LSMpaessler

MutationoftheN-TerminalRegionofChikungunyaVirusCapsidProtein:ImplicationsforVaccineDesignAdamTaylor,aXiangLiu,aAliZaid,aLucasY.
H.
Goh,bJodyHobson-Peters,bRoyA.
Hall,bAndresMerits,cSureshMahalingamaInstituteforGlycomics,GrifthUniversity,GoldCoastCampus,Queensland,Australiaa;AustralianInfectiousDiseasesResearchCentre,SchoolofChemistryandMolecularBiosciences,TheUniversityofQueensland,Queensland,Australiab;InstituteofTechnology,UniversityofTartu,Tartu,EstoniacABSTRACTMosquito-transmittedchikungunyavirus(CHIKV)isanarthritogenical-phavirusoftheTogaviridaefamilyresponsibleforfrequentoutbreaksofarthriticdis-easeinhumans.
Capsidprotein,astructuralproteinencodedbytheCHIKVRNAge-nome,isabletotranslocatetothehostcellnucleolus.
Inencephaliticalphaviruses,nucleartranslocationinduceshostcelltranscriptionalshutoff;however,theroleofcapsidproteinnucleolarlocalizationinarthritogenicalphavirusesremainsunclear.
Usingrecombinantenhancedgreenuorescentprotein(EGFP)-taggedexpressionconstructsandCHIKVinfectiousclones,wedescribeanucleolarlocalizationse-quence(NoLS)intheN-terminalregionofcapsidprotein,previouslyuncharacter-izedinCHIKV.
MutationoftheNoLSbysite-directedmutagenesisreducedef-ciencyofnuclearimportofCHIKVcapsidprotein.
Inthevirus,mutationofthecapsidproteinNoLS(CHIKV-NoLS)attenuatedreplicationinmammalianandmosquitocells,producingasmall-plaquephenotype.
AttenuationofCHIKV-NoLSislikelyduetodisruptionoftheviralreplicationcycledownstreamofviralRNAsynthesis.
Inmice,CHIKV-NoLSinfectioncausednodiseasesignscomparedtowild-typeCHIKV(CHIKV-WT)-infectedmice;lackofdiseasesignscorrelatedwithsignicantlyreducedviremiaanddecreasedexpressionofproinammatoryfactors.
MiceimmunizedwithCHIKV-NoLS,challengedwithCHIKV-WTat30dayspostimmunization,developnodis-easesignsandnodetectableviremia.
SerumfromCHIKV-NoLS-immunizedmiceisabletoefcientlyneutralizeCHIKVinfectioninvitro.
Additionally,CHIKV-NoLS-immunizedmicechallengedwiththerelatedalphavirusRossRivervirusshowedreducedearlyandpeakviremiapostchallenge,indicatingacross-protectiveeffect.
ThehighdegreeofCHIKV-NoLSattenuationmayimproveCHIKVantiviralandrationalvaccinedesign.
IMPORTANCECHIKVisamosquito-bornepathogencapableofcausingexplosiveepi-demicsofincapacitatingjointpainaffectingmillionsofpeople.
Afteraseriesofmajoroutbreaksoverthelast10years,CHIKVanditsmosquitovectorshavebeenabletoex-pandtheirrangeextensively,nowmakingCHIKVahumanpathogenofglobalim-portance.
WithnolicensedvaccineorantiviraltherapyforthetreatmentofCHIKVdisease,thereisagrowingneedtounderstandthemoleculardeterminantsofviralpathogenesis.
Thesestudiesidentifyapreviouslyuncharacterizednucleolarlocaliza-tionsequence(NoLS)inCHIKVcapsidprotein,beginafunctionalanalysisofsite-directedmutantsofthecapsidproteinNoLS,andexaminetheeffectoftheNoLSmutationonCHIKVpathogenesisinvivoanditspotentialtoinuenceCHIKVvaccinedesign.
AbetterunderstandingofthepathobiologyofCHIKVdiseasewillaidthede-velopmentofeffectivetherapeuticstrategies.
Chikungunyavirus(CHIKV)isanarthritogenicalphavirusoftheTogaviridaefamilytransmittedtohumansbymosquitoesoftheAedesgenus.
Urbantransmission(human-mosquito-human)canspreadCHIKVrapidlywithinhumanpopulations,caus-Received26October2016Accepted19December2016Published21February2017CitationTaylorA,LiuX,ZaidA,GohLYH,Hobson-PetersJ,HallRA,MeritsA,MahalingamS.
2017.
MutationoftheN-terminalregionofchikungunyaviruscapsidprotein:implicationsforvaccinedesign.
mBio8:e01970-16.
https://doi.
org/10.
1128/mBio.
01970-16.
EditorDianeE.
Grifn,JohnsHopkinsBloombergSchoolofPublicHealthCopyright2017Tayloretal.
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttribution4.
0Internationallicense.
AddresscorrespondencetoAdamTaylor,a.
taylor1@grifth.
edu.
au,orSureshMahalingam,s.
mahalingam@grifth.
edu.
au.
ThisarticleisadirectcontributionfromaFellowoftheAmericanAcademyofMicrobiology.
Externalsolicitedreviewers:MichaelDiamond,WashingtonUniversitySchoolofMedicine;ScottWeaver,UniversityofTexasMedicalBranch;ThomasMorrison,UniversityofColoradoSchoolofMedicine.
RESEARCHARTICLEcrossmJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org1onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromingoutbreaksofchikungunyafever(CF).
CFisadiseasecharacterizedbyfever,amaculopapularrash,myalgia,anddebilitatingpolyarthralgia,similarinseveritytothatseeninpatientswithrheumatoidarthritis(1).
CFisusuallyself-limitingandnonfatal;however,fatalitiesandsevereformsofCHIKVdiseasehavebeenreportedinrecentoutbreaks,withcomplicationsduringinfectionoftenfoundinpatientswithcomorbidi-ties.
Inaddition,studiessuggestthatahighproportionofpatientsinfectedwithCHIKVexperiencechronicrheumaticsymptomsorrelapsesofCFthatcanoccuryearsaftertheinitialinfection(2,3).
FollowingaseriesofoutbreaksthatbeganinKenyain2004,theregionsofcirculatingCHIKVquicklyexpandedfromlong-establishedareasofende-micityinEast/CentralAfrica,Asia,andWestAfrica,nowmakingCHIKVavirusofglobalconcern(4).
In2013,CHIKVspreadtotheAmericas,wherelocaltransmissionhasbeenidentiedinover40countriesorterritories.
Thisoutbreakhasresultedinmorethan1millionsuspectedcasesofCF.
CHIKVhasapositive-sensesingle-strandedRNA(ssRNA)genomethatcontainstwoopenreadingframes(ORFs):thenonstructuralORF(nsP1,nsP2,nsP3,andnsP4)andthestructuralORF(capsid,E3,E2,6K/TF,andE1).
BothORFsaretranslatedaspolyproteins,whichundergocisandtranscleavagetoformthematureviralproteins.
TheCHIKVinfectiousparticlecomprisesanucleocapsidcorecontaininggenomicRNAsurroundedbyahost-derivedlipidbilayerembeddedwiththeE2-E1glycoproteins.
TheRNAgenomeassociateswith240copiesofthecapsidproteintoformtheicosahedralnucleocapsid(5).
Thecapsidprotein,encodedbythe5=regionofthestructuralORF,iscapableofautoproteolyticcleavagefromthestructuralpolyproteinasthecarboxyl-terminalpartofcapsidproteincontainsaserineprotease(6).
TheN-terminalregionofcapsidproteinisrequiredformultimerizationoftheproteintoformthenucleocapsidandcontainsahighlypositivelychargedRNA-bindingdomainvitalforencapsidationofthegenome(7–9).
SignalmotifsintheNterminushavealsobeenshowntobeimportantinthenuclear/cytoplasmictranslocationofcapsidproteinandnucleolartargeting(10–12).
Inencephaliticalphaviruses,suchasVenezuelanandeasternequineencephalitisviruses,themultifunctionalcapsidproteinisresponsibleforhosttranscrip-tioninhibitionandsubsequentcytopathiceffect(13,14).
Nucleartrafckingofcapsidproteinwasshowntoberequiredforthistranscriptionalshutoffeffect(15).
Inthearthritogenicalphavirusesexamined,hosttranscriptionalshutoffandtranslationalshutoffarethoughttobedependentonnsP2ratherthancapsidprotein(16,17).
