Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes

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Virus Research

jo u r n al h om ep age :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

Review

Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes

Clara C. Posthuma

, Aartjan J.W. te Velthuis

, Eric J. Snijder

aMolecularVirologyLaboratory,DepartmentofMedicalMicrobiology,LeidenUniversityMedicalCenter,Leiden,theNetherlands

bSirWilliamDunnSchoolofPathology,UniversityofOxford,SouthParksRoad,OxfordOX13RE,UnitedKingdom

cClarendonLaboratory,DepartmentofPhysics,UniversityofOxford,ParksRoad,OxfordOX13PU,UnitedKingdom

a r t i c l e i n f o

Articlehistory:

Received9December2016

Receivedinrevisedform24January2017 Accepted26January2017

Availableonline6February2017

Keywords:

Coronavirus Arterivirus

Replicationandtranscriptioncomplex Polymerasefidelity

Processivityfactors SubgenomicmRNAsynthesis

a b s t r a c t

Coronavirusesandarterivirusesaredistantlyrelatedhumanandanimalpathogensthatbelongtothe orderNidovirales.Nidovirusesarecharacterizedbytheirpolycistronicplus-strandedRNAgenome,the productionofsubgenomicmRNAsandtheconservationofaspecificarrayofreplicasedomains,including keyRNA-synthesizingenzymes.Coronaviruses(26–34kilobases)havethelargestknownRNAgenomes andtheirreplicationpresumablyrequiresaprocessiveRNA-dependentRNApolymerase(RdRp)and enzymaticfunctionsthatsuppresstheconsequencesofthetypicallyhigherrorrateofviralRdRps.The arteriviruseshavesignificantlysmallergenomesandformanintriguingpackagewiththecoronaviruses toanalyseviralRdRpevolutionandfunction.TheRdRpdomainofnidovirusesresidesinacleavage productofthereplicasepolyproteinnamednon-structuralprotein(nsp)12incoronavirusesandnsp9 inarteriviruses.Inallnidoviruses,theC-terminalRdRpdomainislinkedtoaconservedN-terminal domain,whichhasbeencoinedNiRAN(nidovirusRdRp-associatednucleotidyltransferase).Althoughno structuralinformationisavailable,thefunctionalcharacterizationofthenidovirusRdRpandthelarger enzymecomplexofwhichitispart,hasprogressedsignificantlyoverthepastdecade.Incoronaviruses severalsmaller,non-enzymaticnspswerecharacterizedthatdirectRdRpfunction,whilea3^-to-5^exori- bonucleaseactivityinnsp14wasimplicatedinfidelity.Inarteriviruses,thensp1subunitwasfound tomaintainthebalancebetweengenomereplicationandsubgenomicmRNAproduction.Understand- ingRdRpbehaviourandinteractionsduringRNAsynthesisandsubsequentprocessingwillbekeyto rationalisingtheevolutionarysuccessofnidovirusesandthedevelopmentofantiviralstrategies.

©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction...59

2. GeneralfeaturesofnidovirusRNApolymerasesubunits:twodomainswithdistinctactivities...60

3. StructuralmodelsofnidovirusRdRps...62

4. InvitroRdRpactivityoftheCoVnsp12...63

5. CoVnsp8:primaseornot? ... 63

Abbreviations:5-AZC,5-azacytidine;5-FU,5-fluorouracil;AV,arterivirus;CBV3,coxsackievirusB3;CoV,coronavirus;EAV,equinearteritisvirus;EM,electronmicroscopy;

ExoN,exoribonuclease;FCoV,felinecoronavirus;FMDV,footandmouthdiseasevirus;HCoV-229E,humancoronavirus229E;IMPDH,inosine-5^-monophosphatedehydro- genase;IVRA,invitroRNAsynthesisassay;kb,kilobases;M2H,mammalian2-hybrid;MERS,MiddleEastrespiratorysyndrome;MHV,murinehepatitiscoronavirus;NiRAN, nidovirusRdRp-associatednucleotidyltransferase;nsp,non-structuralprotein;ORF,openreadingframe;PABP,Poly(A)-bindingprotein;PEDV,porcineepidemicdiarrhea virus;PNS,post-nuclearsupernatant;pp,polyprotein;PRRSV,porcinereproductiveandrespiratorysyndromevirus;RBV,ribavirin;RdRp,RNAdependentRNApolymerase;

RTC,replicationandtranscriptioncomplex;SARS,severeacuteresporiatorysyndrome;sg,subgenomic;ss,single-stranded;TGEV,transmissiblegastroenteritisvirus;TRS, transcription-regulatorysequence;ub,ubiquitin;wt,wild-type;Y2H,yeast2-hybrid;ZBD,zinc-bindingdomain.

∗ Correspondingauthor.

E-mailaddress:c.c.posthuma@lumc.nl(C.C.Posthuma).

1 Theseauthorscontributedequally.

http://dx.doi.org/10.1016/j.virusres.2017.01.023

0168-1702/©2017TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

6. TheRNApolymeraseactivityofnsp12andtheroleofnsp8asco-factor:thensp7+8+12tripartitecomplex .... 64

7. TheelusiveinvitroRNApolymeraseactivityofAVnsp9...65

8. Thenidovirus-specificdomainattheN-terminusoftheRdRp-containingsubunit:NiRAN...66

9. Faithfulnidovirusreplicationandtranscriptioninvitroandtheinvolvementofotherco-factors...66

10. MakingtheRdRpswitchfromcontinuousintodiscontinuousmode:AVnsp1...67

11. PolymerasefidelityandnucleotideexcisionbytheCoVnsp14-ExoNexoribonuclease...68

12. InhibitorsofnidovirusRNApolymeraseactivity...69

13. Conclusionandoutlook...70

Acknowledgements ... 70

References...70

1. Introduction

Positive-strandedRNA(+RNA)virusesthatbelongtotheorder Nidoviralesinfectawiderangeofvertebrates(familiesArteriviridae and Coronaviridae)or invertebrates (Mesoniviridae and Roniviri- dae)(deGrootetal.,2012;Lauber etal.,2012)andcanhave a significanteconomicandsocietalimpact.Forexample,infections withthearterivirus(AV)porcinereproductiveandrespiratorysyn- dromevirus(PRRSV) haveseverelyaffectedthe swineindustry foralmostthreedecadesnow(Holtkampetal.,2013;Perezetal., 2015; Pileriand Mateu,2016), whereas zoonotic coronaviruses (CoVs)havecausedepisodesofsevereacuterespiratorysyndrome (SARS)andMiddleEastrespiratorysyndrome(MERS)inhumans (Grahametal.,2013;PerlmanandNetland,2009)andmaydoso again(Menacheryetal.,2015).AnimalCoVscontinuetoemergeand causegreateconomiclosses,asexemplifiedbytherecentoutbreaks of porcine deltacoronavirus and the porcine epidemic diarrhea virus(PEDV) in Chinaand theUnited States(Choudhuryet al., 2016;Wengetal.,2016;Zhang,2016).Genetically,thenidoviruses constituteamonophyleticgroupthatischaracterizedbycommon ancestryoftheirkeyreplicativeenzymesandassociatedsimilari- tiesingenomeorganizationandexpression(Fig.1A)(deGrootetal., 2012;Lauberetal.,2013).However,nidovirusgenomesizesvary significantly,withAVgenomesrangingfrom13to16kilobases(kb), mesonivirusgenomesfrom20to21kb,andCoVgenomesfrom26 to34kb(Lauberetal.,2013).Ithasbeenpostulatedthatthissize variationreflectsalonghistoryofgradualgenomeexpansion,dur- ingwhichthedifferentnidoviruslineagesadaptedtotheirspecific nichesbyacquiringarangeofnovelfunctions,encodedbydomains thatwereeitherincorporatedasadditionalgenesorintegratedinto thelargenidovirusreplicasegene(Lauberetal.,2013).

