF3couldphysicallyinteractwithprotein8b
b)didnotshowasimilareffect,wehypothesizedthatthiseffectmaybe and8ab.
uniquetothe8bregionofthetwoproteins. Toconfirmthatthisinteractionoccursinthecontextofcoronavirus
infection,totallysatespreparedfromcellsinfectedwithwtIBV,rIBV8b
andrIBV8abtogetherwithmockinfectedcontrolswerealsosubjected
3.3. Proteins8band8abinteractphysicallywithIRF3
toimmunoprecipitationwitheitherpolyclonalantibodiesraisedagainst
SARS-CoV8borIgGcontrols.Immunoprecipitatedproteinswerethen
In view of the central role of IRF3 in regulating IFN activation
analyzedforthepresenceofendogenousIRF3usingantiseraagainstthe
duringvirusinfection,8band8abwithFlagepitope-taggedtotheirN-
protein. The detection of IRF3 in cells infected with rIBV8b and
terminiwereco-expressedwithMyc-taggedIRF3(Fig.3a)inCos-7cells
rIBV8ab,butnotinmock-andwtIBV-infectedcells(Fig.3c)indicates
using the vaccinia/T7 expression system (Anderson et al., 1996; Lim
thatprotein8band8abformedcomplexeswithIRF3duringthevirus
andLiu,2001)forco-immunoprecipitationassaystodetermineifthere
replicationprocess.Itwasnotedthatapartfromthe55kDabandcor-
isanyphysicalinteractionbetweentheproteins.IRF3wasprecipitated
responding to the endogenous monomeric IRF3, an additional band
fromthecelllysatespreparedfromcellsharvestedat20hpost-trans-
with the apparent molecular weight of approximately 85kDa was
fection using anti-Myc antibody-coated agarose beads, followed by
168
H.H.Wongetal. Virology 515 (2018) 165–175
Fig. 3.Interaction ofproteins 8band 8abwith
IRF3.a.Schematicdiagramshowingthefunctional
domains of IRF3 and various IRF3 deletion con-
structs used. The N-terminal DNA-binding domain
(DBD),nuclearexportsignal(NES),proline-richre-
gion(Pro),theIRFassociationdomain(IAD)andthe
C-terminal repeat domain (RD) are shown.
Fragmentsthatimmunoprecipitatedwithprotein8b
aredenotedwith(+).b.Co-immunoprecipitationof
Myc-taggedIRF3withFlag-tagged8band8ab,re-
spectively. Cos7 cells were infected with the re-
combinantvaccinia/T7virusatanMOIofapproxi-
mately5percell.Afterincubationfor1,cellswere
transfected with pMyc-IRF3, pFlag-8b, pMyc-
IRF3+pFlag-8b, pFlag-8ab and pMyc-IRF3+pFlag-
8ab,respectively.Cellswereharvestedat18hpost-
transfection,lysatesprepared,andsubjectedtoim-
munoprecipitation with either the Myc antibody-
conjugated agarose beads (top two panels) or the
Flag-antibody-conjugated beads (bottom two pa-
nels).TheprecipitateswereseparatedonSDS-PAGE
andanalyzedbyWesternblotwitheitheranti-Myc
(topandbottompanels)oranti-Flag(2ndand3rd
panels)antibodies.c.Pulldownoftheendogenous
IRF3proteinwithprotein8b.H1299cellswerein-
fectedwithwtIBV,rIBV8bandrIBV8ab,respectively.
Cells were lysed 20h post-infection and im-
munoprecipitatedwitheitherpolyclonalantibodies
raised against SARS-CoV 8b or control IgG anti-
bodies.Theprecipitateswereprobedwithantibodies
against the full-length IRF3. d. Co-im-
munoprecipitation analysis of the Flag-tagged pro-
tein8bco-expressedwiththeuntaggedIRF3from
1–193,1–375and1–427(full-length).H1299cells
were infected with the recombinant vaccinia/T7
virus at an MOI of approximately 5 per cell.