Indeed,despiteanexclusivelycytoplasmicreplicationcycle,bothencephaliticandarthritogenicalphaviruscapsidproteinshavebeenfoundtotrafctothehostcellnucleus.
Studiesindicatethatnucleartrafckingofcapsidproteininencephaliticalphavirusesimpactsvirulenceandpathogenesisinvivo(15,18).
Incontrast,littleisknownoftheimportanceofcapsidproteinnuclear,orindeedsubnuclear,trafckinginarthritogenicalphavirusessuchasCHIKV.
ImportantlythefunctionalsignicanceofcapsidproteinsubcellulartrafckingduringCHIKVreplicationandpathogenesishasnotbeeninvestigated.
Here,usingsite-directedmutagenesis,weidentifyaminoacidresiduesintheN-terminalpartofCHIKVcapsidproteinrequiredfornucleolarlocaliza-tioninmammaliancellsandperformafunctionalanalysisofmutantcapsidprotein.
Furthermore,weexaminethegrowthkineticsandpathogenicityofaCHIKVcontainingmutationsinthenucleolarlocalizationsequence(NoLS)ofcapsidprotein.
RESULTSLocalizationofCHIKVcapsidproteintothenucleolusandidenticationoftheNoLS.
Previousstudieshaveshownasubstantialamountofalphaviruscapsidproteintranslocatestonucleolus-likestructuresinhostcells(10,11).
ToinvestigatethesubcellularlocalizationofCHIKVcapsidprotein,Verocellsweretransfectedwithenhancedgreenuorescentprotein(EGFP)orEGFP-taggedwild-type(WT)capsidproteinexpressionplasmids.
ToensurecapsidproteinremainedfusedtotheNtermi-nusofEGFP,theC-terminaltryptophanresidue(W261)ofcapsidproteinrequiredforcapsidproteinautoproteolyticcleavagewasremoved(19);thismanipulationhadnoeffectoncapsidproteinsubcellularlocalization(datanotshown).
After24h,cellswereTayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org2onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromxedandpermeabilized,andcapsidproteinlocalizationwasvisualizeddirectlyviaEGFPuorescence.
EGFP-taggedcapsidproteinsubcellularlocalizationwasconrmedwithcapsidprotein-specicantibodiesandindirectimmunouorescence(Fig.
1A).
Asseenpreviously,inadditiontobeingpresentinboththecytoplasmandthenucleus,capsidproteinshowedaprominentlocalizationtonucleolus-likestructures.
Toconrmthatthesesubnuclearstructureswereindeednucleoli,indirectimmunouorescencewasusedtoidentifythenucleolususingnucleolin-specicantibodies.
Nucleolinisanucle-olarphosphoproteinthatinuencessynthesisandmaturationofribosomesandiswidelyusedasanucleolarmarker.
ColocalizationwiththenucleolarmarkernucleolinconrmedthatCHIKVcapsidproteinisindeedlocalizingtothenucleolus(Fig.
1B).
Bioinformatics,deletionanalysisofCHIKVcapsidprotein(datanotshown),andpreviousstudiesofalphaviruscapsidproteinsubcellulartrafckinghaveidentiedaregionintheN-terminalpartofCHIKVcapsidprotein,betweenresidues58and110,richinbasicaminoacidsastheregioncontainingtheputativenucleolarlocalizationsequence(NoLS)(11).
ThebipartiteNoLSofSemlikiForestviruscapsidprotein,be-tweenaminoacidresidues66to83and92to105,sharesbasicaminoacidsequencehomologytoCHIKVcapsidprotein(11).
ToidentifytheNoLSofCHIKVcapsidprotein,site-directedmutagenesiswasperformed,mutatingselectedbasicresiduesofWTproteintoalanines(Fig.
2A).
TodeterminethatmutantformsofcapsidproteinwereexpressedatsimilarlevelstoWTcapsidprotein,transfectedVerocelllysateswereassayedbyWesternblottingusinganEGFP-specicantibody.
TheresultsshowthatthemutantsCapsid-101andCapsid-NoLSwereexpressedatleastatthesamelevelasWTcapsidprotein(Fig.
2B).
UpontransfectionofVerocellswithplasmidsexpressingEGFP-taggedWTormutantcapsidproteins,itwasfoundthatcapsidmutantCapsid-101,althoughstilllocalizingtothenucleolus,accumulatesinthenucleolusatmuchreducedlevelscomparedtoWTcapsidprotein(Fig.
2C).
TheuorescenceintensityofCapsid-101inthenucleoluswascomparabletothatseenintherestofthenucleus.
Capsid-NoLSwasabsentfromthenucleolus,lackingcolocalizationwiththenucleolarmarkernucleolin,andremainedlargelyinthenucleusandcytoplasm.
TheNoLSofCHIKVcapsidproteinthereforeconstitutes10basicaminoacidslocatedintheN-terminalregionoftheprotein.
FIG1CHIKVcapsidproteinlocalizestothenucleolus.
VerocellsweretransfectedwithpEGFPorpCapsid-EGFP.
At24hposttransfection,cellswerexedandpermeabilized,EGFPuorescencewasanalyzedbydirectvisualization,andindirectimmunouorescencewasperformedusingcapsidprotein-specicantibodies(A)andnucleolin-specicantibodies(B)toidentifythenucleolus.
Imagesarerepresentativeofatleast6eldsofview.
DAPI,4=,6-diamidino-2-phenylindole.
Thesizebarsrepresent15m.
CHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org3onMarch16,2021byguesthttp://mbio.
asm.
org/DownloadedfromFLIPindicatesmutationsintheNoLSofcapsidproteinalsoinhibititsnucleartrafckingability.
Previousstudieshaveshownthatcapsidproteinisactivelytrans-portedtoandfromthenucleus(10).
HavingidentiedtheNoLSofcapsidprotein,wesoughttoinvestigatewhethermutationoftheNoLSaffectedcapsidproteinnuclearimporttrafcking.
WTandmutantcapsidproteintrafckingfromthecytoplasmtothenucleuswasinvestigatedusinguorescencelossinphotobleaching(FLIP)tocontinu-ouslyphotobleachadenedportionofthenucleusinliveVerocellsexpressingEGFP-taggedcapsidproteinat24hposttransfection.
Fluorescencelossatthecytoplas-micregionofinterest(ROI)wasnormalizedusingtherelativeuorescenceintensityfromtheLSM510software,andtheinitialuorescenceintensitywassetas1.
Contin-uousphotobleachingofthenucleusofWTcapsidprotein-expressingcellsresultedinthelossofuorescentsignalinthenucleusandsubstantiallossofuorescenceattheROIinthecytoplasm,indicatingthatcytoplasmictonucleartrafckingoccurred(Fig.
3A).
MutantCapsid-101andCapsid-NoLSproteins,however,displayedadistinctinabilitytotrafctothenucleus,asevidencedbythemarginaldecreaseintherelativeuorescenceintensityofthecytoplasmicROI(Fig.
3B).
After280sofphotobleaching,theROIofCapsid-NoLSshowsthesmallestdecreaseinrelativeuorescenceintensity,suggestingCapsid-NoLSnucleartrafckingisdramaticallyinhibited.
Theseresultssuggestthat,inadditiontoinhibitingnucleolarlocalization,mutationoftheNoLSofcapsidproteinalsoinhibitscapsidproteinnuclearimporttrafcking.
MutationoftheNoLSdidnotaffectcapsidproteinautoproteolyticcleavage.
WenextanalyzedifmutationoftheNoLSretainedcapsidproteinautoproteaseactivity.
ConstructsexpressingEGFP-taggedWTCapsid-W,Capsid-101W,andCapsid-NoLS-W,containingtheC-terminaltryptophan(W261)requiredforcapsidproteinautoproteo-lyticcleavage,weregenerated.
Figure4showsthatallconstructslackingW261residuefromtheconservedcleavagesitewereunabletocleaveEGFPfromcapsidprotein.
However,capsidproteaseefcientlycleavedEGFPfromcapsidproteininallconstructsthatcontainedW261,includingtheNoLSmutantsofcapsidprotein(Fig.
4).
Thus,mutationoftheNoLShasnoeffectontheautocatalyticproteaseactivityofCHIKVcapsidprotein.
