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Virus
Research
jo u r n al hom e p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v i r u s r e s
Control
of
human
papillomavirus
gene
expression
by
alternative
splicing
Sheila
V.
Graham
∗,
Arwa
Ali
A.
Faizo
MRC-UniversityofGlasgowCentreforVirusResearch;InstituteofInfection,ImmunityandInflammation;CollegeofMedicalVeterinaryandLifeSciences, UniversityofGlasgow,GarscubeEstate,GlasgowG611QH,Scotland,UK
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received19August2016
Receivedinrevisedform8November2016 Accepted10November2016
Availableonline17November2016
Keywords: Humanpapillomavirus Lifecycle Alternativesplicing E2 SRprotein hnRNP
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HumanpapillomavirusespossesscirculardoublestrandedDNAgenomesofaround8kbinsizefrom
whichmultiplemRNAsaresynthesizedduringaninfectiouslifecycle.Althoughatleastthreeviral
pro-motersareusedtoinitiatetranscription,viralmRNAsarelargelytheproductofprocessingofpre-mRNAs
byalternativesplicingandpolyadenylation.TheHPVlifecycleandviralgeneexpressionaretightlylinked
todifferentiationoftheepitheliumthevirusinfects:thereisanorchestratedproductionofviralmRNAs
andproteins.InthisreviewwedescribeviralmRNAexpressionandtherolesoftheSRandhnRNPproteins
thatrespectivelypositivelyandnegativelyregulatesplicing.WediscussHPVregulationofsplicingfactors
anddetailtheevidencethatthepapillomavirusE2proteinhassplicing-relatedactivities.Wehighlight
thepossibilitythatHPV-mediatedcontrolofsplicingindifferentiatingepithelialcellsmaybenecessary
toaccomplishtheviralreplicationcycle.
©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/by/4.0/).
Contents
1. Introduction...84
2. Thehumanpapillomaviruslifecycle...84
2.1. Humanpapillomavirusentry...84
2.2. Humanpapillomavirusreplicationandepithelialdifferentiation ... 84
2.3. InteractionofHPVswiththeepithelium...84
3. Splicing...85 3.1. Controlofsplicing...85 3.2. Alternativesplicing ... 86 3.3. SRproteins...87 3.4. hnRNPproteins...88 4. SplicingofHPVRNAs...88
4.1. HPVgeneexpressionandsplicing...88
4.2. SRproteinscontrollingHPVgeneexpression...89
4.2.1. E6andE7splicing...89
4.2.2. EarlyRNAsplicing...90
4.2.3. LateRNAsplicing...90
4.3. hnRNPproteinscontrollingHPVgeneexpression...91
4.4. Terminalexondefinition...91
5. HPVregulationofSRproteinactivity ... 92
Abbreviations:SA,spliceacceptorsite;SD’,splicedonorsite;ESE,exonicsequenceenhancer;ESS,exonicsequencesilencer;ISE,intronicsequenceenhancer;ISS,intronic sequencesilencer;PTB,polypyrimidinetractbindingprotein;CPSF,cleavageandpolyadenylationspecificityfactor;CstF,cleavagestimulatoryfactor;snRNP,smallnuclear ribonucleoproteinparticle;hnRNP,heterogenousnuclearribonucleoprotein.
∗ Correspondingauthorat:Rm254,JarrettBuilding,GarscubeEstate,UniversityofGlasgow,Glasgow,ScotlandG611QH,UK. E-mailaddress:[email protected](S.V.Graham).
http://dx.doi.org/10.1016/j.virusres.2016.11.016
5.1. E2asasplicingfactor...92
5.2. SRproteinphosphorylationduringinfection...92
6. Conclusions...93
Acknowledgements ... 93
References...93
1. Introduction
Papillomaviruses comprise an ancient and ubiquitous virus familythatinfectshumansandotheranimals(Bravoand
Félez-Sánchez, 2015). Human papillomaviruses comprise the largest
groupofpapillomaviruses.Thereareover200differentHPV geno-typesidentifiedtodatebasedonfullgenomesequencing(2012). ThemajorityofHPVsubtypesareclassifiedunderthealpha-and beta-HPVgroupswhileafewotherHPVsubtypeshavebeen classi-fiedunderthegamma,muandnugenera(Bernardetal.,2010;de Villiersetal.,2004).Ingeneral,alphaHPVsinfectmucosalepithelia whilebetaHPVsinfectexternalcutaneousepithelia.HPVinfection causesarangeofbenignconditionssuchascondylomaacuminata (genitalwarts),focalepithelialhyperplasia,commonwarts, plan-tarwartsandpigmentedwarts(Cubie,2013;Doorbaretal.,2015). InfectionwithHPVisusuallytransientandthemajorityof infec-tionsareclearedbytheimmunesystem(Stanley,2012).However, inthecaseofsomeHPVs,ifinfectionbecomespersistentthismay leadtotumourprogression (Bodilyand Laimins,2011).Around fortyalphaHPVsinfecttheanogenitalepithelium.Ofthese,upto fif-teengenotypesareso-called“high-risk”HPVs(HR-HPVs)because theyareassociatedwitharangeofcancersincludingcervicaland otheranogenitalcancersandoropharyngealcancers(Cubie,2013). HPVtype16isthemostprevalentHR-HPVresponsiblefor55%of cervicalcancers.Afterchlamydia,itisthesecondmostprevalent sexuallytransmittedinfectiousagentworldwide.Inthedeveloped worldtheincidenceofcertainanogenitalandoropharyngeal can-cershasincreasedsignificantlyoverthelastdecade(Gillisonetal., 2015). Thus, themedical importance of HPV is clear. Vaccines againstHPV16andHPV18,thenextmostprevalentHR-HPVand thegenitalwart-causing,non-oncogenicHPVs6and11,havebeen availableforeightyears.However,theseareprophylacticand can-notprotecttheverylargenumbersofpeopleworldwidewhoare alreadyinfectedandatriskofseriousdisease.Understandingviral generegulationanditsrelationshiptotheinfectedepitheliumisa keygoaltoallowdevelopmentofnovelantiviralstrategiesinfuture. 2. Thehumanpapillomaviruslifecycle
2.1. Humanpapillomavirusentry
Papillomaviruseshavea smallcircular double-stranded DNA genomeof around8kb thatis packagedin anicosahedral pro-teinshell.Thecurrentmodelofthecapsidisthatitcomprises72 pentamersofL1protein,withL2proteinmonomersinsertedat thecentresofthepentamers(BuckandTrus,2012).HPVsenter basalcellsofthecutaneousormucosalepitheliathroughtrauma ormicroabrasions,butparticularlyinthecervicalepitheliuminitial infectionmayoccurinthesinglecelllayerbetweentheectoand endocervix(Herfsetal.,2012;Mirkovicetal.,2015)before trans-fertothemulti-layeredepithelium.FormostHPVsstudied,theL1 capsidproteinattachestoheparansulphateproteoglycansonthe basementmembraneorthebasalepithelialcellsurfaceandvirus entersintothecellbymicropinocytosis(Sappand Bienkowska-Haba,2009).TheentryreceptorsforHPVsarenotfullyunderstood butmayinvolveanumberofproteinsincludingepidermalgrowth factorreceptor(EGFR),integrins,tetraspanin-enrichedmembrane microdomains,lamininsandtheannexin-A2heterotetramer(Raff
etal.,2013).HPVtravelsinthecytoplasmfromendosomestothe trans-golginetworkandreachesthenucleusapproximately24h afterinitialattachmentofvirus.Recentevidencesuggeststhatthe viralgenomeentersthenucleusfollowingbreakdownofthe mem-braneduringmitosis(DiGiuseppeetal.,2016).Insidethenucleus, initialamplificationofthevirusgenometo50–100copiesoccurs throughexpressionofE1andE2viralreplicationproteins(Ozbun, 2002).Duringdivisionofinfectedcells,E2-bindingproteinssuch ascellularBrd4can tetherviralepisomestocellularchromatin toallowequalsegregationofviralgenomesintodaughter epithe-lialcells(WuandChiang,2007).Uponbasalcelldivision,infected daughtercellsmaystayinthebasallayerormaybecometransit amplifyingcellsthatbegintomoveintothesuprabasalepithelial layers(Doorbar,2005).