Immunoprecipitation was performed as described
above.Totallysatesandtheprecipitatesweresepa-
rated on SDS-PAGE and analyzed by Western blot
witheitheranti-Mycoranti-Flagantibodies.e.Co-
immunoprecipitation analysis of the Flag-tagged
protein8bco-expressedwiththeMyc-taggedIRF3
from 1–133, 134–240 and 134–427. H1299 cells
were infected with the recombinant vaccinia/T7
virus at an MOI of approximately 5 per cell.
Immunoprecipitation was performed as described
above.Totallysatesandtheprecipitatesweresepa-
rated on SDS-PAGE and analyzed by Western blot
witheitheranti-Mycoranti-Flagantibodies.
detected(Fig.3c)incellsinfectedbyrIBV-8b.Theidentityofthisband N terminal fragment comprising of only the first 193 residues, thus
isnotcertain,butitmayrepresentamodifiedformofIRF3.IRF3iswell excluding the 47 residues of IAD, abolished the binding (Fig. 3d), in-
documentedtobeheavilytargetedforpost-translationalmodifications dicating that just theNES and Pro regions alone were insufficient for
such as phosphorylation, ubiquitination, SUMOylation, and neddyla- thebinding.Consistently,thefull-lengthIRF3withtheN-terminalMyc-
tion(Bibeau-Poirieretal.,2006;Hiscott,2007;Kubotaetal.,2008;Ran tagandtheN-terminalregioncoveringthefirst375residuescouldbe
etal.,2011). efficientlypulleddownbyprotein8b(Fig.3d).
Sinceproteins8band8abexhibitedcomparableefficiencyinterms
ofpullingdownIRF3,justthe8bregionwasthenusedforsubsequent
pull-downexperimentstopinpointthedomainsinIRF3essentialforthe 3.4. Proteins8band8abreducepoly(I:C)-inducedIRF3andIFNactivation
interaction. Four deletion constructs of IRF3 either with or without a
Myc-tag attheN-termini wereconstructed (Fig.3a)and co-expressed Wenextsoughttotesttheeffectof8band8abexpressiononIRF3
withtheFlag-tagged8b.Co-immunoprecipitationexperimentsshowed activation.CellsweretransfectedwitheitherplasmidencodingFlag-8b,
thattheN-terminalregioncoveringthefirst133residuesofIRF3(Myc- Flag-8ab or a corresponding control vector, before subjected to poly
IRF3(1-133))failedtointeractwithprotein8b(Fig.3e),suggestingthat (I:C) treatment. Western blot analysis coupled with native PAGE re-
the N terminal DNA-binding domain (DBD) is dispensable for the in- vealed that the ectopic expression of proteins 8b and 8ab resulted in
teraction. Immunoprecipitation with fragments covering residues markedly decreased levels of poly (I:C)-induced IRF3 dimerization
134–240 and 134–427, respectively, resulted ineffective pulldown of (Fig.4a).However,analysisofthesamesamplesbySDS-PAGEshowed
8b (Fig. 3e), suggesting that the 134–240 region is likely to be im- noobservabledifferenceinthelevelsofhyper-phosphorylatedIRF3(p-
portantfortheinteraction.Thisregionspansacrossseveralfunctional IRF3) (appearing as more slowly migrating bands) in cells over-ex-
domains,includingthenuclearexportsignal(NES),proline-richregion pressingproteins8band8ab,comparedtothatinthecontrol(Fig.4a).
(Pro), and the first 47 residues of the IRF association domain (IAD). It was also noted that the total amounts of IRF3 were approximately
Interestingly,immunoprecipitationexperimentsrepeatedwithjustthe comparableinthesetransfectedcells(Fig.4a).
The concomitant decreased in IRF3 dimerization with 8b and 8ab
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H.H.Wongetal. Virology 515 (2018) 165–175
Fig. 4.Suppression of poly (I:C)-induced IRF3
activationbyprotein8band8ab.a.Suppressionof
IRF3dimerizationbyproteins8band8ab.Huh7cells
weretransfectedwith4μgofemptyvector,Flag-8b
and Flag8ab, respectively, followed by stimulation
withpoly(I:C)for20h.Wholecelllysatesweresub-
jected to either native PAGE or SDS-PAGE and
probed with anti-IRF3. Tubulin was included as a
loadingcontrol.TheratioofdimericIRF3tomono-
mericIRF3wascalculatedasthebandintensityof
monomerdividedbythebandintensityofdimer.b.