FIG2IdenticationofthenucleolarlocalizationsequenceofCHIKVcapsidproteinusingsite-directedmutagen-esis.
(A)WTaminoacidresiduesthatmakeuptheputativeNoLSweremutatedtoalanines(highlightedinred).
VerocellsweretransfectedwithpEGFP,pCapsid-EGFP,pCapsid-101-EGFP,orpCapsid-NoLS-EGFP.
At24hpost-transfection,cellswereeither(B)lysedandcelllysatesanalyzedbyWesternblottingusingEGFPandactin-specicantibodiesor(C)xedandpermeabilized,withEGFPuorescenceanalyzedbydirectvisualizationandindirectimmunouorescenceperformedusingnucleolin-specicantibodies.
Imagesarerepresentativeofatleast6eldsofview.
Thesizebarsrepresent15m.
Tayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org4onMarch16,2021byguesthttp://mbio.
asm.
org/DownloadedfromCHIKVcontainingtheNoLSmutationincapsidproteinshowsattenuationinvitro.
ToassesstheimportanceofcapsidproteinnucleolarlocalizationonCHIKVreplication,weexaminedtheeffectoftheNoLSmutationinthecontextofafull-lengthCHIKVinfectiousclone-derivedvirus.
CHIKVcontainingtheNoLSmutationincapsidprotein(CHIKV-NoLS)wasrescuedandpropagatedinVerocells.
PlaquepuricationofvirusandSangersequencingoftheentireCHIKVgenomeconrmedtheNoLSmutationwasmaintainedinpassagedvirusintheabsenceofadditionalmutations.
IndirectFIG3MutationofcapsidproteinNoLSinhibitscapsidproteinnucleartrafcking.
FLIPanalysiswasperformedonVerocellstransfectedwitheitherpCapsid-EGFP,pCapsid-101-EGFP,orpCapsid-NoLS-EGFP.
(A)Fluorescencelossattheregionofinterest(ROI[coloredsquares])inthecytoplasmwasmeasuredovera4-minperiodduringcontinualphotobleachingofthenucleus(lightningbolt).
(B)Quanticationofuorescencelossatthecytoplasmicregionofinterestduringa280-speriodofcontinualphotobleachingofthenucleus.
Toconstructuorescencelosscurves,thequanticationoftherelativeuorescenceintensitywascarriedoutusingLSM510software;theinitialuorescenceintensitywassetas1.
Eachdatapointrepresentsthemeanstandarderrorfromatleast3independentreplicates.
FIG4MutationofcapsidproteinNoLSdoesnotaffectcapsidproteinautoproteaseactivity.
VerocellsweretransfectedwithpEGFP,pCapsid-EGFP,pCapsid-101-EGFP,orpCapsid-NoLS-EGFPorplasmidsexpressingEGFP-taggedWTCapsid-W,Capsid-101W,andCapsid-NoLS-W.
EGFP-taggedWTCapsid-W,Capsid-101W,andCapsid-NoLS-WcontaintheC-terminaltryptophan(W261)requiredforcapsidproteinautoproteolyticcleavage.
Cellswerelysedat24hposttransfection,andcelllysateswereanalyzedforcleavageofEGFPfromcapsidproteinbyWesternblottingandEGFP-specicantibody.
CHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org5onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromimmunouorescence,usingcapsid-specicantibodies,wasusedtoanalyzethesub-cellularlocalizationofCHIKVcapsidproteininCHIKV-WT-andCHIKV-NoLS-infectedVerocellsandmosquito(Aedesalbopictus)-derivedC6/36cells.
ResultsshowthatinCHIKV-WT-infectedVerocells,capsidproteinaccumulatesinsubnuclearbodiesremi-niscentofthenucleolusat24hpostinfection(Fig.
5A).
InCHIKV-NoLS-infectedVerocells,thesepunctaareabsent.
Thus,inthecontextofthevirustheNoLSmutationcausessimilardisruptionofcapsidproteinsubnuclearlocalizationininfectedVerocells.
TheNoLSmutationisthereforestableinthevirus,resultinginaphenotypicdisruptionofcapsidproteinsubnuclearlocalization.
Interestingly,inCHIKV-WT-infectedC6/36cells,capsidproteindidnotaccumulateinsubnuclearbodiesandwasfoundpredom-inantlyinthecytoplasmat24hpostinfection(Fig.
5B).
InCHIKV-NoLS-infectedC6/36cells,capsidproteinwasalsofoundtopredominateinthecytoplasm,similartothelocalizationobservedinCHIKV-WT-infectedC6/36cells.
SubnuclearlocalizationofcapsidproteinisthereforenotacharacteristicofCHIKVinfectionininsectcells,andmutationoftheNoLShasnoeffectonthesubcellularlocalizationofcapsidproteinininfectedC6/36cells.
ToexaminethereplicationkineticsofCHIKV-WTandCHIKV-NoLSinmammalian(BHK-21)andmosquito(C6/36)cells,cellswereinfectedatamultiplicityofinfection(MOI)of0.
1PFU/cell,andmultistepgrowthkineticswereanalyzed.
CHIKV-NoLSgrewtosignicantlylowertitersthanCHIKV-WTinbothBHK-21cells(Fig.
6A)andC6/36cells(Fig.
6B).
Furthermore,CHIKV-NoLShadasmall-plaquephenotypeinBHK-21cellsFIG5SubcellularlocalizationofcapsidproteininCHIKV-WT-orCHIKV-NoLS-infectedmammalianandmosquitocells.
Vero(A)orC6/36(B)cellswereinfectedwithCHIKV-WTorCHIKV-NoLSatanMOIof1PFU/cell.
Cellswerexedandpermeabilizedat24hpostinfection,andindirectimmunouorescencewasperformedusingcapsidprotein-specicantibodies.
Imagesarerepresentativeofatleast6eldsofview.
Thesizebarsrepresent15m.
Tayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org6onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfrom(Fig.
6C),indicatingareducedabilityofthevirustospreadfromtheinitialsiteofinfectionandthusattenuation.
TofurtherinvestigatetheattenuationofCHIKV-NoLSinmammalianandinsectcells,complementaryreversetranscription-quantitativePCR(RT-qPCR)analysisofvirusgenomecopynumberintheculturesupernatantsandvirusRNAcopynumberininfectedcellswasperformed.
ResultssuggestthatsynthesisofviralRNAremainsunperturbedbytheNoLSmutationinbothBHK-21(seeFig.
S1Ainthesupplementalmaterial)andC6/36(Fig.
S1B)cells.
Furthermore,by12hpostinfec-tion,thecopynumberofCHIKV-NoLSRNAsininfectedBHK-21(Fig.
S1A)andC6/36(Fig.
S1B)cellssignicantlyexceededthoseinCHIKV-WT-infectedcells.
ThedifferenceispotentiallyduetoincreasedsurvivalofCHIKV-NoLS-infectedcellsallowingprolongedormoreefcientsynthesisofviralRNAand/orduetoreducedcompetitionbetweenviralreplicaseandcapsidproteinforbindingviralgenomicRNAs.
However,thegenomecopynumbersofCHIKV-NoLSinculturesupernatantsdidnotshowanyincreaseupto12hpostinfectioninbothBHK-21(Fig.
S1C)andC6/36(Fig.
S1D)cells,indicatingthatnoorverylittleviruswasreleased.
ThisresultcorrelateswiththedelayedreleaseandreducedtitersofinfectiousCHIKV-NoLSrecoveredfromculturesupernatants(Fig.
6AandB).
Incontrast,forCHIKV-WT-infectedcells,virustitersstartedtoincreaseat8hpostinfection.
TheresultssuggestthatalthoughsynthesisofviralRNAininfectedcellswasnotreduced,mutationofthecapsidproteinNoLScausesadefectininfectiousvirusparticleformation,causingreductionanddelayofreleaseofviralprogeny.
Together,thesedatasuggestthatsubnuclearlocalizationofCHIKVcapsidproteinisnotahallmarkofinfectionacrossdifferenthostcellsandthattheattenuationofCHIKV-NoLSinbothmammalianandinsectcellsislikelytheresultofadefectininfectiousvirusparticleformationduetotheNoLSmutation.
CHIKV-NoLS-infectedmiceshownosignsofacuteCHIKVdisease.
