The Role of the Antiviral APOBEC3 Gene Family in Protecting Chimpanzees against Lentiviruses ...

24 Pages·2015·1.98 MB·English

RESEARCHARTICLE
The Role of the Antiviral APOBEC3 Gene
Family in Protecting Chimpanzees against
Lentiviruses from Monkeys
LucieEtienne1*¤,FredericBibollet-Ruche2,PeterH.Sudmant3,LilyI.Wu1,Beatrice
H.Hahn2,MichaelEmerman1*
1 DivisionsofHumanBiologyandBasicSciences,FredHutchinsonCancerResearchCenter,Seattle,
Washington,UnitedStatesofAmerica,2 DepartmentsofMedicineandMicrobiology,PerelmanSchoolof
Medicine,UniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesofAmerica,3 Department
ofGenomeSciences,UniversityofWashingtonSchoolofMedicine,Seattle,Washington,UnitedStatesof
America
¤ Currentaddress:CIRI,InternationalCenterforInfectiologyResearch,ENS-Lyon,UCBL,INSERMU1111,
CNRSUMR5308,Lyon,France
*[email protected](LE);[email protected](ME)
Abstract
OPENACCESS
Citation:EtienneL,Bibollet-RucheF,SudmantPH,
Cross-speciestransmissionsofvirusesfromanimalstohumansareattheoriginofmajor
WuLI,HahnBH,EmermanM(2015)TheRoleofthe
humanpathogenicviruses.Whiletheroleofecologicalandepidemiologicalfactorsinthe
AntiviralAPOBEC3GeneFamilyinProtecting
ChimpanzeesagainstLentivirusesfromMonkeys. emergenceofnewpathogensiswelldocumented,theimportanceofhostfactorsisoften
PLoSPathog11(9):e1005149.doi:10.1371/journal. unknown.Chimpanzeesaretheclosestrelativesofhumansandtheanimalreservoiratthe
ppat.1005149
originofthehumanAIDSpandemic.However,despitebeingregularlyexposedtomonkey
Editor:ChristopherAiken,VanderbiltUniversity lentivirusesthroughhunting,chimpanzeesarenaturallyinfectedbyonlyasinglesimian
SchoolofMedicine,UNITEDSTATES
immunodeficiencyvirus,SIVcpz.Here,weaskedwhychimpanzeesappeartobeprotected
Received:April22,2015 againstthesuccessfulemergenceofotherSIVs.Inparticular,weinvestigatedtheroleof
Accepted:August13,2015 thechimpanzeeAPOBEC3genesinprovidingabarriertoinfectionbymostmonkeylentivi-
ruses.WefoundthatmostSIVVifs,includingViffromSIVwrcinfectingwestern-redcolobus,
Published:September22,2015
thechimpanzee’smainmonkeypreyinWestAfrica,couldnotantagonizechimpanzee
Copyright:©2015Etienneetal.Thisisanopen
APOBEC3G.Moreover,chimpanzeeAPOBEC3D,aswellasAPOBEC3FandAPO-
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits BEC3H,providedadditionalprotectionagainstSIVVifantagonism.Consequently,lentiviral
unrestricteduse,distribution,andreproductioninany replicationinprimarychimpanzeeCD4+Tcellswasdependentonthepresenceofalenti-
medium,providedtheoriginalauthorandsourceare
viralvifgenethatcouldantagonizechimpanzeeAPOBEC3s.Finally,byidentifyingand
credited.
functionallycharacterizingseveralAPOBEC3genepolymorphismsinbothcommonchim-
DataAvailabilityStatement:Allrelevantdataare
panzeesandbonobos,wefoundthattheseapepopulationsencodeAPOBEC3proteins
withinthepaperanditsSupportingInformationfiles.
thatareuniformlyresistanttoantagonismbymonkeylentiviruses.
Funding:ThisworkwassupportedbyNIHgrants
AI30937(toME),R37AI050529,R01AI058715,P30
AI045008(toBHH),YerkesBaseGrantORIP/OD
P51OD011132forthechimpanzeeprimaycells,and
anamfARMathildeKrimFellowshipinBasic AuthorSummary
BiomedicalResearch(108499-53-RKGN,toLE).The
fundershadnoroleinstudydesign,datacollection Manyhumanpathogensareofzoonoticorigin,meaningtheyoriginatedinanimals.This
andanalysis,decisiontopublish,orpreparationof includesHIV-1,thecauseofthehumanAIDSpandemic,whichistheresultofcross-
themanuscript.