2.2. Humanpapillomavirusreplicationandepithelial differentiation
TheHPVreplicationcycleistightlylinkedtohostcell differen-tiation.Thevirusdisplaysatightlyorchestratedgeneexpression programthatresultsinepitheliumstratum-specificproductionof viralproteins(Doorbar,2005).TheHPVgenomecanbecategorized intothreeparts:thelongcontrolregion(LCR),theearlyregionand thelateregion(Fig.1A).TheLCRcontainspromotersequencesthat directtranscriptionofboththeearlyandlategenes(Bodilyand Laimins,2011)andcis-actingsequencesthatregulate polyadenyla-tionandvirallatemRNAstability(Graham,2008).EarlymRNAsare polyadenylatedattheearlypolyadenylationsite,whilelatemRNAs arepolyadenylatedatoneoftwoalternativepolyadenylationsites intheLCR(Milliganetal.,2007).Controlofread-throughofthe earlypolyadenylationsite seemstoconstitutethemajorswitch signalfromearlytolategeneexpression(JohanssonandSchwartz, 2013).Theearlyregioncontainssevenopenreadingframesthat encodetheproteinsE1,E2,E3,E4,E5,E6,E7,andE8,whichcarry outregulatoryfunctions.OnlyE6andE7,andpossiblyE1andE2 (thereisinsufficientdatatobesureofthesitesofE8expression atpresent)proteinsaretrulyearlyproteinsthatcanbedetected in basal epithelialcells (Doorbar,2005).E1, E2, E4,and E5 are expressedinthesuprabasallayersandcanbeconsidered inter-mediateproteins. (Fig.1B). Infact, maximumexpressionofthe E1and E2viralreplicationandtranscription factorsisfoundin themidtoupperepitheliallayers(Coupeetal.,2012;Xueetal., 2010).E4proteinisthefirst,andmostabundant,lateproteinto beexpressedinthemidtoupperlayersoftheepitheliuminthe replicativestageofHPVinfection(Doorbaretal.,1997;Middleton etal.,2003)anditislikelythatthisisalsothelocationof maxi-mumE5expression(DiMaioandPetti,2013).AtleastforHPV16, thelatestructuralproteinsL1andL2thatformtheviruscapsid areexpressedonlyinthefinalstagesofcellulardifferentiationin theuppermost,granularlayeroftheepitheliumwhereviralDNAis packagedinthecapsidtobereleasedtoinfectothercells(Fig.1B) (Graham,2010).
2.3. InteractionofHPVswiththeepithelium
Inanuninfectedepithelium,thesuprabasalcellsdonotdivide, but undergo differentiation to eventually form the highly ker-atinized squames that comprise the epithelial barrier to the
Fig.1. ASchematicdiagramoftheHPV16genome.ThedoublestrandedDNAgenomeisshownasagrayshadedhoop.Numbersindicatepositionsonthegenome.Promoters P97,P670andPE8areindicatedwithchevrons.OncogenesE6andE7areindicatedasorangecoloredarcs,replicationfactorsE1andE2areinred,regulatoryproteinsE4and E5areinlilacandcapsidproteinsL1andL2areingreenandbluearcsrespectively.LCR(bluecurvedline),longnoncodingregion.pAE,positionoftheearlypolyadenylation site.pALs,positionofthetwolatepolyadenylationsites(Milliganetal.,2007).B.SchematicdiagramoftheHPV16lifecycleinadifferentiatingepithelium.Virusesareshow aslightbluecircles.Keratinocytesareinlightorangecolor.Nucleiarecoloredpink.Thebasementmembraneisdrawnwithagrayline.Thekeyeventsinthevirusreplication cycleareindicatedtotherighthandsideofthediagramoftheepitheliumtogetherwithaschematicdiagramofthegeneexpressionprogramoftheviruswithintheinfected epithelium.Shadingonthearrowsrepresentsthequantityofexpressionofeachproteinsubsetduringthevirusreplicationcycle.
environment(Tayloretal.,2009).HPV-infectedepitheliaalso dis-playdifferentiation,buttheprocessissomewhatabrogatedbythe presenceofthevirus.Inparticular,expressionoftheviralE6and E7proteinsinthelowertomiddleepitheliallayerstriggersthe differentiatingcellsofthesuprabasallayerstore-enterS-phase. Althoughthismisregulationwouldnormallyinduceapoptosis,E6 proteininhibitsthisprocessbydegradingp53(BodilyandLaimins, 2011).Thus,theHPV-infecteddividingcellsofthemidlayersofthe epitheliumcansupportviralDNAreplicationthroughrecruitment ofanE2/E1complextotheviraloriginofreplication.Thisinturn recruitsthecellularDNAreplicationmachinery(McBride,2013). Replicationinthesuprabasallayersgeneratesmanythousandsof copiesofprogenyHPVgenomes.E4seemstoplayaroleinpriming theinfected,differentiatedepithelialcellstoreleasenewlyformed virionsbyrestructuringcytokeratinfilaments(Doorbar,2013).It mayalsocontributedtogenomeamplificationandenhancevirion synthesis(Doorbaretal.,2015).E5isalsoexpressedlatein infec-tion.Itsvariousroles duringtheinfectiouslife cyclehavebeen difficulttoelucidateduetotheverylowlevelsofexpressionofthis smallprotein.However,amajorroleofE5isrepressionofMHC pre-sentationofviralpeptidestohelpavoidimmunedetection(DiMaio andPetti,2013).InteractionswithgrowthfactorreceptorsEGFR (mainlyHPVs)andPDGFR(mainlybovinepapillomaviruses) indi-catesthatE5canfeedinto,andmodify,growthcontrolandcellcycle pathways(DiMaioandPetti,2013).Finally,inthegranularlayerof theepitheliumtheL1andL2capsidproteinsareproduced.They encapsidatenewly synthesizedviral genomesto producemany thousandsof progenyviruseswhich caninitiatenewinfections (Fig.1B)(BuckandTrus,2012).
Owingtothe complexinterplay betweenthe differentiating epitheliumandtheHPVreplicationcycle,pathogenicityofHPVis likelyduetospecificregulatoryinteractionsbetweenviralproteins andhostcellsfactors.Overthelastdecade,ithasbecomeclearthat RNAprocessingfactorsespeciallysplicingfactorsareanintegral partoftheseinteractionsandviralsplicingcontrolisthefocusof thisreview.
3. Splicing
Theprimarytranscript(pre-mRNA)ofagenethatemergesfrom RNA polymeraseII upon transcription undergoesprocessing to
formamaturemessengerRNA(mRNA).Theseprocessingevents occurco-transcriptionally and include capping, polyadenylation andsplicing(MooreandProudfoot,2009).Splicingisabasic cel-lularprocessrequiredforexpressionofthemajorityofmetazoan genes.Duringsplicing,intronsareremovedfromtheprimary tran-script andtheprotein-codingexonsarespliced together(Black, 2003).A macromolecularribonucleoprotein complex called the spliceosomecarriesoutthesereactionsthroughrecognitionof5 and 3-splice sites that markexon/intron junctions in the pre-mRNA, a “branch point” sequence, and a polypyrimidine tract withintheintronupstreamofthe3-splice site(Fig.2A)(Wahl etal.,2009).Thesesequencesarethe“landingpads”forthesmall nuclearribonucleoproteinparticles(snRNPsU1,U2,U4,U5andU6) thatmakeupthespliceosomethatcarriesoutthesplicingreaction (Wahletal.,2009).Splicingoccursthroughasetofwell-defined steps.First U1snRNPbindtothe5-splicesitethrough comple-mentarity betweenthesnRNA of U1 snRNPand thesplice site itself,andthebindingisstabilisedthroughsnRNPproteinssuchas U1C.Nextthebranchpointbindssplicingfactor1(SF1/BBP1),the polypyrimidinetractbindsU2AF65whilethe5-splicesitebinds itsheterodimerpartner,U2AF35.Next,SF1isreplaceduponthe branchpointsequence byU2snRNPwhosebindingisstabilised byU2AFandtheU4.U5.U6tri-snRNPthatjoinsthecomplex.U1 andU4snRNPsarereleasedandU2,U5andU6providetheactive siteforsplicing(Fig.2A).Thetwo-stepenzymaticreactioninvolves releaseoftheupstreamexonandlariatformationoftheintronback tothebranchpointfollowedbyjoiningoftheexonsandrelease anddegradationofthelariatintermediate(Fig.2B)(Papasaikasand Valcárcel,2016).
3.1. Controlofsplicing
Thehugecomplexitiesofthesplicingmachineryfacilitatesplice siterecognition,butotherregulatorymechanismsareimportant forefficientandaccuratesplicesitedetection.Exonscontain cis-actingsequences,calledexonicsequenceenhancers(ESEs),which influence splicing efficiency. They do this by binding splicing enhancing serine-arginine rich proteins (SR proteins). SR pro-teinsareconservedineukaryotesandarepresentmainlyinthe nucleusalthoughsomecanshuttletothecytoplasm(Buschand Hertel,2012).VeryearlyinthesplicingreactionU1andU2snRNP
Fig.2. ABasicfeaturesofapre-mRNArecognisedbytheearlysplicingcomplex.Exonsareshownasgreenboxesandintronthewithablackline.Pinktrianglesindicate5 -and3-splicesites(ss).TheintronbranchpointisindicatedwithA-OH.The3intronicpolypyrimidinetractisshownasYn.U1snRNPisshownasalightblueoval.U2snRNP isshownasalightbeigeoval.TheU4.U5.U6tri-snRNPisshownasred/pinkspheres.U2AF65and35kDadimerisshownasablueovalandacircle.Bproteinscomplexesare notdrawntoscale.Thecatalyticstepsinsplicing.Exonsareshownasgreenboxesandtheintronasablackline.Splicesitesandbranchpointannotationsareasabove.In thefirststepofthesplicingreactionthe2 OHofthebranchpointadenosineattacksandbreaksthephosphodiesterbondintheRNAbackboneatthe5splicesiteanda newbondiscreatedbetweenthe5splicesiteandthebranchpointtoformalariatintermediatestructure.Thesecondstepinvolvesanotherexonucleolyticattackofthe5 splicesite−OHontothe3splicesite.Theexonsaresplicedtogetherandtheintronlariatisdiscarded.