In all nidoviruses, at leasttwo-thirds of thecapacity of the polycistronicgenomeisoccupiedbythetwolargeopenreading frames(ORFs;1aand1b)thattogetherconstitutethereplicasegene (Fig.1A).BothORFsaretranslateddirectlyfromtheviralgenome andbrieflyoverlapwherea −1ribosomalframeshiftdirectsthe expression of ORF1b to facilitate the formation of an ORF1ab- encoded polyprotein (pp1ab). Cleavage of the pp1aand pp1ab polyproteinsby multipleintrinsic protease activities,in combi- nationwith−1and−2frameshifting inthensp2codingregion inmostarteriviruseswiththeexceptionofequinearteritisvirus (EAV),resultsintheproductionof 13to17non-structuralpro- teins (nsps) (Fang et al., 2012; Li et al., 2014b; Snijder et al., 2013;Ziebuhretal.,2000).Thecommonancestryoftheextremely divergednidoviruslineagesisprimarilyreflectedin theconser- vationofanarrayof‘corereplicasedomains’(Gorbalenyaetal., 2006; Lauber et al., 2013; Snijder et al., 2016), which is com- posedoftwo trans-membraneproteins,theviralmainprotease, and–encodeddownstreamoftheORF1a/1bframeshiftsite–the RNA-dependentRNApolymerase(RdRp)-andhelicase-containing subunits (Fig. 1B),with thecanonical RdRp domainresiding in CoVnsp12andAVnsp9.Nidovirusgenomesarethoughttohave expanded graduallyby gene duplication and theacquisition of

noveldomains(Lauberetal.,2013).Duringthisprocess,specific innovationsmayhaveenabledthemtoexploreanunprecedented evolutionary space and adapt toa wide variety of hostorgan- isms,includingmammals,birds,fish,insectsandcrustaceans.In particular, thegeneralgenomesize restrictions ofRNA viruses, commonlyattributedtothepoornucleotideincorporationfidelity oftheviralRdRpdomain,mayhavebeenmitigatedbytheacqui- sition of compensatoryenzymatic functions(Denget al.,2014;

Eckerleetal.,2010;Eckerleetal.,2007;Gorbalenyaetal.,2006;

Snijderetal.,2003).Consequently,somenidovirusnspscontaina uniquesetofactivitiesnotseeninother+RNAviruses(discussed e.g.inSections8and11inmoredetail).Imagingandbiochemical characterizationofnidovirusnspshaveshownthattheyaretar- getedtospecificvirus-inducedmembranestructures(reviewedin (Hagemeijeretal.,2012;Neumanetal.,2014;vanderHoevenetal., 2016))wheretheyassembleintoaso-calledreplicationandtran- scriptioncomplex(RTC;see(Neumanetal.,2014;Snijderetal., 2016;Subissietal.,2014a)forreviews).

The RNA-templatedsynthesis of newRNA bythe viralRNA polymeraseisarguablythekeystepintheinfectioncycleof all RNA viruses. In the case of nidoviruses and theirpolycistronic genomes,RNAsynthesisentailsnotonlygenomeamplificationbut alsothesynthesisofanestedsetofsubgenomic(sg)mRNAs(Fig.1A;

reviewedin(Pasternaketal.,2006;Sawickietal.,2007;Solaetal., 2011).ThesgmRNAsservetomakethegenesdownstreamofthe nidovirusreplicaseORFs1aand1baccessiblefortranslation.These ORFsencodestructuralandso-called ‘accessory’proteins,which areoftendispensableforreplicationinvitro,butimportantfore.g.

immuneevasionandpathogenesisinvivo(Liuetal.,2014;Weiss andLeibowitz,2011).Forthepurposeofthisreview,wewillreferto theprocessofsgmRNAsynthesisas‘transcription’;theunderlying mechanismswillbediscussedinmoredetailinSection10.

Eachnidoviral sg mRNAis producedfrom a complementary subgenome-lengthtemplate.MinusstrandRNAsynthesiscanbe either continuous (producing a full-length minus-strand tem- plate for genome replication) or discontinuous to produce the subgenome-lengthtemplatesfortheproductionofthesgmRNAs (SawickiandSawicki,1995;Sethnaetal.,1989).Inadditiontothe overallRNAstructureofthegenomeandtranscription-specificpro- teinfactors,conservedtranscription-regulatorysequences(TRSs) in the genomic template are thought to be the prime trigger andguidingelementsthatmakethenidovirusRTCsynthesizea subgenome-lengthratherthana full-lengthminusstrand.Thus, theTRSsconstituteoneclassofcis-actingRNAsignalswithwhich thenidovirusRTCneedstointeract,although wenotethatthis featurehasmainlybeenaddressedfromtheRNAside(e.g.,bysite- directedmutagenesisofTRSs)(Dufouretal.,2011;Pasternaketal., 2001;Zheng et al.,2014;Zuniga etal.,2004).Thesame essen- tiallyappliestothemajorcis-actingRNAelementsthatconstitute theinitiationsitesforminusandplusstrandRNAsynthesisnear the3^endoftheplusandminusstrand,respectively.Indeed,in bothCoVandAVgenomes,anumberofprimaryandhigher-order structuralfeatureshavebeenidentifiedandstudied,includingsev-

Fig.1.Nidovirusgenomeorganizationandthecorereplicasedomains.(A)Genomeorganizationofrepresentativesofthefourmajornidoviruslineages(CavV,Cavallyvirus;

GAV,gill-associatedvirus;notethattheEAVgenomeisdrawntoadifferentscale).Foreachgenome,theknownopenreadingframes(ORFs)areindicatedwiththereplicase ORFs1aand1bdepictedingreyandstructuralproteingenesdepictedindifferentcolours.ORFsencoding‘accessoryproteins’(inSARS-CoV)orpoorlycharacterizedproducts aredepictedinwhite.ToillustratetheprincipleofsubgenomicmRNAsynthesis,asemployedbyallnidoviruses,thenestedsetstructureandcompositionofthemRNAs issummarizedforSARS-CoV,withthecommon5^leadersequenceindicatedinredandthetranslatedpartofthegenomeandeachofthesubgenomicmRNAsdepicted ingreen.Seemaintextformoredetails.(B)Domainorganizationofthepp1abreplicasepolyproteinforthefourmajornidoviruslineages(notethattheAVproteinis drawntoadifferentscale).Proteolyticcleavagesandnon-structuralproteinnumberingareindicatedforEAVandSARS-CoV.Theschemehighlightstheconservationofthe so-callednidovirus‘corereplicase’,consistingoftheORF1a-encodedmainprotease(Mpro)flankedbytwotransmembrane(TM)domains,followedbytheORF1b-encoded NiRANnucleotidyltransferase(NT),RNApolymerase(RdRp),zincbindingdomain(Z)andsuperfamily1helicase(HEL1).Accessory(papain-like)proteasedomainsand theircleavagesitesareindicatedforEAVandSARS-CoV(P1,P2,PLpro).Thezinc-fingerdomain(F)inEAVnsp1thatiscrucialforsubgenomicmRNAsynthesis(seetext)is alsohighlighted.TheC-terminalpartofpp1abencodesanumberofenzymaticdomainsthatarenotstrictlyconservedamongallnidoviruslineages:U,endoribonuclease, conservedinvertebratenidoviruses;EN,exoribonuclease(ExoN)conservedinnidoviruseswithgenomesizes>20kb(seetext);N7-and2^-Omethyltransferases(N7and 2O)involvedincapmodification(notidentifiedinAVs).