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 1/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. speciestransmissionsoflentivirusesfromchimpanzeesandgorillas.However,littleis
knownaboutthehostfactorsthatprovidenaturalprotectionagainstviralemergenceina
newspecies.Chimpanzees,whicharehumans’closestrelatives,harboronlyasinglelenti-
virallineage,despitetheirfrequentexposuretolentivirusesthatinfectmonkeysonwhich
theyprey.Here,weinvestigatethecapacityoftheaccessoryproteinViffromdifferentpri-
matelentivirusestoantagonizetheAPOBEC3antiviralgenefamilyfoundinchimpanzees.
WefoundthattheVifproteinfrommostmonkeylentiviruseswasnotabletoantagonize
chimpanzeeAPOBEC3G.Furthermore,otherAPOBEC3proteinsfromchimpanzeeswere
alsoresistanttoVifantagonism.Finally,weshowedthat,despitepolymorphisminthe
APOBEC3genes,commonchimpanzeeandbonobopopulationsareuniformlyresistantto
monkeylentiviralVifantagonism.Ourresultsareconsistentwiththehypothesisthatthe
hostAPOBEC3antiviralproteinsprotectchimpanzeesagainstmanyHIV-relatedviruses
commonlyfoundinmonkeys.
Introduction
AlthoughlentivirusesarewidespreadinAfricanmonkeys,therehaveonlybeenafewdocu-
mentedcasesofcross-speciestransmissionandlentiviralemergenceintohominoids[1].Chim-
panzeesareofparticularinterestbecausetheirlentivirus,SIVcpz,liesattherootofallHIV-1
infections[2].SIVcpzhasacomplexevolutionaryhistoryasitresultedfromthecross-species
transmissionandrecombinationofSIVrcmfromred-cappedmangabeysandSIVmus/mon/
gsnfromguenons[3,4].However,onlycentralandeasternchimpanzeesareinfectedby
SIVcpz,whilewesternandNigerian-Cameroonianchimpanzeesaswellasbonobosseemcur-
rentlyfreeofanylentiviralinfection[1,5,6].Thefactthatchimpanzeesareinfectedbyonlya
singlelentivirallineageissurprisinggiventhattheyareexposedtoSIVsthatarepresentathigh
prevalenceintheirmonkeyprey[6,7].Moreover,therehavebeenmultipleviralcross-species
transmissionsofsimianfoamyvirus(SFV)andsimianT-lymphotropicvirus(STLV)tochim-
panzeesfromtheirmainprey,thewestern-redcolobus[8–10],yet,noinfectionwiththismon-
keyspecies’lentivirus,SIVwrc,hasbeendocumentedinchimpanzees[6,7].Overall,this
suggeststhattherearehostfactors,ratherthansolelyepidemiologicalorecologicalbarriers,
thatprotectchimpanzeesagainsttheemergenceofnewlentiviralinfections.
TherehavebeenfourindependenttransmissionsofHIV-1intohumansthatoriginated
fromSIVcpz;twoofthesetransmissionshadtheirimmediatesourceinchimpanzees(HIV-1
groupsMandN),whiletwootherspassedthroughgorillasbeforeinfectinghumans(HIV-1
groupsOandP)[2,11].HIV-2,ontheotherhand,istheresultofcross-speciestransmissions
ofSIVsmmfromsootymangabeystohumans[2].WhileSIVsmmhasjumpedtohumanson
overnineindependentoccasions,neithertheequivalentSIVsmminfectionofchimpanzeesnor
anyotherSIVotherthantherecombinantvirusthatgaverisetoSIVcpzhasbeenreportedin
apes.Aschimpanzeesaretheclosestrelativesofhumans,themechanismsgoverningtheirsus-
ceptibilityorresistancetolentiviruseshavedirectrelevanceforthepotentialofadditionalpri-
matelentivirusestoadapttohominoidsandsubsequentlyspreadinhumans.
Hostrestrictionfactorsareintrinsicblockstoviralreplication[12,13].Therefore,tocom-
pletetheirlifecycle,virusesencodeantagoniststhattargettheseinnateimmunefactors.These
antagonisticrelationshipshaveledtogeneticconflictsdrivingtheevolutionandspecificitiesof
virus-hostinteractions,whichmayimposepotentspeciesbarrierstocross-speciestransmission
[12].APOBEC3Gisoneoftheantiviralproteinsthathavebeenimplicatedinthespecies-speci-
ficityoflentiviruses,althoughtheroleofAPOBEC3Gasaspeciesbarrierhasbeenmainly
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 2/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
investigatedinexperimentalcross-speciestransmissions[14–17].Moreover,thereareatleast
fourgenesfromtheAPOBEC3genefamily(APOBEC3D,F,G,andH)thatpotentlyblockthe
lentivirallifecycleintheabsenceofthespecificviralantagonistVif(reviewedin[18,19]).Vif
primarilycounteractstheAPOBEC3sbybindingthehostproteinandtargetingitforproteaso-
maldegradationbyrecruitinganE3ubiquitinligasecomplex.Althoughvifishighlydiverse
withinandbetweenSIVandHIVlineages,itispresentinallprimatelentivirusesandhasan
ancientandconservedroleinantagonizingthehostAPOBEC3Gprotein[20].
