‘Wisc e sota’
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‘Wisc‐e‐sota’ 5 th Joint UMN‐UW Virology Training Grant Symposium October 11 th , 2017 7‐10pm Opening reception & keynote lecture October 12 th , 2017 8:00am‐4:00pm Radisson Hotel, La Crosse, WI NIH T32‐supported virology training programs at: University of Wisconsin‐Madison University of Minnesota‐Twin Cities Featuring talks and poster sessions by students, postdocs and faculty Sponsored by NIH grants T32 AI083196, T32 AI078985, and the Department of Microbiology at the University of Wisconsin‐La Crosse
Transcript of ‘Wisc e sota’
‘Wisc‐e‐sota’5th Joint UMN‐UW Virology Training Grant
Symposium
October 11th, 2017 7‐10pm
Opening reception & keynote lecture
October 12th, 2017 8:00am‐4:00pm
Radisson Hotel, La Crosse, WI
NIH T32‐supported virology training programs at:
University of Wisconsin‐Madison
University of Minnesota‐Twin Cities
Featuring talks and poster sessions by students, postdocs and faculty
Sponsored by NIH grants T32 AI083196, T32 AI078985, and the Department of Microbiology at the University of Wisconsin‐La Crosse
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5thAnnualWisc‐e‐sotaVirologySymposiumWednesday‐Thursday,October11‐12,2017
RadissonHotelLaCrosse,Wisconsin
OralPresentations
Talkabstractnumbersinleftcolumn
October11,2017
(Wednesday)6:30‐7:30pm
Registration
7:00‐10:00pm
Openingreception
7:40‐7:45pm
Welcomeandopeningremarks
7:45‐8:45pm
Openingtalk–JamesNeaton(UMN)
October12,2017
(Thursday)
Convenor:RezaDjavadian(UW)
7:30‐8:00am
Check‐inandposterset‐up
8:00am
Breakfast
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9:00am
RobertGeraghty(UMN)
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9:30am
PatrickNyman(UW)
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9:45am
MorganMeissner(UMN)
10:00‐10:30am
Morningbreak
Convenor:LiangGuo(UMN)
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10:30am
AidinTavakoliTameh(UW)
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10:45am
DmitriKotov(UMN)
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11:00am
JamesBruce(UW)
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11:30‐2:00pm
Lunch,postersession,andgrouppicture
Convenor:IsaacAngert(UMN)
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2:00pm
YoshiKawaoka(UW)
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2:30pm
ChristopherRichards(UMN)
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2:45pm
AureliaFaure(UW)
Convenor:MyeongKyunShin(UW)
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3:00pm
TiLuong(UMN)
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3:15pm
TonyDawson(UW)
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3:30pm
RyanLanglois(UMN)
4:00pm
ClosingRemarks
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PosterPresentations1. “HSV‐1ReplicationisInhibitedbytheAntiviralDNACytosineDeaminase”
AdamCheng,ThomasFrost,StephenRice,ReubenHarris(UMN)2.
“GeneCorrelationAnalysisIdentifiesFunctionalPro‐andAnti‐ViralFactorsforInfluenzaVirusInfection”GloriaP.Larson,VyTran,ShuiqìngYú,YíngyúnCaì,DanielleSmith,JensH.Kuhn,andAndrewMehle(UW)
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“HIV‐1AdaptationStudiesRevealaNovelVif‐IndependentMechanismforEvadingLethalRestrictionbyAPOBEC3G”TerumasaIkeda,MenelasoSymeonides,JohnS.Albin,MingLi,MarkusThali,andReubenHarris(UMN)
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“PolymorphismsinRhesusMacaqueTetherinareAssociatewithDifferenceinPeakViremiaDuringAcuteInfectionwithSIV”SanathKumarJanaka,AidinTavakoli‐Tameh,WilliamJ.NeidermyerJr.,RuthSerra‐Moreno,BinJia,JamesA.Hoxie,RonaldC.Desrosiers,PaulJohnson,JeffreyD.Lifson,StevenM.Wolinsky,andDavidT.Evans(UW)
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“APOBEC3BLysineResiduesareDispensableforDNACytosineDeamination,HIV‐1Restriction,andNuclearLocalization”AmyM.Molan,HealthM.Hanson,CynthiaM.Chweya,BrettD.Anderson,GabrielJ.Starrett,ChristopherM.Richards,ReubenS.Harris(UMN)
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“UVBMediatedImmunosuppressionisNecessaryforPapillomavirusMediatedPathogenesis”AayushiUberoi,MeganSpurgeon,TaoWei,JeremiahYe,EdGlover,IanFrazer,ChrisBradfield,PaulLambert(UW)
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“LethalMutagenesisofHIV‐1Inducedby5‐aza‐2‐deoxycytidineinHumanPrimaryCD4+TCells”EmilyJulik,MorganE.Meissner,MeganE.Roth,MarzenaBaran,JerryDaniel,DarylM.Gohl,KennethB.Beckman,JoshuaA.Baller,StevenE.Patterson,LouisM.Mansky(UMN)
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“BacterialViralInfectionInfluencesMetabolicInteractionsofMicrobialCommunityMembers”LisaFazzino,WilliamHarcombe(UMN)
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“PerturbationstotheHIV‐1ProgrammedRibosomalFrameshiftSiteDictatesTranslationalRegulationandViralGenomicRNATranscriptFate”BayleighE.Benner,JordanT.Becker,PabloGarcia‐Miranda,SamuelE.Butcher,andNathanM.Sherer(UW)
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10. “SingleNucleotideEditingUsingChimericAPOBEC‐Cas9Complexes”AmberSt.Martin,DanielSalamango,andReubenS.Harris(UMN)
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“LatentEpstein‐BarrVirusInfectioninaKeratinocyteModelImpairsDifferentiation”MarkEichelberg,ReneWelch,AhmedAli,MakotoOhashi,JosephGuidry,ShannonKenney,SunduzKeles,RonaScott,andEricJohannsen(UW)
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“StructuralBasisforTargetedDNACytosineDeaminationandMutagenesisbyAPOBECEnzymes”K.Shi,M.A.Carpenter,S.Banerjee,N.M.Shaban,K.Kurahashi,D.J.Salamango,J.L.McCann,G.J.Starrett,J.V.Duffy,ÖDemir,R.E.Amaro,D.A.Harki,H.Aihara,andR.S.Harris(UMN)
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“EpsteinBarrVirusTranscriptionFactorsRtaandZtaPlayDistinctlyDifferentRolesinPromotingEBVReplicationinEpithelialCells”AhmedAli,MarkEichelberg,MakotoOhashi,RezaDjavadian,ShannonKenney,andEricJohannsen(UW)
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“TranslationControlofHIV‐1”KathleenBoris‐Lawrie(UMN)
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“HIV,RatioNormalizationandWinningtheGameofTCells”RobStriker,DawitWolday,MitchellKirsh,AjaySethi,JoeMcBride,andIreneOng(UW)
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“TheHepatitisCViralProteinNS5AStabilizedGrowth‐RegulatoryHumanTranscripts”LiangGuo,SureshD.Sharma,JoseDebes,DanielBeisang,BerndRattenbacher,IrinaA.Vlasova‐St.Louis,DarinL.Wiesner,CraigE.Cameron,andPaulR.Bohjanen(UMN)
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“QuantitativeImagingofRhinovirusInfectionSpreadwithSingle‐CellResolution”HuichengShi,BaharInankur,andJohnYin(UW)
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“RegulationofEBVLyticReactivationbyp53andp63inEpithelialCells”NickVanSciver,DhananjayNawandar,DenisLee,KathellenMakielski,PaulLambert,andShannonC.Kenney(UW)
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“CharacterizingGuineaPigCytomegalovirusPathogenesisintheAmnion”DiraS.Putri,CraigJ.Bierle(UMN)
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“DeepSequencingBarcodedZikaVirusfromMacaquesUsingaSmallAmpliconApproach”KatieZarbock,PaolaSilveira,TeamZEST,GregEbel,DavidO’Connor,andShelbyO’Connor(UW)
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21. “Cryo‐EMStructureofPorcineDeltaCoronavirusSpikeProteininthePre‐FusionState”JianShang,YuanZheng,YangYang,ChangLiu,QibinGeng,WanboTai,LanyingDu,YusenZhou,WeiZhang,FangLi(UMN)
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“CXCR5‐TransducedPrimaryRhesusMacaquePBMCsAccumulateinBCellFolliclesinaNovelExVivoBCellFollicleMigrationAssay”GwantwaMwakalundwa,PHaran,SLi,MPampusch,HMAbdeelal,EGRakasz,EConnick,PJSkinner(UMN)
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“EBNA3C‐DeletedEpstein‐BarrVirusCausesLymphomasinaHumanizedMouseModelDespiteBeingSeverelyDeficientforBCellTransformationInVitro”JamesRomero‐Masters,RezaDjavadian,MakotoOhashi,ShidongMa,AndreaBilger,EricJohannsen,andShannonKenney(UW)
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“NovelFeaturesofCRM1‐DependentRetroviralmRNANuclearExportRevealedUsingLiveCellImaging”RyanT.Behrens,ChristinaHiggins,andNathanM.Sherer(UW)
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“AnalysisofProviralandParticleStructurefromDistinctChronicallyInfectedCellLinesDemonstratesthePolymorphicNatureofHTLV‐1ParticleCores”LuizaMendonça,MorganE.Meissner,WeiZhang,LouisMansky(UMN)
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“RoleofIQGAP1inHPV‐PositiveandHPV‐NegativeHeadandNeckCarcinogenesis”TaoWei,SuyongChoi,DaryaBuehler,RichardAnderson,andPaulLambert(UW)
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“SIVVifandHumanAPOBEC3BInteractionsResembleThoseBetweenHIV‐1VifandHumanAPOBEC3G”JiayiWang,NadineM.Shaban,AllisonM.Land,WilliamL.Brown,ReubenS.Harris(UMN)
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“Species‐SpecificRegulationofHIV‐1GeneExpressionandVif‐InducedCellCycleArrest”EdwardL.EvansIII,JordanT.Becker,StephanieS.Fricke,KishanM.Patel,andNathanM.Sherer(UW)
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“VisualizingHTLV‐1GagPunctaBiogenesisbySuper‐ResolutionMicroscopy”JohnEichorst,JohnKohler,JoachimMueller,andLouisMansky(UMN)
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“ComprehensiveAnalysisoftheKineticsofEpstein‐BarrVirusLyticGeneExpression”RezaDjavadian,MitchHayes,andEricJohannsen(UW)
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“IdentificationofCancerCellLineswithaClearAPOBECMutationSignature”MatthewC.Jarvis,DiakoEbrahimi,NuriA.Temiz,andReubenS.Harris(UMN)
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32. “Cryo‐ElectronTomographyRevealsNovelFeaturesofaViralRNAReplicationCompartment”KennethJ.Ertel,DesiréeBenefield,DanielCastaño‐Diez,JaniceG.Pennington,MarkHorswill,JohanA.denBoon,MarisaS.Otegui,andPaulAhlquist(UW)
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“StudiesofNon‐Punctate,Membrane‐BoundHTLV‐1GagbyFluorescenceFluctuationSpectroscopyandFluorescenceLifetimeImagingMicroscopy”IsaacAngert,JohnEichorst,JohnKohler,JessicaMartin,WeiZhang,LouisMMansky,andJoachimDMueller(UMN)
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“Whole‐TranscriptomeSequencingtoIdentifyImmuneGeneVariants”AmeliaK.Haj,JulieA.Karl,RogerW.Wiseman,andDavidH.O’Connor(UW)
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“HumanPapillomavirusPromotesEpstein‐BarrVirusLyticReactivationinImmortalizedOralKeratinocytes”KathleenMakielski,DenisLee,LaurelLorenz,DhananjayNawandar,Ya‐FangChiu,ShannonKenney,andPaulLambert(UW)
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Abstracts–OralPresentations