Suppression of poly (I:C)-induced IFN-β promoter
activity by proteins 8b and 8ab. Huh7 cells were
transfectedwithcontrolvectorpcDNA3.1,pcDNA-8b
andpcDNA-8ab,respectively, together withaluci-
ferasereporterconstructunderthecontrolofIFN-β
promoter.At24hpost-transfection,cellswerethen
furthertransfectedwithpoly(I:C).At24hpost-sti-
mulation, cells were lysed and measured for the
fireflyluciferaseactivity.pRL-TKwasalsoco-trans-
fectedtoserveasaninternalcontrol.Datawerere-
presentedasmeanoftriplicatesfrom3independent
experiments. c-f. Huh7 cells were transfected with
4μgofemptyvector,Flag-8bandFlag8ab,respec-
tively, followed by stimulation with poly(I:C) for
20h.TotalRNAwasthenextractedforquantitative
real-time RT-PCR with specific primers for IFN−β
(c),ISG56(d),RANTES(e)andISG15(f).Theex-
pressionofeachgenewasexpressedrelativetotheir
respective control sample transfected with empty
vector.Datawererepresentedasmeanofreplicates
from2independentexperiments.GAPDHwasusedas
internalcontrol.
expressionleadustoexaminetheimpactofsuchphenomenononthe ISG56(Fig.4d),RANTES(Fig.4e)andISG15(Fig.4f)werereducedto
activation of IFN-β and other known IRF3 downstream effectors. For approximately 40–41, 35–40 and 25–30%, respectively, in poly (I:C)-
thestudyontheeffectonIFN-βpromoteractivity,areporterconstruct treated cells transfected with 8b and 8ab, compared to those in cells
expressing firefly luciferase driven by the IFN-β promoter was co- transfectedwiththeemptyvector.
transfectedwitheither8b,8aboracontrolplasmidpriortopoly(I:C)
stimulation.At16hpostpoly(I:C)treatment,lysateswereassayedfor 3.5. InductionofrapiddegradationofIRF3bySARS-CoVproteins8band
luciferase activity. Relative to cells transfected with control plasmid, 8ab
poly(I:C)-inducedIFN-βpromoteractivationincellsexpressing8band
8ab were reduced to 59% and 57% respectively (Fig. 4b). This data Thenegativemodulationof8band8abexpressionontheactivation
mirrored the results obtained from the analysis of endogenous IFN-β of IRF3 was further verified from the analysis of IRF3 activation and
transcriptlevelsexaminedbyquantitativereal-timeRT-PCRwherere- stabilityinvirus-infectedcellsshowninFig.2a.Intheabsenceofpoly
lativelevelsofIFN-βmRNAinpoly(I:C)-treatedcellsexpressing8band (I:C) treatment, infection by wild type and all the three recombinant
8abwerereducedtoapproximately18–20%(Fig.4c)ofcontrol.Similar viruses did not induce a detectable level of IRF3 dimerization using
to the effect on IFN-β expression, the mRNA levels of other IRF3 nativePAGEanalysis(Fig.5a).WhileIRF3dimerizationwasdetectedin
downstream target genes (Grandvaux et al., 2002), including ISG56, allinfectedcellsinthepresenceofpoly(I:C)treatment(Fig.5a),less
RANTES and ISG15, were significantly lower in cells over-expressing poly(I:C)-inducedIRF3dimerscouldbedetectedincellsinfectedwith
proteins 8b and 8ab than those of the control. The relative levels of rIBV-8b, comparedtothose infectedbywtIBV(Fig.5a).Reduction in
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H.H.Wongetal. Virology 515 (2018) 165–175
Fig.5.RapiddegradationofIRF3incellsinfectedwithrecombinantIBVexpressing
proteins8band8ab.a.WesternblotanalysisofIRF3incellsinfectedwithwildtypeand
recombinantIBVinthepresenceandabsenceofIFNinduction.Cellswereinfectedwith
wtIBV,rIBV8b,rIBv8abandrIBV8a/bfor8hbeforetransfectionwithpoly(I:C).Whole
celllysatecollected10hpost-poly(I:C)treatmentwasanalyzedforIRF3indenaturing
SDS-PAGE.Actinwasprobedasaloadingcontrol.IRF3dimerizationinthesamesamples
wasdetectedbynativePAGE.b.RelativeexpressionofIFN-βmRNAinpoly(I:C)-treated
cellsinfectedwithrecombinantviruseswithrespecttothewildtypevirus,wasanalyzed
usingquantitativereal-timeRT-PCR.Experimentswereperformedintriplicates.Data
wererepresentedasmeanofreplicatesfrom2independentexperiments.GAPDHwas
usedasaninternalcontrol.