Here,weexaminedwhychimpanzeesharboronlyasingleSIVlineagedespitebeingfre-
quentlyexposedtovariousSIVsthatinfecttheirpreyspecies.WeshowthatViffromdiverse
lentivirusesisincapableofantagonizingchimpanzeeAPOBEC3G.Moreover,additionalchim-
panzeeAPOBEC3familymembers,especiallyAPOBEC3D,alsoprovideblockstolentiviral
replication.Consequently,wefindthatthepotentialofalentivirustoreplicateinprimary
chimpanzeeCD4+TcellsisgovernedbyitsaccessoryproteinVif.Ourdatasuggestthatreten-
tionandevolutionoftheAPOBEC3family,whereseveralhostproteinsareantagonizedbya
singleviralproteinatdifferentmotifs,setupadiversebattlegroundagainstviruses,whichmay
overallenhancetheprotectionofthehostagainstviralemergence.Finally,weshowthatthe
APOBEC3genesarepolymorphicincommonchimpanzeesandbonobos,butthatthepopula-
tionsaresimilarlyresistanttolentiviruseswithvariousvif.Overall,weproposethattherestric-
tionimposedbytheAPOBEC3familyofhostrestrictionfactorsisacrucialmechanismby
whichcommonchimpanzeesandbonobosmaybenaturallyprotectedagainstmostlentiviral
cross-speciestransmissions.
Results
Viffrommostmonkeylentivirusesdonothavethecapacityto
antagonizechimpanzeeAPOBEC3G
Chimpanzeeshaveoverlappingrangeswithmanymonkeys[21]thatarewidelyandcommonly
infectedbylentiviruses[1].EachSIVbearsalineage-specificvifwhosesequencevariesgreatly
betweenlentivirallineages(Fig1A).ItwaspreviouslyshownthattheinclusionofSIVmacvif
inHIV-1isnecessarytoexperimentallygenerateasimian-tropicHIV-1inrhesusmacaques
[14,15].Moreover,inpopulationsofAfricangreenmonkeys(AGMs)naturallyinfectedby
SIVs,vifco-evolvedwithAPOBEC3GpolymorphismsinthedifferentAGMspeciestomain-
tainantagonism[17].Inaddition,wepreviouslyfoundthatadaptationofSIVcpztochimpan-
zeesinvolvedtheevolutionofthevifgenetoadaptandcounteractchimpanzeeAPOBEC3G
[4].Therefore,todeterminewhetherAPOBEC3Gcouldberesponsibleforthelackoftransmis-
sionofdiverseSIVstochimpanzees,wetestedanextendedpanelofVifsfromtenlentivirallin-
eagesthatspansthediversityofprimatelentivirusesforitsabilitytoantagonizechimpanzee
APOBEC3G(Fig1A).ThispanelincludedvifgenesfromSIVsthatinfectknownpreysof
chimpanzees(e.g.SIVwrcfromwestern-redcolobus[6,7]).EachSIVvifgenewasclonedinto
anHIV-1backboneinplaceofHIV-1vifaspreviouslydescribed[20].ThecapacityofVifto
antagonizechimpanzeeAPOBEC3Gwasmeasuredinsingle-roundinfectivityassaysbyco-
transfectingtheHIV-1proviruscontaininganSIVvifgenewithaplasmidencodingchimpan-
zeeAPOBEC3G[22].Thesupernatantwasnormalizedforp24gagexpressionandusedtoinfect
aTcellline,SupT1.TheHIV-1provirusencodedadefectiveenvelopegeneandwaspseudo-
typedwithVSV-Gsothatonlyoneroundofinfectionwasassayedandtheprovirusexpressed
aluciferasegeneusedasthereadout.