“TwoViruses(HIVandEbola),TwoResearchNetworks(INSIGHTandPREVAIL),TooBusy!”JamesNeatonTwomajorinternationalcollaborationsinvolvingtheUniversityofMinnesota,theInternationalNetworkforStrategicInitiativesinGlobalHIVTrials(INSIGHT)andthePartnershipforResearchonEbolaVirusinLiberia(PREVAIL),haveresultedinthedesignandconductofseveralmajorclinicaltrials.Inthistalk,wewilldescribetheoriginofeachoftheresearchgroups,challengeswefacedinthedesignandconductofclinicaltrials,someofthetrialresults,andwhatfutureresearchisbeingplanned.1. “InhibitionofHCMVpUL89EndonucleaseActivityandVirusReplication”YanWang,JayakanthKankanala,JingTang,ZhengqiangWangandRobertJ.GeraghtyHumancytomegalovirusinfectioninindividualslackingafullyfunctioningimmunesystem,suchasnewbornsandtransplantpatients,canresultinsevereanddebilitatingconsequences.Approvedanti‐humancytomegalovirusdrugsmainlytargettheviralpolymeraseandresistancetothosedrugshasappeared.Therefore,newdrugsfromnoveltargetsareneededforuseinsteadof,orincombinationwith,currentpolymeraseinhibitors.HumancytomegalovirusterminasecomplexcleavestheconcatemericgenomicviralDNAintounitlengthsduringgenomepackagingandparticleassembly.TerminasecomplexATPaseandendonucleaseactivityisprovidedbytheviralproteinpUL89.pUL89isanattractivedrugtargetbecauseitsactivitiesarerequiredforinfectiousvirusproduction.AdomainlocatedintheC‐terminusofpUL89hasanRNaseH/integrase‐likefoldandendonucleaseactivitythatcanbeinhibitedbycompoundsfeaturingachelatingtriadmotif.WehaveidentifiedavarietyofsmallmoleculechelatingscaffoldsthatinhibitpUL89endonucleaseactivityinvitroanddemonstratecharacteristicsofpUL89inhibitorsincellculture.Takentogether,theresultssubstantiateourpharmacophorehypothesisandvalidateourligand‐basedapproachtowardidentifyingnovelinhibitorsofpUL89endonuclease.2. “ExaminingtheRoleoftheNotchSignalingPathwayandHPVinHeadandNeckSquamousCellCarinoma”PatrickNyman,DaryaBuehlerandPaulLambertHumanPapillomavirus(HPV)isasmallDNAvirusthatinfectsstratifiedsquamousepithelia,anditiswellknowntobetheprimarycauseofcervicalcancer,aswellasasubsetofHeadandNeckSquamousCellCarcinomas(HNSCC).OnepathwayrecentlyimplicatedinHNSCCistheNotchsignalingpathway.TheNotchsignalingpathwayisanimportantpathwayincelldifferentiationdecisionswithdifferentialdownstreamtargetsandeffectsdependingontissuetypeanddevelopmentalstage,andthereceptorproteinNotch1hasbeenfoundtobeoneofthemosthighlymutatedgenesinHNSCC.WehavethereforehypothesizedthattheNotchsignaling
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pathwaywouldactasatumorsuppressorinHPV‐positiveandHPV‐negativeHNSCC.Here,wehaveutilizedamousestrainwithinactivatedNotchsignalingviaexpressionofadominantnegativeformofMAML1(DNMAML1),arequiredtranscriptionalcoactivatorofNotchsignaling.WeutilizedthisgeneinconjunctionwitheitherHPV16E6/E7transgenesoramutatedp53tosimulateHPV‐positiveandHPV‐negativecancerdrivers,respectively,andmonitoredthegroupsforheadandneckcanceruponlong‐termtreatmentwiththecarcinogen4NQO.MicewithinactivatedNotchsignalingexhibitedagreaterdiseaseseverityandtumormultiplicitybothwithHPV16E6/E7andwithmutantp53.WealsofoundthatDNMAML1‐expressingmiceexhibitedanincreasedpresenceofnuclear²‐catenin,amarkerforincreasedtumorigenicpotential.Finally,despitehavingsimilarratesoftumorincidence,HPV16E6/E7micewithlostNotchsignalingdevelopedahighernumberofhigh‐gradecarcinomasthanp53R172HmicewithlostNotchsignaling.TheseresultsindicatethattheNotchsignalingpathwaycanserveinatumorsuppressingfunctioninbothHPV‐positiveandHPV‐negativeHNSCCandthattheremaybeanaddtionalsynergisticeffectbetweenHPVandlostNotchsignalingindevelopinghigh‐gradecarcinomas.3. “MolecularMechanismsofHIV‐2MutagenesisDuringReplication”MorganMeissner,EmilyJulik,andLouisManskyMorethan36millionindividualsareinfectedwithHIVworldwide.Nearly95%oftheseindividualsareinfectedwithHIVtype1(HIV‐1),whichhasahighrateofviralmutationthathelpsdriveimmuneevasion,diseaseprogression,andrapidemergenceofdrugresistance.HIVtype2(HIV‐2)accountsforfewerthan2millioninfectionsoverall,remainsprimarilyrestrictedtoWestAfrica,andexhibitsasignificantlyattenuateddiseasephenotypecomparedtoHIV‐1,characterizedbylowerratesoftransmissibilityandaslowerprogressiontoAIDS.HIV‐2hasrecentlybeenfoundtohaveasignificantlylowerrateofmutationcomparedwithHIV‐1,whichmayberelatedtothedifferencesinviraldiseaseprogressionandpersistence.AlthoughthemaindriverofHIVmutagenesisisthelowfidelityofthevirallyencodedreversetranscriptase,hostfactorssuchastheAPOBEC3(A3)familyofcytosine‐deaminaseshavebeenfoundtocontributetoviralmutationaswell.HerewereportthatHIV‐1andHIV‐2arerestrictedbydifferentsubsetsofAPOBEC3proteinsinvitro.InfectionoftheCEM‐GFPT‐celllinewithHIV‐2doesnotelicitarobustchangeinA3expressionashasbeenseenforHIV‐1.Usingasingle‐cycleinfectivityassayforvirusesproducedinthepresenceofthesevendifferentA3proteins,wedemonstratethatwhileA3D,‐F,‐Gand‐HrestrictHIV‐1infectioninvitro,onlyA3F,‐G,andH(andtoalesserextentA3C)areabletorestrictHIV‐2infectionwhileincreasingmutantfrequency.ThisisconsistentwithpreviouslyreporteddifferencesinthefrequencyandrateofG‐to‐Amutationsbetweenthetwoviruses,andwithdifferencesintheirpackagingintobuddedvirionsintheabsenceofVifproteins.SequencingofproviralsequenceswillbedonetodetermineifrestrictionbythespecificA3proteinscorrelateswithincreasedratesofG‐to‐Amutationandhypermutation,andaccumulationofviralRNAproductswillbeanalyzedtodetermineifreversetranscriptionisinhibited.PreliminaryresultsofthisworksuggestthatHIV‐2experienceslessmutagenesisfromA3proteinscomparedwithHIV‐1,consistentwithpreviousresults.Futurestudieswillexpanduponthisworkinanattempttocorrelatemutation
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frequencywithinfectivityforthevirusesusingnucleosideanalogsandsmallmoleculecompoundstoprovideinsightsintothecontrastingphenotypesobservedbetweentheviruses.4. “MutationsinNefthatSelectivelyDisruptTetherinAntagonismImpairSIVReplicationduringAcuteInfectionofRhesusMacaques”AidinTavakoli‐Tameh,SanathKumarJanaka,LaurenCallahan,KseniaBashkueva,KatieZarbock,ShelbyO’Connor,KristinCrosno,SaverioCapuano,RuthSerra‐Moreno,HajimeUno,JeffreyD.Lifson,David.T.EvansNefpromotesthereleaseofSIVfrominfectedcellsbycounteractingrestrictionofnon‐humanprimatetetherin(BST‐2orCD317).Nefalsodownmodulatescell‐surfaceCD4andMHCclassI(MHCI)moleculesandenhancesinfectivitybypreventingincorporationofSERINC3/5intovirions.WepreviouslydemonstratedthattetherinantagonismbyNefisgeneticallyseparablefromCD4‐andMHCI‐downmodulationandinfectivityenhancement.HereweshowthatselectivedisruptionoftetherinantagonismimpairsvirusreplicationduringacuteSIVinfectionofrhesusmacaques.Separategroupsoffourrhesusmacaqueswereinfectedwitheitherwild‐typeSIVmac239oranSIVmac239mutant(SIVmac239‐NefAAA)withacombinationofthreeaminoacidsubstitutionsintheflexibleloopregionofNefthatimpairtetherinantagonism,butnotCD4‐orMHCI‐downmodulationorinfectivityenhancement.ViralRNAloadsinplasmaweresignificantlylowerduringacuteinfection(weeks1‐4post‐infection)foranimalsinfectedwithSIVmac239‐NefAAAcomparedtoanimalsinfectedwithwild‐typeSIVmac239(p=0.0044,mixed‐effectsmodel).SequenceanalysisoftheviruspopulationinplasmaconfirmedthatthesubstitutionsinNefwereretainedduringacuteinfection;however,changeswereobservedbyweek24post‐infectionthateitherrestoredthewild‐typeresidueorintroducedalternativeresiduesatthesepositions,consistentwithselectivepressureonNeftoregainanti‐tetherinactivity.Theseobservationsprovidethemostdirectevidencetodatethattheabilitytocounteractrestrictionbytetherinisimportantforlentiviralreplicationinprimates.5. “TCRAffinityInfluencesHelperTCellDifferentiationbyBiasingDendriticCellInteractions”DmitriI.Kotov,ThomasPengo,JasonS.Mitchell,JessicaA.Kotov,ChristianeRuedl,SingSingWay,RyanA.Langlois,BrianT.Fife,MarcK.JenkinsCD4+TlymphocytesutilizetheirTcellreceptortodetectmicrobialproteinspresentedbydendriticcellscausingTcellstodifferentiateintovarioussubsets.HoweverthemechanismbywhichTcellreceptoraffinitybiasesTcelldifferentiationisunknown.WedemonstratethatlowaffinityTcellreceptorsbiasedTcellsintoBcellhelperswhilehighaffinityTcellreceptorsskewedtowardsnon‐Bcellhelperfates,suggestingthatthedefaultfateisaBcellhelper.ThisTcellreceptor‐drivenbiasindifferentiationwasidentifiedfollowinginfectionwiththebacteriaListeriamonocytogenesaswellasthePR8strainofinfluenzaAvirus.ToexamineTcell‐dendriticcellinteractions,wecreatedasoftwareforhigh‐throughput,multicolor,quantitativeimagingcalledChrysalis.WithChrysalis,weshowthathighaffinityTcellsmaintainprolongedinteractionswithXCR1+dendriticcellsbiasingTcellsintomacrophagehelpers,whileloweraffinityTcellspreferentiallyinteractedwithSIRPz+dendriticcellsandbecameBcellhelpers.
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Thus,TcellreceptoraffinityinfluencesTcell‐dendriticcellinteractionstherebydictatingTcelldifferentiationduringbacterialandviralinfections.6. “ZBTB2RepressesHIV‐1GeneExpressioninaZASC1‐andATR‐DependentManner”JamesBruce,MeganBracken,GingerPocockandPaulAhlquistPreviously,wereportedthatcellulartranscriptionfactorZASC1facilitatesDNA‐dependent/RNA‐independentrecruitmentofTAT/P‐TEFbtotheHIV‐1promoterandisacriticalfactorinregulatingHIV‐1transcriptionalelongation(PLoSPathe1003712).Subsequently,weidentifiedtranscriptionfactorZBTB2asastrongZASC1interactorandshowedthatZBTB2repressestheHIV‐1promoter,dependentonfunctionalZASC1bindingsitesintheHIV‐1promoterandtheZBTB2POZdomain,whichweshowedtorecruitHDAC1andHDAC4.Accordingly,siRNAknockdownorCRISPR/CAS9knockoutofZBTB2inTcelllinesenhancedexpressionfromWTHIV‐1,butnotfromproviruseslackingZASC1bindingsites.ChIPofinfectedprimaryT‐cellextractsrevealedthatZBTB2isrecruitedtotheWTHIV‐1LTRbutnottoanLTRlackingZASC1bindingsites.Thus,ZASC1recruitsZBTB2andrepressestheHIV‐1promoter.HIV‐1VPRactivatestheviralLTRbyinducingtheATRkinaseDNAdamageresponse.SinceaconsensusATRphosphorylationsiteislocatedinZBTB2sZASC1bindingregion,weinvestigatedifZBTB2functionwasregulatedbyVPRandtheDNAdamageresponse.Co‐expressingZASC1dramaticallyrelocalizedZBTB2fromthecytoplasmtothenucleus.MutationsabolishingZASC1/ZBTB2interactionpreventedZBTB2nuclearrelocalizationandZBTB2‐mediatedrepressionofHIV‐1transcription.MutatingtheputativeATRphosphorylationsiteinZBTB2increaseditsnuclearlocalizationandrepressiveactivity.Conversely,expressingwtVPR,butnotVPRmutantsdeficientinATRactivation,increasedZBTB2cytoplasmiclocalization.Significantly,stimulatingtheATRpathwaywithDNAdamagingagentsreproducedVPRseffectsonZBTB2localization.Moreover,theeffectsofDNAdamagingagentsandVPRonZBTB2localizationcouldbeblockedbyATRkinaseinhibitorsoroverexpressingWIP1phosphatase,whichremovesATRphosphorylations.ThesedatasupportamodelinwhichATR‐phosphorylatedZBTB2ispreferentiallycytoplasmic,whilede‐phosphorylatedZBTB2isnuclearandactivelyrepressesHIV‐1transcriptioninaZASC1‐andATR‐dependentmanner.7. “WorkingWithDeadlyViruses:BattlingEbolaandInfluenza”YoshiKawaokaEveryyear,influenzaepidemicsoccur,causingincreasedmorbidityandmortality,particularlyinvulnerablepopulations,suchastheveryyoungandveryold.Inaddition,worldwideepidemics,suchhasthe1918pandemic,occasionallyoccur.Consequently,influenzahasanenormousimpactontheglobaleconomy.Bycontrast,Ebolavirushasonlybeenrecognizedsince1976,and,untilrecently,outbreaksofthisvirushadcausedrelativelyfewdeathsbecausetheyoccurredinrural,isolatedareas.However,therecentoutbreakinWestAfricaoccurredoveralarge,denselypopulatedurbanareaandchangedourunderstandingofwhatconstitutesanEbolavirusoutbreak.Iwilldiscussourrecentresearchontheseviruses.