Fig.3.SplicingcontrolbySRproteinsandhnRNPs.SRproteinscanenhancesplicingbyaidingtheformationandstabilityofsplicingcomplexes.Inthiscase,forsplicing enhancement,onlyinteractionswithU1andU2snRNPsareshown.However,SRproteinsboundtoexonicsequenceenhancers(ESEs)caninfluenceformationofthevarious U-snRNPcomplexesthatformduringasplicingreaction(HowardandSanford,2015).hnRNPsboundtoexonicsequencesilencers(ESSs)cancounteracttheactivitiesofSR proteins(Eperonetal.,2000).ApossiblerouteofterminalexondefinitionisalsoshownwhereSRproteinsbindtoapolyadenylationupstreamsequenceelement(USE)and createinteractionsfromtheretotheupstream3splicesitetoenhanceU-snRNPrecruitment(HowardandSanford,2015).Greenboxesindicateexons.Lightbluevertical boxesindicateESEs.DarkblueverticalboxesindicateESSs.Intronsand5and3untranslatedregionsareindicatedwithablackline.Pinktrianglesindicate5-and3-splice sites(ss).U1snRNPisshownasalightblueoval.U2snRNPisshownasalightbeigeoval.U2AFdimerisshownasabluecircle.SRproteinsarerepresentedbypinkspheres. hnRNPsareindicatedwithlilacovals.ThemRNAcapisshownasagraybullet.TheCPSFandCstFpolyadenylationcomplexesarerepresentedaslightanddarkorangespheres. AbeigeboxindicatedapolyadenylationUSEinthe3untranslatedregion.Adownwardblackarrowindicatesthepolyadenylationsite.
earlysplicingcomplexescanassembleacrossintronstodirectthe spliceosometothecorrectsplicesites(Fig.2).Earlysplicecomplex formationandstabilisationiscontrolledbySRproteinsbindingto ESEs.Forexample,interactionbetweenSRproteinsandU2AF35 stabilisesU2snRNPboundtothe3 endofanintron.Cross-exon orintronsplicingcomplexesarecooperativelystabilisedby inter-actionsbetween U1 snRNP and U2AF and SR proteins (Fig.3). Moreover, SR proteins are implicated in recruitment of multi-plesplicing factorsduringtheprocessof spliceosomeassembly andarekeyplayersinthecatalyticstepsofthesplicingreaction
(HowardandSanford,2015).Forcomplexgenomes,SRproteins
“define”exonsinpre-mRNAstoestablishexon-intronboundaries. SRproteinsboundtoESEsconnectthe3splicesiteatthe5end ofanexonwiththe5 splicesiteattheotherendoftheintron and mark thesequence as an exon for retention in themRNA (Fig. 3). They also facilitate definition and splicing of terminal exons(HowardandSanford,2015;LongandCaceres,2009).They accomplishthisbybindingdirectly,orinteractingwithotherRNA processingfactorcomplexestetheredinthemRNA5untranslated regionorthe3 polyadenylationregion,toprovidea“feedback” interactionwiththefirst 5-splicesite orterminal 3-splicesite respectivelyin thepre-mRNA(Fig.3)(Berget, 1995;Proudfoot, 2000).
InadditiontoSRproteins,theexon/intronarchitecture,steric hindranceand RNAsecondarystructure mayalsoplay arole in directingtheactivitiesofthespliceosome(DeContietal.,2013).
3.2. Alternativesplicing
Inconstitutivesplicingallintronsareremovedfromthe pre-mRNAandeveryexonispresentinthematuremRNA.Accurate andspecificrecognitionofcorrect5-and3-splicesitesis essen-tialtoensureproductionoftheappropriatesetofmRNAsinacell (Black,2003).However,althoughconsensussequenceshavebeen determined,5-and3-splicesitesarefrequentlyfoundtobe degen-erate.Thisambiguity insplicesite recognitiongivesrise tothe possibilityofmultiplechoicesofsplicesiteswithincomplex pre-mRNAs(Barashetal.,2010)(Rocaetal.,2013).Indeed,wenow knowthatmostmammalianpre-mRNAs canundergoregulated selectionof alternative5 and 3-splicesite. Alternativesplicing resultsindifferentialintronandexonretention, orskipping,or choiceofalternative(pseudo)splicesitestoalterexonsize,and thisprocesscangenerateseveraldifferentmRNAandprotein iso-formsfromeachproteincodinggene(Fig.4)(Black,2003;Irimia andBlencowe,2012;WardandCooper,2010).Themajorityof alter-nativesplicingeventscomprise“cassette”exon removalfroma pre-mRNAbutmutuallyexclusivesplicingisalsocommon(Fig.4). ProbablybecausetheHPVgenomeispolycistronic,severaltypes ofalternativesplicingareusedtogenerateHPVmRNAs.Cassette exonremovalisseeninthecaseofmRNAsthatskiptheE4open readingframe,e.g.E1ˆL1mRNAs.Read-throughversussplicingalso occurs,e.g.E1ˆE4,E5,L2,L1versusE1ˆE4ˆL1mRNAs.Alternativechoice of3spliceacceptorsitesisseeninthecaseoftheE6E7RNAs.
How-Fig.4.AsinglegenecangiverisetoseveralalternativemRNAs.Atthetopisshownthestructureofahypotheticalthree-exon,two-introngene.Exonsareillustratedingreen andintronsand5and3untranslatedregionsasblacklines.TwoalternativepromotersPwtandPaltareshownasforwardfacingblackarrows.Twoalternativepolyadenylation sites(Poly(A)wtandPoly(A)alt)areshownasdownwardfacingblackarrows.Analternative(pseudo)3splicesiteisindicatedwithablueupwardarrow.Somealternative mRNAstructuresthatcanarisefromthehypotheticalgeneareshown.Thethreeisoformsinthetoprowhaveundergonecompleteconstitutivesplicing.Thethreeisoforms inthemiddlerowaretheproductsofalternativesplicingofthegene.Thebottomrowshowsthepossiblesplicingpatternsofathreeexongenewherethemiddleso-called “cassette”exoncanberetainedorsplicedoutandofafourexongenewheremutuallyexclusivesplicingcantakeplace.Inthiscaseeitherexon2orexon3isincludedinthe finalmRNAisoformsproduced.
ever,thereisnoevidenceasyetfortruemutuallyexclusivesplicing. AlthoughothercellularstrategiesexistbywhichdifferentmRNA isoformscanbeexpressedfromasinglegene,includingalternative promoterusageandalternativepolyadenylation(Fig.4), alterna-tivesplicingmakesthegreatestcontributiontomaximisingprotein production fromthegenomesof highereukaryotes andviruses
(Hernandez-Lopez andGraham,2012;WardandCooper,2010).
Collectivestudieshaveshownthatmosthumanpre-mRNAs nor-mallyundergoextensivealternativesplicing.Over90%ofhuman RNAsarealternativelysplicedandgiverisetoacellularmRNA pop-ulationthatcanencodearound4–5foldmoreproteinsthanthere areprotein-codinggenesinthegenome(Halleggeretal.,2010). Alternativesplicingisessentialfordevelopmentand differentia-tionandorganfunction(IrimiaandBlencowe,2012).Mis-splicing however,ispossibleand itcangiverisetoserioushealth prob-lemsincludingcancersandgeneticdiseases(ScottiandSwanson, 2016).
Controlofalternativesplicingisexertedbythestrengthof5 and3-splicesites,theorder inwhich exonsemergefromRNA polymeraseIIduringtranscription, thepatternof RNA process-ingfactorsbindingthepre-mRNA,therateatwhichthegeneis transcribed,and cellsignaling.Asmentioned aboveSR proteins canactpositivelytocontrolconstitutivesplicing,andthisisalso true for alternative splicing (Fig. 5).Splicing can becontrolled negativelybyalargefamilyofheterogeneousribonucleoproteins (hnRNPs).hnRNPs can blockexon/intron definitionby interfer-ingwithassemblyoftheexondefinitioncomplex(Hertel,2008). Therefore,theSRandhnRNPproteinfamiliescanact antagonis-tically in controlling splicing (Eperonet al., 2000).Apart from ESEs, other splicing regulatory cis-active signals exist: intronic sequence enhancers (ISEs) and exonic and intronic sequence silencers (ESSs, ISSs). SR proteins generally bind the enhancer sequenceswhilehnRNPsbindthesilencers.Thissequence-specific
controlofsplicinghasbeentermedthe“splicingcode”(DeConti etal.,2013).