IRF3 dimerization, although to a lesser extent, was also observed in
cellsinfectedwithrIBV8ab(Fig.5a).Interestingly,analysisofthesame
lysates by denaturing SDS-PAGE revealed that hyper-phosphorylated
IRF3wasinvariantlydetectedinthepoly(I:C)treatedcellsregardless
ofwhethertheyaremockinfected,infectedwithwildtypevirusorthe
recombinantIBVs(Fig.5a).Theseobservationswereinagreementwith
the data we described in Fig. 4a. However, we did observe that the
overall level of IRF3 expression was significantly reduced in cells in-
fected with rIBV8b, both in the presence and absence of poly (I:C)
Fig.6.DegradationofIRF3by8binaubiquitin/proteasome-dependentmanner.a.
treatment (Fig. 5a). A more moderate reduction of IRF3 was also de-
DegradationofIRF3by8bbutnotbyalysine-knockoutmutant8b.Alysineknockout
tectedincellsinfectedwithrIBV8ab(Fig.5a). mutantofrIBV,rIBV8bm,wascreatedbysubstitutingthethreelysineresidues(K4,K26
WhenthemRNAlevelsofIFN-βfromthesesampleswereanalyzed, andK81)witharginine.Cellsweretheninfectedupto32hwithrIBV-8bandrIBV8bm,
negligible IFN-β induction was observed in virus-infected cells in the respectively,beforeanalyzingforIRF3expression.Thesamemembranewasalsoprobed
absenceofpoly(I:C)stimulation(datanotshown).Amongthesamples withanti-IBVN,SARS-CoVprotein8bandactinantibodies.TherelativeamountofIRF3
treated with poly (I:C), IFN-β induction in cells infected with rIBV8b wascalculatedasthebandintensityoftheproteindividedbythebandintensityofactin.
b.Ubiquitin-dependentdegradation ofIRF3.CellstransfectedwitheitherMyc-tagged
andrIBV8abinfectionwassuppressed(25%and75%relativetocontrol
ubiquitin alone or co-transfected with Flag-tagged 8b, in the presence or absence of
wtIBV,respectively)(Fig.5b).Takentogether,thesedatasuggestthat MG132, were subjected to immunoprecipitation with antibodies against Myc.
the observed enhanced replication of rIBV8b and rIBV8ab in the pre- Immunoprecipitateswerethenprobedforpull-downofendogenousIRF3withspecific
senceofpoly(I:C)maybeduetothediminishedIFNactivationowing antibodies. The relative amount of IRF3 was calculated as the band intensity of the
to the down-regulation of IRF3 levels coupled to reduced IRF3 dimer proteindividedbythebandintensityofactin.
formationandIFN-βinduction.