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8. DifferentialExpressionofAPOBEC3HSpliceVariantsandaPotentialHIV‐1ProteaseDefenseMechanismChristopherRichards,DiakoEbrahimi,MichaelCarpenter,JiayiWang,BrettAnderson,NadineShaban,AdamCheng,TerumasaIkeda,andReubenS.HarrisAPOBEC3enzymesprovideinnateimmuneprotectionfromavarietyofpathogensincludingtheAIDSvirusHIV‐1.APOBEC3D,APOBEC3F,APOBEC3G,andAPOBEC3Hcombinetopotentlyrestrictviralreversetranscriptionbydeamination‐dependentand‐independentmechanisms.Inhumans,APOBEC3HisthemostdiversememberoftheAPOBEC3family,asitisencodedbyatleastsevendifferenthaplotypes(whicharecomprisedofdifferentcombinationsof5SNPs)andfourdifferentisoforms(SV154,SV182,SV183,SV200).UsingRNA‐Seqfromthe1000GenomesDatabase,wehavefoundthatHapIisprevalentoutsideofAfrica,whileHapIIisdominantinAfricaandthatthesetwodifferenthaplotypeshavesignificantlydifferentalternativesplicingofAPOBEC3HmRNA,whichmanifestsashigherexpressionofSV200inHapIIindivualscomparedtosignificantlylowerexpressioninHapIindividuals.Unexpectedly,whilecharacterizingtheA3HsplicevariantsinHIV‐1infectivityassays,weobservedthatHIV‐1proteasewasspecificallyinteractingwiththeSV200formofA3H.TheseresultssuggestthatHIV‐1mayhaveadaptedtouseproteasetocounteract/modulateA3HSV200.9. “HowDoesEpstein‐BarrVirusLikelyContributetoPrimaryEffusionLymphomas?”AureliaFaure,BillSugdenPrimaryEffusionLymphoma(PEL)istheonlyhumancancerknowntobeassociatedwithtwotumorviruses.Kaposi'ssarcoma‐associatedherpesvirus(KSHV)ispresentin100%ofPELsand90%ofthemareco‐infectedwithEpstein‐Barrvirus(EBV).Wewanttounderstandthelikelyco‐dependenceofPELsonthesetwoviruses.KSHVencodesanti‐apoptoticgenesessentialforthesurvivalofPELcells.WehavethereforeaskedwhatEBVgenesprovideselectiveadvantagestoPELcells.IntroductionofvectorsexpressingvariousEBVgenesintoBC‐1cellsshowedthattheBARTmiRNAsweresufficienttoleadtothelossofEBVfromthesecells.WeconcludethatEBVsBARTmiRNAssupportBC‐1cellsandlikelyotherPELstoo.WehypothesizethatEBV,likelyinpartviatheBARTmiRNAs,providesproliferativesignalsinitiallytoBcellsthatevolveintoPELswhenco‐infectedwithKSHVbecauseKSHVhasfailedtosupportlong‐termproliferationofBcellsinvitro.Totestthishypothesis,wehaveinfectedperipheralBcellswithKSHVBAC16,whichencodesaGFPmarker(Bruloisetal.J.Virol.2012),plusorminusEBV.ThenumberofcellsexpressingGFPwasmeasuredbyflowcytometryfromday4through42followinginfectionwithKSHV.PeripheralprimaryBcellscouldnotbedetectablyinfectedwithKSHVwhereasco‐infectionwithEBVincreasedtheefficiencyofinfectionupto3%.WehavefoundthatEBVinfectionhastwoopposingeffectsonKSHVinfection:earlyeventsofEBVinfectionincreasetheefficiencyofinfectionwithKSHVwhiletheefficiencyofinfectionofEBV‐infectedcellswithKSHVdecreasesovertime.KSHVinfectsthelargestfractionofprimaryBcellswhenitenterscellspriortoEBVinfection.InvitroactivationofBcellswithIL‐4andCD40ligandpartiallymimicsEBVinfectionbyallowingdetectableinfectionofBcellswithKSHV.TheseexperimentsrevealthatEBVinfectionisessentialforKSHVtoinfectperipheralBcells
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detectablyandthattherequirementsforthisco‐infectionarecomplex.10. “ExomsomesSecretedFromVirus‐InfectedMicrogliaCanActivateanInflammatoryResponseintheCentralNervousSystem”Nhungoc(Ti)LuongandJulieK.OlsonExosomesaremembrane‐boundnanovesiclesreleasedfromcellsthatcanbetakenupbyothercells,thusmediatingintercellularcommunication.ExosomescontainmiRNA,mRNA,andproteinsthatarespecifictothecellsfromwhichtheywerereleased.Theilersmurineencephalomyelitisvirus(TMEV)induceddemyelinatingdiseaseisamousemodelformultiplesclerosis.TMEVinfectionofsusceptiblemiceleadstoapersistentviralinfectionofmicrogliainthecentralnervoussystem(CNS)whichcontributestothedevelopmentofachronicprogressivedemyelinatingdiseaseassociatedwithaninflammatoryimmuneresponse.WehavepreviouslyshownthatTMEVinfectionofmicrogliaactivatesaninnateimmuneresponsewithexpressionoftypeIinterferons,cytokines,chemokines,andeffectormoleculesassociatedwithdevelopmentandprogressionofdemyelinatingdisease.Next,wewantedtodeterminewhethermicrogliainfectedwithTMEVsecreteexosomesandwhethertheseexosomescouldinfluenceuninfectedCNSresidentcells,suchasmicroglia,astrocytes,andneurons.TheexosomessecretedfromTMEV‐infectedmicrogliacontainviralRNAthatcouldbetransferredtobystanderuninfectedCNScells.Theexosomessecretedfromvirus‐infectedmicrogliaactivatedtheinnateimmuneresponseinbystanderuninfectedmicroglia,astrocytes,andneuronsbyincreasingtheexpressionoftypeIinterferonsandpro‐inflammatorycytokines.TheviralRNAwassensedbyinnateimmunereceptorsintherecipientcellsandactivatedaninnateimmuneresponseintheuninfectedbystandercells.Mostinterestingly,exosomessecretedfrommicrogliainthebrainofTMEV‐infectedmicecouldbetransferredtouninfectedmiceandactivateaninflammatoryimmuneresponseinthebrainofuninfectedmice.TheseresultsshowthatexosomessecretedbyTMEV‐infectedmicrogliacanactivatetheimmuneresponseinbystanderuninfectedCNScellswhichmaycontributetotheinflammatoryresponseassociatedwithpersistentvirusinfectionanddemyelinatingdisease.ThisresearchwassupportedbygrantsfromUMNComparativeMedicine(R21742)andNIH(T32DA007097).11. “Phospho‐RegulationoftheInfluenzaVirusReplicationMachinery”AnthonyR.Dawson,ArindamMondal,GregoryKPotts,ElyseCFreiberger,StevenFBaker,LindseyAMoser,KristenABernard,JoshuaJCoon,AndrewMehleInfluenzavirusexpressestranscriptsearlyininfectionandtransitionstowardsgenomereplicationatlatertimepoints.Thisprocessrequiresdenovoassemblyoftheviralreplicationmachinery,largeribonucleoproteincomplexes(RNPs)composedoftheviralpolymerase,genomicRNAandoligomericnucleoprotein(NP).DespitethecentralroleofRNPsduringinfection,thefactorsdictatingwhereandwhentheyassemblearepoorlyunderstood.HerewedemonstratethathumanproteinkinaseC(PKC)familymembersregulateRNPassembly.ActivatedPKC´interactswiththepolymerasesubunitPB2andphospho‐regulatesNPoligomerizationandRNPassemblyduringinfection.ConsistentwithitsroleinregulatingRNP
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assembly,knockoutofPKC´impairsvirusinfectionbyselectivelydisruptinggenomereplication.However,primarytranscriptionfrompre‐formedRNPsdepositedbyinfectingparticlesisunaffected.Thus,influenzavirusexploitshostPKCstoregulateRNPassembly,asteprequiredforthetransitionfromprimarytranscriptiontogenomereplicationduringtheinfectiouscycle.12. “CellularResistanceandSusceptibilitytoInfluenzaVirusInfections”LouisSjaastad,ElizabethFay,JessicaFiege,BarbaraWaring,MarissaMacchietto,StevenShen,andRyanLangloisInfluenzaAvirus(IAV)infectsabroadrangeofcelltypeswithintherespiratorytract.Thecellsinitiallytargetedbythevirusinanaïvehost,foundercells,arethesiteofprimaryreplicationandvirusspread.FoundercellsaredifficulttodetectusingreplicationcompetentIAVasthevirusspreadstooquicklytoisolatethecellsthatwereinfectedfirst.ToovercomethisobstacleweutilizeafluorescenceexpressingsinglecycleIAVtocharacterizefoundercellsinthemouselung.Usingthistoolwedeterminedthatseveraldistrictcelltypesindifferentanatomicallocationsintheairwayareinfectedduringthefirstroundofreplication.Usingtheintensityofthefluorophoreasasurrogateforviruspolymeraseactivitywealsodiscoveredtwodistinctpopulationsofcellswithhighandlowofvirus.Thesedatasuggestthatsomecellswithinthelungarehighlypermissivetovirusreplicationwhileothersareabletobluntreplication.TodeterminethedegreeofprotectioninfectedfoundercellscanconfertopotentialsecondaryinfectedcellsweusedsequentialsinglecycleIAVsthatexpressdistinctfluorophorestolabelprimaryandsecondaryinfectedcells.Asexpectedtherewasasignificantreductionintheoverallnumberofsecondaryinfectedcells,likelyduetotheantiviralenvironmentestablishedbythefirstroundofreplication.Surprisinglythisreductionwasonlyinobservedinthecellswithlowlevelsofpolymeraseactivity.Cellswithhighlevelsofpolymeraseactivitywerenotreduced.Togetherthesedatademonstratethattherearecellswithinthelungthatarehighlypermissivetovirusreplication.WepostulatethatthesecellsarethevirusfactoriesthatamplifyviruseveninthefaceofpotentIFNresponses.