IntheabsenceofVif,chimpanzeeAPOBEC3Gwasabletoblocklentiviralinfection(Fig1B,
whitebar).Aspositivecontrol,thiswasrescuedbySIVcpzVif,whichfullyantagonizedchim-
panzeeAPOBEC3G(Fig1B,blackbars,SIVcpzPtsandSIVcpzPttVifs)[4].BytestingtheVif
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 3/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
Fig1.APOBEC3GprotectschimpanzeesfrommostSIVcross-speciesinfections.A,PhylogeneticanalysisofVifproteinsfromdifferentprimate
lentivirusesasdescribedintheMethods.B,Single-roundinfectivityassayperformedinthepresenceorabsenceofchimpanzeeAPOBEC3G;infectivityin
theabsenceofAPOBEC3Gwasnormalizedto100%.Thegraphsshowtheinfectivityvaluesfortheaverageofsixtonineinfections;errorbarsindicatethe
SDfromthemeanofthesereplicates.TheinfectivityofHIV-1ΔVif(white,negativecontrol),andHIV-1ΔVifΔEnvLuc2plasmidwithviffromSIVcpzPtsTan3or
SIVcpzPttGab1(black,positivecontrols),orviffromSIVsfromthegivenprimatespecies(greybars)weretested.EachoftheVifproteinswasfullycapableof
antagonizingatleastoneAPOBEC3proteinusingidenticalproviralexpressionconstructstothoseinFig1B([4,17,20]andFig2).
doi:10.1371/journal.ppat.1005149.g001
proteinfromeightdifferentSIVcpzisolates,wefoundthatallofthemwerealsoabletoantago-
nizechimpanzeeAPOBEC3G(S1BFig).However,ViffromotherSIVlineageshaddifferential
capacitiestoantagonizechimpanzeeAPOBEC3G(Fig1B,greybars).Aspreviouslyshown[4],
ViffrombothSIVrcmandSIVmuswereabletopartiallyrestoreinfectivityinthepresenceof
chimpanzeeAPOBEC3G(17–25%rescueofinfectivity),whichmayhavefacilitatedtheirevo-
lutionandadaptationtochimpanzees.Amongsttheothermonkeylentiviralvifgenes,allbut
onelackedthecapacitytorescueviralinfectioninthepresenceofchimpanzeeAPOBEC3G
(lessthan4%infectivityrelativetoSIVcpzVif)(Fig1B).Inparticular,theViffromSIVwrcwas
unabletocounteractchimpanzeeAPOBEC3Grestriction(Fig1B).Thisresultalonemay
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 4/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
Table1. AllvirusesthatnaturallyjumpedthespeciesbarrierhadsomecapacitytoantagonizethenewspeciesAPOBEC3G. SensitivityofAPO-
BEC3G(fromtherecipienthostspecies)totheSIVVifprotein(fromthevirusthatcrossedthespeciesbarrier);infectivityofvirusesproducedinthepresence
ofAPOBEC3Gisreportedasapercentage,relativetoinfectivityintheabsenceofAPOBEC3G(100%).
SIVVifIsolatesa APOBEC3Gb Relativeinfectivityofvirusesc Originof...d Referencee
SIVrcm Chimpanzee 17% SIVcpz Fig1B
SIVmus Chimpanzee 25% SIVcpz Fig1B
SIVcpz Human 73% HIV-1 Etienneetal.2013
SIVcpz Gorilla Minimal SIVgor Letkoetal.2013,D’Arcetal.2015
SIVgor Human ~85% HIV-1 Letkoetal.2013
SIVsmm Macaques 90% SIVmac Comptonetal.2013
SIVsmm Human 100% HIV-2 Comptonetal.2013
SIVver Baboon 86% SIVver-bab S2Fig
SIVsab Patas 71% SIVsab-pat S2Fig
a,Virusfromwhichthevifgenewastaken.Vifproteinsarefromvirusesthatcrossedtoanewhostspecies.
b,HostspeciesfromwhichAPOBEC3Gwastaken.APOBEC3Gproteinsarefromrecipienthostspecies.
c,InfectivityofviralconstructsproducedinthepresenceofAPOBEC3G(relativetonoAPOBEC3G).
d,Virusthatresultedfromthecross-speciestransmissionevent(s).
e,References:Etienneetal.2013[4],Comptonetal.2013[20],Letkoetal2013[56],D’Arcetal.2015[11],othersaredatafromthisstudy(Figs1Band
S2).
doi:10.1371/journal.ppat.1005149.t001
explainwhychimpanzeesarenotinfectedbySIVsfromoneoftheirmostcommonprey,the
western-redcolobus.
Ascontrols,wealsoexaminedtheVif-APOBEC3Gantagonismintwocasesofknown
cross-speciestransmissions,thatofSIVagm.ver(orSIVver)intobaboons[23]andSIVagm.sab
(orSIVsab)intoPatasmonkeys[24].Inbothcases,theViffromthedonorspecies(i.e.SIVver
VifandSIVsabVif)wascapabletoovercometheAPOBEC3Goftherecipientspecies(i.e.