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H.H.Wongetal. Virology 515 (2018) 165–175
3.6. DegradationofIRF3mediatedbyproteins8band8abisubiquitin-
dependent
We had previously demonstrated that 8b binds to both poly- and
mono-ubiquitin(Leetal.,2007).Toaddressifthedown-regulationof
IRF3inrIBV8b-infectedcellsislinkedtoitsubiquitin-bindingactivities,
lysineknockoutmutantof8b,8bmwasgeneratedbymutatingallthree
lysineresidues(K4,K26andK81)in8btoarginine.Mutationofthese
lysine residues enhanced the stability of the 8b protein. As shown in
Fig.6a,whilethemutant8bmproteinwasreadilydetectedinrIBV8bm-
infectedcellsat20and32hpost-infection,protein8bwasnotdetected
under the same conditions (Fig. 6a). This is consistent with previous
reportsoftheinstabilityof8bproteinandthatprotein8bcouldonlybe
detected in rIBV8b-infected cells in the presence of proteasome in-
hibitors (Le et al., 2007). IRF3 was down-regulated in cells infected
with rIBV8b at both 20 and 32h post-infection (Fig. 6a). Infection of
cellswithrIBV-8bmdidnotresultinsimilarreductionsinIRF3protein
levels, despite a similar replication efficiency of the two recombinant
viruses(Fig.6a).
As mentioned previously, IRF3 levels are regulated by ubiquitina-
tionduringvirusinfection.However,somevirusesexploitthismodeof
regulation by expressing viral proteins that promote untimely protea-
somaldegradationofIRF3(Z.Chenetal.,2007;Sairaetal.,2009;Sen
et al., 2009). To affirm the involvement of the ubiquitin-proteasome
pathway in the down-regulation of IRF3 observed with 8b, IRF3 sta-
bilitywasstudiedincellsoverexpressingprotein8binthepresenceof
ubiquitin. Over-expression of8b together with ubiquitin resulted ina
reduction of detectable levels of IRF3 compared to cells expressing
ubiquitinalone(Fig.6b).Thisdegradationwaspartiallysuppressedin
the presence of proteasome inhibitor MG132 (Fig. 6b). In a previous
study,wefoundthatproteasome-mediatedrapiddegradationofprotein
8b could be much more efficiently inhibited by NLVS than did lacta-
cystin (Le et al., 2007). As the supply of NLVS was discontinued, we
chosetouseMG132,aproductshownsimilarlymildinhibitoryeffectas
lactacystin in the suppression of proteasome-mediated degradation of
protein8b,inthisstudy.
Fig.7.SuppressionofIRF3-5D-inducedIFN-βexpressionbyproteins8band8ab.a.
ControlvectorpcDNA3.1,pcDNA-8borpcDNA-8abtogetherwithaluciferasereporter
3.7. SuppressionofIRF3-5D-inducedinterferonresponsebyproteins8band
constructunderthecontroloftheIFN-βpromoterweretransfectedintoHuh7cells.At
8ab
24hpost-transfection,cellsweretransfectedwithpIRF3-5Dandco-transfectedwithpRL-
TKtoserveasaninternalcontrol.At24hpost-stimulation,cellswerelysedandmeasured
Finally,toaddresstheobservationofthenegligibleimpactexerted forthefireflyluciferaseactivity.Datawererepresentedasmeanoftriplicatesfrom3
by8band8abonIRF3phosphorylationdespitetheircapacitytosup- independentexperiments.b.Cellswereco-transfectedwith200ngofpxj40-IRF3-5Dand
pressIRF3dimerformation,westudytheeffectof8bonactivatedIRF3. either0,50,100,200ngofpKTO-8b.ThetotalDNAtransfectedwasmadeupto400ng
usingemptypKTOvector.Lysatesharvested20hpost-transfectionwereeithersubjected
AconstructexpressingthephosphomimeticformofIRF3(IRF3-5D)was
tonativeorSDSPAGEandprobedwithanti-IRF3antibodies.Actinwasalsoprobedto
constructed,whichcontainsaminoacidsubstitutionsatpositions396,
serveasaloadingcontrol.c.IRF3-5Dwasco-transfectedpKTO-8b.20hposttransfection,
398,402,404,and405bythephosphomimeticasparticacid.IRF3-5D co-transfectedcellswaseitherleftuntreatedortreatedwithMG1322hpriortoharvest.
undergoes spontaneous dimerization leading to IFN-β induction LysateswerethenprobedforIRF3,8bandactinexpression.TherelativeamountofIRF3-
(Grandvauxetal.,2002;Linetal.,1999).Over-expressionofproteins 5Dwascalculatedasthebandintensityoftheproteindividedbythebandintensityof
8b and 8ab was able to reduce the IRF3-5D-induced IFN-β promoter actin.