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PosterAbstracts1. “HSV‐1ReplicationisInhibitedbytheAntiviralDNACytosineDeaminaseAPOBEC3G”AdamCheng,ThomasFrost,StephenRice,ReubenHarrisAPOBEC3(A3)proteinsbelongtoafamilyofDNAcytosinedeaminaseenzymesthatfunctionininnateimmunityandcancerpathogenesis.OfthesevenhumanA3proteins,APOBEC3G(A3G)isawell‐studiedrestrictionfactorthatinhibitsthereplicationofretroviruseslikeHIVandHTLV‐1bycatalyzingtheconversionofcytosinestouracilsinviralcDNAreplicationintermediates,whichresultsinviralDNAdegradationorhypermutation.Onlyonestudyto‐datehasaddressedtheeffectofA3sonHSV‐1infectivityandmutagenesis.Interestingly,onlyAPOBEC3C(A3C),butnotA3GwasfoundtorestrictHSV‐1.TofurtherinvestigatethebiologicalrelevanceofA3sonHSV‐1replication,wegeneratedapanelofstableA3‐expressing293Tcells,infectedcellswithHSV‐1KOS1.1,andmonitoredvirusreplicationbyplaqueassays.WefoundthatA3Ginducesalog‐folddecreaseinHSV‐1replicationatalowMOIsandadampened,butconsistentreductioninreplicationathighMOIs.Additionally,HSV‐1doesnotappeartohavethecapacitytodegradeA3s,whichisincontrasttotheHIV‐1Vif‐mediateddegradationofA3sviaaubiquitin‐proteasomepathway.FurtherworkisrequiredtoelucidatethemechanismHSV‐1restriction,butoutinitialstudiessuggestthatA3GisanovelbiologicallyrelevantinnateimmunemoleculethatfunctionsininhibitingHSV‐1replication.2. “GeneCorrelationAnalysisIdentifiedFunctionalPro‐andAnti‐ViralFactorsforInfluenzaInfection”GloriaP.Larson,VyTran,ShuiqìngYú,YíngyúnCaì,DanielleSmith,JensH.Kuhn,andAndrewMehleVirusesbothutilizeandareantagonizedbyhostcellularfactors,pathways,andpost‐translationalmodifications.Severalscreeningmethodologieshavemappedhost‐cellinteractionswithinfluenzaAviruses(IAV).ThesescreensidentifiednumeroushostfactorsthatregulateIAVinfection,butrelyonendpointphenotypes,manipulationofthehostcell,andstableinteractionsbetweenviralandcellularproteins.Toovercometheselimitations,weutilizedagenecorrelationanalysisapproachthatcorrelatesIAVinfectionsusceptibilitywithnaturallyoccurringdifferencesingeneexpressionamongdifferentcelllines.ThisapproachidentifiedseveralhostfactorsthatstronglyenhanceorsuppressIAVreplication.Weinvestigatedtopcandidates,includingthepotentialenhancerepidermalgrowthfactorreceptor(EGFR)pathwaysubstrate8(EPS8)andsuppressorpoly‐ADP‐ribosepolymerase8(PARP8).OurdatasuggestedEPS8isupregulateduponinfection,andoverexpressingEPS8increasesviralgeneexpressionandviraltiter,validatingitsroleasapro‐viralhostfactor.ProbingstepsthroughoutthevirallifecyclesuggestedEPS8functionspost‐attachmentduringviralentry.EPS8isinvolvedinactinremodelingandendocytosisprocesses,andmutantanalysessuggestedthesefunctionsareimportantforviralentryenhancement.Theotherhostfactor,PARP8,isamemberofthepoly‐ADP‐ribosepolymerase(PARP)familyofenzymes.PARPsfacilitatethepost‐translationalmodificationADP‐ribosylation,whichhasbeenshowntobeupregulatedin
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responsetodiverseviralinfections.WeshowedPARP8inhibitsIAVpolymeraseactivityinreporterassays,reducesviralgeneexpressionduringinfection,anddecreasesoverallviralreplication.OurdatafurtherdemonstratethatPARP8andADP‐ribosylationmaydirectlytargettheviralpolymerasetorepressIAVreplication,supportingourscreenresults.Byexploringanalternativescreenapproachgenecorrelationanalysisweidentifiedpreviouslyunknownviralhostfactorswithopposingeffectsonviralinfectionoutcome.TheseresultsexpandourknowledgeofhowIAVco‐optshostcellularmachinerytosupportsuccessfulinfectionandpotentialanti‐viraldrugtargets.3. “HIV‐1AdaptationStudiesRevealaNovelVif‐IndependentMechanismforEvadingLethalRestrictionbyAPOBEC3G”TerumasaIkeda,MenelaosSymeonides,JohnS.Albin,MingLi,MarkusThali,andReubenSHarrisHIV‐1replicationrequiresVif‐mediatedneutralizationofAPOBEC3antiviralenzymes.ViruseslackingVifsuccumbtodeamination‐dependentand‐independentrestrictionprocesses.Here,HIV‐1adaptationstudiesareleveragedtoaskwhetherviruseswithanirreparablevifdeletioncoulddevelopresistancetorestrictivelevelsofAPOBEC3G.SeveralresistantviruseswererecoveredwithmultipleaminoacidsubstitutionsinEnv,andthesechangesalonearesufficienttoprotectVif‐nullvirusesfromAPOBEC3G‐dependentrestrictioninTcelllines.Envadaptationscausedecreasedfusogenicity,whichallowsforhigherlevelsofGag‐Polpackaging.IncreasedconcentrationsofpackagedPolinturncorrelatewithfastervirusDNAreplicationandreducedAPOBEC3G‐mediatedhypermutationofviralreplicationintermediates.Takentogether,thesestudiesrevealanovelEnv‐andPol‐dependentmechanismthatHIV‐1canusetoescaperestriction.Thismechanismmayprovidetransmittingviruseswithanadditionallayerofprotection,andmayalsoberelevanttoothervirusesthatlackobviousVif‐likedefensemechanisms.4. “PolymorphismsinRhesusMacaqueTetherinareAssociatedwithDifferencesinPeakViremiaduringAcuteInfectionwithSIV”SanathKumarJanaka,AidinTavakoli‐Tameh,WilliamJ.NeidermyerJr.,RuthSerra‐Moreno,BinJia,JamesA.Hoxie,RonaldC.Desrosiers,PaulJohnson,JeffreyD.Lifson,StevenM.Wolinsky,andDavidT.EvansTetherin(BST‐2orCD317)isaninterferon‐inducibleproteinthatinhibitsvirusreleasefrominfectedcells.TodeterminetheextentofsequencevariationandtheimpactofpolymorphismsinrhesusmacaquetetherinonSIVinfection,full‐lengthtetherincDNAclonesweresequencedfrom139rhesusmacaques,including69animalsinfectedwithwild‐typeSIVmac239and47animalsinfectedwithSIVmac239 nef.SinceNefistheviralgeneproductofSIVthatcounteractsrestrictionbytetherin,thesegroupsaffordacomparisonoftheeffectsoftetherinpolymorphismsonSIVstrainsthatare,andarenot,resistanttotetherin.Weidentified14allelesofrhesusmacaquetetherinwithdimorphicresiduesat9positionsthroughouttheprotein.Therelationshipbetweentheseallelesandpeakplasmaviralloadswascompared
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duringacuteinfection,priortotheonsetofadaptiveimmunity.Peakviremiadidnotdiffersignificantlyamongthewild‐typeSIV‐infectedanimals;however,differencesinpeakviralloadswereassociatedwithpolymorphismsintetherinamongtheanimalsinfectedwithSIV nef(Kruskal‐Wallistest,p<0.05).Inparticular,polymorphismsatpositions14,43and111(D14,P43andH111)wereassociatedwithsignificantlylowerpeakviralloadsforSIV nefinfection(Kruskal‐Wallistest,p<0.05).Theseobservationsrevealextensivepolymorphisminrhesusmacaquetetherin,maintainedperhapsasaconsequenceofvariabilityintheselectivepressureofdiverseviralpathogens,andidentifytetherinallelesthatmayhaveaninherentlygreatercapacitytorestrictSIVreplicationintheabsenceofNef.5. “APOBEC3BLysineResiduesareDispensableforDNACytosineDeamination,HIV‐1Restriction,andNuclearLocalization”AmyM.Molan,HeatherM.Hanson,CynthiaM.Chweya,BrettD.Anderson,GabrielJ.Starrett,ChristopherM.Richards,ReubenS.HarrisTheAPOBEC3familyofDNA‐cytosinedeaminasesarewellcharacterizedasinnatedefensemechanismsagainstviralinfectionandareperhapsbestknownfortheirrestrictionofHIV‐1.TheAPOBEC3srestrictHIV‐1bymutatingproviralssDNAcytosinestouracilsdisruptingtheviralmessage.APOBEC3shavealsobeenimplicatedinrestrictionofotherviruses,aswellastransposableelements,andrepresentacriticalpartofthehumaninnateimmunesystem.Asthesedefendersarethemselvesmutagens,whatistopreventthemfromturningagainstthecellstheyevolvedtoprotect?RecentworkhasshownthatforAPOBEC3B(A3B),theregulatorymechanismscanfailandhypermutationofthehumangenomeresults.ThereisamultitudeofevidencethatA3Bisregulatedthroughitsexpressionlevel,nuclearlocalization,celltype‐dependentrestrictionofHIV‐1.Manyproteinsaresubjectedtopost‐translationalmodifications(PTMs)aspartoftheirregulatorymechanisms.Oftheover400knownPTMs,manyoccuratlysineresiduesincludingacetylation,ubiquitination,andmethylationtonameafew.ToinvestigatewhetherornotlysineresiduesarepartofA3Bspost‐translationalregulatorymechanismweconstructedalysine‐less(K‐free)A3Bwhereall10ofthelysineresiduesweremutatedtoarginine.Theimpactofthesemutationsonproteincatalyticactivity,viralrestriction,SIV‐Vifmediateddegradation,andlocalizationwasassessed.SurprisinglywefoundthatA3BlysineresiduesaredispensableforallmeasurableactivitiesexceptsensitivitytoSIVVif‐mediateddegradation.6. “UVBMediatedImmunosuppressionisNecessaryforPapillomavirusMediatedPathogenesis”AayushiUberoi,MeganSpurgeon,TaoWei,JeremiahYe,EdGlover,IanFrazer,ChrisBradfield,andPaulLambertPapillomaviruses(PVs)arespeciestropicdouble‐strandedDNAvirusesthathavebeenimplicatedincausingseveralformsofcancerinhumansandotheranimals.PVscaninfectmucosalorcutaneousepitheliumandtheirlifecycleisdependentupontheepithelialdifferentiationprogramthuslimitingourabilitytomonitorandstudytheprogressionofthe
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diseaseintheirnaturalhostsindepth.Ourlabandothershaveextensivelystudiedmucosotropichigh‐riskHumanpapillomaviruses(HPVs)implicatedinanogenitalandheadandneckcancers.However,thebiologyandprogressionofskindiseasesassociatedwiththeircutaneousPVsisrelativelylesswellunderstood.Epidemiologicalstudieshaveimplicatedthatultravioletradiation(UVR)fromsunlightdrivepapillomavirus‐induceddiseaseinhealthyaswellasimmunocompromizedhumans.Inordertostudythisphenomenon,wehavedevelopedapapillomavirus‐basedinvivoinfectionmodelusingtherecentlydiscoveredmurinepapillomavirus(MmuPV1),whichisthefirsteverpapillomavirustonaturallyinfectthelaboratorystrainofmice.WhileMmuPV1failedtoinduceskinpapillomatosisinmostimmunecompetentstrainsofmice,wediscoveredthatUVRmakessuchmicehighlysusceptibletoMmuPV1‐inducedskindisease,includingformationofwartsandsquamouscellcarcinoma(Uberoietal.,PLoSPathogens,2016).WealsofoundacorrelationbetweenUVR'sabilitytomakemicesusceptibletoinfectionanditscapacitytocausesystemicimmunosuppression(ibid).WehavenowmadeeffortstotestwhetherUVR'sabilitytocausesystemicimmunosuppressionisnecessarytomakemicesusceptibletoMmuPV1‐inducedskindisease.SeveralfactorsmediatethecapacityofUVRtocausesystemicimmunosuppressioninmice,including:a)theactivationofthearylhydrocarbonreceptor(AHR),atranscriptionfactorthatmodulatesseveralarmsoftheimmunesystemandb)theelicitationoftheimmune‐suppressivecytokine,IL10.WefirstconfirmedthatUVRinducesactivationofAHRandelicitationofIL10uponUV‐irradiation.WethendeterminedthatUVR‐inducedsusceptibilitytoMmuPV1‐inducedskindiseasewasabolishedinbothAHR‐nullandIL10‐nullmice,aswastheabilityofUVRtocausesystemicimmunosuppression.TheseresultsdemonstratethatUVR'sabilitytocausesystemicimmunosuppressionisnecessarytomakemicesusceptibletoMmuPV1‐inducedskindisease.ThesestudiesalsoshedfurtherlightintotheroleofUVRinskindiseasecausedbycutaneousHPVs.7. “LethalmutagenesisofHIV‐1Inducedby5‐aza‐2‐deoxycytidineinHumanPrimaryCD4+TCells”EmilyJulik,MorganE.Meissner,MeganE.Roth,MarzenaBaran,JerryDaniel,DarylM.Gohl,KennethB.Beckman,JoshuaA.Baller,StevenE.Patterson,andLouisM.ManskyHIV‐1replicateswithanintrinsicallyhighmutationrateduetotheerror‐pronenatureofitsreversetranscriptaseenzyme.Theexperimentalantiviralstrategyoflethalmutagenesis,whichhasnotyetenteredclinicaluse,exploitsandincreasesthishighmutationratetothepointofcatastrophicerrorandviraleliminationduetoexcessiveaccumulationofdeleteriousmutationsinducedbysmallmoleculemutagens.Previously,ourgroupdemonstratedthatadivalerateprodrugformofthedeoxcytidineanalog5‐aza‐2‐deoxycytidinewasabletoeliminateHIV‐1infectivityinU373‐MAGIcells.Here,weextendthesefindingsintoamorephysiologicallyrelevantcelltype,humanprimaryCD4+Tcellsisolatedfromwholeblood.Asincelllines,non‐cytotoxicdosesof5‐aza‐2‐deoxycytidinearecompetenttoeliminateviralinfectivityintheseprimarycells.Thedeclineinviralinfectivityisaccompaniedbyanincreaseintheviralmutationrateand,inparticular,elevationofG‐to‐Ctransversionlevels,consistentwiththeproposedmechanismofactionofthisdrug.Wearefurtherevaluatingtheantiviralspecificityof5‐aza‐2‐