3.3. SRproteins
Therearenine classicalSR proteins(SRSF1-9)inaddition to othernon-classical proteinssuchas SRp38and Tra2ˇ (SRSF10) (ManleyandKrainer,2010).EachSRproteiniscomposedofatleast onecopyofeachoftwodomains:1)anRNArecognitionmotifand 2)a serine/arginine-richdomain(RSbindingdomain)(Longand Caceres,2009).Asdiscussed above,in constitutiveand alterna-tivesplicingSRproteinscontrolrecruitmentofcomponentsofthe basicsplicingmachineryatexon-intronboundariesandmediate exon/introndefinition.SRproteinsboundtoESEscanincreasethe efficiencybywhichU-snRNPsdetectsplicesites,andthisis par-ticularlyimportantifthesesitesarepoorlyconservedandliable tobeskippedbythesplicingmachinery(Fig.5)AlthoughSR pro-teinsusuallyacttoenhancesplicing,theyhavealsobeenshown toinhibitsplicing.Forexample,SRSF9caninhibitrecognitionofa 3 splicesite(leadingtoexonskipping)ofexon7BinthehnRNP A1pre-mRNAthroughbindinganISSelement(SimardandChabot, 2002).
ThefunctionsofSRproteinsarecontrolledbyphosphorylation of theirRS domains. Several kinases are knownto phosphory-late SR proteins including Chk1, Topoisomerase (TOPO) 1 and Serine/Arginine-protein kinases (SRPK) 1 and 2 (Giannakouros et al., 2011). Phosphorylation is essential for SR protein func-tionsinconstitutiveandalternativesplicing,butbothhypoand hyper-phosphorylatedSRproteinscaninhibitsplicing(Zhouand Fu, 2013)meaning that site-specific or temporal alterations in phosphorylationmustbeamajorpointofcontrol.Theexact phys-iologicalrolesofphosphorylatedformsofSRproteinsarestillto beaddressed.However,thesuggestedimportanceofSRprotein
Fig.5.SRproteinsdirectalternativesplicing.Theredcentralexoninthishypotheticalgeneisa“cassette”exonthatcanbeomittedfromoneofthemRNAisoformsgenerated fromthegenebyafailureofthespliceosometorecognisetheexonboundariesefficiently.A.ThemRNAisoformproductofsplicingistheconstitutiveisoformbecausethe twointronsaresplicedoutandthethreeexonsaresplicedtogether.SRproteinsbindingtoexonicsequenceenhancers(ESEs:lightblueverticalboxes)actinadominant positivemannertorecruitU-snRNPsand/orincreaseefficiencyofU-snRNPrecognitionof3and5splicesitesand/ortoantagonisetherepressiveactivityofhnRNPsbound toexonicsequencesilencers(ESS:darkblueverticalboxes).ThenumberandrangeofSRproteinsbinding(usually)multipleESEsontheexoncanmodulatethelevelofthe positiveeffect.B.ThemRNAisoformproductofsplicingisthealternativeisoformbecausethecentralexonhasnotbeenrecognisedefficientlyforsplicingintothemRNA andonlythetwoflankingexonsaresplicedtogether.InthiscasehnRNPsboundtoexonicsequencesilencersmayexertrepressiveactivityonU-snRNPrecruitmentandSR proteinsenhancingactivities.Greenorredboxesindicateexons.Intronsand5and3untranslatedregionsareindicatedwithablackline.Pinktrianglesindicate5-and3 -splicesites(ss).U1snRNPisshownasalightblueoval.U2snRNPisshownasalightbeigeoval.SRproteinsarerepresentedbypinkspheres.hnRNPsareindicatedwithlilac ovals.
phosphorylationincludesintracellularlocalizationandtrafficking, protein–proteininteractionsandcontrolofalternativesplicingof mRNAs(Giannakourosetal.,2011;LongandCaceres,2009).
SR proteins play a range of other roles in regulating gene expressionincludingregulationoftranscriptionelongation,mRNA nuclear export, stability and translation (Howard and Sanford, 2015).Indeed, it seemslikelythat SR proteinsmayhavemuch broader relevance to normal cellular metabolism than simply theirroleinsplicingregulation.Asdocumentedfortheparadigm SR protein SRSF1, other functionsof SR proteins include chro-matinremodelling,genomestabilitymaintenance,nucleolarstress response,cellcycle progressionand apoptosiscontrol (Das and Krainer,2014).CurrentresearchhasdescribedsomeoftheSR pro-teinsasoncogenicastheyhavebeenfoundtobeoverexpressed inarangeofcancers(Dasand Krainer,2014).Moreoverseveral havebeenshowntopossessoncogenicactivityincludingSRSF1 (ASF/SF2),SRSF2 (SC35),SRSF3(SRp20) andSRSF9(SRp30c) (Fu etal.,2013;Jiaetal.,2010;Karnietal.,2007;McFarlaneetal.,2015). OncogenicactivityofSRproteinsisduelargelytotheir deregu-lationofalternativesplicingofRNAswhoseproteinproductsare involvedinkeycellularpathways.Insummary,increasedSRSF lev-elscanresultinproductionofalternativelysplicedRNAisoforms thatencodekeyanti-apoptotic,cellproliferationand epithelial-mesenchymaltransition(EMT)-inducingproteins(DasandKrainer, 2014).
3.4. hnRNPproteins
ThehnRNPfamilyislargerandmorecomplexthantheSR pro-teinfamily.Inhumans,therearethirteenhnRNPproteinfamilies eachofwhichcontainseveralsubtypes(BuschandHertel,2012). ExactdetailsofhowhnRNPscontrolsplicingareunderstoodinonly afewcases.Theycanbindcooperatively,multimerize,andspread alongexonstorepressassemblyofthespliceosomeacrossadjacent introns(Fig.3)(BuschandHertel,2012).Inalternativesplicing,they mayblocksnRNPbindingtoadjacentsplicesites.Importantly,SR proteinscanantagonisethenegativeeffectsofhnRNPproteinson splicingperhapsbysterichindranceofhnRNP/RNAprotein inter-actions(Fig.5)(Eperonetal.,2000).
4. SplicingofHPVRNAs
DNAvirusessuchashumanpapillomavirus(HPV)require con-stitutive and alternative splicing to generate mRNAs encoding
themany essentialproteinsthat arerequired toinitiate, main-tainand complete theirlife cycles.During HPVinfectionofthe epitheliumatleasttwentydifferentmRNAsareexpressed,some ofwhicharetheproductsofalternativesplicing(BakerandCalef, 1997;Chenetal.,2014;Chowetal.,1987a,b;Doorbaretal.,1990;
Isok-Paasetal.,2015;OzbunandMeyers,1997,1998;
Palermo-Dilts et al., 1990; Stoler et al., 1992, 1989; Tan et al., 2012; Toots etal., 2014; Wangetal., 2011).Transcript mapsmaybe viewed at (https://pave.niaid.nih.gov/#explore/transcriptmaps). SRandhnRNPproteinscontrolviralRNAprocessingduring
infec-tion (Graham, 2010;Johansson and Schwartz, 2013).A map of
known HPV16 mRNAs is shown in Fig. 6. In addition to this infection-relatedcontrol,SRandhnRNPproteinsareoverexpressed inHPV-associatedcervicalpre-cancers andcancers(Moleetal., 2009a;Fayetal.,2009)andthereforehavethepotentialtoimpact HPVgeneexpressionintumorigenesis.
4.1. HPVgeneexpressionandsplicing
Mostinformation onregulationof viralgeneexpression has beengatheredfromstudiesonHPV16,orthemostcloselyrelated HPV,HPV31.EarlyintheHPV16infectiouslifecycle,transcription initiatesfromtheviralearlypromoter locatedatP97,and
poly-cistronic mRNAsencoding E6and E7 E1, E2, E8E4 and E5 are synthesized.(Fig.6).ThereisextensivesplicingintheE6E7region ofthepre-mRNAswithatleastfourspliceisoformspossiblethat havebeenconfirmedinpatienttissues(Chenetal.,2014;Schmitt etal.,2011).E6full length(E6fl)is anunsplicedtranscriptthat includestheE6andE7openreadingframes.E6*I,E6*IIandE6*X (alsotermedE6ˆE7orE6*III)aremRNAsalternativelysplicedfrom one5-splicesitetooneofthreealternative3-splicesitesinthe primarytranscript(Fig.6).Tworarespliceisoformswithalternate 5-splicesiteshavealsobeendetectedinHEK293cellstransfected withanE6E7expressionconstruct(Ajiroetal.,2012).Therearefour spliceisoformsproducedfromtheE1E2regionofthegenome,E1ˆE2, E1ˆE2C,E8ˆE1,E8ˆE2C(Chenetal.,2014;Coupeetal.,2012;Milligan etal.,2007;Schmittetal.,2011).Theseuseasplicedonorateither genomeposition880or1302andoneoftwospliceacceptorsat 2582,and2709(Fig.6).