eralRNAhairpinsandpseudoknots.Theimportanceofmultipleof theseelementsissupportedbybioinformatics(conservation),bio- chemicalprobing,andsite-directedmutagenesis.Foranoverview ofthestructureandfunctionoftheRNAelementsthathavebeen implicatedinCoVorAVreplicationandtranscription,thereader isreferredtodetailedreviewsthathavebeenpublishedelsewhere (Madhugirietal.,2016;Masters,2007;Snijderetal.,2013;Sola etal.,2011;YangandLeibowitz,2015).However,ithasremained largelyunclearwhetherthese elementsinteractwiththeRdRp domain-containingsubunitdirectlyorwithothercomponentsof theRTC,asforexamplereportedbyZüstetal.whoobservedthat second-sitemutationsinthesmallnsp8andnsp9subunitsofthe RTCcouldcompensateformutationsinconservedRNAsequences nearthegenomic3^endofthemurinehepatitiscoronavirus(MHV) genome(Züstetal.,2008).Inthecaseofthe5^-proximaldomainof thegenomicRNA(oritsminus-strandedcounterpart),functional studieshavebeen complicatedby thefactthat RNAsignals for replication,transcription,translationand(potentially)packaging maybeoverlappinganddifficulttoseparate,bothphysicallyand functionally.

Inthis review, we willfocusmainly on‘theproteinside’ of nidovirusRNAsynthesis,andwhatisknownabouttheinitiation

of RNAsynthesis by nidovirus RNA polymerases.The ability of thenidovirusRTCtodirectthevariousprocessesofRNAsynthesis outlinedabovecriticallyreliesontheactivityofacanonicalRdRp domain,whichresidesinnsp12inCoVsandinnsp9inAVs.These replicasesubunits,whichalsocarryauniqueandnidovirus-specific N-terminalextension,arebelievedtocatalysethenucleotidecon- densationreactionthatdrivesreplicationandtranscription.Given theirpivotalroleinviralreplicationandtheefficacyofusingpoly- meraseinhibitorstocombatothervirusinfections,AVnsp9and CoVnsp12are alsoconsideredan importanttarget for rational drug design. Unfortunately, these nsps have remained refrac- torytostructuralanalysessofar,butsignificantadvancesinour understandingof theseenzymesand theirco-factorshavebeen madeusingbioinformatics,biochemicalandmolecularvirological approaches,inparticularforAVsandCoVs,aswewillsummarize below.

2. GeneralfeaturesofnidovirusRNApolymerasesubunits:

twodomainswithdistinctactivities

In all nidoviruses, the RdRp-containing subunit consists of at least two domains (Fig. 2A): a nidovirus-specific N-terminal

Fig.2.Schematicrepresentationofnsp12fromSARS-CoVandnsp9fromEAV.A)ThepositionofNiRAN(lightgrey),theRdRpdomain(darkgrey)andtherespective motifs(whiteboxes;subscript“N”wasaddedtoNiRANmotifstodicriminatethemfromRdRpmotifs)areindicatedinthefigure.TheexactC-terminalborderofNiRAN, aswellastheN-terminalborderoftheRdRparenotdefinedyetandareindicatedwithdashedlines.Thepositionofribosomalframeshiftingisindicatedwithatriangle;

translationoftheprecedingORF1aproductsterminatesshortlydownstreamtheframeshiftsite(SARS-CoVnsp11within4aminoacids,EAVnsp8within1aminoacid,not indicatedinthefigure).NiRANandRdRpmotifsaredisplayedaswhiteboxesinthefigure,basedon(Lehmannetal.,2015a)forNiRANmotifs,and(Xuetal.,2003)(SARS-CoV) or(Beerensetal.,2007)(EAV)fortheRdRpmotifs.NotethatMotifDwasnotdefinedforEAVnsp9inBeerensetal.andthattheapproximatepositionisindicatedasadashed box.Therulersindicateaminoacidpositionsintheproteins.Questionmarkindicatespartofthenspthatmayrepresentalinkingdomainoradomainwithanadditional (unknown)function.B)AlignmentofNiRANmotifsA,BandCfromeightrepresentativenidovirusesfromall4families(Modifiedfrom(Lehmannetal.,2015a)).C)Alignment ofRdRpmotifsA,BandCfromthesamenidoviruses.Completelyconservedresiduesareindicatedingreyboxes.SARS-CoV,SARScoronavirusFrankfurt1(AY291315;

Coronaviridae);MERS-CoV,MiddleEastrespiratorysyndromecoronavirusEMC/2012(JX869059.2;Coronaviridae);GAV,Gill-associatedvirus(AF227196;Roniviridae);YHV, yellowheadvirus(EU487200;Roniviridae);CAVV,Cavallyvirus(HM746600;Mesoniviridae);MenoV,Menovirus(JQ957873;Mesoniviridae);PRRSV-1,porcinereproductive andrespiratorysyndromevirus,Europeangenotype(GU737264.2;Arteriviridae).EAV,Equinearteritisvirus(DQ846750;Arteriviridae).

domainisfollowed bya canonical RdRpdomainoccupyingthe C-terminaltwo-thirdsoftheprotein(Gorbalenyaetal.,1989;Xu etal.,2003).AlthoughthepresenceofthisN-terminalextension wasfirstrecognizedwhenthereplicasepolyproteincleavagesites werepredictedover25yearsago(Gorbalenyaetal.,1989),anenzy- maticactivity(nucleotidylation)wasassignedtoitonlyrecently.

ThedomainisnowreferredtoasthenidovirusRdRp-associated nucleotidyltransferase (NiRAN)domain (Lehmann et al.,2015a) (Fig.2B),anditwillbediscussedinmoredetailinSection8.AstheC- terminalborderofNiRANisdifficulttodefine,yetanotherdomain (approximately50–175aminoacidsforAVandCoV,respectively;

Fig.2A)mayturnouttoconnecttheNiRANand RdRpdomains (Lehmannetal.,2015a).

RdRpdomainsincludesixcanonicalconservedmotifs,named AtoF(Figs.2Cand3A),thatareinvolvedintemplateandsub- strate recognitionand the catalysis of nucleotidecondensation (Pochetal.,1989;teVelthuis,2014).Nucleotidecondensationpar- ticularlydependsonmotifsAandC,whichassistincoordinating thetwometalionsintheactivesite(AlphonseandGhose,2017;

teVelthuis,2014).NidovirusRdRpdomainswerefirstidentified bysequencecomparisonwithknownRdRpdomainsfromother

virusgroups,revealinganSDDsequence(insteadoftheusualGDD signaturefoundinotherpositive-strandRNAvirusRdRpdomains) asthekeyresiduesofmotifC(denBoonetal.,1991;Gorbalenya etal.,1989;Snijderetal.,1990)(residues759-761inSARS-CoV)and D618andD623astheconservedaspartateresiduesofmotifAof SARS-CoVnsp12.TheseresiduescorrespondwithD445andD450 inmotifAandSDDatpositions559-561innsp9oftheAVprototype EAV.Twodecadeslater,themutationalanalysisofconservedRdRp domainresiduesconfirmedthatthecatalyticaspartatesofmotifsA andCareimportantforRNApolymeraseactivityandviralRNAsyn- thesis(Ahnetal.,2012;Lehmannetal.,2016;Subissietal.,2014b;

teVelthuisetal.,2010a).InadditiontotheessentialRdRpdomain motifsmentionedabove,CoVnsp12hasamotifG,whichisconsid- eredasignaturesequenceforprimer-dependentRNApolymerases (Gorbalenyaetal.,2002;Xuetal.,2003).Remarkably,thismotifis onlypartiallyconservedintheAVnsp9(Beerensetal.,2007).