baboonsandPatasmonkeys)aswellasitovercametheAPOBEC3Gofitsnaturalhost(S2
Fig).Atabulationofothercross-speciestransmissionsofprimatelentiviruses(Table1)indi-
catesthatallknownnaturalhostswitchesthatoccurredwerefromprimatelentivirusesthat
hadsomeorfullcapacitytoantagonizetheirnewhostAPOBEC3G(Table1andS2Fig).This
suggeststhatatleastpartialantagonismofAPOBEC3Gmaybeapre-requisitetonatural
cross-speciesinfection.Ontheotherhand,APOBEC3Gantagonismisnotsufficienttoallow
cross-speciestransmission,asotherbarriersmaybeinvolved.Forexample,SIVsmmVifwas
theonlymonkeylentiviralproteinabletocompletelyantagonizechimpanzeeAPOBEC3G
(Fig1B),showingthatAPOBEC3Gcannotexplainthelackofinfectionofchimpanzeepopula-
tionswiththisvirus(seebelow).Insummary,thelackofantagonismofAPOBEC3Gbylenti-
viralVifscouldexplainthelackofSIVemergenceintochimpanzeesofmost,butnotallSIVs
frommonkeys.
TheVifproteinsfrommostmonkeySIVspoorlyantagonizetheantiviral
activityofchimpanzeeAPOBEC3proteins
BecauseotherAPOBEC3genesmayalsobeimplicatedintheVif-dependentrestrictionoflenti-
viruses,wetestedifvifgenesfromthevariousSIVlineagesarecapableofantagonizingchim-
panzeeAPOBEC3D,APOBEC3F,andAPOBEC3H.APOBEC3Disofparticularinterest,
becausethechimpanzeeversionofthisproteinishighlyactiveagainstlentiviruses,whilethe
humanversionhaslesssuchactivity[25].Usingthesingle-roundassaywhereexogenousAPO-
BEC3genesaretransfectedinto293TcellsasdescribedinFig1B,weconfirmedthatinthe
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 5/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
absenceofVif,chimpanzeeAPOBEC3DismorepotentthanthehumanAPOBEC3Din
restrictingalentivirus(S3AFig,delta-Vifcondition;similarexpressionlevelofchimpanzee
andhumanAPOBEC3Dinadose-dependentmanner,S3BFig)[25].Despitetheincreased
activityofchimpanzeeAPOBEC3Drelativetothehumanprotein,theVifproteinfromtwo
divergentSIVcpzisolateswasabletoantagonizechimpanzeeAPOBEC3Dandtorescueviral
infection(Fig2A,blackbars).Ontheotherhand,theVifproteinfromsixmonkeySIVline-
ages,includingSIVwrcVif,wasnotabletoantagonizechimpanzeeAPOBEC3D(Fig2A).
Moreover,itwasalsonotablethat,althoughitcouldreadilyantagonizechimpanzeeAPO-
BEC3G,SIVsmmVifwasonlypoorlyactiveagainstchimpanzeeAPOBEC3D(Fig2A,15%
capacityversusSIVcpzVif).
ChimpanzeeAPOBEC3FandAPOBEC3Halsoreducedlentiviralinfectivityintheabsence
ofVif,althoughAPOBEC3FrestrictionwasnotasstrongastheotherAPOBEC3s(Fig2Band
2C,whitebars).MostmonkeySIVlineagesencodeaVifthathadamoderateactivityagainst
chimpanzeeAPOBEC3F(Fig2B),whilemostVifswerefullyequippedtoantagonizechimpan-
zeeAPOBEC3H(Fig2C,levelsofinfectivitysimilartoSIVcpzVif).Thissuggeststhatchim-
panzeeAPOBEC3HandAPOBEC3FontheirownmaynotbemajorspeciesbarrierstoSIVs
ingeneral,althoughsomeSIVsweremoreefficientthanothersatantagonizingthechimpanzee
APOBEC3Finparticular.
Overall,onlytheVifproteinfromSIVcpzwasabletoantagonizealltestedmembersofthe
chimpanzeeAPOBEC3family.TheantagonismofSIVcpzVifcorrespondstodecreasesinthe
levelsofchimpanzeeAPOBEC3D,F,G,andH(S4Fig),consistentwiththeknownmecha-
nismsofVif-mediateddegradationofAPOBEC3proteins[18].TheproteinViffromdifferent
lentivirallineageshaddifferentspecificitiesforagivenAPOBEC3substrate(Fig2D,readthe
heatmapvertically).Forexample,chimpanzeeAPOBEC3GcouldbeantagonizedbySIVsmm,
butnotbySIVwrcorotherSIVs(Fig2D).Importantly,wealsofoundthatagivenvifhaddif-
ferentspecificitiesamongsttheAPOBEC3genes(Fig2D,readtheheatmaphorizontally).