Atlatetimesofinfectionthevirallatepromoter(P670)is
acti-vated(BodilyandLaimins,2011)togetherwithapromoterlocated atthe5endoftheE1openreadingframetermedtheE8promoter (Straubetal.,2015)(Fig.6).DespitethefactthattheE8ˆE2Cprotein isaninhibitorofE2intranscriptionandreplication,thisresults
E6 E7 E1 E2 E4 E5 L2 7154 7265 Late poly(As) P97 P670 Early poly(A) 1 PE8 L1
A
E8 SD226SA409 SA526 SD880 SA3358 SD226 SA409 SA526 SD880 SA2709 SD226 SA409 SA526 SD880 SA2582 P97B
Early mRNAs
SD3632 SD880 SA5638 SA3358 SA3358 SA409 SA743 SA526 SD3632 SA3358 SA5638 SA5638 SA2709 P670 P97 SD226 SD1301 PE8C
Late mRNAs
Fig.6.A.DiagramofthelinearisedHPV16genomeshowingthenineopenreadingframes(coloredboxes)thethreecharacterisedpromoters(forwardfacingarrows)andthe earlyandlatepolyadenylationsites(thickblackverticallines).B.SchematicdiagramoftheknownHPV16earlymRNAsplicesites(adaptedfrom(ZhengandBaker,2006)). Thediagramdoesnotindicatethe3endsoftheRNAslistedandisnottoscale.OrangecoloredboxesindicateE6E7codingregions.Red/pinkcoloredboxesindicateE1E2 codingregions.Lilac/purplecoloredboxesindicateE4E5codingregions.C.SchematicdiagramoftheknownHPV16latemRNAsplicesites(adaptedfrom(Milliganetal., 2007)and(Chenetal.,2014)).Thelatepolyadenylationsitesarenotindicated.ThecolorschemeisthesameasforB.withtheadditionofL1andL2codingregionsindicated inblue.SA,spliceacceptor.SD,splicedonor.Arrowheadsindicatesplicesites.Graydottedlines,intronsequences.
inincreasedexpressionoftheHPVreplication/transcription fac-torsE1andE2thatinitiatevegetativeviralgenomeamplification. Theviralcapsidproteinsareexpressedfromtwoclassesof poly-cistronictranscriptstranscribedfromthelatepromoter.Thefirst oneincludestwospliceeventstogiveE1ˆE4ˆL1mRNAsthatare con-sideredtoencodeE4andL1proteinsandthesecondoneincludes onlytheE1ˆE4spliceeventandread-throughfromtheearlyregion toyieldanE4,E5,L2,L1polycistronicmRNA.Thepackednature of theHPV genomein terms of signalsequences that regulate transcription,splicingandpolyadenylationsuggeststhatcontrol ofmRNAproductioniscomplex.Moreover,itisclearthat alterna-tivesplicingplaysamajorroleingeneratingtherangeofmRNAs requiredtoencodeviralproteins,andthatviralsplicingmaybe regulatedinadifferentiation-stagespecificmanner.
4.2. SRproteinscontrollingHPVgeneexpression
Theextentandcomplexityofalternativesplicingrequiredto produceHPVmRNAssuggeststhatSRandhnRNPproteinscould bekeyregulatorsoftheHPVreplicationcycle.Alargebodyof evi-dencehasbeenamasseddetailingtheSRandhnRNPproteinsthat contributetoHPVmRNAalternativesplicingandwearebeginning tounderstandsomeofthecontrollingmechanisms.
4.2.1. E6andE7splicing
In the case of E6 and E7 RNA isoforms, theroles of SRSF1, SRSF2 andSRSF3 havebeeninvestigated. Intwo studies,SRSF1 wasnot foundtocontrolE6E7 RNAsplicing whiledepletionof SRSF3resultedinsomereductioninE6E7mRNAexpression(Jia etal.,2009;McFarlaneetal.,2015).However,averysignificant reductioninE6E7RNAexpressionwasobservedintheabsenceof SRSF2(McFarlaneetal.,2015).Althoughthis splicingfactorisa majorpositiveregulatorofviraloncoproteinexpression,the evi-dencesuggeststhatSRSF2regulatesE6E7RNAstabilityratherthan splicing.NonsensemediateddecayisamechanismwherebyRNAs suchastheshortE6E7mRNAs,containing3-splicesitescloseto stopcodonsarerecognisedasaberrantandsubjecttodegradation (PoppandMaquat,2014).SRSF2maybeinvolvedinE6E7splicing andprotectthemRNAisoformsagainstdecay.AlloftheE6E7mRNA isoformsweresimilarlyaffectedintheaboveexperiments.Ofthe mainE6E7RNAisoforms,E6*Iappearstobethemostabundant intumourcellslinesandinpatienttissues(Schmittetal.,2011; SchmittandPawlita,2011).E6*ImRNAmayencodeanadditional viralproteinexpressedbyHR-HPVs(Yuanetal.,2012)andindepth studieshavedemonstratedthattheputativeE6*Iproteinappears tohaveantagonisticpropertiestoE6itself.Forexample,E6*Ican promoteapoptosisbycounteractingE6fl-mediateddegradationof p53(Yuanetal.,2012).Ontheotherhand,datafrominvitro
stud-ieshasindicatedthatE6*1mRNAmayallowtranslationofE7by reinitiationonadownstreamAUG(Staceyetal.,2000;Tangetal., 2006).InHPV16infectedkeratinocytes,thebalanceofE6fl (intron-containing)versusE6*I(intronremoved)RNAisoformexpression wasdemonstratedtoberegulatedbyEGF(Rosenbergeretal.,2010). EGFsignalingresultedinintroninclusion(SD226–SA409)togive predominantlyE6flmRNAswhileEGFdepletionshiftedthebalance towardsintronsplicingandE6*Iproduction.Inagreementwiththe studiesmentionedabove(Jiaetal.,2010;McFarlaneetal.,2015), SRSF1didnotplayaroleinregulatingE6alternativesplicing. How-ever,twotranscriptionfactorsthatcanalsocontrolsplicing,Brm andSam68wereimplicated.BrmisacomponentoftheSWI/SNF chromatinremodelerandisproposedtoregulatesplicingby con-trollingtherateofRNApolymeraseIIelongationwhileSam68isa memberoftheSTARproteinfamilythatcontrolssplicingthrough signaltransduction.Interestingly,itwasproposedthatEGF lev-elsintumourcellsmightallowaswitchtoproductionoftheE6*I mRNAisoformfromwhichE7proteinwouldbemoreefficiently translated viareinitiation at theE7 AUG. Increased E7 expres-sionwouldensureenhancedcellcycleprogression,ahallmarkof HPV-associatedtumourprogression(RomanandMunger,2013). Conversely,ininfectednormalkeratinocytesEGFsignalinginthe basalepithelialcellswouldfavourE6fulllengthproductionand inhibitionofapoptosisoftheinfectedcell(Rosenbergeretal.,2010). Finally,anotherstudyhasshownthatE6isoformproductionfrom HPV18iscontrolledbyanothertranscriptionfactorthatcanalso haverolesintranscription-linkedsplicing,CCCTC-bindingfactor (CTCF).CTCFbindstoamotifintheE2regionoftheHPV18genome andinitiatesapauseinRNApolymeraseIItranscriptionthatfavours correctsplicingoftheE6E7RNA.DeletionoftheCTCFbindingsite withintheviralgenomeledtoasignificantincreaseinlevelsofthe E6andE7oncoproteins(Parisetal.,2015).Itwillbeinterestingin futuretodiscoverifthereisanylinkbetweenCTCFactivityandEGF signaling.