AstructuralpredictionfortheSARS-CoVRdRpdomainwaspub- lishedasearlyas2003(Xuetal.,2003)(Fig.3B),yetexperimental studiestosupportthismodelhavebeengreatlyhamperedbydif- ficultieswithattemptstostablyexpressandpurifyCoVnsp12.As aresult,thecrystalstructureoftheCoVRNApolymeraseremains

Fig.3. ModelsofthenidovirusRdRps.A)StructureoftheFMDVRdRp(pdb2E9R).Inthetoppanel,conservedpolymerasemotifsA-Fareindicated.Inthebottompanel, thetemplateentry,templateexitandNTPentrychannelsareindicated.B)ModelsoftheSARS-CoVnsp12RdRpgeneratedbySwiss-ModelandPhyre2superposedonthe model1O5S(Xuetal.,2003).AllthreemodelsarebasedontheC-terminalpolymerasedomainandexcludedtheN-terminalNiRANdomain.Allmodelsshowanoverall similarfold.Differencesexistinthefingerssubdomainandsurfaceloopsofthethumbsubdomain.C)SuperposedmodelsoftheEAVnsp9RdRpgeneratedbyuseofI-TASSER, Swiss-ModelandPhyre2.Overall,allthreemodelsshowaverysimilarfold,withsmalldifferencesinthefingerssubdomain.D)MotifsofthepolymerasedomainofSARS-CoV nsp12indicatedonthestructuralmodel1O5S(Xuetal.,2003).E)MotifsofthepolymerasedomainofEAVnsp9thatwasgeneratedusingPhyre2.F)Keyactivesiteand fidelityresiduesindicatedonthemodelofpolymerasedomainofSARS-CoVnsp121O5S.

tobesolvedanditsbiochemicalcharacterizationinaveryearly stage.Unfortunately,thesituationisnotmuchdifferentfortheAV RNApolymerase.Mostofourcurrentunderstandingofthestruc- turalandenzymaticfeaturesofthenidovirusRNApolymerase/RTC isbasedexclusivelyonstudieswithSARS-CoV,whichwastargeted byanumberoflaboratoriesfollowingthe2003outbreak.

3. StructuralmodelsofnidovirusRdRps

Todate,nocryo-EMorcrystallographicstructuresofCoVnsp12 orAV nsp9areavailable. However,usingsequence alignments, secondary structure predictions and homology modelling, the molecularstructureofthensp12subunitofSARS-CoV(Xuetal., 2003)(Fig.3B)andMHV(Sextonetal.,2016)hasbeenpredicted.

Incaseoftheformer,amodelcouldbereliablygeneratedforitscon- servedRdRpdomain,basedonacarefulalignmentofaminoacids 388–890toknownpolymerasesequences(Xuetal.,2003);Fig.3B.

SimilarstructuresforSARS-CoVnsp12arepredictedbyonlinetools likeSwiss-Model(Biasinietal.,2014)andPhyre2 (Kelley etal., 2015)(Fig.3B).However, nostructural predictionsarepossible fortheN-terminaldomainofnsp12,which containstheNiRAN domain,andtheC-terminaltailoftheRdRpdomain(aminoacids 891–932inSARS-CoVnsp12).Likewise,onlytheC-terminalRdRp

domainoftheAVnsp9sequencecanbemodelledatpresent,as depictedinFig.3CforEAV.

Ananalysis of the predictedCoV RdRp domain architecture usingthreemodels(Fig.3B)revealedaright-handedfoldthatcon- sistsofthumb, palmand fingerssubdomains, similartoknown crystalstructuresofRdRpdomainsorcompleteRNApolymerases (shownforfootandmouthdiseasevirus(FMDV)inFig.3A).The modelsalsopredictthepresenceofasingle-stranded(ss)RNAentry channelatthetopofthepolymeraseandanNTPentrychannelat therear(Fig.3).TheduplexthatisformedastheRNApolymerase catalysesnucleotidecondensation,thusconsistingofthenascent RNAproductandtheviraltemplate,likelyleavestheenzymevia arelativelywideexitchannelatthefrontofthemolecule.This predictedthree-channelarchitecturewouldmake thenidovirus RNApolymerasecomparabletoother+RNA-viralRNApolymerases, butdifferentfromtheRNApolymerasesofnegative-strandedRNA viruses,whicharebelievedtohaveseparateexitchannelsforthe template and nascent strand(Pfluget al., 2017;Regueraet al., 2016;teVelthuisandFodor,2016).Putativechannelssimilarto thosepredictedfortheCoVnsp12RdRpdomaincanbeseeninthe modelsoftheEAVnsp9RdRpdomaingeneratedwiththepredic- tiontoolsSwiss-Model, Phyre2andI-TASSER(Yangetal.,2015) (Fig.3C),in linewithearlier sequenceanalysessuggesting that

theoverallmoleculararchitectureisconservedamongnidovirus RdRpdomains(Beerensetal.,2007).InneithertheCoVnortheAV RdRpdomainmodelthereisevidenceforthepresenceofapriming- looporsimilarinitiationplatform,whichistypicallyrequiredfor denovoinitiationonthe3^terminusoftheviralRNA(teVelthuis, 2014).Thisisinlinewiththepresenceof(apartial)motifG(see above)andtogetherthesefeaturesmayhaveimportantfunctional implicationsfortheinitiationofviralRNAsynthesis.

In boththeCoV nsp12and EAV nsp9RdRpdomainmodels, thethreesubstratechannelsconverge atthebetasheetsofthe palmsubdomain.HerethecatalyticaspartatesofmotifsAandC coordinatethecatalyticionsthatareessentialforthenucleotide condensation reaction (te Velthuis, 2014) (Fig. 3D and E). As describedforotherRdRpdomains,thekeylysineofmotifD,which likely actsasgeneralacidduringcatalysis (Castro et al.,2009), is locatednear theentrance of the NTP channel. Furtherfunc- tionalpredictionsabouttheCoVRNApolymerasecomefroman alignmentofaPhyre2-basedmolecularmodelofMHVnsp12with thecrystalstructuresoftheRdRpdomainsofcoxsackievirusB3 (CVB3)andpoliovirus(Sextonetal.,2016).Inparticular,thisstudy identifiedV553andM611(homologsofSARS-CoVnsp12residues V557 and M615, Fig. 3F) as putative equivalents of the CVB3 RdRpresiduesI176andI230,whichareknowntobeinvolvedin RdRpfidelity.Subsequentmutationoftheseresiduestoisoleucine (V553I)andphenylalanine(M611F)inthevirusconferredresis- tanceto5-fluorouracil(5-FU)and5-azacytidine(5-AZC),or5-FU only,respectively(Sextonetal.,2016).

