Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap ...

27 Pages·2016·9.61 MB·English

RESEARCHARTICLE
Systems Biology-Based Investigation of
Cellular Antiviral Drug Targets Identified by
Gene-Trap Insertional Mutagenesis
FeixiongCheng1,2☯¤a*,JamesL.Murray3☯¤b,JunfeiZhao4,JinsongSheng5,
ZhongmingZhao2,4,6,7*,DonaldH.Rubin5,8,9*
1 StateKeyLaboratoryofBiotherapy/CollaborativeInnovationCenterforBiotherapy,WestChinaHospital,
WestChinaMedicalSchool,SichuanUniversity,Chengdu610041,Sichuan,China,2 Departmentof
BiomedicalInformatics,VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee,UnitedStatesof
a11111 America,3 Zirus,Incorporated,Buford,Georgia,UnitedStatesofAmerica,4 CenterforPrecisionHealth,
SchoolofBiomedicalInformatics,TheUniversityofTexasHealthScienceCenteratHouston,Houston,
Texas,UnitedStatesofAmerica,5 DivisionofInfectiousDisease,DepartmentofMedicine,Vanderbilt
UniversitySchoolofMedicine,Nashville,Tennessee,UnitedStatesofAmerica,6 DepartmentofPsychiatry,
VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee,UnitedStatesofAmerica,7 Departmentof
CancerBiology,VanderbiltUniversitySchoolofMedicine,Nashville,Tennessee,UnitedStatesofAmerica,
8 DepartmentofPathology,MicrobiologyandImmunology,VanderbiltUniversitySchoolofMedicine,
Nashville,Tennessee,UnitedStatesofAmerica,9 ResearchMedicine,VeteransTennesseeValley
HealthcareSystem,Nashville,Tennessee,UnitedStatesofAmerica
OPENACCESS
☯Theseauthorscontributedequallytothiswork.
Citation:ChengF,MurrayJL,ZhaoJ,ShengJ,Zhao
¤a Currentaddress:CenterforCancerSystemsBiology,Dana-FarberCancerInstitute,HarvardMedical
Z,RubinDH(2016)SystemsBiology-Based
School,Boston,MA02215,USA,andCenterforComplexNetworksResearch,NortheasternUniversity,
InvestigationofCellularAntiviralDrugTargets
Boston,MA02115,USA.
IdentifiedbyGene-TrapInsertionalMutagenesis. ¤b Currentaddress:GeneTAGTechnology,Inc.,Atlanta,Georgia,USA.
PLoSComputBiol12(9):e1005074.doi:10.1371/ *[email protected](FC);[email protected](ZZ);[email protected](DHR)
journal.pcbi.1005074
Editor:GregTucker-Kellogg,NationalUniversityof
Singapore,SINGAPORE Abstract
Received:April5,2016
Virusesrequirehostcellularfactorsforsuccessfulreplication.Acomprehensivesystems-
Accepted:July22,2016
levelinvestigationofthevirus-hostinteractomeiscriticalforunderstandingtherolesofhost
Published:September15,2016
factorswiththeendgoalofdiscoveringnewdruggableantiviraltargets.Gene-trapinser-
Copyright:Thisisanopenaccessarticle,freeofall tionalmutagenesisisahigh-throughputforwardgeneticsapproachtorandomlydisrupt
copyright,andmaybefreelyreproduced,distributed,
(trap)hostgenesanddiscoverhostgenesthatareessentialforviralreplication,butnotfor
transmitted,modified,builtupon,orotherwiseused
hostcellsurvival.Inthisstudy,weusedlibrariesofrandomlymutagenizedcellstodiscover
byanyoneforanylawfulpurpose.Theworkismade
availableundertheCreativeCommonsCC0public cellulargenesthatareessentialforthereplicationof10distinctcytotoxicmammalian
domaindedication. viruses,1gram-negativebacterium,and5toxins.Wehereinreported712candidatecellular
DataAvailabilityStatement:Allrelevantdataare genes,characterizingdistincttopologicalnetworkandevolutionarysignatures,andoccupy-
withinthepaperanditsSupportingInformationfiles. ingcentralhubsinthehumaninteractome.Cellcyclephase-specificnetworkanalysis
Funding:ThisworkwassupportedbytheNational showedthathostcellcycleprogramsplayedcriticalrolesduringviralreplication(e.g.MYC
NaturalScienceFoundationofChina(81573020). andTAF4regulatingG0/1phase).Moreover,theviralperturbationofhostcellularnetworks
ThisworkwasalsopartiallysupportedbyNational reflecteddiseaseetiologyinthathostgenes(e.g.CTCF,RHOA,andCDKN1B)identified
InstitutesofHealth(NIH)grants(R01LM011177)to
werefrequentlyessentialandsignificantlyassociatedwithMendelianandorphandiseases,
ZZ.Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or orsomaticmutationsincancer.Computationaldrugrepositioningframeworkviaincorporat-
preparationofthemanuscript. ingdrug-genesignaturesfromtheConnectivityMapintothevirus-hostinteractomeidenti-
CompetingInterests:JLMwaspreviouslyemployed fied110putativedruggableantiviraltargetsandprioritizedseveralexistingdrugs(e.g.