4.2.2. EarlyRNAsplicing
Most viral early RNAs are spliced from a 5-splice site at nucleotide880intheE1generegiontoa3-splicesiteatnucleotide 3358toretaintheE4openreadingframe(Fig.6).TheE4genedoes notcontainastartcodonbutthisisprovidedthroughsplicingofthe regionencodingthefirstfiveaminoacidsofE1ontoE4(Roberts, 2006).The3-splicesitelocatedatthe5endoftheE4openreading frameissuboptimalduetolackofagoodupstreampolypyrimidine tractmeaningthatitshouldbeusedatlowefficiency(Kajitaniand Schwartz,2015).Despitethis,thesplicedtranscriptE1ˆE4that con-tainstheE4openreadingframeisthemostabundantHPVmRNA expressedduringan infection(Chenet al.,2014;Schmittetal., 2011).AnalysisofSRproteinbindingtotheHPV16E4exonhas shownthatthe3-splicesiteatnucleotideposition3358(SA3358) iscontrolledbyacomplexESEcontaininganinsilico-predictedten clustersofSRSF1bindingmotifs(SombergandSchwartz,2010). Inall,fifteenSRSF1bindingsiteswereidentified,andmutationof theseresultedinaredirectionofsplicingfromSA3358toa down-stream3-splicesiteatnucleotideposition5639atthe5endofthe L1openreadingframe(Fig.6).Asubsequentstudyrevealedthatthe majorityoftheESEactivitywasduetoasingleSRSF1bindingsite (Lietal.,2013a).Similarmotifsarepredictedinverysimilarregions ofE4openreadingframesoflowandhighrisk,mucosaland cuta-neousHPVssuggestingaubiquitousSRSF1-mediatedmechanism forcontrollingE4splicing.IntheabsenceofSRSF1enhancementof the33583-splicesite,therewascompetitionfromthedownstream SA5639thatisusedtoproduceL1-encodingmRNAs(Sombergand Schwartz,2010).Thesedata suggestthat SRSF1 controlsuseof SA3358atthe5 end oftheE4openreading frameandinhibits latemRNAproduction.SRSF1alsohadalowlevelrepressiveeffect onthesplicesiteatthe3endofE4openreadingframe(SA3632)
thatwouldalsoresultininhibitionoflatemRNAsplicing(Somberg
and Schwartz,2010).These observationsdemonstratethe
posi-tiveand negativeeffects that a singleSR proteincan exert on mRNAsplicing.SRSF3alsobindsanESEwithintheE4open read-ingframeandenhancessplicingatSA3358.Moreover,SRSF3could alsoinhibitvirallatemRNAexpressionbut thistime by stimu-latingpolyadenylationattheearlypolyadenylationsite(Jiaetal., 2009).
SplicingofthevariousRNAsarisingfromtranscriptionoftheE1 andE2geneshasbeenreportedbutitisasyetunclearhowthese splicingeventsarecontrolled.However,becausetheRNAsusea5 -splicesiteatgenomeposition880or1302withoneoftwosplice acceptorsat2582and2709,selectionofonesiteoveranothermust beacontrolledevent(Straubetal.,2015).ComparedtoE6E7and E4-containingRNAstheseseemtoberareRNAspecies,whichmay hampertheiranalysis.
4.2.3. LateRNAsplicing
Analyses of the early splice isoforms and their regulation were mostly carried out in tumour cells such as HeLa cells thatmimicundifferentiatedepithelialcellsorinundifferentiated keratinocytes. These cell systems only support HPVearly gene expressionbecausekeratinocytedifferentiationisrequiredforviral lateproteinexpression.TobegintoexaminehowSRproteins con-tributetolatemRNAproductionthroughalternativesplicingour laboratoryusedsiRNAstodepleteSRproteinsinHPV16-infected, differentiatedkeratinocytestodiscoverwhichwereresponsiblefor controllingcapsidmRNAandproteinexpression.HPVcapsid pro-teinexpressionisreadilydetectedinkeratinocytesthatmaintain wildtypeepisomalHPVgenomes(Klymenkoetal.,2016).Among SRSFs1–3,5,and7,depletiononlyofSRSFs1and3causedachange inL1capsidproteinexpression.SRSF1knockdownresultedinonly asmallreductioninL1expressionbutSRSF3knockdowncaused agreaterthan50%reductioninL1levelsinthecells.Conversely, SRSF3overexpression inanundifferentiatedkeratinocyte popu-lationresultedininductionofL1 proteinexpression(Klymenko etal.,2016).AnalysisofthemajorsplicedRNAsencodingthe cap-sidproteinsrevealedthatSRSF3wasrequiredforproductionof thesplicedlateE4ˆL1mRNAthatencodestheL1majorcapsid pro-teinbecauseareductioninlevelsofSRSF3causedadecreasein E4ˆL1mRNAlevelswithacorrespondingincreaseintheunspliced L2L1mRNAthatencodestheL2minorcapsidprotein(Klymenko etal.,2016).In agreementwithapreviousstudy(Sombergand Schwartz,2010),SRSF1alsocontributedtomaintaininglevelsof the E4ˆL1spliced mRNA, but had a much more significant and inhibitoryeffectonL2L1RNAlevels.ThedataimplicateSRSF3asa keydirectregulatorofvirallategeneexpressionindifferentiating keratinocytes.
SRSF9(SRp30c)hasalsobeenimplicatedinenhancedsplicing ofHPV16latetranscripts(Sombergetal.,2011).In undifferenti-atedHeLacells,SRSF9inhibitedsplicingatSA3358atthe5 end oftheE4openreadingframeresultinginredirectionofsplicing downstreamtoSA5639atthe5endoftheL1openreadingframe. SRSF9 wasalsoshown to overcomesuppressionof SA5639via neutralisationofsplicingsilencersintheL1openreadingframe (Sombergetal.,2011)resultinginL1RNAproduction.Finally, over-expressionofSRSF9inducedlevelsofararemRNAcalledL1i(E1ˆL1) (Sombergetal.,2011),whichcanbedetectedindifferentiated ker-atinocytes(Milliganetal.,2007)bypromotingskippingoftheE4 exon(Sombergetal.,2011).TheL1codingregioncontainsanESE whosepositiveeffectonsplicingtoSA5639canbeoverriddenby hnRNPA1.Whiletheproteinsthatbindarenotyetelucidatedthey couldbeanessentialregulatorofL1mRNAsplicing(Zhaoetal., 2007a).
RNAbindingproteinsandtheireffectsontheirtargetHPV16RNAs.Ifithasbeenidentified,thetargetspliceacceptor(SA)orsplicedonor(SD)siteislisted.
RNAbindingprotein TargetRNA Effect Reference
SRSF1 E4 E4 LRE SA3358enhancement
SD3632suppression
Repression
Lietal.(2013a);Sombergand Schwartz(2010)
Lietal.(2013a);Sombergand Schwartz(2010)
McPhillipsetal.(2004)
SRSF2 E6E7 Enhancementvia
RNAstability
McFarlaneetal.(2015)
SRSF3 E6E7 E4 Enhancement
SA3358suppression
Jiaetal.(2009);McFarlane etal.(2015)
Jiaetal.(2009,2010)
SRSF9 E4 SA3358suppression
SA5639activation
Sombergetal.(2011)
Sam68 E6E7 E6exoninclusion Rosenbergeretal.(2010)
Brm E6E7 E6exoninclusion Rosenbergeretal.(2010)
hnRNPA1 E6E7L1LRE E6exonexclusion
SA5639suppression Repression
Rosenbergeretal.(2010) Zhaoetal.(2007a);Zhaoetal. (2004);ZhaoandSchwartz (2007)
Chuen-Imetal.(2008)
hnRNPA2/B1 E6E7 E4 E6exonexclusion
Suppression/enhancement
Rosenbergeretal.(2010) Lietal.(2013b);Orrùetal. (2012)
hnRNAPC1/C2 E4Early3UTR SD3632enhancement
Activation/repression
Dhanjaletal.(2015) Dhanjaletal.(2015)
hnRNPD E4 SD3632suppression Dhanjaletal.(2015)
hnRNPH L2 Enhancesearly
polyadenylation
Öbergetal.(2003);Öbergetal. (2005)
hnRNPI(PTB) Early3UTR Enhancesearly
polyadenylation
RelievesSD3632suppression
Sombergetal.(2008);Zhao etal.(2005)
Sombergetal.(2008)
CTCF E2 RNAPolII-relatedcontrolof
earlyregionalternative splicing
Parisetal.(2015)
4.3. hnRNPproteinscontrollingHPVgeneexpression
ThehnRNPproteinfamilyhasalsobeenshowntocontrolHPV16 mRNAsplicing.BothearlyandlatemRNAsareunderhnRNP con-trol.ExpressionoftheviraloncoproteinsiscontrolledbyhnRNPA1 whichactivatessplicingbetweenthefirstpairofsplicesites(SD226 and SA409)in viralE6E7 pre-mRNAs.As describedabove E6E7 mRNAisoformexpressionisregulatedbyEGF-controlled alterna-tivesplicing(Rosenbergeretal.,2010).EGFdepletionislinkedto theactivitiesofhnRNPsA1andA2andfavoursintronsplicing lead-ingtoexpressionoftheE6*Iisoform(Rosenbergeretal.,2010). Evidencealsopointstoa roleforhnRNPA1 invirallate mRNA expression.hnRNPA1canbindAG-richsplicingsilencerelements intheHPV16L1codingregion(Table1)andcounteracttheactivity ofSRproteinsboundatL1ESEstosuppresstheuseoftheHPV16 late3-splicesiteSA5639(Zhaoetal.,2007a;Zhaoetal.,2004). hnRNPC1appearstobindtheviralearly3 untranslatedregion andactivateuseofthe5-splicesiteSD3632atthe3endofthe E4openreadingframeresultinginlatemRNAproduction(Dhanjal etal.,2015).Conversely,hnRNPDhasbeenshowntobindtotwo AUAGUAmotifsinanESSelementadjacenttoSD3632thatcontrols latemRNAsplicing(Lietal.,2013b).Indeed,ithasbeenproposed thathnRNPC1,togetherwithhnRNPDandhnRNPA2/B1,forma complexonthissplicingsilencer,buthnRNPC1activityis dom-inantandcounteractsthehnRNPDand hnRNPA2/B1-mediated ESS-inducedsuppressionofSD3632leadingtolatemRNAsplicing (Dhanjaletal.,2015;Lietal.,2013b).Polypyrimidinetractbinding protein(PTB,hnRNPI)hasalsobeenreportedtoactivatesplicing fromSD3632,perhapsbycompetingwiththeotherhnRNPproteins thatsuppressuseofthissplicesite(Sombergetal.,2008).hnRNP HhasbeenimplicatedinstimulatingHPV16earlypolyadenylation throughaG-richenhancerelementintheL2codingregion(Öberg etal.,2005)andlimitingvirallategeneexpression.hnRNPHcould
alsoantagonisevirallatemRNAsplicing,especiallyifitpromoted cooperativebindingofhnRNPproteinsontheL2L1exons,butthis possibilityremainstobeinvestigated.Finally,hnRNPsE1,E2and hnRNPKwerefoundtobindHPV16L2mRNAs.Althoughsplicing wasnotaffectedbythesehnRNPproteins,theyinhibitedlatemRNA translationininvitrostudies(Collieretal.,1998).