4. InvitroRdRpactivityoftheCoVnsp12

ThefirstpublishedattemptstopurifyaCoVnsp12employed anN-terminalGST-fusionwithSARS-CoVnsp12,whichyieldeda recombinantproteinwithpoorsolubilityandstability(Chengetal., 2005).Sincethen,advanceshavebeenmadetoimprovethesta- bilityandyieldofSARS-CoVnsp12byusingcodonoptimization, differentpurificationtagsattheN-orC-terminus,ortheaddition ofanN-terminal,cleavableubiquitin(ub)fusionpartner(incom- binationwithaC-terminalHis₆-tag)(Ahnetal.,2012;Subissietal., 2014b;teVelthuisetal.,2010a).Bacterialexpressionofthelat- ter(i.e.,ub-nsp12-His6)stillresultedinanunstableprotein,but co-expressionoftheubiquitinproteaseUbp1,whichcanhydrol- ysetheub-nsp12fusionsitetoproducearecombinantnsp12-His₆ containingthenaturalnsp12N-terminus,significantlyimprovedits stability(teVelthuisetal.,2010a).TheC-terminalHis₆-tagproved suitabletopurifynsp12fromE.coliwithoutasignificanteffecton thestabilityoftheenzyme.Overall,theseresultssuggestedthat,as forthepoliovirusRNApolymerase3D^pol(ThompsonandPeersen, 2004),theN-terminusofnsp12or,alternatively,theproperfold- ingoftheNiRANdomainwhichisnotpresentin3D^pol,isimportant forthestabilityandpossiblyalsotheactivityofnsp12(teVelthuis etal.,2010a).

AnalysisoftheCoVnsp12aminoacidsequence usingalign- mentsandmolecularmodellingpredictedthattheenzymelacks apriminglooporotherinitiationplatformthatwouldpromotede novoinitiationofRNAsynthesis(seeabove).SincemotifG,apre- sumed hallmark of primer-dependentpolymerases (Gorbalenya etal.,2002;Xuetal.,2003),wasidentifiedinthensp12sequence and structural model (Figs. 2A and 3B), it wasassumed that a primer would be required for the initiation of RNA synthesis bynsp12. Studiesfromboth Chenget al.and te Velthuiset al.

showedthattheactivityofrecombinantSARS-CoVGST-nsp12or nsp12-His6wasindeedprimer-dependentinthepresenceofMg²⁺

(Cheng et al.,2005; teVelthuis et al., 2010a).By contrast,Ahn etal.foundthatrecombinantSARS-CoVnsp12withanN-terminal His6-tag(His6-nsp12)requiredhighconcentrationsofMn²⁺.Under

theseconditions,theenzymewasalsoabletoinitiatedenovoon homopolymerictemplatesandasequencerepresentingthe3^ter- minusoftheviralgenome(Ahnetal.,2012).Likewise,His₆-nsp12 wasabletosynthesizerelativelylongRNAproductsinthepresence of Mn²⁺ (Ahn etal., 2012).The activityof nsp12-His6 on tem- plateslongerthan20nucleotideswasnottested(teVelthuisetal., 2010a).Curiously,themostrecentstudyonrecombinantSARS-CoV nsp12(containingaC-terminalstrep-tag)didnotfindanyprimer extension,denovoinitiationactivity,or binding(using1␮Mof nsp12-strep)toaprimedRNAtemplate(Subissietal.,2014b).By contrast,thensp12-His6 variantwasfoundtobindRNAwithan apparentKdof∼0.1␮M(teVelthuisetal.,2010a).

Thelimitedreproducibilityofthesebiochemicalobservations and thescant supportfor substantial processivityof nsp12are in starkcontrastwiththefactthat theCoVRNApolymeraseis requiredtoreplicateandtranscribea∼30-kbgenomeduringinfec- tion.Technicaldifferencesbetweenthesestudies(e.g.regarding theconstructsandtemplatesused)mayhavecontributedtothe mostlycontradictoryresults.Nevertheless,togethertheobserva- tionsteachusthattheactivityofnsp12alonemustberelatively weakandsensitivetothepresenceofpurificationtags,buffercon- ditionsandthesimplicityofthetemplate.ItislikelythatCoVnsp12 requiresadditionalfactorsthatimproveitsRNApolymeraseactiv- ityandthereisindeedevidencethatothernspsfulfilthisrole(see below)(Subissietal.,2014b).

5. CoVnsp8:primaseornot?

In astudyaimedat findingputativeinteraction partnersfor nsp12, a second RNA polymerase activity was reported to be associated with SARS-CoV nsp8, a 22-kDa protein encoded in ORF1a(Imbertetal.,2006).Thisactivity,whichwouldbeunique amongRNAviruses,wasobservedtoinitiateRNAsynthesisina primer-independentmannerontemplatescontainingcytidine-rich sequencesinvitroandtogenerateproductsofapproximately6nt inlength(Imbertetal.,2006).Togetherwiththeassumptionbased oncomparativegenomicsthatnsp12isaprimer-dependentRNA polymerase(seeabove),nsp8wasthusproposedtofunctionasa primase,presumablygeneratingtheinitiationsubstratefornsp12.

Insubsequentbiochemicalanalyses,itwasfoundthattheN- terminus of SARS-CoV nsp8 modulates its oligomerization and polymeraseactivity (te Velthuis et al.,2012; Xiaoet al.,2012).

Moreover,proteolyticcleavageofaninactivensp8containingan N-terminal ubiquitin- and C-terminal His₆-tag showed that its nucleotidecondensationabilities (i.e.denovosynthesisbut also primerextensionactivity)canbeactivatedbytheremovalofthe terminal tags. These observations are in linewithexperiments showingthatnsp8appearstobelargelyinactiveinthecontextof theprecursorpolyproteinsnsp7-8andnsp7-10(teVelthuisetal., 2012;Xiaoetal.,2012)andtogethertheysuggestthatthe(faithful) processingofnsp8canaffectandpotentiallyregulatetheactivity ofnsp8inthecomplexesinwhichitresidesininfectedcells.We note,however,thattheabilityofnsp8toextendprimer-template duplexeswith[␣-³²P]ATPcouldnotbeconfirmedinasubsequent studyusingaradiolabeledprimer(Subissietal.,2014b).Thedenovo RNAsynthesisactivityofnsp8wasnotaddressedinthisstudy.

Thestructureofnsp8aloneispresentlyunknown,butitislikely thatitformsoligomersbasedongelshift,electronmicroscopy(EM) andgelfiltrationstudies(teVelthuisetal.,2012;Zhaietal.,2005).

The involvement of conserved nsp8 residues in its polymerase activityhassofarbeenprobedintwoinvitrostudies(Imbertetal., 2006;teVelthuisetal.,2012).Inbothstudies,aconservedlysine (K58inSARS-CoV)wasfoundtobeessentialforpolymeraseactiv- ityandcrucialforthebindingofnsp8toRNA(Imbertetal.,2006;te Velthuisetal.,2012).Anotherconservedlysine(K82)andseveral,

amongCoronavirinae conserved aspartates,including aD/ExD/E motifwithD50andD52askeyresiduesinSARS-CoVnsp8,affected nsp8’sabilitytosynthesizeRNAinvitro(Imbertet al.,2006;te Velthuisetal.,2012).Thecriticalroleoftheaboveresidueswassub- stantiated,inpart,byobservationsthatnsp8mutationsD52Aand K82AcrippleSARS-CoVRNAsynthesisincellcultureandthatmuta- tionK58Aislethalforthevirus(Subissietal.,2014b).However, nsp8sharesnostructuralhomologywithotherprimasesorRNA polymerases(seeSection6),sointheabsenceofatertiarystruc- tureofnsp8boundtoRNAandnucleotides,itispresentlyunclear howtheseputativeactivesiteresiduesmayassistthenucleotide condensationactivityofnsp8invitro(Imbertetal.,2006;teVelthuis etal.,2012).Moreover,asforSARS-CoVnsp12,contradictoryresults havebeenobtainedinthehandfulofnsp8studiesperformedby differentlaboratories,whichmayhavebeenduetodifferencesin experimentaldesignand/orthefactthatnidovirusRdRpsappear tobetechnicallychallengingproteins.Unfortunately,wetherefore havetoconcludethatthequestionwhethernsp8functionsaspri- masestillremainswideopen.Inthenextsection,wewillelaborate ontheroleofnsp8asacofactorforRNAreplicationbynsp12in moredetail.