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deoxycytidineusingasinglecellnextgenerationsequencingapproachwhereinsingleprimaryCD4+TcellsinfectedwithanHIV‐1vectorandtreatedwith5‐aza‐2‐deoxycytidineatits50%effectiveconcentrationareisolatedonanintegratedfluidiccircuitforwholegenomeamplificationandsequence‐basedgenotypingofhostandproviralDNA.Weexpecttoobserveincreasedmutationalload,skewedtowardG‐to‐Ctransversions,inproviralbutnothostDNAincellstreatedwith5‐aza‐2‐deoxycytidinecomparedtountreatedcells,demonstratingthatthisdrugisspecificallyaviralmutagenwithoutoff‐targetmutageniceffectsforpatientDNA.Theseresultswillpromotetheclinicaltranslationofantivirallethalmutagens,whichhassofarprovedelusive.8. “BacterialViralInfectionInfluencesMetabolicInteractionsofMicrobialCommunityMembers”LisaFazzinoandWilliamHarcombeBacteriophage(phage)areanunderstudiedmemberofnaturalmicrobialcommunities.Phageinfectspecificbacterialspecies,whichcanevolvephage‐resistance.Infectionisinfluencedbythehostcell’sphysiologicalstate,whichisaffectedbymetabolicinteractionsbetweenhostcellsandothermembersofthemicrobialcommunity.Furthermore,competitionwithotherbacterialcommunitymembersinfluenceahostcellsabilitytoevolvephageresistance.OursyntheticbipartitemicrobialcommunityofEscherichiacoliandSalmonellaentericaisgeneticallyengineeredtocooperatebysharingmetabolicbyproducts,orcompeteforcarbonsources,dependingonprovidedmetabolites.Bygrowingthisco‐culturesystemcooperativelyorcompetitivelyinthepresenceorabsenceofanE.coli‐specificphage(T7),wecandeterminehowphageinfectionaffectscommunitymembersindifferentmetaboliccontextsbytrackingspecies‐specificpopulationdynamicsandevolutionofphageresistance.ResultsindicatethatcooperationfavorstheevolutionofT7‐resistantE.coli,whilecompetitionsuppressestheevolutionofT7‐resistantE.coli.Thisisalsopredictedbyourresource‐explicitODEmathematicalmodel.However,mathematicalmodelsdonotpredicttheobservedincreaseinS.entericayieldwhengrowninthecooperativecommunitywithphagecomparedtowithoutphage.Thisshowsthatphageattackcanbreakmetabolicinteractionsofmicrobialcommunitymembers.WehypothesizethatS.entericaiseitherconsumingburstE.colicellularcontents,orburstE.colilysatecontainsextracellularlyactiveenzymesthatbreakdownsuppliedlactoseintomonosaccharidecomponentsthatS.entericacanmetabolize.Determiningtheeffectthatphagehasonmicrobialcommunitieswillallowustomoreaccuratelymodelcommunity‐phageinteractions,andwillprovideanecologicalframeworktounderstandphageinfectionandevolutionofphageresistancewithinamicrobialcommunitycontext.9. “PerturbationstotheHIV‐1ProgrammedRibosomalFrameshiftSiteDictatesTranslationalRegulationandViralGenomicRNATranscriptFate”BayleighE.Benner,JordanT.Becker,PabloGarcia‐Miranda,SamuelE.Butcher,andNathanM.ShererTheHIV‐1genomicRNA(gRNA)secondarystructureisakeyregulatorofviralreplication
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utilizingcis‐actingfeaturestomodulateproductionofviralproteins.Aconservedstem‐loopwithinthegagreadingframedictatestheproportionsofessentialpolyproteinsGagandGag‐Pol.Thepolgeneistranslatedviaa‐1programmedribosomalframeshift(PRF)regionwithinthegaggene,producingtheGag‐Polpolyproteinapproximately5%ofthetime.Gag‐Polcontainstheviralenzymesnecessaryforproductionofmaturevirions.Previouslywehaveshownthatperturbingframeshiftingefficiency2‐3foldgreatlyreducesviralinfectivity.Hereweinvestigatethemechanismunderpinningthislossofinfectivity,andhowalteredframeshiftingaffectsmRNAtraffickingandstability,subsequentlyimpactinggRNApackaging.UtilizingRT‐PCRonourpanelofPRFmutantswithalteredframeshiftingefficiency,weprovideevidencesuggestingthatmutantswhichframeshiftgreaterorlessthan5%exhibitasignificantdecreaseinpackagingofviralgRNAintovirions.Wefurtherdemonstrateusingfluorescenceinsituhybridization(FISH)andlive‐cellimaging,thatinhibitingframeshiftingreducesthecytoplasmicviralgRNApool,resultinginlessgRNAavailableforpackaging.WehypothesizethatinhibitingHIV‐1frameshiftingresultsindecreasedviralgRNAstabilitythroughcellularmRNAdegradationmechanisms.Lastly,bycomplementingourpanelofframeshiftsitemutantswithwild‐typevirusprovidedintrans,weseeapartialrescuetoinfectivityinmutantswithabolishedframeshiftingbutnotmutantswithincreasedframeshifting.ThesedatasuggestthatGag‐PolmayhelpstabilizetheunsplicedviralgRNA,andrescueitfrommRNAdegradation.Thus,frameshiftingfidelityiscriticalnotonlyforregulatingGag:Gag‐Polratiosbut,unexpectedlyforHIV‐1gRNAstabilization,packagingandinfectivity.10. “SingleNucleotideEditingUsingChimericAPOBEC‐Cas9Complexes”AmberSt.Martin,DanielSalamango,andReubenS.HarrisBaseeditingisanexcitingnewapplicationforgenomeengineeringtechnology.C‐to‐TmutationsingenomicDNAhavebeenachievedusingribonucleoproteincomplexescomprisedofratAPOBEC1single‐strandedDNAdeaminase,Cas9nickase(Cas9n),uracilDNAglycosylaseinhibitor(UGI),andguide(g)RNA.Here,wereportthefirstreal‐timelentiviral‐basedsystemforquantificationofbaseeditinginlivinghumancellsaswellasnext‐generationeditingconstructsthatachievehighereditingfrequencies.MutationofanAPOBEC‐preferredtrinucleotide,5‐TCA‐to‐TTA,restoresmCherryfluorescenceinareportermarkedbyeGFP,andeditingfrequenciesarequantifiedthroughratiosofmCherry‐positivetoeGFP‐positivecells.Usingthissystemasbothanepisomalandachromosomaleditingreporter,weshowthathumanAPOBEC3AandAPOBEC3BbaseeditingconstructsaremoreefficientthanaratAPOBEC1construct.Wealsodemonstrateanenrichmentofeditingeventsataheterologouschromosomallocusinreporter‐activated,mCherrypositivecells.Thecombinationofarapid,fluorescence‐basedbaseeditingreportersystemandmoreefficient,structurallydefinedDNAeditingenzymesexpandstheversatilityofthispowerfulnewtechnology.
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11. “LatentEpstein‐BarrVirusInfectioninaKeratinocyteModelImpairsDifferentiation”MarkEichelberg,ReneWelch,AhmedAli,MakotoOhashi,JosephGuidry,ShannonKenney,SunduzKeles,RonaScott,andEricJohannsenEpstein‐Barrvirus(EBV)isahumanherpesvirusassociatedwiththedevelopmentofspecificformsoflymphoma,nasopharyngealcarcinoma,andgastriccarcinomas.Althoughcarcinomasaccountforalmost90%ofEBVassociatedcancers,progresshadbeenlimitedduetolackofaphysiologicinvitromodelofEBVepithelialinfection.Recently,EBVinfectionofnormaloralkeratinocytes(NOKs)hasemergedasamodelthatrecapitulatesaspectsofEBVinfectioninvivo,suchasdifferentiation‐associatedviralreplication.UsingNOKandNOK‐EBVcellswecharacterizedgeneexpressionchangesbyRNA‐seqduetoEBVinfectionanddifferentiation.AlthoughlatentEBVinfectioninundifferentiatedcellsresultedinmoresubtlechangesingeneexpression,NOK‐EBVgenechangesduringdifferentiationweremarkedlydifferentthanthoseobservedinNOK.Thedown‐regulationcellulargrowthandmetabolismpathwaysassociatedwithdifferentiationwereattenuatedinNOK‐EBVcomparedtoNOK.ToassesswhichEBVgeneproductswereresponsiblefortheseeffects,weinfectedNOKcellswithEBVmutantsdefectiveformicroRNAexpression,replication,andEBERexpression.Inaddition,wehavegrownNOKs,NOK‐EBV,andNOK‐EBVmutantsinorganotypicraftculturesandarecharacterizingthemfordifferencesindifferentiation,viralreplication,andBrdUincorporation.WeexpecttodefinethemechanismbywhichEBVlatentinfectionalterskeratinocytedifferentiationandprovideabasisforunderstandingtheroleofEBVinepithelialcancers.12. “StructuralBasisforTargetedDNACytosineDeaminationandMutagenesisbyAPOBECEnzymes”K.Shi,M.A.Carpenter,S.Banerjee,N.M.Shaban,K.Kurahashi,D.J.Salamango,J.L.McCann,G.J.Starrett,J.V.Duffy,Ö.Demir,R.E.Amaro,D.A.Harki,H.Aihara,R.S.HarrisAPOBEC‐catalyzedcytosine‐to‐uracildeaminationofsingle‐strandedDNA(ssDNA)hasbeneficialfunctionsinimmunityanddetrimentaleffectsincancer.APOBECenzymeshaveintrinsicdinucleotidespecificitiesthatimparthallmarkmutationsignaturesinmutatedviralDNAandincancer.Althoughnumerousstructureshavebeensolved,mechanismsforglobalssDNArecognitionandlocaltarget‐sequenceselectionremainunclear.HerewereportcrystalstructuresofhumanAPOBEC3AandachimeraofhumanAPOBEC3BandAPOBEC3AboundtossDNAat3.1‐Åand1.7‐Åresolution,respectively.ThesestructuresrevealaU‐shapedDNAconformation,withthespecificity‐conferring1thymineflippedoutandthetargetcytosineinserteddeepintothezinc‐coordinatingactivesitepocket.The1thyminebasefitsintoagroovebetweenflexibleloopsandmakesdirecthydrogenbondswiththeprotein,accountingforthestrong52‐TCpreference.ThesefindingsexplainbothconservedanduniquepropertiesamongAPOBECfamilymembers,andtheyprovideabasisfortherationaldesignofinhibitorstoimpedetheevolvabilityofvirusesandtumors.Shi,Carpenteretal.NSMB2017
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13. “EpsteinBarrVirusTranscriptionFactorsRtaandZtaPlayDistinctlyDifferentRolesinPromotingEBVReplicationinEpithelialCells”AhmedAli,MarkEichelberg,MakotoOhashi,RezaDjavadian,ShannonKenney,andEricJohannsenEpsteinBarrVirus(EBV)isahumanherpesvirusassociatedwithlymphomasandspecificepithelialmalignancies.EBVreplicationiscontrolledbytheviraltranscriptionfactorsZtaandRta.AlthoughbothZtaandRtaarerequiredforthelyticcascade,dissectingthespecificroleplayedbyRtaorZtaischallengingbecauseeachactivatestheotherexpression.Tocircumventthis,wegeneratedanEBVmutantdeletedforbothRtaandZtaintheAkatastrainBACmid(designatedAkata‐dRdZ).StablelatentinfectionofAkata‐dRdZwasestablishedintelomerase‐immortalizednormaloralkeratinocytes(NOKs).TheresultantNOK‐Akata‐dRdZcellsweretransfectedwithRta,Zta,orRta+ZtaandEBVlyticgeneexpressionexaminedbyRNA‐seqat48hoursposttransfection.OurdatarevealedthatthemajorityoflytictranscriptswereinducedbyRta,butsurprisingly,Ztaactivatedonlythetwotranscripts(LF3andBHLF1)drivenbytheOriLytpromoters.Thissuggeststhat,atleastinthisepithelialinfectionmodel,EBVreplicationmaybemuchmoresimilartothatofKSHVthatpreviouslyappreciated.InKSHV,theRtaortholog(ORF50)drivesexpressionofalllyticpromoterswhereastheZtaortholog(K8)servesonlyasanOriLytbindingproteinforDNAreplication.WearecurrentlyinvestigatingwhetherthislimitedroleforZtaisspecifictoepithelialcellsorholdstrueinBcells,butwaspreviouslyunappreciated(e.g.,obscuredbyZtainductionofRtaexpression).Inaddition,weareinvestigatingthemechanismsbywhichZtasynergizeswithRtatoactivatelyticpromotersinNOK‐Akata‐dRdZthatitisunabletoactivatewhenexpressedalone.OurresultshaveimportantimplicationsforlyticinductiontherapyasameansofkillingEBVpositivecarcinomas.14. “TranslationcontrolofHIV‐1”KathleenBoris‐LawrieIn‐solution,HIV5UTRformstwointerchangeablelong‐rangent‐pairings,onesequestersthegagstartcodonpromotingdimerizationwhiletheothersequestersthedimerinitiationsignalpreventingdimerization.Whiletheeffectofthesent‐pairingsondimerizationandpackaginghasbeendocumentedtheireffectonauthenticHIVtranslationincellulohasremainedelusiveuntilnow.HIVNL4‐35UTRsubstitutionsweredesignedtoindividuallystabilizethedimer‐proneormonomer‐proneconformations,validatedin‐solution,andintroducedtomolecularclones.Theeffectof5UTRconformationonribosomeloadingtoHIVunsplicedRNAandrateofGagpolypeptidesynthesiswasquantifiedincellulo.Monomer‐anddimer‐prone5UTRsdisplayedequivalent,basalrateoftranslation.Gain‐of‐functionsubstitutionU103,inconjunctionwithpreviouslydefinednt‐pairingsthatreorientAUGtoflexiblent‐pairing,significantlyactivatedthetranslationrate,positingbasaltranslationrateisunderpositiveselection.Theobservedtranslationup‐regulationisattributabletont‐pairingsatthejunctionofRandU5upstreamofpreviouslycharacterizedHIVriboswitchanddemonstratesthebasaltranslationrateofauthenticHIVRNAisregulatedindependentlyofmonomer:dimer
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equilibriumofthe5UTR.15. “HIV,RatioNormalizationandWinningtheGameofTCells”RobStriker,DawitWolday,MitchellKirsh,AjaySethi,JoeMcbrideandIreneOngCurrentlyHIVtherapycandroptheviralloadtoundetectableandallowadepletedCD4responsetorecover.TherapydoeslesstoreversethestimulationofCD8s.Whiletherapydramaticallyreducesmortality,itdoesnotcompletelyreversethenegativehealthconsequencesofHIV.Forexample,patientlivingwithHIVstillhavehigherratesofcancerandcardiovasculardiseasethanagematchedcontrols.RecentdatasuggeststhatthisexcessmorbidityislargelyconcentratedinpatientswithCD4/8ratiosbelow0.4,butitisunclearifnormalizationoftheratioispossiblewiththerapy,andifsohowlongittakes.Furthermoreimmunedefectscoexistwhentheratiodoesntriseabove1.0.Tobegintoaddresstheseandotherquestionswehaveconstructedlongtermfollow‐upofthreedistinctcohortsofpeoplelivingwithHIV.One,fromEthiopia,isoneofthefewcohortstocontainmorethanadecadeoffollow‐upinwomenlivingwithHIV.AsecondcohortisfromourMadisonVA,andathirdisfromtheWisconsinDepartmentofCorrections.Normalizationisvariableinallthreecohortsanddoesnotoccuruniversallyinthesecohortsdespiteadecadeoftherapyormore.IntheEthiopiancohortwhichisonly35%womenthough,8%offemalesreachaCD4/CD8ratioof1.5,whileonly4%ofmenreach1.5.Thelengthoftherapyincreasesthedifferencebetweenmenandwomen.Factorsassociatedwithlackofratiorecoverywillbediscussedandaswellascohortspecificaverageratiorises.RationormalizationmaybeamoreusefulmeasurementofimmunerecoverythanthemoretraditionalCD4countinthemodernantiviraltherapyera.16. “TheHepatitisCViralProteinNS5AStabilizesGrowth‐RegulatoryHumanTranscriptsLiangGuo,SureshD.Sharma,JoseDebes,DanielBeisang,BerndRattenbacher,IrinaA.Vlasova‐St.Louis,DarinL.Wiesner,CraigE.Cameron,andPaulR.BohjanenNumerousmammalianproto‐oncogeneandothergrowth‐regulatorytranscriptsareupregulatedinmalignancyduetoabnormalmRNAstabilization.InhepatomacellsexpressingahepatitisCvirussubgenomicreplicon,wefoundthattheviralNS5A,aproteinknowntobindtoviralRNA,alsoboundspecificallytohumancellulartranscriptsthatencoderegulatorsofcellgrowthandapoptosis,andthisbindingcorrelatedwithtranscriptstabilization.AnimportantsubsetofhumanNS5A‐targettranscriptscontainedGU‐richelements,sequencesknowntodestabilizemRNA.WefoundthatNS5AboundtoGU‐richelementsinvitroandincells.MutationoftheNS5AzincfingerabrogateditsGU‐richelement‐bindingandmRNAstabilizingactivities.Overall,weidentifiedamolecularmechanismwherebyHCVmanipulateshostgeneexpressionbystabilizinghosttranscriptsinamannerthatwouldpromotegrowthandpreventdeathofvirus‐infectedcells,allowingthevirustoestablishchronicinfectionandleadtothedevelopmentofhepatocellularcarcinoma.