Studiesexaminingtheroleofeasternequineencephalitisvirus(EEEV)capsidproteinintheinhibitionofhostgeneexpressionduringinfectionidentied,throughdeletionanaly-sis,aminoacidsintheN-terminalpartofcapsidproteinimportantforvirulenceinvivo(18).
ToevaluatethevirulenceofaCHIKVunabletotrafccapsidproteintothenucleolus,C57BL/6micewereinfectedsubcutaneouslyintheventral/lateralsideoftheFIG6CHIKVcontainingtheNoLSmutationincapsidproteinshowsattenuationinvitro.
MultistepgrowthkineticsinBHK-21(A)andC6/36(B)cellswereobtainedbyinfectingcellswithCHIKV-WTorCHIKV-NoLSatanMOIof0.
1PFU/cell.
Supernatantswerecollectedattheindicatedtimepoints,andinfectiousviruswasquantiedbyplaqueassay.
**,P0.
01,and***,P0.
001,bytwo-wayanalysisofvariance(ANOVA)withBonferroniposttests.
Eachsymbolrepresentsthemeanstandarderrorfrom3independentexperiments.
(C)Plaquesize(millimeters)ininfectedBHK-21cells.
***,P0.
001,byStudent'sunpairedttests.
Eachsymbolrepresentsthediameterofasingleplaque.
CHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org7onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromfootwith104PFUCHIKV-WTorCHIKV-NoLSormockinfectedwithphosphate-bufferedsaline(PBS)aloneandmonitoreddailyforsignsofCHIKV-inducedfootpadswelling.
TwopeaksoffootpadswellingwereobservedinCHIKV-WT-infectedmice,atapprox-imatelydays2and6postinfection,andswellingwasfullyresolvedbyday17postin-fection(Fig.
7A).
CHIKV-NoLSinfectedmicedevelopednofootpadswelling(Fig.
7A).
ViremiainCHIKV-NoLS-infectedmicewassignicantlyreducedatdays1and2postin-fection,comparedtothatinCHIKV-WT-infectedmiceandwasbelowthelimitofdetectionatdays3and4postinfection(Fig.
7B).
Theamountofinfectiousvirusrecoveredfromtheankletissue,closetothesiteofinoculation,ofCHIKV-NoLS-infectedmicewasalsosignicantlyreducedcomparedtothatinCHIKV-WT-infectedmiceatday3postinfection(Fig.
7C).
Also,theactivationofexpressionofkeyinammatorymedi-atorsofCHIKVdisease,monocytechemoattractantprotein-1(MCP-1),interferongamma(IFN-),andtumornecrosisfactoralpha(TNF-),wassignicantlylesspromi-nentintheankletissueofCHIKV-NoLS-infectedmicecomparedtoCHIKV-WT-infectedmiceatday3postinfection(Fig.
7D).
Together,theseresultsdemonstratethatCHIKV-NoLSishighlyattenuatedinmiceandthatmutationsintheNoLSofcapsidproteindiminishCHIKVvirulenceinvivo.
CHIKV-NoLS-immunizedmiceareprotectedfromCHIKVdiseasewhenchal-lengedwithCHIKV-WT.
AsmutationoftheNoLSofcapsidproteindramaticallyreducedCHIKVvirulenceininfectedmice,thehighlyattenuatedCHIKV-NoLSmayhavepotentialasaCHIKVvaccinecandidate.
ToexaminetheprophylacticefcacyofCHIKV-NoLS,miceweresubcutaneouslyimmunizedintheventral/lateralsideoftheFIG7CHIKV-NoLS-infectedmiceshownosignsofacuteCHIKVdisease.
Twenty-one-day-oldC57BL/6micewereinfectedsubcutaneouslyintheventral/lateralsideofthehindfootwith104PFUofCHIKV-WTorCHIKV-NoLSormockinfectedwithPBSalone.
(A)CHIKV-inducedfootpadswellingwasassesseddailybymeasuringtheheightandwidthoftheperimetatarsalareaofthehindfoot.
Eachsymbolrepresentsthemeanstandarderrorfrom5to6mice.
(B)Atdays1,2,3,and4postinfection,serumwasharvestedandviremiadetermined.
(C)Ankletissueswereharvestedatday3postinfectionandhomogenizedtodetermineamountofinfectiousvirusbyplaqueassayonVerocells.
Eachbarrepresentsthemeanstandarderrorfrom5to6mice.
***,P0.
001,bytwo-wayANOVAwithBonferroniposttests.
(D)ExpressionofproinammatoryfactorsofacuteCHIKVdiseaseinankletissuewasanalyzedusingRNAextractionandRT-qPCR.
ResultswerenormalizedtothelevelofthehousekeepinggeneHPRT1andexpressedasfoldchangescomparedtothelevelsinthemockcontrolsamples(horizontaldottedline).
*,P0.
05,and**,P0.
01,byStudent'sunpairedttests.
Tayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org8onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromfootwithonedoseof104PFUCHIKV-NoLS;controlmicewereinfectedwiththesamedoseofCHIKV-WTormockimmunizedwithPBSalone.
At30dayspostimmunization,micewerechallengedwith104PFUCHIKV-WTintheventral/lateralsideofthefootandmonitoreddailyforsignsofCHIKV-inducedfootpadswelling.
Twopeaksoffootpadswelling,atdays2and6postinfection,wereobservedinmock-immunizedmicechallengedwithCHIKV-WT(Fig.
8A).
SimilartoCHIKV-WT-infectedcontrolmice,miceimmunizedwithCHIKV-NoLSshowednosignsoffootpadswellinguponchallengewithCHIKV-WTanddevelopednodetectableviremiafromdays1to3postchallenge(Fig.
8B).
Thus,CHIKV-NoLSimmunizationefcientlyprotectedCHIKV-WT-challengedmicefromthedevelopmentofCHIKV-induceddiseasesignsanddetectableviremia.
Asprotectionfromalphaviralinfectionhasbeenreportedtobelargelyantibodydriven,theneutralizingcapacityofCHIKV-NoLS-inducedantibodieswasexamined.
Serafrommiceimmunizedwith104PFUCHIKV-NoLS,infectedwiththesamedoseofCHIKV-WTormockimmunizedwithPBSalone,werecollectedatday30postimmuni-zation.
TheneutralizingcapacityofseraagainstCHIKVwasrathersimilarinseracollectedfrommiceimmunizedwithCHIKV-NoLSorinfectedwithCHIKV-WTatday30postimmunization(seeFig.
S2inthesupplementalmaterial).
Thehighestconcentrationofserum(101)frombothofthesegroupsofmicepreincubatedwithCHIKVeffectivelyneutralizedinfectivityinvitro.
TheeffectofserafromCHIKV-NoLSimmunizedmicewas,however,somewhatlessprominentthanthatofserafromCHIKV-WT-infectedmice,mostprobablyduetolesseffectivespread(andcorrespondinglyreducedamountsofantigen-producingcells)ofthemutantvirus.
Nevertheless,antibodiesinducedbyCHIKV-NoLSimmunizationwereshowntoefcientlyneutralizeCHIKV.
CHIKV-NoLSimmunizationreducesearlyandpeakviremiainRRV-challengedmice.
Anumberofstudieshavehighlightedthecross-protectiveeffectsofbroadlyFIG8MiceimmunizedwithCHIKV-NoLSareprotectedfromdiseasewhenchallengedwithCHIKV-WT30dayspostimmunization.
Twenty-one-day-oldC57BL/6micewereimmunizedsubcutaneouslyintheventral/lateralsideofthehindfootwith104PFUofCHIKV-NoLS,infectedwiththesamedoseofCHIKV-WT,ormockinfectedwithPBSalone.
At30dayspostinfection,micewerechallenged(Ch)intheventral/lateralsideofthehindfootwith104PFUofCHIKV-WT.
AcuteCHIKVdiseaseuponchallengewasassessedbymeasuringtheheightandwidthoftheperimetatarsalareaofthehindfootevery24h(A).
Eachsymbolrepresentsthemeanstandarderrorfrom5to6mice.
(B)Viremiawasmeasuredatdays1,2,and3postchallenge.
Eachbarrepresentsthemeanstandarderrorfrom5to6mice,andthehorizontaldottedlinerepresentsthelimitofdetection.
CHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org9onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromneutralizingantialphavirusantibodies.
Toexaminethecross-protectiveeffectofCHIKV-NoLSagainstmedicallyimportantalphavirusesotherthanCHIKV,miceimmunizedwith104PFUofCHIKV-NoLS,infectedwiththesamedoseofCHIKV-WTormockimmunizedwithPBSalone,werechallengedwith104PFUofrelatedarthritogenicalphavirusRossRivervirus(RRV)subcutaneouslyinthethorax,andviremiawasmeasured.
CHIKV-NoLS-immunizedmiceshowsignicantlyreducedpeakandearlyviremia,days1and2postchallenge,uponchallengewithRRV(Fig.
9).
Coherentwithdatafromtheneutral-izationassay,theprotectiveeffectofCHIKV-NoLSimmunizationwaslessprominentthanthatobservedinCHIKV-WT-infectedmice.
Nevertheless,byreducingviremia,anindicatorofdiseaseoutcome,CHIKV-NoLShasthepotentialtooffercross-protectionagainstdiseasecausedbyotherarthritogenicalphavirusessuchasRRV.
DISCUSSIONAspositive-strandRNAviruses,alphavirusesreplicatewithinthecytoplasmofhostcells.
Despitethis,severalalphavirusestargetanumberofviralproteinstothenucleus.
Therationaleforthisisnotimmediatelyobvious;however,previousstudieshavehighlightedhostcellshutoffasonefacetofcapsidproteinnuclearlocalizationinencephaliticalphaviruses(20).
Compartmentalizationofviralproteinswithinsubcellularstructuresofthehostcellisthereforeamechanismusedbyalphavirusestoaugmentproteinfunctionandoptimizeinfectivity,particularlyforaproteinwithmultiplerolesduringinfectionsuchasCHIKVcapsidprotein.
Thenucleolusisahighlydynamicsubnuclearstructure.
Typicallythoughtofasthesiteofribosomebiogenesis,thenucleolusparticipatesinnumerouscellularprocesses,includingtranscriptionalregulation,cellsignaling,andresponsetostress(21,22).
Virusesfromavarietyofdifferentfamiliesarenowknowntointeractwiththenucleolusduringinfection.
Althoughthefunctionalsignicanceofviralnucleolartropismisgenerallynotwellunderstood,agrowingbodyofevidencesuggeststhatvirusessequestercellularnucleolarproteinsortargetviralproteinstothenucleolustofacilitateviralreplication(23–25).
MutationsintheNoLSofporcinereproductiveandrespiratorysyndromevirusNproteinandJapaneseencephalitisviruscoreproteinhavebeenshowntoattenuatevirusreplicationandreducepathogenesisinvivo,makingthesemutantsidealliveattenuatedvaccinecandidates(26,27).
DeletionanalysishasidentiednuclearimportandexportsignalswithinCHIKVcapsidprotein,andhighlightstheimportanceoftheN-terminalpartoftheproteininnuclearimport(10).
Furthermore,deletionstudiesoftheN-terminalpartofeasternequineencephalitisvirus(EEEV)capsidproteinshowthatthisregioniscriticalforEEEVpathogenesis(18).
Theintracellularlocalizationofcapsidproteinhasbeenlinkedtoencephaliticalphavirusattenuationpreviouslyinbothinvitroandinvivosettings(15,FIG9MiceimmunizedwithCHIKV-NoLSshowreducedviremiawhenchallengedwithRRV.
Twenty-one-day-oldC57BL/6micewereimmunizedsubcutaneouslyintheventral/lateralsideofthehindfootwith104PFUofCHIKV-NoLS,infectedwiththesamedoseofCHIKV-WT,ormockinfectedwithPBSalone.
At30dayspostinfection,micewerechallenged(Ch)subcutaneouslyinthethoraxwith104PFUofRRV.
Viremiawasmeasuredatdays1and2postchallenge,andtheamountofinfectiousvirusinchallengedmouseserawasquantiedbyplaqueassay.
Eachbarrepresentsthemeanstandarderrorfrom5to6mice,andthehorizontaldottedlinerepresentsthelimitofdetection.
*,P0.
05,and***,P0.
001,bytwo-wayANOVAwithBonferroniposttests.
Tayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org10onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfrom28).
Here,weconrmnucleolarlocalizationofCHIKVcapsidproteinusingrecombinantEGFP-taggedproteins.
Usingsite-directedmutagenesis,weidentifyaminoacidresidueswithintheN-terminalpartofCHIKVcapsidproteinresponsiblefornucleolarlocaliza-tion.
Furthermore,disruptionoftheNoLSofCHIKVcapsidproteinwasalsofoundtodramaticallyinhibitcapsidproteinnucleartrafcking.
ItislikelythatthereisfunctionaloverlapbetweenthetrafckingmotifsandNoLSlocatedwithintheN-terminalportionofcapsidprotein.
AsubstantialnumberofaminoacidresiduesthatmakeuptheNoLScharacterizedhereinweredeletedinthepreviouslydenednuclearlocalizationsignalofCHIKVcapsidprotein(10).
Furthermore,thedecreasednucleartrafckingabilityofEGFP-Capsid-NoLScomparedtoEGFP-Capsid-101seenatlatertimepoints(Fig.
3B)maybeanaccumulativeeffectfromtheadditionalmutationspresentinCapsid-NoLS.
HavingidentiedtheNoLSofcapsidprotein,weexaminedtheimportanceoftheregioninvolvedincapsidproteinnucleolarlocalizationforCHIKVreplication.
Immu-nouorescenceanalysisestablishedthatinCHIKV-WT-infectedVerocells,capsidproteinwasaccumulatinginasubnuclearbody,reminiscentofthenucleolus.
InCHIKV-NoLS-infectedVerocells,suchlocalizationwasnotobserved,suggestingtheNoLSmutationwasmaintainedintheinfectiousvirusgenome.
However,inCHIKV-WT-infectedmos-quito(C6/36)cells,capsidproteinsubcellularlocalizationwaslargelycytoplasmic,withnosubnuclearpunctaobserved.
LocalizationofCHIKVcapsidproteintosubnucleardomainsisthereforenotacharacteristicofinfectioninallcelltypes.
ThereplicationofCHIKV-NoLSinmammalian(BHK-21)andmosquito(C6/36)cellswasattenuated,pro-ducingsignicantlylowertitersthanCHIKV-WT.
TheoverallreducedreplicationofCHIKV-NoLSobservedinbothIFN-/-decientBHK-21cellsandmosquitocellssug-geststhatmutationoftheNoLSresultsinadefectininfectiousvirionformationandnotinvirusRNAproduction.
Thisislikelylinkedtodisruptionofanotheressentialfunctionofcapsidprotein—forexample,RNAbindingand/orhomomultimerization.
Wehaveshownthatnucleolarlocalizationisnotrequiredfortheautoproteolyticcleavageofcapsidprotein;however,theN-terminalregionofcapsidproteinisalsothoughttobeimportantfornucleocapsidformation(8).
FurthermolecularstudiesarerequiredtodeterminewhethertheregionresponsiblefornucleolarlocalizationofCHIKVcapsidprotein,orevennucleolarlocalizationoftheproteinitself,isrequiredforRNAbindingand/orhomomultimerization.
Viralproteinnucleolarlocalizationisthoughttobeintimatelylinkedtoviralprotein-RNAandprotein-proteininteractionsinanumberofvirusesfromvariousfamilies.
Attenuatedreplicationininsectcellsis,however,animportantsafetyfeatureforanyliveattenuatedarbovirusvaccinecandidate,toavoidinfectionofviralvectors.
CHIKV-NoLS-infectedmiceshownosignsofacuteCHIKVdisease,highlightingtheimportanceoftheregionresponsibleforcapsidproteinsubcellularlocalizationtothereplicationandpathogenicityofanarthritogenicalphavirus.
ThedramaticeffectoftheNoLSmutationincapsidproteinonCHIKVvirulenceinmiceledustoinvestigatethesuitabilityofCHIKV-NoLSasaliveattenuatedvaccinecandidate.
MiceimmunizedwithCHIKV-NoLSshowednosignsoffootpadswellinguponchallengewithCHIKV-WTatday30postimmunizationanddevelopednodetectableviremiafromdays1to3postchallenge.
ImmunizationwithCHIKV-NoLSprotectedmicefromCHIKVchallengeforupto30days,andantibodiesinducedbyCHIKV-NoLSimmunizationefcientlyneutralizeCHIKVinvitro,indicatingCHIKV-NoLSisimmunogenicafteronedose.