Indeed,whilemostoftheVifproteinsretainedthecapacitytoantagonizechimpanzeeAPO-
BEC3FandAPOBEC3H,onlyafewofthemwerecapabletocounteractchimpanzeeAPO-
BEC3DandAPOBEC3G(Fig2D).Thissuggeststhattheevolutionandretentionofmultiple
antiviralAPOBEC3proteinsinchimpanzeesprovideapotentrestrictiontoabroaddiversity
oflentiviruses,andthereforelikelyconferanadvantagetothespeciesagainstcross-species
infections.OurdataalsoshowthatthevariousSIVlineageshavedifferentsusceptibilityto
chimpanzeerestrictionfactors.Forexample,SIVsmmstrainswouldbeexpectedtoadaptand
antagonizechimpanzeeAPOBEC3proteinsmorereadilythanstrainsofSIVwrc,whichwould
needtoadapttoantagonizethreeAPOBEC3members.Indeed,SIVwrcwasunabletocause
degradationofchimpanzeeAPOBEC3G,F,andDproteins(S4Fig).Hence,inadditiontothe
strongbarrierconferredbyAPOBEC3G,otherAPOBEC3members,especiallyAPOBEC3D,
alsoposeanobstacletowardstransmissionandadaptationofdiverseSIVsharboredbymon-
keystochimpanzees.
Vif-dependentrestrictionofalentivirusinprimarychimpanzeeCD4+T
cells
WewishedtodetermineiftheVif-dependentrestrictionsobservedintheAPOBEC3over-
expressionassays(Figs1and2)couldberecapitulatedininfectionsofprimarychimpanzee
CD4+Tcells.WemodifiedtheprovirusesusedinFigs1and2byreplacingtheHIV-1env
gene,sothattheHIV-1backbonecontainingdifferentSIVvifgeneswouldbereplication-com-
petentwithanX4envelopefromHIV-1.Theadvantageofthissystem,asopposedtoinfection
ofchimpanzeecellswithdifferententireSIVs,isthatwecouldcontrolfortheotherhostfactors
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 6/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 7/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
Fig2.ChimpanzeeAPOBEC3D,APOBEC3FandAPOBEC3HalsohaveantiviralcapacitiesthatmonkeySIVVifsdifferentiallyantagonize.Single-
roundinfectivityassayperformedinthepresenceorabsenceofchimpanzeeAPOBEC3D(A),APOBEC3F(B),andAPOBEC3H(C),asdescribedinFig1B.
D,HeatmapsummarizingtheantagonisticpotentialofViffromvariouslentiviruses(shownontheleft)againstchimpanzeeAPOBEC3genes(shownatthe
top).TheintensityofthecolorcorrespondstothelevelthatagivenSIVVifcouldantagonizethecorrespondingchimpanzeeAPOBEC3protein(darkeris
moreantagonism,lighterislessantagonism).ThecolorsweredeterminedaccordingtotheinfectivityvalueofthegivenviralVifconstructrelativetothe
infectivityofthepositivecontrolbearingSIVcpzVif:lightestgreen,lessthan10%relativeinfectivity;intermediategreen,between10%and60%relative
infectivity;darkgreen,morethan60%relativeinfectivity.
doi:10.1371/journal.ppat.1005149.g002
thatmayinteractinaspecies-specificmannerwiththevirus,asonlythevifgenewasdifferent
betweenthereplication-competentviruses.Moreover,certainHIV-1strainshavebeenshown
toreplicateinprimarychimpanzeecells[26].
Becauseofthelimitingamountsofprimarychimpanzee(Pantroglodytesverus)CD4+T
cells,weusedfourHIV-1clones,eachcontainingadifferentSIVVif;twoSIVVifsthatfully
antagonizedchimpanzeeAPOBEC3Gintheover-expressionsystem(SIVcpzVif,whichserved
asapositivecontrol,andSIVsmmVif),oneVifthatpartiallyantagonizedchimpanzeeAPO-
BEC3G(SIVrcmVif),andonethatfailedtoantagonizechimpanzeeAPOBEC3G(SIVsabVif).
AnHIV-1deletedinvifservedasanegativecontrol.Thisassaydiffersintwoimportantways
fromthesingle-roundassayusedinFigs1and2.First,sincethevirusesarereplication-compe-
tent,wemeasuredp24gagproductionratherthanareportergene.Second,andmoreimpor-
tantly,viralinoculawereinitiallyproducedintransfected293Tcellsintheabsenceofany
addedAPOBEC3gene.BecauseAPOBEC3Gisactiveinthetargetcell,ratherthanthepro-
ducercell,thismeansthatthefirstroundofinfectionoftheprimarycellswillproceeduninhib-
itedbyAPOBEC3GandtheeffectsofendogenouschimpanzeeAPOBEC3Gwouldbe
observedonlyinthesecond(andsubsequent)roundsofinfection.