4.4. Terminalexondefinition
Sofar,nodataexistonhowthe5-mostexoninanyHPV tran-scriptisdefined.Itisentirelypossiblethatsomeoftheproteins alreadydiscoveredtobindviralRNAscouldplaythissortofrole insplicingregulation.Forexample,forHPV16,proteinsboundto thecap,or5 untranslatedregion,ofviralmRNAscouldforma cross-exoncomplexwiththefirstE6exon/intronjunction(SD226) intranscriptssynthesizedfromP97,ortheE4exonintranscripts
initiatedfromP670.Moreinformationisavailableregarding
possi-blemechanismsof3terminalexondefinition,bothfortheearly mRNAsthatterminateattheearlypolyadenylation,andthoselate mRNAsthatterminateatthelatepolyadenylationsitesofHPV16. Theearlypolyadenylationsiteisinherentlyweakbecausethe cis-acting sequences that bind the CPSF and CstF polyadenylation complexesareofpoorconsensus.However,polyadenylation com-plexformationisstrengthenedbyRNAbindingproteinsthatform acomplexona57nucleotideU-richregionintheearly3 untrans-latedregion.ProteinsthatbindthisregionincludehnRNPC1/C2, PTB(hnRNPI) andthepolyadenylationfactorsCPEB1andhFip1 (Zhaoetal.,2005).Recently,HPVE2hasalsobeenshowntobind theCPSF-CstFpolyadenylationcomplextoreduceefficiencyofHPV earlypolyadenylationleadingtotranscriptionread-throughtothe lateregionandproductionofvirallatemRNAs(Johannsonetal., 2012).SinceE2canbindSRproteinsincludingSRSF1(Jangetal., 2015;Mulleretal.,2012),itispossiblethatE2candefinetheearly
terminalexon,E4,throughanE2-containingpolyadenylation com-plexevenifthecomplexhasthepotentialofinhibitoryactivityfor earlypolyadenylation.Moreover,E4ESEsequencesthatbindSRSF1 areknowntoinfluenceefficientuseoftheearlypolyadenylation site(Rushetal.,2005;SombergandSchwartz,2010)thus high-lightingtheconnectionbetweenterminalexon-boundproteinsand polyadenylation.
AttheendoftheL1openreadingframe,andspanningthestart ofthelate3untranslatedregion,isa79ntRNAelementtermed thenegativeregulatoryelement(NRE)orlateregulatoryelement (LRE)(Graham,2008).Theelementisaconservedfeatureof papil-lomaviruses(Zhaoetal.,2007b),andinhibitslategeneexpression inundifferentiatedepithelial cells. For HPV16,it hasbeen pro-posedthattheLREmayenhancelatepolyadenylation,butanother mechanismmayinvolveformationofanexondefinitioncomplex composedofU1 snRNP,U2AFand SRSF1 ontheelement.Such acomplexcouldmimicamini-intronandnegativelyregulateL1 exondefinition(Furthetal.,1994;McPhillipsetal.,2004). Interest-ingly,theelementalsobindshnRNPA1whichmightbeexpected tocounteracttheactivityoftheSRSF1complex(Chuen-Imetal., 2008).Theeffectofthebalancebetweensplicingstimulatoryand inhibitoryfactorsinHPVmRNAterminalexondefinitionrequires furtherinvestigation.
5. HPVregulationofSRproteinactivity
Demonstratingtheir key rolesin HPV infection,SR proteins appeartobeupregulatedduringtheHR-HPVlifecycleinan epithe-lialdifferentiation-specificmanner.Forexample,SRSF1,2and3 levelsare significantly increased in themid toupper layers of infectedkeratinocytesand intissuesamplesfrompatientswith lowgradecervicallesionsthatrepresenttransientHPVinfection (Moleetal.,2009a).ThisiscontrolledbytheHPVE2transcription factor(Moleetal.,2009a),whichbindsandtrans-activatesthe pro-motersoftheSRproteingenes(Klymenkoetal.,2016;Moleetal., 2009b).TheobservedhighlevelsofSRproteinsinthenucleiofcells ofthemidtoupperlayersoftheinfectedepitheliumcorrelatewith peaklevelsofE2thatarealsodetectedinthesecells(Coupeetal., 2012;Klymenkoetal.,2016;Xueetal.,2010).Itcouldbeargued thatE2activationofSRproteinsinthemidtoupperepithelial lay-erswouldbedetrimentaltoviralreplication.HighlevelsofSRSF1 shouldactivatealternativesplicingfromSD880toSA3358atthe 5endoftheE4openreadingframe,thusprecludingexpressionof mRNAsencodingE2.ItispossiblethatotherSRproteinsorhnRNPs thatbindtheE2regionofviralpre-mRNAscompetewithE4splice siteselectiontoallowexpressionofE2,butE2splicingregulatory factorshavenotyetbeenreported.Itisworthnotingthatverylow levelsofE2mRNAscomparedtoE4mRNAsaredetectedin HPV-infectedpatienttissues(Schmittetal.,2010;Chenetal.,2014). InalternativesplicingtheruleseemstobethatformRNAs sub-jecttoalternativesplicing,thefirstpairof5-and3-splicesites intronthatemergefromRNApolymeraseIIarepreferentially cho-senforsplicingoversubsequentsites.Thus,inmRNAsinitiatingat P670ofHPV16theintronbetweenSD880andSA2582orSA2709
shouldberemovedin preference totheintronbetweenSD880 andSA3358.Leakysplicingcontrolcouldresultinread-through totheE4splicesiteandcompetitionbetweenarchitecture (first-splice-first)andadominantE4ESEcouldyieldtheobservedlow levelsofE2mRNAsandhighlevelsofE4-encodingmRNAs.E2 con-trolofSRproteinscouldbebeneficialtocompletionofthevirus replicationcycle.SRproteinexpressionisgreatestinbasal epithe-liallayersbutexpressionlevelsdecreasetoalowlevelinnormal, uninfected,differentiatedkeratinocytes(Fayetal.,2009;Jiaetal., 2010;Moleetal.,2009a).Thischangeisexpectedbecause differen-tiatedepithelialcellsarebeginningtoshutdownnuclearfunctions
suchassplicing.However,ininfected,differentiatedkeratinocytes thevirallateproteinssuchasE4andL1areexpressfromspliced mRNAs.Therefore,HPV-mediatedupregulationofkeysplicing fac-tors,forexamplefactorsthatbindtheL1ESE(Zhaoetal.,2007a), couldfacilitateefficientandaccuratesplicingintheinfected differ-entiatingepithelialcell.SRSF3seemstobeakeySRproteindriving lategeneexpression(Klymenkoetal.,2016),butSRSF3also regu-latesotherSRproteinsandhasbeendesignatedamasterregulator ofsplicing(Ajiroetal.,2016;Änköetal.,2012).ThismeansthatHPV up-regulationofSRSF3couldhavequiteglobaleffectson constitu-tiveandalternativesplicingindifferentiatedkeratinocytes,evento theextentofinducingade-differentiationorpre-neoplastic phe-notype.AnintriguingpossibilityemergesthatHPVE2controlofSR proteinexpressionduringandinfectiouslifecyclecouldcontribute toHPV-associatedtumourprogression.