activitytoapproximately58%and49%tothatofcontrol,respectively
(Fig. 7a). This suggests that 8b can act on IRF3 at step(s) that is functions that may enhance SARS-CoV pathogenesis in cells. By de-
downstream of its activation. When protein 8b is co-expressed with monstratingthatrecombinantIBVexpressing8bor8abreplicatesmore
IRF3-5D, 8b reduces the expression of IRF3-5D in a dose-dependent efficientlyinthebackgroundofIFNactivation,data presentedinthis
manner (Fig. 7b). For reasons yet to be known, we observed that the study demonstrate that expression of SARS-CoV ORF8b and ORF8ab
expressionofamonomericformofIRF3-5Dseemedtobemoreaffected contributespositivelytoviralpathogenesis.
by the presence of 8b than its homodimeric counterpart. Similar to As the expression of 8b helped to overcome partially the potent
results presented earlier with endogenous IRF3, IRF3-5D levels was inhibitory effect of IFN induction on coronavirus replication in cell
partiallyrescuedwiththeadditionofMG132,indicatingtheroleofthe culture, it suggests that 8b and 8ab are novel IFN antagonists. ORF8
ubiquitin-proteasomepathway(Fig.7c). failedtoshowupinthescreenforinterferonantagonistinaprevious
study (Kopecky-Bromberg et al., 2007), likely due to the incomplete
4. Discussion inhibition of IFN activation exhibited by ORF8. Other reasons could
includethelowexpressionof8bintheirsystemowingtotheinherent
The SARS-CoV genome encodes an exceptionally high number of instabilityoftheprotein(Leetal.,2007),and/orthattheantagonistic
accessorygenesthatbearlittleresemblancetootherknowncoronavirus activityof8bisnotapparentintheirNDVmodelbecausetheIFNan-
accessoryproteins.Itisbelievedthatwhiletheseuniqueproteinsmay tagonisticactivityofthisproteinmaybespecifictocoronavirusinfec-
not participate directly in viral replication, they possess biological tion due to the requirement of other viral proteins. However, this is
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H.H.Wongetal. Virology 515 (2018) 165–175
quiteunlikelybecausetheinhibitoryeffectcouldalsobeobservedwith Hence, we hypothesize that the growth advantage conferred by the
ectopicexpressionof8band8abintheabsenceofviralreplication. expressionof8binrecombinantIBVwasduetotheroleof8binlate-
Theothersignificantfindingfromthisstudyisthat8band8abdi- stageviralpathogenesis,whentheexpressionof8baidsindampening
rectly physically interact with IRF3 and that 8b-IRF3 interaction in- theactivation ofIRF3thatmayoccurduringthelaterphaseofinfec-
volvespartoftheIADdomainthatisresponsiblefortheformationof tion.Nevertheless, we donotruleoutthepossibility that8b and8ab
IRF3 homodimers. Over-expression of 8b and 8ab appears to have a mayregulateIRF3viamechanismsindependentofitsubiquitin-binding
moreprofoundeffectonIRF3dimerizationthanonitsphosphorylation activity as it has also been shown to down-regulate E protein via a
status.Althoughitisstillunclearwhetherthisisaconsequenceofassay ubiquitin-independentproteasomalpathway(Kengetal.,2006,2011).
insensitivityorthattheinhibitoryactionsof8band8abbypassthestep Using an infectious clone system based on the Urbani strain of
of phosphorylation and targets specifically on the event of IRF3 di- SARS-CoV,the29-ntdeletionisinsertedtofuseORF8a/bbackintothe
merization,weareinclinedtobelievethatitisthelatterowingtothe single ORF8. Compared with the wild type control, this recombinant
ability of 8b to suppress IFN-β induced by constitutively active phos- virusreplicatessimilarlyinbothcellcultureandinthemurinemodel
phomimetic IRF3-5D. While disruption to the dimerization event asa (Yountetal.,2005).Theoretically,onlyprotein8abisproducedincells
resultofdirectstericinterferencebroughtaboutby8binteractionatthe infectedwiththisvirus,whereasbothproteins8aand8bareproduced
IADdomainisanattractivemodel,theexactmechanismscanonlybe inthewildtypecontrol.Becauseboth8band8abcanantagonizeIFN
ascertainedthroughmorein-depthstudiessuchasstructuralanalysis. signalingbymediatingIRF3down-regulation,itisnosurprisethatthe
Wepreviouslyreportedthatthe8bregionofSARS-CoVproteins8b recombinantvirusreplicatessimilarlyasthewildtypecontrol.