Furtherlong-termstudieswillassesswhetherCHIKV-NoLS-inducedimmunityislong-lived.
Agrowingbodyofevidence,includingourownstudies,indicatescross-reactivityofalphavirusantibodieswithbroadlyneutralizingeffectsbothinvitroandinvivo(29–32).
Cross-protectionagainstanumberofotherarthritogenicalphavirusesisadesirablefeatureofaCHIKVvaccinecandidate.
CHIKV-NoLS-immunizedmiceshowsignicantlyreducedpeakandearlyviremiauponchallengewithrelatedalphavirusRRV.
Byreducingviremia,anindicatorofdiseaseoutcome,CHIKV-NoLShasthepotentialtooffercross-protectionagainstdiseasecausedbyotherarthritogenicalphaviruses.
TheidenticationofmutationsabletospecicallyinhibitcapsidproteinnucleolarlocalizationisofgreatimportanceinevaluatingtherolesofnucleolarinteractionsCHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org11onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfromduringalphaviralinfection,aswellasdemonstratingthepotentialoftheseinteractionsastargetsforthedevelopmentofnovelantiviraltherapiesthattargetviralproteinsubcellulartrafckingoraidthedesignofnovelliveattenuatedvaccinecandidatesagainstCHIKVandotherarthritogenicalphaviruses.
Additionalstudieswillberequiredtoinvestigatethesafetyprole,levelofattenuation,andlong-termvaccineefcacyofCHIKV-NoLSorsimilarmutantvirusestoallowfurtherdevelopmentofaliveattenuatedvaccinecandidate.
MATERIALSANDMETHODSOligonucleotides,plasmids,andantibodies.
TogeneratepCapsid-EGFP,cDNAcorrespondingtoCHIKVcapsidproteinwasampliedbyPCRusingprimersCHIKCprotF(5=GCGGCGCAAGCTTATGGAGTTCATCCCAACCC3=)andCHIKCprotR(5=CGCGGATCCGACTCTTCGGCCCCCTCG3=)andclonedintopEGFP-N1(TaKaRaBio,Inc.
,USA).
TogeneratepCapsidW-EGFPcontainingthetryptophanresiduerequiredforcapsidproteinautoproteolyticcleavageattheC-terminalpartofcapsidprotein,primersCHIKCprotFandCHIKCprotWR(5=CGCGGATCCGACCACTCTTCGGCC3=)wereused,andtheobtainedfragmentwasclonedintopEGFP-N1.
pSP6-CHIKV-ZsGreen,aplasmidcontainingcDNAofaCHIKVvariantexpressingtheZsGreenmarkerprotein,wasconstructedusingafull-lengthinfectiouscDNAcloneoftheLaReunionCHIKVisolateLR2006-OPY1asdescribedpreviously(33).
Theoligonucleotidesusedinsite-directedmutagenesisarelistedinTableS1inthesupplementalmaterial.
MutantsweregeneratedusingaQuikChangeIIsite-directedmutagenesiskit(AgilentTechnologies,Inc.
,USA).
Antibodiestonucleolin(SantaCruzBiotech,Inc.
,USA),EGFP(BDBiosciences,USA),andactin(SantaCruzBiotech,Inc.
,USA)werepurchasedfromtherespectivesuppliers.
Monoclonalcapsidproteinantibodywasmadein-houseandcharacterizedasdescribedpreviously(34,35).
Acocktailofanticapsidmonoclonalantibodies(1.
7B2and4.
1H11)wasusedforimmunouorescence.
Cellculture,transfection,andviruspropagation.
VeroandBHK-21cellswereculturedinOpti-MEM(Gibco,ThermoFisherScientic,Australia),supplementedwith3%fetalcalfserum(FCS).
C6/36cellswereculturedinLeibovitz'sL-15medium(Gibco,ThermoFisherScientic,Australia),supplementedwith10%tryptosephosphatebrothand10%FCS.
PlasmidtransfectionswerecarriedoutwithLipofectamine2000(ThermoFisherScientic,Australia)accordingtothemanufacturer'sinstructions.
Mice.
C57BL/6WTmicewereobtainedfromtheAnimalResourcesCentre(Perth,Australia)andbredin-house.
AllanimalexperimentswereperformedinaccordancewiththeguidelinessetoutbyTheGrifthUniversityAnimalEthicsCommittee.
Twenty-one-day-oldC57BL/6maleandfemalemice,inequaldistribution,wereinoculatedintheventral/lateralsideofthefootwith104PFUCHIKV-WTorCHIKV-NoLSdilutedinPBStoavolumeof20l.
Mock-infectedmicewereinoculatedwithPBSalone.
Micewereweighedandscoredfordiseasesignsevery24handsacricedbyCO2asphyxiationatexperimentalendpoints.
CHIKV-inducedfootpadswellingwasassessedbymeasuringtheheightandwidthoftheperimetatarsalareaofthehindfoot,usingKincromedigitalverniercalipers.
At30dayspostinfection,micewerechallengedintheventral/lateralsideofthefootwith104PFUCHIKV-WT,weighed,andscoredfordiseasesignsevery24h,withviremiameasuredatdays1,2,and3postchallenge,or104PFURossRivervirus(RRV)subcutaneouslyinthethorax,withviremiameasuredatdays1and2postchallenge.
Neutralizationassay.
TheneutralizingcapacityofantibodyfromCHIKV-WT-orCHIKV-NoLS-infectedmiceatday30postinfectionwasanalyzedbyimmunouorescence-basedcellinfectionassaysusingVerocellsandCHIKV-ZsGreen.
Infectiousvirus,takenatanamountsufcientforamultiplicityofinfection(MOI)of0.
4,wasmixedwithdiluted(101,102,and103),heat-inactivated(56°Cfor30min)pooledmouseserum,followedbyincubationfor2hat37°C.
Virus-antibodymixtureswereaddedtoVerocellsandincubatedat37°Cfor1h.
Thevirusinoculumwasremoved,cellswerewashedwithPBS,andOpti-MEMcontaining3%FCSwasadded,followedbyincubationfor6hat37°C.
Cellsweregentlyresuspended,stainedwithLIVE/DEADnear-infraredcellstain(ThermoFisherScientic,Australia),andxedin4%paraformaldehyde.
InfectivitywasmeasuredaspercentageofZsGreen-positivelivecellsusingaBDLSRIIFortessacellanalyzerandquantiedwithFlowJosoftware(Treestar,Inc.
,USA).
ImmunouorescencemicroscopyandFLIP.
Cellsgrownonpolylysine-treatedcoverslipswerexedin4%paraformaldehydeandpermeabilizedin1%TritonX-100.
Cellswerethenblockedin1%bovineserumalbumin(BSA)madeinPBSandincubatedat37°Cfor1h.
Primaryantibodieswerediluted1:100in1%BSAandincubatedwiththecellsfor1hat37°C.
AlexaFluor647-conjugatedsecondaryantibody(Invitrogen,ThermoFisherScientic,Australia),wasdiluted1:500in1%BSAandincubatedwiththecellsfor1hat37°C.
CoverslipsweremountedinVectorshieldmountingmedium(VectorLaboratories,USA),andstainingwasvisualizedonanOlympusFluoViewFV1000confocalmicroscope.
ForFLIPanalysis,Verocellswereplatedonglass-based33-mmculturedishesandimagedat24hposttransfectionusinganLSM510Metaconfocalmicroscope(Zeiss,Oberkochen,Germany).
Cellsweremaintainedat37°C,andduringimaging,thecellculturemediumwasexchangedforCO2-independentmedium(Invitrogen,ThermoFisherScientic,Australia).
Fluorescencelossattheregionofinterest(ROI)wasnormalizedusingtherelativeuorescenceintensityfromtheLSM510software;initialuorescenceintensitywassetas1.
Invitroviralreplicationkinetics.
BHK-21andC6/36cellswereinfectedwithCHIKV-WTorCHIKV-NoLSatanMOIof0.
1andallowedtoincubatefor1hat37°Cina5%CO2incubatorbeforeviruswasremovedandthecellswerewashedwithPBSandoverlaidwithOpti-MEMcontaining3%FCS.
Atvarioustimespostinfection,supernatantaliquotswereharvestedandviraltitersmeasuredbyplaqueassayasoutlinedbelow.