Allviralconstructsreplicatedwithcomparablekineticsina“permissive”Tcellline,SupT1,
whichdoesnotexpressendogenousAPOBEC3G(S5AFig)[27,28].Thisshowsthatnoneof
theviralconstructshadaninherentreplicationdefect.InprimaryCD4+Tcellsfromthree
chimpanzeedonors,wefoundthatthedelta-Vifconstructreplicatedaftertheinitialinfection
(asexpectedsinceitwasproducedincellswithoutAPOBEC3G;Fig3A,greylines).However,
itdidnotreplicatebeyondthefirsttime-pointmeasured(Fig3A,greylines).Ontheother
hand,theconstructthatencodedanSIVcpzVifreplicatedbetweenonetotwoordersofmagni-
tudebetterthanthatofthedelta-Vifconstruct(Fig3A,blackversusgreylines).Thesedata
showthatthevifgeneisessentialforefficientviralreplicationinprimarychimpanzeeCD4+T
cells.
WefoundsimilarreplicationpatternsinallthreechimpanzeeCD4+Tcellculturesamong
virusescontainingdifferentvifgenes(Fig3A).TheviralconstructcontainingSIVsabvifrepli-
catedtosimilarlevelsasthenegativecontrol(absenceofvif),showingthatSIVsabVifwasnot
activeinchimpanzeecells(Fig3A,orangeline).ThisresultisconsistentwiththedatathatSIV-
sabVifisunabletoovercomechimpanzeeAPOBEC3G(Fig1B).Ascontrol,theinfectiouspro-
viralconstructwithSIVsabVifwasfullycapableofovercomingAGMAPOBEC3Ginan
infectionassay(S6Fig).Moreover,theseresultsindicatethatoftheAPOBEC3proteins,APO-
BEC3GaloneiscapableofblockingvirusreplicationsinceSIVsabVifwasabletoantagonize
chimpanzeeAPOBEC3D,F,orH(Fig2D).
IncontrasttovirusesencodingSIVsabVif,virusesthatencodedSIVrcmVifandSIVsmm
Vifhadintermediatecapacitiestoreplicateinthechimpanzeedonorcells(Fig3A,blueand
greenlines).TheintermediatereplicationofthevirusencodingSIVrcmVif(Fig3A)isconsis-
tentwiththeintermediateabilityofSIVrcmViftoovercomechimpanzeeAPOBEC3G(Fig
1B).However,theintermediatereplicationofthevirusencodingSIVsmmVif(Fig3A)isnot
consistentwiththefullactivityofSIVsmmVifagainstchimpanzeeAPOBEC3G(Fig1B),but
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 8/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
Fig3.Vif-dependentrestrictionoflentiviralreplicationinprimarychimpanzeeCD4+Tcells.A,PrimaryCD4+Tcellsfromthreechimpanzeedonors
wereinfectedwithreplication-competentHIV-1clonescontainingeithernoVif(ΔVif)orViffromdifferentSIVlineages(SIVcpz,SIVsab,SIVrcm,orSIVsmm)
asdescribedinthemethods.ViralreplicationwasevaluatedbymeasuringHIVp24titersevery48hovera9-or10-daycourseofinfection.B,Thesame
experimentwasperformed,butcellsweretreatedwith500U/mlofIFNα(left)and100U/mlofIFNβ(right)24hpriorinfection(dataareshownhereforcells
fromdonor1;datafordonor2areshowninS5BFig).
doi:10.1371/journal.ppat.1005149.g003
couldbeexplainedbythepooractivityofSIVsmmVifagainstchimpanzeeAPOBEC3D(Fig
2Aand2D).Therefore,whiletheVif-dependentrestrictionofSIVrcmandSIVsabinprimary
chimpanzeeCD4+TcellscouldbeexplainedbyAPOBEC3G,theVif-dependentrestrictionof
SIVsmmmustbeduetoanothercellularfactor,potentiallyAPOBEC3D.
TheAPOBEC3proteinsarenotinducedbyinterferon(IFN)inactivatedhumanCD4+T
cells[27].However,otherVif-dependentpotentialrestrictionfactormightbeinducedbyIFN.
Thus,wealsoperformedtheseexperimentsinthepresenceofIFNtodetermineifwewould
seedifferentpatternsofvirusgrowth.WefoundthattreatmentofprimarychimpanzeeCD4+
TcellswithIFNαorIFNβloweredtheoverallamountoflentiviralreplication,butdidnot
changetherelativeVif-dependency(Figs3BandS5B).Thesedatasuggestthatthenon-IFN
inducedAPOBEC3proteinsarethemajorViftargetsinactivatedprimarychimpanzeeCD4+
Tcells.