byZirus,IncandiscurrentlyemployedbyGeneTAG
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
Technology,Inc.Theauthorsconfirmtheyhaveno ajmaline)thatmaybepotentialforantiviralindication(e.g.anti-Ebola).Insummary,this
competinginterests. workprovidesapowerfulmethodologywithatightintegrationofgene-trapinsertionalmuta-
genesistestingandsystemsbiologytoidentifynewantiviraltargetsanddrugsforthedevel-
opmentofbroadlyactingandtargetedclinicalantiviraltherapeutics.
AuthorSummary
Infectiousdiseasesresultinmillionsofdeathsandcostbillionsofdollarsannually.Hence,
thereisurgencyfordevelopingmoreinnovativeandeffectiveantiviraltherapeutics.Inthis
study,weusedlibrariesofrandomlymutagenizedcellstodiscovercellulargenesthat
areessentialforthereplicationof10distinctcytotoxicmammalianviruses.Weherein
reportedover700candidatecellulargenes,over20%ofwhichwereindependentlyselected
bymultiplevirusesinoneormorecelltypes.Usingsystemsbiology-basedanalysis,we
foundthathostgenesassociatedwithviralreplicationtendedtooccupycentralhubsin
thehumanproteininteractomeandtobeancientgeneswithlowevolutionaryrates,com-
paredtonon-virus-associatedgenes.Cellcyclephase-specificsub-networkanalysis
showedthathostcellcycleprogramplayedimportantrolesduringviralreplicationbyreg-
ulatingspecificcellcyclephases.Moreover,wepresentednovelevidencestosuggestthat
hostgenessupportingviralreplicationwerefrequentlyimplicatedinMendelianand
orphandiseases,orplayedcriticalrolesincancer.Importantly,wefoundapproximately
110newputativedruggableantiviraltargetsbymerginggenome-widegene-trapinser-
tionalmutagenesis,drug-genenetwork,andbioinformaticsdata.Furthermore,wehave
demonstratedtheuseofacomputablerepresentationofgenetictestingtoeffectivelyiden-
tifynewpotentialantiviralindicationsforexistingdrugs.Insummary,thisstudypresents
newandimportantmethodologiesfordevelopingbroadlyactiveantiviraltherapeutics.
Introduction
Infectiousdiseasesresultinmillionsofdeathsandcostbillionsofdollarsannually[1].Asof
2012,35.3millionpeopleworldwidewerelivingwithhumanimmunodeficiencyvirus(HIV),
andanestimated1.6millionacquiredimmunodeficiencysyndrome(AIDS)-relateddeaths
werereportedin2012[2].InMarch2014,theWorthHealthOrganizationreportedamajor
EbolavirusoutbreakinthewesternAfricannationofGuinea.AsofMarch25,2015,over
26,000suspectedEbola-infectedcaseshadbeenidentified,withover10,000deaths,andthese
numbersmaybevastlyunderestimated[3].InfectionsbytheEbolaandMarburgfiloviruses
causearapidlyfatalhemorrhagicfeverinhumansforwhichnoapprovedantiviralagentsare
available[4–6].Traditionalantiviraldrugdiscoverypipelineshaveyieldednotablesuccessesin
recentyears.However,twofactorscontinuetoprovidecommercialandmedicalincentivesfor
developingmoreinnovativeandeffectiveantiviraltherapeutics,namelythepropensityof
virusestodevelopdrugresistanceandthesideeffectscausedbyantiviralagents[7,8].With
fasterdevelopmenttimes,increasedsafety,anddecreasedpharmacokineticuncertainty,the
prospectofdrugrepositioning(findingnewindicationsforexistingFDA-approveddrugs)is
emergingasapromisingalternativetotraditionaldrugdesignandoffersanimprovedrisk-
benefittrade-offincombatinginfectiousdiseases[9–11].