TodeterminethevirusRNAgenomecopynumberinculturesupernatantsandviruspositive-strandRNAcopynumberininfectedcells,supernatantwascollectedandmonolayerswashedTayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org12onMarch16,2021byguesthttp://mbio.
asm.
org/DownloadedfromthreetimesinPBS.
RNAextractionwasperformedusingTRIzol(Invitrogen,ThermoFisherScientic,Australia),accordingtothemanufacturer'sinstructions.
ExtractedRNAwasreversetranscribedusingrandomnonamerprimersandMoloneymurineleukemiavirus(MMLV)reversetranscriptase(Sigma-Aldrich,Inc.
,USA)accordingtothemanufacturer'sinstructions.
Thestandardcurvewasgeneratedusingserialdilutionsofafull-lengthinfectiouscDNAcloneoftheLaReunionCHIKVisolateLR2006-OPY1.
QuanticationofviralloadwasperformedusingSYBRGreenreal-timePCRreagentin12.
5-lreactionvolumetodetecttheE1region.
PrimersCHIKVE1F(5=CCCGGTAAGAGCGGTGAA3=)andCHIKVE1R(5=CTTCCGGTATGTCGATG3=)wereusedtodetectCHIKVgenomic,antigenomic,andsubgenomicRNAs.
AllreactionswereperformedusingaCFX96Touchreal-timePCRsystem.
Thestandardcurvewasplotted,andcopynumbersofampliedproductswereinterpolatedfromastandardcurveusingGraphPadPrismsoftwaretodetermineviralRNAcopynumber.
Viraltiterassay.
Miceweresacricedatdays1,2,3,and4postinfection,withtheanklejointandserumcollectedandassayedforviraltiterusingaplaqueassay.
Tissuesampleswerehomogenizedin1mlofPBS,and10-foldserialdilutionsofhomogenateandserawereaddedintriplicatetoVerocells.
Viruswasallowedtoincubatefor1hat37°Cina5%CO2incubatorbeforetheviruswasremovedandthecellsoverlaidwithOpti-MEMcontaining3%FCSand1%agarose(Sigma-Aldrich,Inc.
,USA)andincubatedfor48hina5%CO2incubator.
Cellswerexedin1%formalin,andvirusplaquesweremadevisiblebystainingwith0.
1%crystalviolet.
ResultsareexpressedasPFUpermilliliterorPFUpergramoftissue.
RT-qPCR.
RNAwasextractedfromtissuesusingTRIzol(Invitrogen,ThermoFisherScientic,Austra-lia),accordingtothemanufacturer'sinstructions.
OnemicrogramoftotalRNAwasreversetranscribedusingrandomnonamerprimersandMMLVreversetranscriptase(Sigma-Aldrich,Inc.
,USA)accordingtothemanufacturer'sinstructions.
Reversetranscription-quantitativePCR(RT-qPCR)wasperformedwith50ngoftemplatecDNA,QuantiTectprimerassaykits(Qiagen,Hilden,Germany),andSYBRGreenreal-timePCRreagentinaCFX96Touchreal-timePCRsystemusingastandardthree-stepmeltprogram(95°Cfor15s,55°Cfor30s,and72°Cfor30s).
DatawerenormalizedtotheHPRT1housekeepinggene,andthefoldchangeinmRNAexpressionrelativetomock-infectedPBS-treatedsamplesforeachgenewascalculatedusingthethresholdcycle(ΔΔCT)method:ΔΔCTΔCTofvirusinfectedΔCTofmockinfected,whereΔCTCTofgeneofinterestCTofhousekeepinggene.
Thefoldchangeforeachgeneiscalculatedas2ΔΔCT.
Statisticalanalysis.
Two-wayANOVAwithBonferroniposttestswasusedtoexamineinvitroviralgrowthkineticdataandviremia.
Student'sunpairedttestswereusedtoanalyzeRT-qPCRandankletitersatday3postinfection.
One-wayANOVAwithBonferroniposttestswasusedtoexaminetheneutraliza-tionassay.
APvalueof0.
05wasconsideredtobesignicant.
SUPPLEMENTALMATERIALSupplementalmaterialforthisarticlemaybefoundathttps://doi.
org/10.
1128/mBio.
01970-16.
FIGS1,TIFle,0.
4MB.
FIGS2,TIFle,0.
1MB.
TABLES1,DOCXle,0.
1MB.
ACKNOWLEDGMENTSThisstudywassupportedbygrantsfromtheEstonianResearchCouncil(20-27)toA.
M.
andAustralianNationalHealthandMedicalResearchCounciltoS.
M.
(APP1033068andAPP1047252).
S.
M.
istherecipientofanNHMRCseniorresearchfellowship(APP1059167).
Theauthorsdeclarethattheyhavenoconictsofinterest.
REFERENCES1.
WinMK,ChowA,DimatatacF,GoCJ,LeoYS.
2010.
ChikungunyafeverinSingapore:acuteclinicalandlaboratoryfeatures,andfactorsassoci-atedwithpersistentarthralgia.
JClinVirol49:111–114.
https://doi.
org/10.
1016/j.
jcv.
2010.
07.
004.
2.
BorgheriniG,PoubeauP,JossaumeA,GouixA,CotteL,MichaultA,Arvin-BerodC,PaganinF.
2008.
PersistentarthralgiaassociatedwithChikungunyavirus:astudyof88adultpatientsonReunionIsland.
ClinInfectDis47:469–475.
https://doi.
org/10.
1086/590003.
3.
SissokoD,MalvyD,EzzedineK,RenaultP,MoscettiF,LedransM,PierreV.
2009.
Post-epidemicchikungunyadiseaseonReunionIsland:courseofrheumaticmanifestationsandassociatedfactorsovera15-monthperiod.
PLoSNeglTropDis3:e389.
https://doi.
org/10.
1371/journal.
pntd.
0000389.
4.
JosseranL,PaquetC,ZehgnounA,CaillereN,LeTertreA,SoletJL,LedransM.
2006.
Chikungunyadiseaseoutbreak,ReunionIsland.
EmergInfectDis12:1994–1995.
https://doi.
org/10.
3201/eid1212.
060710.
5.
JoseJ,SnyderJE,KuhnRJ.
2009.
Astructuralandfunctionalperspectiveofalphavirusreplicationandassembly.
FutureMicrobiol4:837–856.
https://doi.
org/10.
2217/fmb.
09.
59.
6.
AggarwalM,SharmaR,KumarP,ParidaM,TomarS.
2015.
Kineticcharacterizationoftrans-proteolyticactivityofChikungunyaviruscapsidproteaseanddevelopmentofaFRET-basedHTSassay.
SciRep5:14753.
https://doi.
org/10.
1038/srep14753.
7.
PereraR,OwenKE,TellinghuisenTL,GorbalenyaAE,KuhnRJ.
2001.
Alphavirusnucleocapsidproteincontainsaputativecoiledcoilalpha-heliximportantforcoreassembly.
JVirol75:1–10.
https://doi.
org/10.
1128/JVI.
75.
1.
1-10.
2001.
8.
HongEM,PereraR,KuhnRJ.
2006.
AlphaviruscapsidproteinhelixIcontrolsacheckpointinnucleocapsidcoreassembly.
JVirol80:8848–8855.
https://doi.
org/10.
1128/JVI.
00619-06.
9.
OwenKE,KuhnRJ.
1996.
IdenticationofaregionintheSindbisvirusnucleocapsidproteinthatisinvolvedinspecicityofRNAencapsidation.
JVirol70:2757–2763.
CHIKVCapsidProteinNucleolarLocalizationJanuary/February2017Volume8Issue1e01970-16mbio.
asm.
org13onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfrom10.
ThomasS,RaiJ,JohnL,SchaeferS,PützerBM,HerchenrderO.
2013.
Chikungunyaviruscapsidproteincontainsnuclearimportandexportsignals.
VirolJ10:269.
https://doi.
org/10.
1186/1743-422X-10-269.
11.
FavreD,StuderE,MichelMR.
1994.
TwonucleolartargetingsignalspresentintheN-terminalpartofSemlikiForestviruscapsidprotein.
ArchVirol137:149–155.
https://doi.
org/10.
1007/BF01311181.
12.
JakobR.
1994.
NucleolaraccumulationofSemlikiForestvirusnucleo-capsidCprotein:inuenceofmetabolicstatus,cytoskeletonandrecep-tors.
JMedMicrobiol40:389–392.
https://doi.
org/10.