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 9/24
APOBEC3asaBarriertoSIVInfectionofChimpanzees
TheAPOBEC3proteinsmediatetheirantiviraleffectsthroughthehypermutationofthe
newlysynthesizedviralgenomebytheircytidinedeaminaseactivity,aswellasotherproposed
mechanisms[19,29].AsoneofthehallmarksoftheAPOBEC3restrictionistheinductionof
G-to-Ahypermutationinviruses(reviewedin[18]),welookedforevidenceofsuchhypermu-
tationinintegratedviralgenomesninedaysafterinfectionofprimarychimpanzeeCD4+T
cells.GenomicDNAwasextractedfrominfectedcellsandtwofragments(of~1,200bpandof
~600bp)encompassingthevifregionwereamplified,cloned,andsequenced(seeMethods).
Twomethodswereusedtodeterminethesignificanceofhypermutationsignatures,Hypermut
[30]andHyperfreq[31],whichbothdeterminethatasequenceishypermutatedwhenG-to-A
mutationsinagivenhypermutation-associatedcontextaremorelikelythanmutationsina
controlcontext.ThefirstmethodusestheFisherexacttest,whilethesecondoneusesaBayes-
ianapproachandcanevaluatethestrengthofvarioushypermutationcontexts[31](see
Methods).
WefoundthattheviralconstructthatlackedavifgeneaccumulatedmanyG-to-Amuta-
tionsinitsgenome(G-to-Amutationrateof0.65%,versus0.05%forothermutations;Table2,
ΔVifcolumn)and50%ofthesequenceswerefoundtobesignificantlyhypermutatedinan
APOBEC3-context(p<0.05)(Table2).ThesemutationsoccurredprimarilyintheGGcontext,
whichischaracteristicofAPOBEC3Gactivity(85%ofthemutationswereintheGGcontext
and,usingHyperfreq,hypermutationofsequenceswasmostfrequentlyassociatedwiththeGG
context,Table2).However,G-to-AmutationsalsooccurredintheGAcontext,whichmaybe
asignatureofAPOBEC3F,APOBEC3D,and/orAPOBEC3Hactivity(Table2)[25,32].Incon-
trast,theviralconstructthatexpressedtheSIVcpzVifhadnoevidenceofhypermutation
(Table2,cpzVifcolumn).ThissuggeststhattheexpressionoftheAPOBEC3proteinsinacti-
vatedprimarychimpanzeeCD4+Tcellswasabletohypermutatetheviralgenomeinthe
absenceofVifantagonism,withAPOBEC3Gbeingthemaindriver,andthatSIVcpzVifcould
counteractthisAPOBEC3-mediatedhypermutation.
Table2. G-to-AhypermutationsignaturesinviralgenomesafterninedaysofinfectioninchimpanzeeprimaryCD4+TcellsaredependentonVif.
PrimaryCD4+Tcellsfromthechimpanzeedonor1wereinfectedwithreplicationcompetentvirusesHIV:ΔVif,sabVif,rcmVif,smmVif,orcpzVif.Cellswere
harvestedafterninedaysofinfectionandgenomicDNAwasextracted.Viralfragmentsandcloneswereretrievedasdescribedinthemethods.Sequences
wereanalyzedforG-to-A(“G>A”)hypermutationsignificanceusingHypermut[30]andHyperfreq[31],asdescribedinthemethods.
ΔVif sabVif rcmVif smmVif cpzVif
Sequencedclones 16 9 21 25 21
Totalbpsequenced 9169 7688 21446 27577 23891
G>AinGGcontexta 51 62 2 1 0
G>AinGAcontext 8 0 6 1 1
TotalnumberofG>A 60 62 10 2 1
G>Amutationrate(%) 0.65 0.81 0.05 0.01 0.00
IntactVifORFb NA 2/9 all all all
Hypermut:Hypermutantclonesp<0.05c 6/16 6/9 0/21 0/25 0/21
Hyperfreq:Hypermutantclonesd(strongestpattern)e 8/16(7GG,1GR) 6/9(6GG) 2/21(2GA) 0/25 0/21
Othermutations 5 5 14 13 15
Othermutationrate(%) 0.05 0.07 0.07 0.05 0.06
a,numberofG-to-AmutationsintheGGcontext
b,numberofintactVifopenreadingframe(ORF),NA,notapplicable
c,Numberofclonesthataresignificantlyconsideredashypermutant(p<0.05)usingHypermut2.0[30]
d,Numberofclonesthatthatwereconsideredaspositiveforhypermutationatthesignificancelevelof0.05[31]
e,Strongestpattern,patterninwhichtheevidenceofhypermutationappearedtobethestrongest[31].
doi:10.1371/journal.ppat.1005149.t002
PLOSPathogens|DOI:10.1371/journal.ppat.1005149 September22,2015 10/24