Virusesrequirehostcellularfactorsforsuccessfulreplication.Acomprehensivesystems-
levelinvestigationofthevirus-hostinteractomeiscrucialforunderstandingtherolesofhost
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
factorswiththeendgoalofdiscoveringnewdruggableantiviraltargets[8,12].Inthisregard,
quantitativetemporalviromics[13]andviralopenreadingframes[14]canbeusefulinstudy-
ingthevirus-hostinteractome[12–15].However,theincorrectassignmentofbiologicalactivi-
tiestoviralandhostfactors,andthelimitedscaleofexperimentaltechniqueshavelimited
theseapproaches[8,16].Gene-trapinsertionalmutagenesisisapromisingapproachforeluci-
datinghostcellularnetworkperturbationsduringviralreplication[17,18].Gene-trapinser-
tionalmutagenesisisahigh-throughputforwardgeneticsapproachtorandomlydisrupt(trap)
hostgenesanddiscovercellulargenesthatareessentialforviralreplication,butnotforhost
cellsurvival[19].Thisapproachisbasedontwoimportantprinciples:(i)viralinfectionmust
betoxictothechosenhostcellline,and(ii)disruptingagenecriticalforcompletingtheviral
lifecycleconferssurvivabilityduringsubsequentviralselection,providedthatthehostcellcan
survivefollowingreducedorabolishedexpressionofthemutagenizedgene.
Inthisstudy,weincorporatedgene-trapinsertionalmutagenesis,knowndrug-genesigna-
tures,andbioinformaticsanalysisintoanintegratedantiviraldrugdiscoverypipeline(Fig1).
Weusedlibrariesofrandomlymutagenizedcellstodiscoverhostcellulargenesthatareessen-
tialforthereplicationof10distinctcytotoxicmammalianviruses,1gram-negativebacterium,
and5bacterialtoxins.Wepresentnovelevidencetosuggestthathostgenessupportingviral
infectionarefrequentlyimplicatedinMendelianandorphandiseases,orplayrolesincancer.
Furthermore,weidentifiedantiviraltargetsthatarelikelytobeinhibitedbyknowndrugs,
allowingustopredictseveralnewantiviralindications(i.e.,anti-Ebola)forexistingdrugs.In
summary,wepresentapowerfulapproachforidentifyingpotentialdruggabletargetsandexist-
ingdrugswithgoodpharmacokineticsprofilesforthedevelopmentofbroadlyactiveantiviral
therapeutics.
Results
Expandingtheknownvirus-hostinteractomebygene-trapinsertional
mutagenesis
Anintegratedantiviraldrug-discoverypipelinewasdevelopedthatinvolvesgene-trapinser-
tionalmutagenesis,consolidateddrug-genesignatures,andbioinformaticsanalysistorank
candidateantiviraltargetsandidentifypotentialantiviralindicationsforexistingdrugs(Fig1).