6. TheRNApolymeraseactivityofnsp12andtheroleof nsp8asco-factor:thensp7+8+12tripartitecomplex

In2005,theSARS-CoV22-kDansp8wasshowntoformaring- likecomplextogether withthe12-kDansp7(Zhaietal., 2005) (Fig.4A–C).Thiscomplexconsistedof8copiesofeachsubunit,ori- entedsuchthattheinnercavityoftheringwaspositivelycharged andcapableofbindingRNA(Zhaietal.,2005)(Fig.4A).Giventhe structuralsimilarityofthishexadecamertotheeukaryoticPCNA slidingclampandthe␤-subunitoftheE.coliDNApolymeraseIII, itwasproposedthatitcouldfunctionasaprocessivityfactorfor theRNApolymerasefunctionofnsp12(Zhaietal.,2005).Interest- ingly,nsp7andnsp8fromthedistantlyrelatedfelinecoronavirus (FCoV)werefoundtoformadifferentnsp7+8complexinwhich nsp7andnsp8formaheterotrimerconsistingof2copiesofnsp7 and1copyofnsp8(Fig.4D)(Xiaoetal.,2012).Althoughthehet- erotrimerwasfoundtobethemostlikelycomplexinsolution,it maybepossiblethatthistrimerformshigherorderstructures,as itcanself-interactundercrystallizationconditionsandformahet- erohexamer(Xiaoetal.,2012).Togetherwiththeobservationsfrom Zhaietal.,thissuggeststhatthensp7+8complexisrelativelyplastic andthatitmayperformmultiplefunctions.Indeed,severalnsp7+8 complexeshavebeenobservedacrossanumberofexperimentsand studies(teVelthuisetal.,2012;Xiaoetal.,2012;Zhaietal.,2005).

Alternatively,thedifferencebetweenthevariousnsp7+8complex structurescanbeattributedtothepositionofthepurificationtags, thegeneticbackgroundoftheprotein(FCoVversusSARS-CoV)or thecrystallizationprocedures(Xiaoetal.,2012).

Incontrasttotheiroligomericform,thestructureoftheindi- vidualnsp7andnsp8subunitsisconsistentacrossthestructural studies.Nsp7adoptsaglobularstructurethatconsistsmostlyof alpha-helices,bothinsolutionandinthensp7+8crystal(Johnson etal.,2010;Zhaietal.,2005).Nsp8canadoptagolfclub-likeshape thatispresentina‘straight’(nsp8-I)and‘bent’(nsp8-II)conforma- tion.Bothformsarepresentinthehexadecamericnsp7+8complex (Fig.4BandC),butonlythe‘straight’formofnsp8isseeninthe FCoVnsp7+8complex(Xiaoetal.,2012)(Fig.4D).Insidethering structure,SARS-CoVnsp7andnsp8interactextensively,withnsp7 likelystabilisingtheframeworkofinterlinkingnsp8subunits(Zhai etal.,2005).AlthoughSARS-CoVnsp8canassembleintooligomers intheabsenceofnsp7(teVelthuisetal.,2012;Zhaietal.,2005), (Johnsonetal.,2010;Xiaoetal.,2012;Zhaietal.,2005),nsp7+8 complexeswerefoundtohaveahigherRNAbindingaffinitythan

nsp8oligomersalone(teVelthuisetal.,2012)andmutationsin nsp7affecttheRNAbindingabilityofthensp7+8complex(Subissi etal.,2014b).

Inmultipleearlierstudies,nsp7andnsp8werealsoshowntoco- purifyorinteractwithnsp12(Imbertetal.,2008;vonBrunnetal., 2007).In2014,Subissietal.discoveredthatanunknownformof thensp7+8complexcaninteractwithnsp12-strepandincrease theprocessivityofitsprimer-dependentpolymeraseactivitysev- eralordersofmagnitude(Subissietal.,2014b).Inthepresenceof Mg²⁺,thensp7+8+12tripartitecomplexwasalsoabletoinitiate RNAsynthesisdenovoona339-ntlongfragmentrepresentingthe 3^-terminalpartoftheviralgenome(Subissietal.,2014b).Interest- ingly,theefficiencyofthepolymerasereactionwasimprovedby creatingaphysicallinkbetweenthensp7andnsp8subunitsinthe formofa6-or12-aminoacidlinker(nsp7L8),whichsuggeststhat nsp7+8complexformationonthetemplateisarate-limitingstep (Subissietal.,2014b).Subissietal.alsofoundthatthensp7L8+12- strepcomplexhadahigherRNAbindingaffinitythannsp7L8or nsp12-strepalone,ofwhichthelatterwasunabletobindtotheRNA template(Subissietal.,2014b).Unfortunately,nodirectcompari- sonwasmadebetweennsp7+8andnsp7L8,soitisunclearwhether thensp7+8+12-strepcomplexbehavesinasimilarfashion.

Mutagenesis of SARS-CoV nsp8 within the context of the nsp7+8+12complexresultedintheidentificationofresiduesthat areimportantfortheinteractionbetweennsp8andnsp12(nsp8 P183andnsp8R190).Asinstudiesonnsp8aloneandthensp7+8 complex,residueK58,whichisfullyconservedamongmembersof theCoronaviridaefamily,wasfoundtobeinvolvedinthebind- ing of the RNA template. In line with their observed roles in thecomplex,each ofthesethree mutationsalsoinactivatedthe primer-dependentanddenovoactivityofthensp7+8+12complex invitroandresultedinanon-viablevirusphenotypewhenreverse- engineered into the SARS-CoV genome (Subissi et al., 2014b).

By contrasttotheabovethree mutations, only limitedcorrela- tionbetweenthebiochemicalandthereversegeneticsdatawas observedforothermutationsinnsp8,suggestingthattheroleof nsp7andnsp8intheRTCismorecomplexthantheinvitroexper- imentswillhaveusbelieve.Nsp8mutantsD52AorK82A,which werefoundtocripplethensp7+8activityinvitro(teVelthuisetal., 2012)andviralRNAsynthesisincellculture(Subissietal.,2014b), didnotaffectnsp7+8+12complexformationortheprimerexten- sionactivityofthensp7+8+12complex.ThissuggeststhattheRdRp activityofnsp7+8asobservedbyteVelthuisetal.isnotinvolved intheprimer-extensionactivityofthensp7+8+12complex,which providessupportforthehypothesisthatnsp7+8actsasaproces- sivityfactor.However,theabilityofnsp8mutantsD52AandD82A tosupportdenovoinitiationonthe3^terminusoftheviralgenome inthecontextofthensp7+8+12complexhasnotyetbeentested.

MutagenesisofD760inmotifCofSARS-CoVnsp12RdRpdomain (Fig.2C)resultedinaninactivecomplexthatshowednoprimer- extensionand,rathersurprisingly,nodenovoinitiationactivityon the3^ terminusoftheviralgenome.Inlightofthepreviousbio- chemicalstudiesonnsp8andnsp12,itisdifficulttointerpretthis result.Ontheonehand,itsuggeststhatonlynsp12canactively catalyseRNAsynthesis,whilensp7andnsp8aremerelypresentas co-factors.Ontheotherhand,itispresentlyjustaslikelythatnsp12 isadominantfactorinthensp7+8+12complex,whichregulatesthe primaseandelongationactivitiesofnsp8,orthatnsp7+8+12isstill anincompleteorincorrectlyassembledcomplex.Inturn,thelack ofoneormoreadditional(proteinorRNA)factorsmaypreventus fromseeingconsistencybetweenthebiochemicalresponsesofthe polymerasecomplexinvitroandthefunctionalityoftheRTCinthe infectedcell.Clearly,boththeroleofnsp8(primaseand/orproces- sivityfactor?)andtheinitiationmechanismemployedbynsp12 requirefurtherstudy.