1099/00222615-40-6-389.
13.
GarmashovaN,GorchakovR,VolkovaE,PaesslerS,FrolovaE,FrolovI.
2007.
TheOldWorldandNewWorldalphavirusesusedifferentvirus-specicproteinsforinductionoftranscriptionalshutoff.
JVirol81:2472–2484.
https://doi.
org/10.
1128/JVI.
02073-06.
14.
AguilarPV,WeaverSC,BaslerCF.
2007.
Capsidproteinofeasternequineencephalitisvirusinhibitshostcellgeneexpression.
JVirol81:3866–3876.
https://doi.
org/10.
1128/JVI.
02075-06.
15.
GarmashovaN,AtashevaS,KangW,WeaverSC,FrolovaE,FrolovI.
2007.
AnalysisofVenezuelanequineencephalitisviruscapsidproteinfunctionintheinhibitionofcellulartranscription.
JVirol81:13552–13565.
https://doi.
org/10.
1128/JVI.
01576-07.
16.
FrosJJ,vanderMatenE,VlakJM,PijlmanGP.
2013.
TheC-terminaldomainofChikungunyavirusnsP2independentlygovernsviralRNAreplication,cytopathicity,andinhibitionofinterferonsignaling.
JVirol87:10394–10400.
https://doi.
org/10.
1128/JVI.
00884-13.
17.
GorchakovR,FrolovaE,FrolovI.
2005.
InhibitionoftranscriptionandtranslationinSindbisvirus-infectedcells.
JVirol79:9397–9409.
https://doi.
org/10.
1128/JVI.
79.
15.
9397-9409.
2005.
18.
AguilarPV,LeungLW,WangE,WeaverSC,BaslerCF.
2008.
Ave-amino-aciddeletionoftheeasternequineencephalitisviruscapsidproteinattenuatesreplicationinmammaliansystemsbutnotinmosquitocells.
JVirol82:6972–6983.
https://doi.
org/10.
1128/JVI.
01283-07.
19.
ThomasS,RaiJ,JohnL,GüntherS,DrostenC,PützerBM,SchaeferS.
2010.
Functionaldissectionofthealphaviruscapsidprotease:sequencerequirementsforactivity.
VirolJ7:327.
https://doi.
org/10.
1186/1743-422X-7-327.
20.
AtashevaS,FishA,FornerodM,FrolovaEI.
2010.
VenezuelanequineencephalitisviruscapsidproteinformsatetramericcomplexwithCRM1andimportinalpha/betathatobstructsnuclearporecomplexfunction.
JVirol84:4158–4171.
https://doi.
org/10.
1128/JVI.
02554-09.
21.
YunCW,WangYG,MukhopadhyayD,BacklundP,KolliN,YergeyA,WilkinsonKD,DassoM.
2008.
NucleolarproteinB23/nucleophosminregu-latesthevertebrateSUMOpathwaythroughSENP3andSENP5proteases.
JCellBiol183:589–595.
https://doi.
org/10.
1083/jcb.
200807185.
22.
RubbiCP,MilnerJ.
2003.
Disruptionofthenucleolusmediatesstabili-zationofp53inresponsetoDNAdamageandotherstresses.
EMBOJ22:6068–6077.
https://doi.
org/10.
1093/emboj/cdg579.
23.
YuY,JiH,DoudnaJA,LearyJA.
2005.
Massspectrometricanalysisofthehuman40Sribosomalsubunit:nativeandHCVIRES-boundcomplexes.
ProteinSci14:1438–1446.
https://doi.
org/10.
1110/ps.
041293005.
24.
SuPY,WangYF,HuangSW,LoYC,WangYH,WuSR,ShiehDB,ChenSH,WangJR,LaiMD,ChangCF.
2015.
Cellsurfacenucleolinfacilitatesenterovirus71bindingandinfection.
JVirol89:4527–4538.
https://doi.
org/10.
1128/JVI.
03498-14.
25.
OksayanS,NikolicJ,DavidCT,BlondelD,JansDA,MoseleyGW.
2015.
Identicationofarolefornucleolininrabiesvirusinfection.
JVirol89:1939–1943.
https://doi.
org/10.
1128/JVI.
03320-14.
26.
PeiY,HodginsDC,LeeC,CalvertJG,WelchSKW,JolieR,KeithM,YooD.
2008.
Functionalmappingoftheporcinereproductiveandrespiratorysyndromeviruscapsidproteinnuclearlocalizationsignalanditspatho-genicassociation.
VirusRes135:107–114.
https://doi.
org/10.
1016/j.
virusres.
2008.
02.
012.
27.
MoriY,OkabayashiT,YamashitaT,ZhaoZJ,WakitaT,YasuiK,HasebeF,TadanoM,KonishiE,MoriishiK,MatsuuraY.
2005.
NuclearlocalizationofJapaneseencephalitisviruscoreproteinenhancesviralreplication.
JVirol79:3448–3458.
https://doi.
org/10.
1128/JVI.
79.
6.
3448-3458.
2005.
28.
LundbergL,PinkhamC,BaerA,AmayaM,NarayananA,WagstaffKM,JansDA,Kehn-HallK.
2013.
NuclearimportandexportinhibitorsaltercapsidproteindistributioninmammaliancellsandreduceVenezuelanequineencephalitisvirusreplication.
AntiviralRes100:662–672.
https://doi.
org/10.
1016/j.
antiviral.
2013.
10.
004.
29.
FoxJM,LongF,EdelingMA,LinH,vanDuijl-RichterMK,FongRH,KahleKM,SmitJM,JinJ,SimmonsG,DoranzBJ,CroweJE,Jr,FremontDH,RossmannMG,DiamondMS.
2015.
Broadlyneutralizingalphavirusan-tibodiesbindanepitopeonE2andinhibitentryandegress.
Cell163:1095–1107.
https://doi.
org/10.
1016/j.
cell.
2015.
10.
050.
30.
GardnerJ,AnrakuI,LeTT,LarcherT,MajorL,RoquesP,SchroderWA,HiggsS,SuhrbierA.
2010.
Chikungunyavirusarthritisinadultwild-typemice.
JVirol84:8021–8032.
https://doi.
org/10.
1128/JVI.
02603-09.
31.
PartidosCD,PaykelJ,WegerJ,BorlandEM,PowersAM,SeymourR,WeaverSC,StinchcombDT,OsorioJE.
2012.
Cross-protectiveimmunityagainsto'nyong-nyongvirusaffordedbyanovelrecombinantchikun-gunyavaccine.
Vaccine30:4638–4643.
https://doi.
org/10.
1016/j.
vaccine.
2012.
04.
099.
32.
TaylorA,MeltonJV,HerreroLJ,ThaaB,Karo-AstoverL,GagePW,NelsonMA,ShengKC,LidburyBA,EwartGD,McInerneyGM,MeritsA,Ma-halingamS.
2016.
Effectsofanin-framedeletionofthe6kgenelocusfromthegenomeofRossRivervirus.
JVirol90:4150–4159.
https://doi.
org/10.
1128/JVI.
03192-15.
33.
PohjalaL,UttA,VarjakM,LullaA,MeritsA,AholaT,TammelaP.
2011.
Inhibitorsofalphavirusentryandreplicationidentiedwithastablechikungunyarepliconcelllineandvirus-basedassays.
PLoSOne6:e28923.
https://doi.
org/10.
1371/journal.
pone.
0028923.
34.
GohLYH,Hobson-PetersJ,ProwNA,GardnerJ,Bielefeldt-OhmannH,SuhrbierA,HallRA.
2015.
MonoclonalantibodiesspecicforthecapsidproteinofChikungunyavirussuitableformultipleapplications.
JGenVirol96:507–512.
https://doi.
org/10.
1099/jgv.
0.
000002.
35.
GohLYH,Hobson-PetersJ,ProwNA,BakerK,PiyasenaTBH,TaylorCT,RanaA,HastieML,GormanJJ,HallRA.
2015.
TheChikungunyaviruscapsidproteincontainslinearBcellepitopesintheN-andC-terminalregionsthataredependentonanintactC-terminusforantibodyrec-ognition.
Viruses7:2943–2964.
https://doi.
org/10.
3390/v7062754.
Tayloretal.
January/February2017Volume8Issue1e01970-16mbio.
asm.
org14onMarch16,2021byguesthttp://mbio.
asm.
org/Downloadedfrom