Specifically,weusedgenome-widegene-trapinsertionalmutagenesistoidentifynewvirus-
hostinteractionsbythefollowing6steps:(i)randomintegrationofaninsertionalmutagen
shuttlevectorcontainingapromoterlessneomycin-resistancegene;(ii)neomycinselectionof
cellsexpressingneomycinaminotransferase;(iii)cytotoxicviralinfection;(iv)resistancecon-
firmationbyre-infectingsurvivingclonesata10-foldhighermultiplicityofinfection(MOI);
(v)shuttlevectorrecoveryfromresistantclones(genomicDNAdigestion,self-ligation,bacte-
rialtransformation,andampicillinselection);and(vi)sequencingoftrappedgenesfrombacte-
rialcolonies(Fig1Aand1B).Inthismanner,weidentifiedapproximate700candidatehost
genes(Fig1CandS1Table)mediatingthecytotoxiceffectsof10viruses(cowpoxvirus,Ebola
virus,HIV-1,Herpessimplexviruses(HSV)-1andHSV-2,Marburgvirus,poliovirus(Polio),
reovirus,andrhinovirus-2and-16),ofwhich20%wereidentifiedinstudieswithmultiple
virusesinoneormorecelltypes.Followingthesamegeneralmethodforgene-trapstudiesout-
linedabove,wealsoidentified97hostgenes(Fig1DandS1Table)mediatingthelyticeffects
ofFrancisellatularensis(tularensis)and5toxins(ClostridiumdifficileTcdBtoxin,Clostridium
perfringensεtoxin,Helicobacterpylorivacuolatingtoxin,Staphylococcusaureusαtoxin,and
ricintoxin).Encouragedbythesefindings,wethendevelopedasystemsbiology-basedpipeline
tocharacterizethecandidatecellularantiviraltargetsthroughnetworkapproachesandbioin-
formaticsanalysis.Finally,wecomputationallypredictedseveralnewantiviralindicationsfor
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
Fig1.Diagramoftheintegrativeantiviraldrugdiscoverypipeline.(A)Thegene-trapinsertionalmutagenesisapproachemploysanMMLV-
basedshuttlevectorthatrandomlyintegratesintohostcellchromosomesandcontainsapromoterlessneomycin-resistancegene.Shuttle
vectorintegrationbetweenahost-cellpromoterandanearlyexondisrupts(traps)thegene,allowingneomycinselectionandderivationofa
gene-traplibrary.(B)Hostgenesmediatingthetoxiceffectsoflyticviralreplicationorexposuretotoxinswereidentifiedby:(i)selectinggene-
traplibrariesinneomycin;(ii)exposinggene-traplibrarycellstoalyticvirusoratoxin;(iii)isolatingsurvivingclones;(iv)resistanceconfirmation
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
insurvivingclonesfollowingexposuretoa10-foldhigherdoseofthevirusortoxinstudied;and(v)identificationofthetrappedgenebydigesting
genomicDNAtoliberateshuttlevectors,self-ligation,bacterialtransform,ampicillinselection,andsequencingtrappedgenesintherecovered
plasmids.(C)Distributionofnewlydiscoveredvirus-hostinteractionpairsfor10viruses,1bacterium,and5toxins.(D)Globalpathogen-host
interactionnetworkidentifiedbygenome-widegene-trapinsertionalmutagenesis,wheretoxinsandbacteriaarerepresentedbyredandcyan
squaresrespectively.Thehostcellgeneproducts(circles)arecoloredbasedontheirsubcellularlocationscollectedfromtheLocDB(https://
www.rostlab.org/services/locDB/).(E)Identificationofcandidatesforantiviraldrugrepositioningapproachbyincorporatingdrug-gene
signaturesfromtheConnectivityMapintotheglobalvirus-hostinteractome.
doi:10.1371/journal.pcbi.1005074.g001
existingdrugsbyincorporatingdrug-genesignaturesfromtheConnectivityMap(CMap)[20]
intotheglobalvirus-hostinteractome(Fig1E).
Wenextplottedthe900newlydiscoveredpathogen-hostinteractionsnewlydiscoveredin
thisstudyusingtwobipartitegraphs:atoxin-hostinteractionnetwork(Fig1D)andavirus-
hostinteractionnetwork(Fig2),wherenodesrepresent712hostgenes(circles),10viruses
(orangesquares),1gram-negativebacterium(cyansquare),and5toxins(redsquares),and
whereedgesrepresentinteractionsidentifiedbygene-trapinsertionalmutagenesis.Thehost
genesaregroupedbasedonhumanproteinsubcellularlocations,suchasmembranes,thecyto-
plasm,organelles,andthenucleuscollectedfromtheLocDB[21].Adetailedlistofthesedata
isprovidedinS1Table.Identifyingtoxin-hostinteractionsmayprovideimportanttargetsfor
preventingtoxin-inducedcytotoxicityintoxin-producingbacteria.Recently,ourgroupidenti-
fiedpoliovirusreceptor-like3(PVRL3)asacellularfactornecessaryforClostridiumdifficile
TcdB-inducedcytotoxicity,usinggene-trapinsertionalmutagenesis[22].Thus,thetoxin-host
interactionsidentifiedbygene-trapinsertionalmutagenesisshowninFig1Dcouldprovide
usefulresourcesfordevelopingnovelantibiotictherapies.