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17. “QuantitativeImagingofRhinovirusInfectionSpreadwithSingle‐CellResolution”HuichengShi,BaharInankur,andJohnYinDuetothecomplexityofviralandhostcellularprocessesinvolved,itisgenerallynotknownhowthekineticsofviralgeneexpressionwithinindividualcellsrelatestotheoveralldynamicsofinfectionspreadthroughapopulationofsusceptiblehostcells.Toaddressthischallenge,HeLacellswereinfectedwithhumanrhinovirus(HRV)engineeredtoexpressgreenfluorescentprotein(GFP)asabyproductofviralgeneexpression.Fluorescentsignalwastrackedoverseveralroundsofinfectionandkineticparameterswereestimatedfromreporterexpressionatsingle‐cellresolution.Speed‐upofdetectedgeneexpressionbetweentheinitialandsubsequentinfectedcellsprovidespreliminaryevidenceforamechanismofnon‐lyticinfectiousparticlerelease.Shorterdurationsofreportersignalacrossfourinfectioncyclescorrelatewithcellaging.Thisworkshowshowsingle‐cellimagingcanrevealsubtlechangesinthedynamicsofvirus‐cellinteractionduringinfectionspread.18. “RegulationofEBVLyticReactivationbyp53andp63inEpithelialCells”NickVanSciver,DhananjayNawandar,DenisLee,KathleenMakielski,PaulLambert,andShannonC.KenneyEpstein‐BarrVirus(EBV)infects95%oftheadultpopulationworldwideandisassociatedwithanumberofmalignanciessuchasBurkitt’slymphoma,Hodgkin’slymphoma,nasopharyngealcarcinoma(NPC),and10%ofgastriccancers.p53isatumorsuppressorandismutatedinapproximately10%ofallNPCs.Whetherap53mutationconfersanyalterationtotheEBVlifecycleinepithelialcellsisnotclear.OurlabpreviouslyshowedthatEBVlyticreactivationinraftedEBV‐infectedoralkeratinocytesislimitedtothedifferentiatedepitheliallayers.Inordertoinvestigatewhetherp53regulatesEBVlyticreactivationduringdifferentiationofnormalkeratinocytes,wehavecreated(incollaborationwiththeLambertlab)EBV‐infectedisogenicnormaloralkeratinocytelines(NOKs)thathaveeitherwild‐typep53,nop53,oramutantp53(K120E).Ourpreliminaryresultscomparingcellswithwild‐typeversusmutantp53revealamarkedincreaseinEBVlyticreactivationandcellulardifferentiationinthep53mutantcells(incomparisontowildtypecells)duringorganotypicraftculture.Wearecurrentlyexamininghowcompleteknockoutofp53affectsepithelialcelldifferentiationandlyticEBVreactivation.Inaddition,wearedeterminingifarelatedp53familymember, Np63,whichisknowntoregulateepithelialdifferentiationandproliferation,alsoaffectsEBVreactivationinnormalkeratinocytes.Ourpreliminaryresultssuggestthatlossof Np63expressionusingeithersiRNAsorshRNAsinEBVlatentlyinfectedepithelialcelllinesinduceslyticEBVreactivation.Additionally,when Np63isover‐expressedintheaconstitutivelylyticEBV‐infectedgastriccarcinomacellline(whichnormallyexpressesno Np63),AGS‐AkatawefindthatlyticEBVexpressionisreduced.Togethertheseresultssuggestthatbothp53and Np63regulatelyticEBVreactivationduringdifferentiationofnormaloralkeratinocytecells.
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19. “CharacterizingGuineaPigCytomegalovirusPathogenesisintheAmnion”DiraS.PutriandCraigJ.BierleHumancytomegalovirus(CMV)ismostsignificantinfectiouscauseofcongenitaldiseaseandpreventablesensorineuralbirthdefects.CongenitalCMVinfectionoccursinupto2%ofpregnanciesintheUnitedStates,andanestimated8,000childrenaredisabledbytheviruseachyear.RecentstudieshaveobservedthatpersistenthumanCMVinfectionoftheamnionisassociatedwithprematurebirth,intrauterinegrowthrestriction,andcongenitalCMVdisease.Astheinnermostlayerofthefetalmembranes,theamnionisthefinalbarrierseparatingthefetusfromtheoutsideworld.Asinglelayerofamnioticepithelialcellsprotectsagainstviralandbacterialpathogensbysecretingcytokinesandotherfactorstomodulatetheinnateandadaptiveimmuneresponses.HowhumanCMVestablishesinfectionintheamnionandhowpersistentviralinfectionimpactsthemembranesnormalfunctionarepoorlyunderstood.WesoughttodeterminewhetherguineapigCMV,awell‐establishedanimalmodelofinuteroCMVtransmission,alsoinfectstheamnionduringnaturalinfection.Afteramid‐gestationviralchallenge,guineapigCMVwasdetectedintheplacentaandamnionsofpupscollectedat21dayspost‐infection.Guineapigamnioticepithelial(CPAE)cellswereisolatedfromlate‐termamnionsandimmortalizedwithHPV16E6/E7.BothprimaryandimmortalizedCPAEcellssupportedGPCMVreplication.ComparedtoguineapigCMVinfectionsinlungfibroblasts,CMVreplicationinCPAEcellsislesslytic,sustainedforalongerduration,andresultsinmarkedlylessextracellularvirusproduction.Additionally,experimentshaverevealedthatCPAEcellssecretetrans‐actingantiviralfactorsofunknownidentity.Together,theseresultsindicatedthattheguineapigisanappropriateanimalmodelforstudyingviralinfectionoftheamnioninvivoandinvitro.20. “DeepSequencingBarcodedZikaVirusfromMacaquesUsingaSmallAmpliconApproach”KatieZarbock,PaolaSilveira,TeamZEST,GregEbel,DavidO’Connor,andShelbyO'ConnorSequencingZikavirus(ZIKV)hashistoricallybeenchallengingbecauseviraltitersarelowandtransient.ItisdifficulttoisolatesufficientvirustemplatenumberstosuccessfullyamplifyviralcDNAfordeepsequencing.WeadaptedaprocessbyQuicket.al.tosequencetheZIKVgenome.AmpliconsaretaggedandsequencedonanIlluminaMiSeqinstrument.WeusedthistechniquetosequenceauniqueZika‐barcodestock,anditscomparablewildtypeclone.Innonpregnantanimals,barcodediversitymirrorsthestockatdays3and5postinfection.Inonepregnantanimal,thereisadramaticreductioninbarcodediversitybetweendays7and8thatcorrespondstodecreasedviremia.Futurestudiesareneededtodeterminethemechanismleadingtothisbottleneck.AsecondversionoftheZikabarcodevirushasalsobeengeneratedandismorediversethanversion1.WewillinfectnonpregnantanimalswiththeZikabarcodeversion2stocksoon.
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21. “Cryo‐EMStructureofPorcineDeltaCoronavirusSpikeProteininthePre‐FusionState”JianShang,YuanZheng,YangYang,ChangLiu,QibinGeng,WanboTai,LanyingDu,YusenZhou,WeiZhang,andFangLiOronavirusspikeproteinsfromdifferentgeneraaredivergent,althoughtheyallmediateviralentryintocellsbybindingtohostreceptorsandfusingviralandcellmembranes.Herewedeterminedthecryo‐EMstructureofporcinedeltacoronavirus(PdCoV)spikeproteinat3.3‐angstromresolution.Itcontainsthreereceptor‐bindingS1subunitsandatrimericmembrane‐fusionS2stalk.S1containstwodomains,N‐terminaldomain(S1‐NTD)andC‐terminaldomain(S1‐CTD).PdCoVS1‐NTDhasthesamegalectinfoldasalpha‐andbeta‐coronavirusS1‐NTDs,andrecognizessugarasitspotentialreceptor.PdCoVS1‐CTDhasthesamestructuralfoldasalpha‐coronavirusS1‐CTDs,whichdiffersfromthestructureofbeta‐coronavirusS1‐CTDs;itbindstoanunidentifiedreceptoronhostcellsurfaces.PdCoVS2islockedinthepre‐fusionconformationbystructuralrestraintofS1fromadifferentsubunit.PdCoVspikepossessesseveralstructuralfeaturesthatmayfacilitateimmuneevasionbythevirus,suchasitscompactstructure,concealedreceptor‐bindingsites,andshieldedcriticalepitopes.Overall,thisstudyrevealsthatdelta‐coronavirusspikesarestructurallyandevolutionallymorecloselyrelatedtoalpha‐coronavirusspikesthantobeta‐coronavirusspikes;italsohasimportantimplicationsforthereceptorrecognition,membranefusion,andimmuneevasionbydeltacoronavirusesaswellascoronavirusesingeneral.22. “CXCR5‐TransducedPrimaryRhesusMacaquePBMCsAccumulateinBCellFolliclesinaNovelExVivoBCellFollicleMigrationAssay”GwantwaMwakalundwa,HaranP,LiS,PampuschM,AbdeelalHM,RakaszEG,ConnickE,andSkinnerPJBackground:CD8Tcellsarecrucialforcontrollingbothhumanandsimianimmunodeficiencyvirus(HIV/SIV)infections.Lowlevelsofvirus‐specificCD8TcellswithinBcellfolliclespermitongoingreplicationofHIVandSIV.TheBcellhomingmolecule,CXCR5,isrequiredforhomingtoBcellfollicles.Wehypothesizethatengineeringvirus‐specificCD8TcellstoexpressCXCR5willenablethemtohometoBcellfolliclesandsuppressviralreplication.TobegintotestthishypothesiswedevelopedanovelexvivoBcellmigrationassay.Method:Wefirstdeterminedtheoptimaltimethatlymphnodetissueslabscouldbeincubatedandmaintainfollicularmorphology.NextweevaluatedwhetherCD8TcellsengineeredtoexpressCXCR5,accumulatedwithinBcellfollicles.WetransducedCD8TcellsfromSIVinfectedrhesusmacaques,withacontrolvectororaCXCR5transducingvector,labeledwithalivecelldyeCTV,layeredthesecellsonto300umthickfreshtissueslabsandincubatedforsixhours.SectionswerethenfixedandstainedviaIHCwithanti‐CD20tostainBcellfollicles.SectionswereimagedusingaconfocalmicroscopeandCTVlabeledcellswerequantifiedinsideandoutsideofBcellfollicle.Results:Wedeterminedthatfollicularmorphologywasmaintainedafter4and6hoursofincubation,butwaslostafterovernightincubation.Preliminarymigrationresultsshowedafivefoldincreaseinthefolliculartoextrafollicularratio(F:EF)ofCTVlabeledcellsintheCXCR5transducingvectorsamplescomparedtocontrolsamples.Conclusion:Wesuccessfullycreated
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anexvivoBcellmigrationassay.PreliminaryresultsusingthisassaysuggestthatCXCR5transductioninducesTcellmigrationintoBcellfollicles.23. “EBNA3C‐DeletedEpsteinBarrVirusCausesLymphomasinaHumanizedMouseModelDespiteBeingSeverelyDeficientforBCellTransformationinVitro”JamesRomero‐Masters,RezaDjavadian,MakotoOhashi,ShidongMa,AndreaBilger,EricJohannsen,andShannonKenneyEBNA3CisanEBV‐encodedlatentproteinessentialforBcelltransformationinvitro.AcriticalfunctionofEBNA3CduringEBV‐mediatedtransformationinvitroisdown‐regulationofthetumorsuppressorgeneproducts,p14ARF,p16INK4a,andthepro‐apoptoticprotein,BIM.Herewehaveusedacord‐bloodhumanizedmousemodeltoexaminethephenotypeofanEBNA3C‐deleted(EBNA3C)EBVmutantinvivo.WefindthatEBNA3Cinduceslymphomasinapproximately40%ofanimals,whilewildtype(WT)virusinduceslymphomasinvirtuallyallanimals.EBNA3C‐inducedlymphomashaveadelayedonset(60‐90days)comparedtoWTtumors(30‐40days),butoncepresentareaslargeastheWTtumorsandhighlyinvasive.BothWT‐andEBNA3C‐infectedlymphomasareCD20‐positiveactivateddiffuselargeBcelllymphomas.IncomparisontoWTtumors,EBNA3Ctumorshaveincreasednumbersoflyticallyinfectedcells.EBNA3Ctumorsalsohavegreatlyenhancedexpressionofp16.However,EBNA3CtumorsexpresshighlevelsofCyclinE,c‐Myc,andBCL2,whichmayallowcellstobypassp16‐mediatedcellcycleinhibitionandapoptosis.Unexpectedly,wealsofindthatEBNA3CtumorshavegreatlyincreasedTcellinfiltrationincomparisontoWTtumors.RNAseqandqPCRanalysisofWT‐andEBNA3C‐infectedlymphomasrevealsasignificantlyincreasedexpressionoftheTcellcytokines,CCL5andCCL20,intheEBNA3Ctumors;EBNA3Ctumorsalsohaveincreasedexpressionofinterferonalphaandmultipleinterferon‐responsivegenesresponse.Together,theseresultsrevealthatEBNA3CexpressionisnotabsolutelyessentialforEBV‐inducedlymphomagenesisincord‐bloodhumanizedmice,andsuggestthatpreviouslyunappreciatedrolesofEBNA3CincludeinhibitingtheTcellresponsetoEBV‐infectedBcells,aswellastheinterferonresponse.24. “NovelFeaturesofCRM1‐DependentRetroviralmRNANuclearExportRevealedUsingLiveCellImaging”RyanT.Behrens,ChristinaHiggins,andNathanM.ShererPrimatelentivirusesincludingHIV‐1,HIV‐2,andSIVandalsodeltaretrovirusessuchasHTLV‐1havenecessarilyadaptedtoovercomeprofoundcell‐and/orspecies‐specificbarrierstothenuclearexportofessentiallate‐stage,intron‐retainingviralmRNAs.Thesebarrierscriticallyinfluenceviruscelltropismandarekeydeterminantsofhostimmunedetectionandpathogenicoutcomes.BothlentivirusesanddeltaretrovirusesovercomethesebarriersusingRevorRexproteins,respectively,thatrouteunsplicedorincompletelysplicedviralmRNAsoutofthenucleususingthehighlyspecializedcellularCRM1/RanGTPnuclearexportmachinery.Rev/Rex‐equivalentproteinsfromdiverseretroviralpathogens(e.g.,HIV‐1,HIV‐2,SIV,andHTLV)areunifiedinthattheyalllinkviralRNAstoCRM1.However,whetherRev/Rex‐encoded