Fig.4.ThestructureoftheCoVnsp7-8complex.A)ThehollowhexadecamericringoftheSARS-CoVnsp7-8complexhasapositivelychargedchannel(bluesurfaceshading) thatislikelyimportantforRNAbinding.Theoutsideofthehexadecamerispredominantlynegativelycharged(redsurfaceshading).B)TheSARS-CoVnsp8crystalstructure (pdb2AHM)resemblesa‘golfclub’withalongstickattheN-terminus(N)andahead-likeshapeattheC-terminus(C).Thensp8structurecanadopttwoconformations, hereshadedgreen(nsp8-I)andorange(nsp8-II).C)Inthehexadecamereachofthetwonsp8structuresispresentfourtimesandcomplementedbyeightnsp7subunitsthat actasmortar.Twoorientationsofthensp7subunitareindicated(orangeandgreen).D)TheFCOVheterotrimericnsp7-8complex(pdb3ub0)consistsofonensp8(shaded green)subunitandtwonsp7subunits(orangeandblue).Thensp8subunitadoptsannsp8-Iconformation.

7. TheelusiveinvitroRNApolymeraseactivityofAVnsp9

Toourknowledge,attemptstoachieveinvitroactivityofan AV RdRp haveonly beenmade usingEAV nsp9.By contrastto SARS-CoVnsp12,recombinant EAVnsp9witha C-terminalHis- tagcouldbestablyexpressedtohighlevelsinE.coliandreadily

purified(Beerensetal.,2007;Lehmannetal.,2016).Beerensetal.

reportedthatrecombinantEAVnsp9iscapableofdenovoinitia- tionofRNAsynthesisonhomopolymerictemplates(poly(C)and poly(U)).However,noactivitywasfoundwhenanRNAtemplate representingthe3^-terminalpartoftheviralgenomewasprovided orwhenaprimerwashybridizedtothetemplate(Beerensetal.,

2007).Onthehomopolymerictemplates,RNAsynthesisrequired thepresenceofMn²⁺,butitcouldbestimulatedbyMg²⁺ifMn²⁺

waspresent.Althoughnoactivesitemutantwastestedintheinitial EAVnsp9study,thedenovoinitiationonthepoly(U)templatewas repeatedinasubsequentstudywithawild-type(wt)EAVnsp9 andD445AmotifA mutant.In theseassays,theD445Amutant showedasignificantlyreducedactivitycomparedtothewtcon- trol(teVelthuisetal.,2010b).Unfortunately,morerecentefforts usingthe sameT7-driven expression construct failedto repro- ducetheseobservationsregardingtheactivityofnsp9(Lehmann et al.,2016).Sufficient C-terminally His6-taggednsp9 couldbe purified,but afterextensivemutationof theconservedmotif A andCaspartatestheproteinpreparationscontinuedtoshowde novoandprimer-dependentpolymeraseactivitiesonbothRNAand DNAtemplates(Lehmannetal.,2016).Itwasthusconcludedthat contaminatingtraceamountsofT7RNApolymerasemayhavepro- ducedtheseresults.Toalleviatethisproblem,nsp9wasexpressed inaT7polymerase-freeE.colisystemthathadpreviouslybeenused toexpressaC-terminallytaggednsp12withanativeN-terminus (te Velthuis etal.,2010a).Unfortunately, this recombinantver- sionofnsp9showednodenovopolymeraseactivity,whilethe primer-dependentandterminaltransferaseactivitieswereinsen- sitivetoreplacementoftheconservedmotifAandCaspartates (Lehmannetal.,2016).Itmustthereforebeconcludedthat,asfor SARS-CoVnsp12,theactivityofnsp9likelyisveryweakandsen- sitivetothepurificationorassayconditions,andthat–byanalogy withCoVnsp12–otherco-factorsmayberequiredtostimulateits activity.However,preliminaryexperimentsinwhichRdRpassays withrecombinantEAVnsp9weresupplementedwithseveralsmall productsfromthensp6-8regionof pp1afailedtoactivateRNA synthesisinvitro(Lehmannetal.,2016).

8. Thenidovirus-specificdomainattheN-terminusofthe RdRp-containingsubunit:NiRAN

Ithaslongbeenrecognizedthatthenidovirusreplicasesubunit thatharbourstheRdRpdomainhasanunusuallylargeN-terminal extensionthatdoesnotseemtobepartoftheRdRpdomainitself.

Noviralorcellularhomologueshavebeenidentifiedthusfar,and evenwithintheorderNidoviralesthelevelofconservationofthis domainisverylimited,whichlonghamperedattemptstodeducea possiblefunctionand/oractivitybyusingbioinformaticstools.Ina recentstudythatcombinedextensivebioinformaticsanalysis,bio- chemicalstudiesandreversegenetics,anucleotidylationactivity wasassignedtothisdomain,whichwasnamedNiRAN(Lehmann etal.,2015a).Thedomainisonlypresentinnidovirusesand,in additiontoauniquezinc-bindingdomain(ZBD)thatisassociated withthenidovirushelicase(Dengetal.,2014),isconsideredto beaseconduniversalgeneticmarkerforthisvirusorder,andthe firstwithanenzymaticactivity.Despitelimitedsequenceconser- vationandsignificantsizedifferences,from∼220residuesinAV toover300residuesinCoV,threemotifswereidentified:AN,BN

andCN(thesubscriptNfortheNiRANdomainwasaddedtodis- criminatethemfromtheconservedRdRpdomainmotifs).Together thesecontainonlysevenresiduesthat areabsolutelyconserved amongnidoviruses(Fig.2B).Thereisamarkedsizedifferenceinthe spacingbetweenNiRANdomainmotifsB_NandC_Nwhencompar- ingAVwithallothernidoviruslineages:thesemotifsareadjacent inAV,butseparatedby40–60residuesinothernidoviruses.The C-terminalborderofthedomaincouldnotbedefinedwithcer- taintyleavingroomforthepresenceofathirddomainof∼50or

∼175aminoacidsforAVandCoV,respectively,thatmayconnect theNiRANandRdRpdomains(Lehmannetal.,2015a).

NucleotidylationactivitywasshownforrecombinantEAVnsp9, expressedinandpurifiedfromE.coli.Incubationoftheproteinwith

[␣-³²P]GTP or [␣-³²P]UTP, but not [␥-³²P]-labelled nucleotides, resultedin labellingof nsp9itself, whichwasattributedtothe covalentbindingof monophosphatenucleotideswhilereleasing pyrophosphate(Lehmannetal.,2015a).ThisreactionwasMn²⁺- dependentanddisplayeda5-foldstrongeraffinityforUTPoverGTP asasubstrate.Mostlikelyaphosphoamidebondisformedbetween thenucleotideandaconservedlysineinmotifAN(K54inEAVnsp9).