5.1. E2asasplicingfactor
HPVE2proteinplaysacrucial roleintheHPVlifecycleand pathogenicityduetoitsinvolvementinviralgenomereplication, transcriptionandsegregation(McBride,2013).Itconsistsofthree functional regions, an N-terminus which is the transactivation domain,aC-terminalDNAbindingdomainandahingeregionthat linkstheN-andC-termini(Hegde,2002).TheinteractomeofE2 proteinswithcellularproteinshasrecentlybeenanalysedtogive aclearer insightinto thewide rangeof E2activities(Jangetal.,
2015;Mullerand Demeret,2012).E2 caninteractwithSR
pro-teinsSRSF1,2,4,5and7(Bodaghietal.,2009;Jangetal.,2015; Laietal.,1999;MullerandDemeret,2012).E2proteinalso inter-actswithkeycomponentsofthespliceosomeandothercellular RNAprocessingfactors(Graham,2016).Whileearlystudiesonthe lowriskHPV5E2serine-arginine-richhingedomainshowedthatit couldfacilitatesplicing(Laietal.,1999),alaterstudyfoundthatit couldnotandsuggestedinsteadthatHPV16E2mayhavesplicing repressiveactivity(Bodaghietal.,2009).Thisstudyalsoshowed thatE2canbindRNAdirectlyviaitsC-terminaldomain(Bodaghi etal.,2009).E2appearstohavemanyofthepropertiesofaprotein thatcannucleateprotein–protein andprotein-RNAinteractions. AlthoughfurtherstudiesarerequiredtoelucidatetheroleofE2 insplicingregulation,itisclearthatE2proteincouldaffect splic-ingintheinfectedcellsimplythroughitsabilitytorecruitcellular splicingfactorstoRNAinasimilarmannertoitsrecruitmentof polyadenylationfactors(Johannsonetal.,2012).Indeed,astudy usingexonarrayanalysisrevealedthat overexpressionof E2in U2OSosteosarcomacellsresultedinsignificantchangesincellular alternativesplicing(Gausonetal.,2014).Thiseffectcouldbedue toE2transcriptionalupregulationofSRproteinexpression. How-ever,increasedSRproteinlevelswerenotobservedinthestudy. Infact,U2OScells,likemanycancercelllines,alreadyexpresshigh levelsofSRproteins(Graham,unpublisheddata)perhapsnegating thetranscriptionaltrans-activationeffectofE2.Themostlikelyand excitingexplanationisthatE2altersregulationofcellular alterna-tivesplicing.
5.2. SRproteinphosphorylationduringinfection
SRprotein activityis controlledthroughphosphorylationby serine-arginineproteinkinases(SRPK)1and2,Chk1and Topoiso-merase1(ZhouandFu,2013).Phosphorylation/dephosphorylation cyclesarecrucialinsplicingandnuclearexportofSRproteins.As wellasSRSFproteins, SRproteinkinasesmayalsoberegulated duringHPVinfection.Forexample,HPV1E4colocaliseswithand regulatesSRPK1 ininfectedkeratinocytes(Prescottetal.,2014). E4bindingtoSRPK1altersitsabilitytophosphorylateSRproteins invitrosuggestingthatHPVinfectioncancontrolnotonlySR pro-teinlevels,butalsotheirvariouscellularactivities(Prescottetal.,
toalterHPV16mRNAproductiontofavourproductionoftheviral E4ˆL1spliced latemRNA(Sombergetal.,2009).E4orf4interacts withSRproteinsbutcanalsobindthephosphatasePP2A.Indeed, E4orf4overexpressioninHeLacellsresultedinlossofSRprotein phosphorylation(Kanopkaet al.,1998).Using HPVsubgenomic expressionplasmidsinHeLacells,itwasfoundthat overexpres-sionofunder-phosphorylatedSRproteinsinducedviralsplicing tothemajorlate3splicesiteSD5639andproductionofthelate E4ˆL1mRNA(Somberget al.,2009).The phosphorylationstatus of SR proteinsduring HPVinfection of theepithelium remains tobestudiedin detail.At leastforSRSF1, anincrease in phos-phorylationwasdetectedupondifferentiationofHPV16-infected W12 cervical epithelial cells, but we have not yet determined thedownstreameffectsofthisalterationonSRSF1andtheviral lifecycle(McPhillipsetal.,2004).Somedrugsareavailablethat inhibitSRPK1.Thesesmallmoleculeinhibitorshavebeenshownto successfullyinhibitreplicationofhumanimmunodeficiencyvirus (HIV),hepatitisCvirus(HCV)andSindbisvirus(Hernandez-Lopez andGraham,2012).Itwillbeinformativetousethesecompounds toinvestigateanyeffectsonHPVsplicingpatterns.
6. Conclusions
Thisreviewdetailsmany studieswhoseconclusionssupport thehypothesisthatcellularsplicingregulatorymechanisms,and splicingfactorssuchasSRproteinsandhnRNPs,areessentialfor controllingHPVgeneexpression.Moreover,anemerging hypoth-esis is that HPVinfection controls cellular splicing in order to completethevirallifecycleinthedifferentiatingepithelium.The necessarylinkbetweentheHPVlifecycleandepithelial differen-tiationmustbeconsideredimportantinelucidatingmechanisms regulatingviralmRNAproduction.ActivityofanyRNAregulatory elementshouldberesponsivetothecomponentsoftheprotein complexthatformsuponit.Thepositiveandnegativeeffectsofthe variousRNA-bindingfactorsonthemultiplepapillomavirus ele-mentsthatregulateviralRNAprocessingcouldbealteredduring theepithelialdifferentiationprogramduetochangedlevelsofthese factorsbetweenundifferentiatedandfullydifferentiatedepithelial cells.Forexample,indifferentiatedkeratinocytesanHPV-induced increaseinlevelsofkeySRproteinscouldalterthecompositionor efficiencyofformationofsplicingcomplexes(orpolyadenylation complexes)leadingtoappropriatelate splicingevents, stimula-tionoflateterminalexondefinitionandlatepolyadenylation.This wouldleaddirectlytoefficientvirallateproteinproductioninthe appropriate(upper)epitheliallayers.
Alternativesplicingisessentialfor thelife cyclesof nuclear-replicatingvirusesbecauseitallowsexpressionofmultipleproteins fromasmallgenome.Itisclearthatalternativesplicingisrequired fortheHPVreplicativelifecyclebecauseitisonlythrough alterna-tivesplicingthatmRNAsencodingtheE1andE2viralreplication factorsareexpressed,thecorrectbalanceofE6,E6isoformsand E7proteinsaresynthesized,andcapsidproteinsynthesisis coor-dinatedwithepithelialdifferentiation.Alternativesplicingisalso implicatedinHPV-associatedcancerprogressionduetoexpression ofthevariousE6mRNAisoformsthatencodetheviraloncoproteins whoseoverexpressionleadstotumorigenesis.Regulationofviral geneexpressionatthelevelofalternativesplicingstillrequires fur-therstudyandnumberofimportantunansweredquestionsremain tobeaddressed.For example,howdoesthearchitectureofthe variousviralpre-mRNAsallowalternativesplicinggiventhe pos-sibility of sterichindrance betweensplicing complexes formed atintron-exonjunctionsonshortintronssuchasthosefoundin E6E7isoformRNAs?ThevirallatemRNAscontainunusuallylong exons(L1exon: 1.5kb,L2L1bicistronicexon:2.9kb)that likely
How are theseexonsdefined for accurate splicing?Some viral mRNAs(e.gE6flmRNA)arepredictedtocontainintronicsequences whichwould normallyprecludetheirnuclearexportand trans-lation.Othervirusesexpressproteinsthatensureefficientexport ofviralintron-containingtranscripts(HarrisandHope,2000)but there islittle information onhowHPVensures exportof these mRNAs.Further,therole of theHPVE2 protein,and its poten-tialroles inregulating viraland cellularsplicing, hasyetto be fullyelucidated.UnderstandingHPVsplicingcouldleadto devel-opmentofnoveltherapeuticapproachestoinhibitviralreplication or virally-induced tumour formation in future (Graham, 2010;
Hernandez-LopezandGraham,2012).
Acknowledgements
A.A.AFaizoissupportedbyaPhDstudentshipfromKing Abdu-lazizUniversity,SaudiArabia.Weacknowledgefundingfromthe MedicalResearchCouncilascorefundingfortheMRCUniversity ofGlasgowCentreforVirusResearch.StudiesintheGrahamlabon HPVlifecycleandRNAprocessingwerefundedbytheWellcome Trust,grantnumberWTd004098.
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