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activitieshaveevolvedtocircumventidenticalselectivepressuresor,instead,governmultipleuniquemodesofRNAtransportand/orotherfeaturesofregulatedgeneexpressionisunknown.Live‐cellimagingrepresentsapowerfulnewstrategyfordirectcharacterizationofthesepathways.Here,forthefirsttime,wepresentcomparativeimagingofRev/RexequivalentproteinsandRNAtraffickingdynamicsforHIV‐1,SIV,HIV‐2,HTLV,and6additionalretroviruses(BIV,EIAV,FIV,Visna,HTLV‐2andBLV)over>30hoursofcontinuousimaginginsinglelivingcells.WeshowthattheHIV‐1Revproteinexhibitsstriking,highlyuniquesubcellulartransportdynamicsanddemonstratethatRevregulatesanovelCRM1‐dependent,burst‐likeRNAexportbehavior,activatingrapid,late‐stageupregulationofgeneexpressionjustpriortotheonsetofvirusparticleproduction.OurcomparativeexperimentsrevealbothdistinctfeaturesofRev/Rex‐regulatedexportmechanismsand,insomeinstances,remarkableinteroperabilityofviralelementsrelevanttonaturalco‐infections(e.g.,forHIV/HTLVorBIV/BLV)and,potentially,cross‐speciestransmission.Moreover,wediscussaddressthepotentialutilityofnext‐generation,low‐toxicityCRM1inhibitorsasbroad‐spectruminhibitorsoftheseviralpathogens,andhowwehaveexploitedspecies‐specificfeaturesofCRM1toexposeadditionalvulnerabilities.25. “AnalysisofProviralandParticleStructurefromDistinctChronicallyInfectedCellLinesDemonstratesthePolymorphicNatureofHTLV‐1ParticleCores”LuizaMendonça,MorganEMeissner,WeiZhang,LouisManskyHumanT‐cellleukemiavirustype1(HTLV‐1)wasthefirstdescribedhumanretrovirus.ItistheetiologicalagentofadultT‐cellleukemia(ATL)andHTLV‐1‐associatedmyelopathy/tropicalspasticparaparesis(HAM/TSP).HTLV‐1particlestructureisstillpoorlyunderstood,andpreviousstudieshaveanalyzedvirusesproducedbytheMT‐2cellline,atransformedlymphocyticcellchronicallyinfectedwithHTLV‐1,whichharborstruncatedprovirusesandexpressestruncatedGagproteins.Inthisstudy,wedemonstratethatthechronicallyinfectedSPcelllineharborsarelativelylownumberofproviruses,makingitapromisingexperimentalsystemforthestudyofHTLV‐1particlestructure.Weidentifiedthegenomicsitesofintegrationandcharacterizedthegeneticstructureofthegagregionineachprovirus.WedeterminedthatdespiteencodingforatruncatedGag,onlyfulllengthproteinisincorporatedintobuddingviralparticles.Moreover,cryo‐EManalysisofthepurifiedparticlesproducedbySPcellsrevealedthatparticlescanbedividedintothreeclassesregardingthecorestructure:viruseswithcompletecores,viruseswithincompletecores,andparticleswithnocoresbutwithhomogeneouslydistributedelectrondensity.Observedcoreswerelooselypolygonalinshape,andvirusparticlesaveraged115nmindiameter.Studiesofretroviralparticlecoremorphologyhavedemonstratedacorrelationbetweencapsidcorestabilityandrelativeinfectivityofthevirus.Inthisstudy,wedemonstratethatHTLV‐1particlesproducedfromdistinctchronicallyinfectedcelllinesarepolymorphicinnature,withmanyparticlesexhibitingalackoforganizedparticledensityassociatedwithacompletecorestructure.EnsuringthatthisphenotypeisatruecharacteristicofHTLV‐1particlesisthefirststepincorrelatingthediversityinparticlestructuresobservedforthisviruswiththelowlevelsoftransmissibilityassociatedwithHTLV‐1infection.ThesedatacorroboratetheresultsseenforparticlesproducedfromMT‐2cellsand
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furtherindicatethatHTLV‐1particlesexhibithighlypolymorphiccores,whichmaycontributetotheremarkablylowinfectivitythatischaracteristicofthevirus.26. “RoleofIQGAP1inHPV‐PositiveandHPV‐NegativeHeadandNeckCarcinogenesis”TaoWei,SuyongChoi,DaryaBuehler,RichardAndersonandPaulLambertHigh‐riskhumanpapillomavirus(HPVs),suchasHPV16,18and33,cause5%ofhumancancers,includingcervical,analandanincreasingpercentageofheadandnecksquamouscellcarcinoma(HNSCC).PI3K/Aktsignaling,alongwithitsdownstreameffectormTOR,isoneofthemostfrequentlyalteredpathwaysinbothHPV‐positiveandHPV‐negativeHNSCCwithPIK3CA,thegeneencodingthecatalyticsubunitofPI3K,beingthemostfrequentlyalteredgeneintheHPV‐positiveHNSCC(~56%).Thesealterationsleadtoanincreasedactivityofthispathway.Recently,IQmotif‐containingGTPaseactivationprotein1(IQGAP1),a190‐kDaproteinthatcontainsmultipleprotein‐interactiondomains,hasbeenreportedtoscaffoldthePI3K/Aktpathway.ThisgeneisincreasedinitsexpressioninhumanHNSCCs,andwefoundthatinterferingwiththeabilityofIQGAP1toscaffoldPI3KinhibitsthegrowthofhumanHNSCCcelllinesinvitro.AdditionallywefoundthatIQGAP1associateswithHPV16E6&E7,twoviraloncoproteinsimplicatedincarcinogenesis,andthattheseinteractionscorrelatewithanupregulationofthePI3K/AKT/mTORsignalingincellculture.ThesedatahaveledustotestthehypothesisthatIQGAP1contributestoheadandneckcarcinogenesis.Totestthishypothesiswemadeuseofawell‐validatedmousemodelforHNSCC,inwhichasyntheticoralcarcinogen,4‐nitroquinoline1‐oxide(4NQO),isusedtodrivetumorigenesis,andgeneticallyengineeredmicethataredeficientintheexpressionofIQGAP1(IQGAP1‐/‐mice).Utilizingthelatter,wefirstverifiedthatIQGAP1isnecessaryforefficientPI3KsignalinguponEGF‐stimulationinvivo.WethenpursuedHNSCCstudies.Aftertreatingmicewith4NQOintheirdrinkingwaterfor16weeksfollowedby5weeksonnormaldrinkingwater,Iqgap1+/+miceshowedsignificantlyhighercancermultiplicityaswellassignificantlyhighernumbersofhighgradecancersthanIqgap1‐/‐mice.TheseresultssupporttheabovestatedhypothesisthatIQGAP1contributestoheadandneckcarcinogenesis.FuturestudiesfocusonwhetherIQGAP1isnecessaryspecificallyforHPV‐positiveHNSCC,andutilizeourHPV‐transgenicmousemodelforHNSCC.ExperimentalmicegeneratedbycrossingIqgap1‐/‐miceandK14‐E6/E7willbeputunder4NQOtreatmenttodeterminewhetherlossofIQGAP1affectsheadandneckcarcinogenesisinthecontextofHPV.27. “SIVVifandHumanAPOBEC3BInteractionsResembleThoseBetweenHIV‐1VifandHumanAPOBEC3G”JiayiWang,NadineM.Shaban,AllisonM.Land,WilliamL.Brown,andReubenS.HarrisSeveralmembersoftheAPOBEC3familyofDNAcytosinedeaminasescanpotentlyinhibitHIV‐1replicationbycatalyzingextensivecytosinedeaminationinviralcDNAandimpedingreversetranscription.HIV‐1counteractsrestrictionwiththevirallyencodedVifprotein,whichtargetstheAPOBEC3proteinstoacellularubiquitinligaseforproteasomaldegradation.HIV‐1VifishighlyoptimizedfordegradingthehumanAPOBEC3repertoireand,ingeneral,lentiviralVif
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proteinsspecificallytargettherestrictingAPOBEC3enzymesofeachrelevanthostspecies.However,SIVmac239VifelicitscuriouslywiderangeofAPOBEC3degradationcapabilitiesthatincludesseveralhumanAPOBEC3sandevenhumanAPOBEC3B,anon‐HIV‐1restrictingAPOBEC3enzyme.TobetterunderstandthemoleculardeterminantsoftheinteractionbetweenhumanAPOBEC3BandSIVmac239Vif,weanalyzedanextensiveseriesofmutants.WefoundthatSIVmac239VifinteractswiththeN‐terminaldomainofhumanAPOBEC3Band,interestingly,thisoccurswithinastructuralregionhomologoustotheHIV‐1VifinteractionsurfaceofhumanAPOBEC3G.AnalaninescanofSIVmac239VifrevealedseveralresiduesrequiredforhumanAPOBEC3Bdegradationactivity.TheseresiduesoverlappedwithHIV‐1VifsurfaceresiduesthatinteractwithhumanAPOBEC3G,andaredistinctfromthosethatengageAPOBEC3ForAPOBEC3H.Overall,thesestudiesindicatethatthemoleculardeterminantsofthehumanAPOBEC3B‐SIVmac239VifinteractionstronglyresemblethoseofhumanAPOBEC3G‐HIV‐1Vifinteraction,andmaybearemnantofanancestralVifactivityusedforcross‐speciestransmission.
28. “Species‐SpecificRegulationofHIV‐1GeneExpressionandVif‐InducedCellCycleArrest”EdwardL.EvansIII,JordanT.Becker,StephanieS.Fricke,KishanM.Patel,andNathanM.ShererCellsderivedfrommiceandotherrodentsexhibitprofoundblockstoHIV‐1virionproductionreflectingspecies‐specificincompatibilitiesbetweenviralTatandRevproteinsandtheessentialhostfactorsCyclinT1(CCNT1)andExportin‐1(XPO1,alsoknownasCRM1),respectively.HereweaskedifCCNT1andXPO1representthefinalbarrierstoHIVspost‐integrationstagesinmousecellsengineeredtoconstitutivelyexpressHIV‐compatibleversionsofCCNT1andXPO1(3T3.CXcells).3T3.CXcellswereinfectedwithsingleround(Env‐/Vpr‐/Nef‐)HIV‐1/NL4‐3,andviralgeneexpressionandvirusparticleproductionweremonitoredforuptotwoweekspost‐infection.3T3.CXcellsyielded>40‐foldincreasestoHIV‐1virusparticleoutputrelativetoparental3T3cells,butexhibitedan~24hlagtotheonsetofearlyandlateviralgeneexpressioncomparedtopermissivehumancells(HeLa).Unexpectedly,long‐term(>6day)monitoringofinfectionrevealed3T3.CXcellstoberemarkablytolerantofHIV‐1‐associatedcytopathiceffectsrelativetohumancells,aneffectwemappedtothecapacityoftheviralaccessoryproteinViftoinduceG2/Mcellcyclearrestinhumancellsbutnotinnon‐humancelltypes.VifretainedthecapacitytodegradeknownCullin5‐targetedsubstratesincludingAPOBEC3GandaB56subunitofthePP2Aholoenzymeinbothmouseandhumancells,thusindicatingthatVifsroleinthecellcyclecanbeuncoupledmechanisticallyfromitsotherdefinedrolesinproteinturnover.Combined,ourresultsreinforcethatCCNT1andXPO1arethepredominantblockstoHIV‐1slatestagesinmousecellsandexposeadditionalnovelcell‐orspecies‐specificHIV‐hostinteractionsinfluencingviralRNAmetabolismandcellcyclecontrol.