Tofurtherevaluatethequalityofhostgenesidentifiedbygene-trapinsertionalmutagenesis,
wecomparedournetworktothreepreviouslyindependentnetworks.Intotal,weassembled
2,855knownvirus-hostinteractionsconnecting2,443hostgenesand55pathogensidentified
fromRNAinterference(RNAi),579hostproteinsmediating70innateimmune-modulating
viralopenreadingframes(viORFs)[14],and1,292hostgenesmediatinginfluenza-hostinter-
actionsidentifiedbyco-immunoprecipitationandliquidchromatography-massspectrometry
(Co-IP+LC/MS)[23],respectively(S1Table).Severalcriticalviralreplication-relatedpath-
ways,suchasviralmRNAtranslation,influenzaviralRNAtranscriptionandreplication,
influenzainfection,andinfluenzalifecycleweresignificantly(adjustedp-value[q]<0.01)
enrichedamongthehostgenesidentifiedinourgene-trapinsertionalmutagenesis,viROFs,
andCo-IP+LC/MSstudies,butnotintheRNAigeneset(Fig3A,S2andS3Tables).
Networkcentralityofpathogen-targetgenesinthehumanprotein
interactionnetwork
Of712hostgenesidentifiedbygene-trapinsertionalmutagenesis,therewasenrichmentfor
genesassociatedwithinnateimmunity(P=4.7×10−3,Fisher’sexacttest,S1AFig),suggesting
thattheidentifiedhostgenesetmaymediateimmuneresponses[14].Essentialgenes,whose
knockoutresultinlethalityorinfertility,areimportantforstudyingtherobustnessofabiologi-
calsystem[24].Furthermore,therewasalsoasignificantenrichmentforessentialgenes
(P=1.0×10−5,S1BFig).
Tofurtherinvestigatethebiologicalfunctionsoftheidentifiedvirus-targetgenes,wefurther
examinedtopologicalnetworkfeaturesforvirus-targetgeneproducts(proteins)inthehuman
proteininteractome.Consideringthatthecurrentpublicly-availablehumanproteininteraction
databasesarestillincomplete,weconstructed5different,yetcomplementaryhumanprotein
interactionnetworks:aglobalphysicalproteininteractionnetwork(PIN),anatomicresolution
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
Fig2.Thenewlyidentifiedvirus-hostinteractionnetworksbygene-trapinsertionalmutagenesis.Thenodes(squares)areviruses,hostcellgene
products(circles)arecoloredbasedontheirsubcellularlocationscollectedfromLocDB(https://www.rostlab.org/services/locDB/),andedges(lines)
denoteinteractionsidentifiedbygene-trapinsertionalmutagenesis.
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
Fig3.Bioinformaticsanalysisandnetworktopologicalandevolutionarycharacteristicsofhostgenesmediatingviral
replication.(A)Reactomepathwayenrichmentanalysisoffourdifferenthostcellulargenesetsidentifiedbygene-trapinsertional
mutagenesis(trappedgenes),previousRNAinterference(RNAi)screeningstudies,viralopenreadingframes(viORFs),andco-
immunoprecipitationandliquidchromatography-massspectrometry(Co-IP+LC/MS)(S1Table).(B)Boxplotsshowingthe
connectivitydistributionofvirushostgenes(red)versusnon-virus-hostgenes(lightblue)inthephysicalproteininteraction
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IdentificationofAntiviralDrugTargetsbyInsertionalMutagenesis
network(PIN)andlarge-scalecomputationallypredictedproteininteractionnetwork(CPIN).(C)and(D)Evolutionary
characteristicsofvirus-hostgenes(red)versusnon-virus-hostgenes(lightblue).(E)Nodeconnectivitydistributionofhostgenes
identifiedbygene-trapinsertionalmutagenesisandthreepublishedgenesetsandallproteins(Whole)inPIN.(F)GenedN/dS
ratiocumulativedistributionforfourdifferentgenesetsandwholehumangenome(Whole).Mya:millionyearsago.Pvaluesin
B-DwerecalculatedviaWilcoxonrank-sumtest.