and 8ab consists of domains that allow for ubiquitin binding, ubiqui- Deletionofaccessoryproteins6,7,7b,8a,8band9baltogetherin
tinationandglycosylation(Leetal.,2007).Basedonthesefinding,we recombinant virus rSARS-CoV-Δ[6-9b] (Dediego et al., 2008) showed
proposedthatthe8bregionmaymediatethebindingof8band8abto that the recombinant virus replicates as well as wild type control in
ubiquitinatedcellularproteins,suchasp53andIκBα(Leetal.,2007). bothcellcultureandtransgenicmiceexpressingtheSARS-CoVreceptor
HereweshowedthatIRF3,anotherproteinregulatedbyubiquitination human angiotensin converting enzyme-2 (hACE-2). Since IFN antago-
(Siu et al., 2009; Spiegel et al., 2004), interacts with proteins 8b and nist function is also possessed by other SARS-CoV proteins (such as
8ab,suggestingthattheubiquitin-bindingpropertiesof8bregioncould nsp1,PLpro,MandN),itispossiblethatlossoffunctional8binrSARS-
allowthemtointeractwithmultiplecellularproteins.Itwouldbein- CoV-Δ[6-9b] is compensated, and thus the recombinant virus is not
terestingtofindoutwhatothercellulartargetsbindto8band8aband attenuatedinvivo.FurtherstudiesusingrecombinantSARS-CoVwith
whether they have a regulatory role during SARS-CoV infection. Fur- only8bor8abdeletedshouldbeperformedincellcultureandinap-
thermore,expressionof8band8abappearstoregulatethestabilityand propriate animal models, to better characterize the detailed mechan-
function of IRF3 by promoting degradation of IRF3 in a ubiquitin/ isms of their involvement in modulating viral replication and patho-
proteasome-dependent manner. Several viral proteins have been re- genesis.
portedtocauseproteasomaldegradationofIRF3(Z.Chenetal.,2007; Finally, Poly (I:C) is known to be able to induce both IFN and a
Sairaetal.,2009;Senetal.,2009).IRF3degradationistypicallytrig- subset of IFN-stimulated genes through activation of IRF3 in the ab-
geredpost-infection,whenviralinfection-inducedIRF3activationleads senceofIFN.Webelievethattheobservedantiviraleffectsofpoly(I:C)
to the ubiquitination of the protein targeting it for proteasomal de- onwildtypeandrecombinantIBVinthisstudywouldbethecombined
gradation (Higgs and Jefferies, 2008; Liu et al., 2005). This serves to action of poly (I:C)-induced IFN-stimulated genes either dependent
regulate type I IFN production as excessive IFN is detrimental to the uponorindependentofIFNinduction.
cells.Inthisstudy,weobservedIRF3degradationevenintheabsence
of strong IRF3 activation in cells infected with rIBV8b and rIBV8ab, Acknowledgements
suggestingthatthe8b-and8ab-mediatedIRF3degradationisnotthe
resultofatypicalnegativefeedbackmechanismtobringtheIFNlevel ThisworkwaspartiallysupportedbyanAcademicResearchFund
backtothephysiologicallevelattheendofviralinfectionasobserved (AcRF)Tier2grant(ACR47/14),MinistryofEducation,Singaporeand
withothervirusinfection,butmaybeanactivestepundertakenbythe by Guangdong Province Key Laboratory of Microbial Signals and
virustolimitIRF3activationduringitscourseofreplication. DiseaseControlgrantsMSDC-2017-05andMSDC-2017-06,Guangdong,
Atthisstage,whileweconfirmtheinvolvementoftheproteasome, People'sRepublicofChina.
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