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29. “VisualizingHTLV‐1GagPunctaBiogenesisbySuper‐ResolutionMicroscopy”JohnEichorst,JohnKohler,JoachimMueller,andLouisManskyHumanT‐cellleukemiavirustype‐1(HTLV‐1)particleassemblyoccursattheplasmamembraneandisdrivenbyGagoligomerization.However,theprecisemechanism(s)thatinitiatesGagpunctabiogenesisisnotwellestablished.Here,wesoughttoestablishquantitativeimagingapproachestoanalyzethisprocessatsinglemoleculeresolutionbyusingsuper‐resolutionmicroscopy.Todothis,HTLV‐1GagwaslabeledwiththefluorescentproteinmEos3.2andmonitoredincellsusingthesuper‐resolutionapproachofphotoactivationlocalizationmicroscopy(PALM).InPALM,thelateral(xy)spatialresolutionoftheimageoutputisimprovedtonearly40nmbylocatingthepositionofeachsinglefluorescently‐labeledmolecule,independentofanyotherinthecell.ThisresultsinimagesthatcontainintricatedetailsaboutthedistributionandorganizationofHTLV‐1Gagproteinattheplasmamembrane,whicharenotreadilyvisibleusingconfocalorepi‐fluorescenceimaging.Nonetheless,PALMrequiresdetailedcorrectionsandcontrolsinordertotranslatethedataintoabsolutedensitiesandconcentrations.Here,wedescribetheongoingstudiestoimprovetheamountofquantitativeinformationavailablePALMtostudyHTLV‐1particleassembly.Usingmultiplecontrolmethods,includingmeasurementsoffastfluorescencefluctuations,theeffectsofthenon‐idealphotophysicsofthefluorescentproteinsutilizedinPALMonthesubsequentquantitativeanalysiswillbediscussed.Furthermore,withtheimprovedspatialresolutionofPALM,weareconductingstudiestodeterminewhethernon‐punctateHTLV‐1Gagrepresentsanassemblyintermediate.Inparallel,ongoingexperimentsseektodetermineifthespatialorganization,usingpairedcorrelationmethods,ofnon‐punctateHTLV‐1Gagattheplasmamembranearerequisiteforvirusparticlebiogenesis.TheseexperimentalstrategiesareextendingtheinformationcontentavailablefromPALMmeasurementsandaidinprovidingnewinsightsintoHTLV‐1particleassembly.30. “ComprehensiveAnalysisoftheKineticsofEpstein‐BarrVirusLyticGeneExpression”RezaDjavadian,MitchHayes,andEricJohannsenWeandotherspreviouslydemonstratedthat6Epstein‐Barrvirus(EBV)genesthathaveorthologsinbeta‐andgamma‐,butnotalpha‐herpesvirusesmediatelategenetranscriptioninavirallyticDNAreplication‐dependentmanner.Weproposedamodelinwhichthesebeta‐gammagenesencodeaviralpre‐initiationcomplex(vPIC)thatmediatestranscriptionfromnascentviralDNA.SubsequentlyitwasreportedthatsomelategenetranscriptsdonotrequirevPIC.Torigorouslydefinewhichtranscriptionstartsites(TSS)aredependentonvirallyticDNAreplicationorvPIC,weperformedCapAnalysisofGeneExpression(CAGE)‐SEQoncellsinfectedwithwildtypeEBVorEBVmutantsdefectiveforDNAreplication,vPICfunction,orlackinganoriginoflyticreplication.Usingconservativecriteria,wedefined18true‐lateand32earlyTSSaswellas12TSSthatareactiveatlowlevelsearlyandareupregulatedinavPICandlyticDNAreplication‐dependentmanner.Ourresultsrepresentthemostcompletecharacterizationoflyticgeneexpressionkineticsreportedtodateandsuggestthatmost,but
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notallEBVlategenesarevPICdependent.31. “IdentificationofCancerCellLineswithaClearAPOBECMutationSignature”MatthewC.Jarvis,DiakoEbrahimi,NuriA.Temiz,andReubenS.HarrisMutationsfueltumorevolutionandcontributetomultipleprocessesincludingimmuneevasion,metastasis,andtherapyresistance.MultipleendogenousandexogenoussourcesofDNAdamagecontributetotheoverallmutationburdenincancer,withdistinctandoverlappingcombinationscontributingtoeachcancertype.Eachmutationsourceresultsinacharacteristicsignature,whichcanbededucedfromtumorgenomicDNAsequences.GenomicDNAcytosinedeaminationbycellularAPOBECenzymesisthemostabundantnewlyappreciatedsourceofmutationinmanycancers,includingcertaincancertypeswithaviralassociation.HPV‐positivecancers,suchashead/neckandcervicalcancershavebeenlinkedwithmechanismsleadingtoenrichedAPOBECmutagenicsignature,definedasC‐to‐TandC‐to‐Gmutationsoccurringin5‐TCWmotifs.Resolvingthecomplexmutationalprofilesinthesecancertypesiscrucialtounderstandingpathogen‐relatedAPOBECmutagenesisinthesecellsystems.HereweidentifycancercelllineswithastrongAPOBECmutationsignaturebyanalyzingthebasesubstitutionmutationspectrafromover1000cancercelllinesthroughtheCOSMICdatabase.ThesecelllinesmayprovidemodelsystemsforfundamentalAPOBECresearchandforadvancingclinicalstrategiestotargettumorevolvabilityinHPV‐positivecancers.32. “Cryo‐ElectronTomographyRevealsNovelFeaturesofaViralRNAReplicationCompartment”KennethJ.Ertel,DesiréeBenefield,DanielCastaño‐Diez,JaniceG.Pennington,MarkHorswill,JohanA.denBoon,MarisaS.Otegui,andPaulAhlquistPositive‐strandRNAvirusesarethelargestgeneticclassofvirusesandincludemanyestablishedandemerginghumanpathogenssuchasZikavirus.ThesevirusesinvariablyreplicatetheirRNAgenomesonrearrangedintracellularmembranes,frequentlyinsidevirus‐inducedmembraneinvaginations.TheorganizationofviralproteinsandRNAsinthesecompartmentsremainslargelyunknown.Here,wereportstrikingnewresultsfromthefirstcryo‐EMtomographystructuralanalysesofapositive‐strandRNAvirusRNAreplicationcompartment.Usingtheadvancedflockhousenodavirusmodelsystem,wehavevisualizedmanypreviouslyunrecognizedfeaturesofmembrane‐associatedviralRNAreplicationcomplexes.TheimagesrevealthattheFHV‐inducedspherularinvaginationsofoutermitochondrialmembranesaredenselypackedwithcoiledtemplateRNAsandthatadramaticringedstructurecrownstheneckedapertureofeachcomplextothecytoplasm.Subtomogramaveragingofthesecrownsrevealsadetailedtwelve‐foldsymmetricalstructurewithconcentricflankingprotrusionsandacentralelectrondensity.ImmunogoldlabelingshowsthatthecrownscontainproteinA,themajormultifunctionalviralRNAreplicationprotein.Manycrownsareassociatedwithlongextrudingcytoplasmicfibrils,likelytobeexportedprogenyRNA.WeshowthatthevolumedistributionsofthereplicationcompartmentsarecloselycorrelatedwiththesizeandcomplexityoftheviralRNAtemplatesbeingreplicated.Takentogether,these
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structuralfindingsandthecomplementarygeneticandbiochemicalresultsprovideimportantnewmechanisticinsightsandimplydynamicnewmodelsforthestructure,assemblyandfunctionofpositive‐strandRNAvirusRNAreplicationcomplexesand,byextension,importantleadstodesignnewantiviraltreatmentapproaches.33. “StudiesofNon‐Punctate,Membrane‐BoundHTLV‐1GagbyFluorescenceFluctuationSpectroscopyandFluorescenceLifetimeImagingMicroscopy”IsaacAngert,JohnEichorst,JohnKolher,JessicaMartin,WeiZhang,LouisM.Mansky,andJoachimDMuellerHumanT‐cellleukemiavirustype1(HTLV‐1)particleassemblyoccursattheplasmamembraneandisdrivenbytheGagprotein.HTLV‐1particlebiogenesisispoorlyunderstood.Previousstudiessupportamodelinwhichnon‐punctate,plasmamembrane(PM)‐boundGagactsasareservoirforHTLV‐1Gagpunctabiogenesis.Inordertogainfurtherinsightintotheroleofnon‐punctatePM‐boundGaginHTLV‐1particleassembly,fluorescencefluctuationspectroscopy(FFS)hasbeenutilizedtocharacterizethedensityofnon‐punctateGagattheplasmamembraneoflivingcells.Thedensityofnon‐punctate,PM‐boundGagwasfoundtovarywithcytoplasmicGagconcentrationinamannerconsistentwithaLangmuir‐isothermbindingmodel.Tofurtherinvestigatenon‐punctateGagatthePM,wearecurrentlyemployingfluorescencelifetimeimagingmicroscopy(FLIM)measurementsoffluorescenceresonanceenergytransfer(FRET)betweenredandgreenlabeledfluorescentGagproteinsco‐expressedincells.Theshortlength‐scalesensitivityprovidedbyFLIM/FRETmeasurementsofnon‐punctateGagatthePMprovidescomplementarydatatothatofongoingFFSandsuper‐resolutionmicroscopystudies.Anupdateofthesestudieswillbepresented.Thecharacterizationofnon‐punctate,membrane‐boundHTLVGagwillprovideimportantcluesforthebiogenesisofHTLV‐1Gagpuncta,whichiscrucialfordrivingvirusparticleproduction.SuchprocessesarevitalforinfectiousHTLV‐1spread,andadetailedunderstandingshouldaidinthediscoveryofnoveltargetsforantiretroviralintervention.34. “Whole‐TranscriptomeSequencingtoIdentifyImmuneGeneVariants”AmeliaK.Haj,JulieA.Karl,RogerW.Wiseman,andDavidH.O’ConnorTherhesusMHCclassIalleleMamu‐B*08isassociatedwithcontrolofviralreplication,butislikelyonlyoneofmanygeneticmodifiersofsusceptibilitytoHIV.Otherimmunegenes,includingkillercellimmunoglobulin‐likereceptorandFcgammareceptorgenes,arealsolikelytobeimportantindeterminingtherateofHIVdiseaseprogression.Muchofthecurrentworkinmacaquegenomicsemployswhole‐exomeorwhole‐genomesequencing;however,theseapproachesrelyonflawedreferencegenomesandannotationsforvariantcalling,andperformespeciallypoorlyincompleximmunegenes.Long‐readRNAsequencingallowsforidentificationoffull‐lengthtranscriptisoformsandphasedSNPsfrommultipleimmunegenefamiliessimultaneously,withoutareferencegenome.Herewedescribewhole‐transcriptomesequencingofperipheralbloodmononuclearcellsfromonerhesusandonecynomolgusmacaqueusingPacificBioscienceslong‐readRNAsequencing(Iso‐Seq)technologyperformed
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onaPacBioSequelmachine.Weobtainedover800,000consensusreadsperanimal,withanaveragereadlengthofapproximately3000bases.UsingdataproducedfromthePacBioIso‐Seqanalysispipeline(version4.0.0),weidentifiedreadsaligningtoexpectedMHCclassIallelesaswellastospecificKIRallelesandFcgammareceptorisoformsequences.Weanticipatethatthistechnologywillbeusefulincharacterizingcelltype‐specificexpressionofimmunegeneisoformsinmacaquesduringthecourseofSIVinfection.35. “HumanPapillomavirusPromotesEpstein‐BarrVirusLyticReactivationinImmortalizedOralKeratinocytes”KathleenMakielski,DenisLee,LaurelLorenz,DhananjayNawandar,Ya‐FangChiu,ShannonKenney,andPaulLambertEpstein‐Barrvirus(EBV)andhumanpapillomaviruses(HPVs)arehumantumorvirusesthatcauseheadandneckcancers.Bothvirusesinfectandreplicateinupperaerodigestivetractepithelia.Somestudieshavedetectedco‐infectioninbothoropharyngealandnasopharyngealcancersraisingthepossibilitythatthesevirusescouldpotentiallyaffecteachother’slifecyclesand/oroncogenicpotential.OurlabhasestablishedaninvitromodelsystemusingorganotypicraftculturestotesttheeffectsofEBVandHPVoneachotherinstratifiedsquamousoralepithelialcells.WefoundthatthepresenceofHPVpromotesEBVlyticreactivationinsuprabasallayersoftheoralepithelium.Specifically,HPVpromotesEBVgenomeamplificationandexpressionofanEBVimmediate‐earlyprotein,Z,bothofwhicharehallmarksofEBVlyticreactivation.HPV’sabilitytopromoteEBVlyticreactivationisdrivenbyHPVoncoproteinE7,andmorespecifically,itsabilitytobindpocketproteins.Inaddition,HPVpromotesinfectiousEBVproductioninsquamousoralepithelium.StudyingtheeffectsofHPVonEBVlyticreactivationandvirusproductioncouldelucidatemechanismsbywhichco‐infectionmightcontributetoviralinfectionandheadandneckcarcinogenesis.