doi:10.1371/journal.pcbi.1005074.g003
three-dimensionalstructuralproteininteractionnetwork(3DPIN),akinase-substrateinterac-
tionnetwork(KSIN),aninnateimmunityproteininteractionnetwork(INPIN),andabroad
contextcomputationallypredictedproteininteractionnetwork(CPIN),basedontwoprevious
studies[25,26].Fig3Bshowsthattheconnectivityofvirus-targetproteinswassignificantly
strongerthannon-virustargetproteinsinPIN(P<2.2×10−16,Wilcoxontest)andCPIN
(P<2.2×10−16),respectively.Inaddition,wedefined“hubs”asthosenodesthatrankedinthe
top20%oftheconnectivitydistribution,asdonepreviously[25,26].Wefoundthatvirus-target
proteinsweresignificantlyenrichedinhubsinall5humanproteininteractionnetworks(S4
Table).Moreover,thevirus-targetproteinsshowedatendencyforgreaterenrichmentofhubs
ininnateimmunityPIN[27]thaninCPIN(P<0.01),suggestingthattheimmunesystem
playsanimportantroleduringviralreplication,consistentwiththeenrichmentforcellular
genesassociatedwithinnateimmunityshowninS1AFig
Inaddition,weinvestigatedtheconnectivitydistributionofour712hostgeneswiththree
publishedhostgenesets.Wefoundacomparableconnectivitydistributionofour712host
geneswiththeRNAigeneset,althoughtheyweremarginallylowerintermsofsignificance
thanthatobservedwiththeviORFsandCo-PI+LC/MSgenesets(Fig3E).Theseobservations
suggestthereliabilityofgene-trapinsertionalmutagenesis,relativetoresultsobtainedusing
othertechnologiessuchasRNAi,Co-IP+LC/MS,andviORFs.
Purifyingselectionandevolutionaryoriginsofvirus-targetgenes
Toprovideinsightintotheevolutionaryfactorsunderlyingtheselectionofhostgenesusedby
viruses,weexaminedtheselectivepressureandevolutionaryratesofthevirus-targetgenes
identified.Wecomputednon-synonymousandsynonymoussubstitutionrateratios(dN/dS
ratios)usinghuman-mouseorthologousgenepairs(seeMethods).AdN/dSratioof1signifies
neutralevolution,aratioof<1indicatespurifyingselection,andaratioof>1indicates
positiveDarwinianselection.TheboxplotsinFig3Dshowthatvirus-targetgenestendto
undergopurifyingselection(i.e.,theselectiveremovalofallelesthataredeleterious)inhuman
proteinevolutionaryhistories.Moreover,virus-targetgenesdisplayedstrongerpurifying
selection(lowerdN/dSratiosandevolutionaryrateratios)thandidnon-virustargetgenes
(P<2.2×10−16,Wilcoxonrank-sumtest),asshowninFig3D.Forexample,severalgenes
withthelowestdN/dSratios(0)suchasRAB1A[28],PCBP1[29],PCBP2[30],andARF6[31]
werepreviouslyreportedtobeinvolvedinviralreplicationorantiviralsignalingpathways.
However,onlyonegene(DEFB118),whichwasalsoimplicatedinviralreplication-related
pathway[32]hadadN/dSratiolargethan1(1.1).
Theevolutionaryhistoryofaproteinsequenceoftenreflectsitsfunctionalevolution.We
nextinvestigatedtheevolutionaryoriginofvirus-targetgeneproducts.Theaveragetimeof
divergence(1348.6±20.0millionyearsago[Mya])forvirus-targetgeneproductswassignifi-
cantlylongerthanthatofnon-virustargetgeneproducts(1131.3±8.7Mya,P<2.2×10−16;
Fig3D).Furthermore,theaverageevolutionarydistanceofvirus-targetgeneproductswasalso
significantlyhigherthanthatobservedfornon-virustargetgeneproducts(P<2.2×10−16;
Fig3D).WenextcomparedthedN/dSratiodistributionforour712hostgeneswiththatof
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threepublishedhostgenesets.Comparedwithoursetof712hostgenes,asimilartrendwas
observedwiththeRNAigeneset(Fig3F).
Regulatingthehostcellcycleprogram
Mostvirusesareknowntoregulatehostcellcycleprogram[33,34].Weassembled986human
hostcellcyclegenesmediatingG0/1,S,andG2phasesfromapreviousstudy[35].Wefound
thatthe712hostgenesidentifiedbygene-trapweresignificantlyenrichedintermsofhuman
cellcyclegenes(P=6.5×10−5,Fisher’sexacttest).Wenextbuiltacellcyclephase-specific
sub-networktoexaminethecellcycleprogramingmechanismforourhostgeneset.Fig4A
showsthatseveralgenesimportantforviralreplicationalsomediateprogressionthoughG0/
G1phase,includingMYC,ARF4,SRSF3,TAF4,XPO5,andEIF5.ARF4promotesenterovirus
71replication[36],susceptibilitytoChlamydiatrachomatisandShigellaflexneri[37],andden-
gueflavivirussecretion[38].TwopreviousstudieshavesuggestedthatTAF4playscriticalroles
inherpessimplexvirustype1infection[39]andtranscriptionalactivationofEpstein-Barr
virus[40].Here,weidentifiedthatTAF4mightmediateHSV-2replicationbyregulatingG0/1
phaseidentifiedviagene-trap.Furthermore,cellcycle-specificexpressionanalysisusingCyclo-
base[41]furtherconfirmedthatTAF4regulatescellcycleinG1phaseinFig4C.MYC,encod-
ingc-Myc,isinvolvedinthereplicationofmultipleviruses,suchasEpstein-Barrvirus[42,43].
Here,wefoundthatMYCmaymediaterhinovirus-16replicationbyregulatingG0/G1phase
(Fig4Aand4B).InadditiontoG0/1phase,wealsofoundthatseveralgenes(e.g.RPL17and
RPS16)regulateSorG2phasetransition,inadditiontoviralreplication(Fig4A).RPL17,
encoding60SribosomalproteinL17importantforproteinsynthesis,playscriticalrolesinthe
replicationofseveralviruses[44],suchashepatitisCvirusandHSV-1.Collectively,these
observationsfurthersuggestedthatthehostcellcycleprogramplaysimportantrolesduring
viralreplicationbyregulatingspecificcellcyclephases.
Viralperturbationsofcellularnetworksreflectdiseaseetiology
Understandingtheinterrelationsbetweencellularhostgenestargetedbyviralproteinsanddis-
ease-susceptibilitygenesmayrevealcriticalinformationfordiseaseetiology[45,46].Weinves-
tigatedtheoverlapbetweenvirus-targetgenesandthegenesetsimplicatedinMendelian
diseases,orphandiseases,andcancer(Fig5A).Fig5Bshowsthatvirus-targetgenesaresignifi-
cantlyenrichedinMendeliandiseasegenes(MDG;P=1.9×10−7),orphandisease-mutated
genes(ODMG;P=2.4×10−5),andthoseinthecatalogueofcancergenes(CCG
P=3.0×10−51).
Apreviousstudyhassuggestedthatgenomicvariationsandtumorvirusesmightcausecan-
certhroughrelatedmechanisms[45].Thus,weexaminedhowvirus-targetgenespromote
tumorigenesisorareinvolvedincanceretiology.Wecompiled384genesthataresignificantly
mutatedincancer(cancer-drivergenes)fromseverallarge-scalecancergenomeprojects(S5
Table).Interestingly,asignificantassociation(P=7.3×10−14)wasobservedbetweenthecan-
cer-relatedgenesandgenesimplicatedinviralinfectionidentifiedbyourgene-trapstudiesand
priorRNAiscreens(Fig5B).AsshowninFig6andS5Table,26ofthe384cancerdrivergenes
wereidentifiedingene-trapstudieswithlyticviruses(suchasCTCF,RHOA,CDKN1B,and
CUX1),while66ofthe384geneswerepreviouslyidentifiedinRNAiscreens(suchasPIK3CA,
HRAS,EGFR,AKT1,andIDH1).However,theoverlapwiththecancergenesetmaybecon-
foundedbythefactsthatmultiplespeciesofcellswereusedinthisstudyandthatbothimmor-
talizationandviralinfectionperturbedcellularpathwaysrelatedtogrowth.Thisphenomenon
wasalsodiscussedinapreviousstudy[45].
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Fig4.Humancellcyclephase-specificvirus-hostgenenetwork.(A)Humancellcyclephase-specific
virus-hostgenenetworkforhostgenesidentifybygene-trapinsertionalmutagenesis.Theconciseoverview
ofcellcycleregulationforgeneMYC(B)andTAF4(C).Darkcolorrepresentshighexpressionacross
differentcellcyclephases.ImagesinBandCarepreparedbyCyclebase3.0(http://www.cyclebase.org).
doi:10.1371/journal.pcbi.1005074.g004
ThehumanCTCFgeneencodestheCTCFtranscriptionalrepressorbymediatingtranscrip-
tionalregulation,insulatoractivity,andtheregulationofchromatinarchitecture[47].Data
fromseveralrecentcancergenomeprojectsshowedthatCTCFmutationsaresignificantly
associatedwithbreastcancer[48],headandneckcancer[49],anduterinecancer[50].Interest-
ingly,CTCFisinvolvedinreovirusreplicationidentifiedbygene-trap(Fig6).Pre-clinical
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