MutagenesisofconservedresiduesinmotifsA_N,B_NorC_Nresulted in lessthan 10% invitroactivity forall but theK106Amutant, whichconfirmedtheassociationofthenucleotidylationactivity withtheN-terminaldomainofnsp9.AD445Amutationinmotif Aofthensp9RdRpdomainonlymoderatelyaffectednucleotidyla- tion,strengtheningthenotionthatthisactivityisassociatedwith theNiRANdomain.For both EAV nsp9andSARS-CoV nsp12,it wasshownthattheNiRANdomainisessentialforviralreplica- tion:reverseengineeredmutantswithsubstitutionsinthemotifs oftheNiRANdomainwereeithernon-viableorcrippled,inwhich casetheymostlyrevertedbacktothewtsequence(Lehmannetal., 2015a).

Despiteconvincingevidenceforthenucleotidylationactivityof EAVnsp9,thisactivityisyettobedemonstratedforCoVsandother membersoftheorderNidovirales.Inaddition,theroleofthisactiv- ityinthenidovirusreplicativecycleremainsunknown.Givenits covalentlinkagetotheRdRpdomain,ithasbeensuggestedthat NiRANisimportantforRNAsynthesis,inanalogytotheN-terminal domainof theRdRpof double-strandedRNAviruses(Taoetal., 2002;Xuetal.,2003).ItmayalsoaddtothestabilityoftheNiRAN andRdRp-containingreplicasesubunit,assuggestedbytheinabil- itytoexpressEAVnsp9whentheN-terminaldomainisdeleted (Beerensetal.,2007).WhileanRdRpmutationmoderatelyinflu- encedNiRANnucleotidylation,itwasunfortunatelynotpossibleto studytheeffectofNiRANmutationsonAVRNApolymeraseactiv- ity,asarobustinvitroassayremainstobedeveloped(seeabove).

Yet,giventheintegrationofthetwodomainsinasinglereplicase subunit,structuraland/orfunctionalcrosstalkbetweentheNiRAN andtheRdRpdomainisalikelyscenario.

Sofar, three possible functionshave been proposedfor the nucleotidylationactivityoftheNiRANdomaininnidoviralrepli- cation(Lehmannetal.,2015a).Thefirstisaroleintheligationof yettobeidentifiedRNAmoleculesbasedonthedomain’sabilityto covalentlybindnucleotidemonophosphates,oneofthestepsofthe universalligationmechanism(ShumanandLima,2004).Asecondis apossibleroleinthe5^cappingofmRNAs,whileathirdpossibility isthattheNiRANdomainisinvolvedinfacilitatingprotein-primed RNAsynthesis,similartotheuseofauridylatedVpGprimerby picornaviruses(Pauletal.,2000).Eachofthesehypotheseshasbeen discussedinmoredetailbyLehmannetal.(2015a),butsinceall threerequireadditionalassumptionsaboutnidovirusRNAsynthe- sistheyarestillhighlyspeculative.Moreover,basedontheextentof divergentevolutionwithintheNiRANdomainitcannotbeexcluded thattheenzymeperformsdifferentrolesindifferentnidoviruses (Lehmannetal.,2015a).

9. Faithfulnidovirusreplicationandtranscriptioninvitro andtheinvolvementofotherco-factors

ThecoreofthenidovirusRTClikelyconsistsoftheRdRpdomain- containingnspandotherviralRNA-bindingproteins,likensp7-10 andnsp13-16inthecaseofCoVs.Intheinfectedcell,thesesub- unitsassembleintoamembrane-boundcomplexthatisassociated withanetworkofmodifiedhostmembranes,presumablyderived fromtheendoplasmicreticulum(reviewedin(Hagemeijeretal., 2012;Neumanetal.,2014;vanderHoevenetal.,2016)).Analter- nativeapproachtoassaynidovirusRNAsynthesisinvitro,without theuseofpurifiedrecombinantproteins,isbasedontheisolation

ofthemembrane-associatedRTCsfrominfectedcells(vanHemert etal.,2008a,2008b).ToprepareRTCsforthisinvitroRNAsynthesis assay(IVRA),infectedcellsaremechanicallydisruptedandfrac- tionatedbycentrifugation.Lowspeedcentrifugationstepsyield apost-nuclearsupernatant(PNS),fromwhichnuclei,largedebris andremainingintactcellshavebeenremoved.Thesynthesisof SARS-CoVandEAVgenomeandsgmRNAscanbereadilyrepro- ducedusingsuchaPNSsupplementedwith[␣-³²P]-CTP,Mg²⁺and anATP-generatingsystem.

Additional fractionation of the PNS, during which the RTC- containingmembraneswereseparatedfromthecytosolfraction byahigh-forcecentrifugationstep,revealedthatasolublehostfac- torisrequiredforCoVandAVinvitroRNAsynthesis(vanHemert etal.,2008a,b).Furthercharacterizationofthishostfactorusing EAVRTCsshowedthatithasamassrangingfrom59to70kDa,that itisconservedamonganimals,butnotlowereukaryotes,andthat itcanbeaddedtoinactiveRTCpreparationsintheformofacytoso- licfractionthatwasextractedfromuninfectedcells.Presently,the exactnatureofthishostfactorremainsunknown(vanHemertetal., 2008a)anditmayalsonotbetheonlyhostfactor requiredfor nidovirusRNAsynthesis.Protein-protein(Pfefferleetal.,2011)and protein-RNAinteractionstudies,focusingonthe5^and3^termini ofthegenomeasputativetargets(Galanetal.,2009;Spagnoloand Hogue,2000;Tanetal.,2012),havesuggestedthatanumberofhost factors,suchaspoly(A)-bindingprotein(PABP),arecriticalcompo- nentsoftheCoVRTCaswell.Similarly,yeast2-hybrid(Y2H)and mammalian2-hybrid(M2H)experiments(Imbertetal.,2008;Pan etal.,2008;Prenticeetal.,2004;vonBrunnetal.,2007),pulldown assaysorco-purifications(e.g.(Brockwayetal.,2003;Imbertetal., 2008;Subissietal.,2014b;Suttonetal.,2004;vonBrunnetal., 2007))andco-crystallizationstudies(e.g.(Decrolyetal.,2011;Ma etal.,2015;Zhaietal.,2005))haveimplicatedseveraladditional CoVnspsandthenucleocapsid(N)proteinaspotentialviralco- factorsoftheRNApolymerase.FortheAVPRRSV,usingdifferent technicalapproaches,multiplehostproteinsthatappeartointer- actwithnsp9wereidentified,althoughitremainstobestudied whethertheydirectlybindtotheRdRpdomainoftheproteinand affectitsfunction(s)inRNAsynthesis(Dongetal.,2014;Lietal., 2014a;Liuetal.,2016).Sincetheexactroleofthese(presumed) hostandviralco-factorsofthenidovirusRNApolymeraseremains tobeexplored,wewillnowfocusontwoproteinswhoserolein nidovirusRNAsynthesishasbeendefinedinmoredetail:AVnsp1 andCoVnsp14.

10. MakingtheRdRpswitchfromcontinuousinto discontinuousmode:AVnsp1

Ininfectedcells, thenidoviral genomeandsgmRNAsareall producedwithaspecificrelativeabundanceanditispoorlyunder- stoodhowtheRNA polymeraseiscontrolledtomaintainthese ratios.Asoutlinedintheintroduction,eachsgmRNAisproduced fromasubgenome-lengthcomplement(Fig.5),whichderivesfrom aprocessofdiscontinuousminusstrandRNAsynthesis(Sawicki and Sawicki, 1995; Sethna et al., 1989). Whereas continuous minusstrandsynthesisproduces thefull-lengthtemplate(anti- genome)for genomereplication,discontinuous minus-stranded RNAsynthesisyieldsanestedsetofshortertemplatesforsgmRNA production(reviewedin(Pasternaketal.,2006;Sawickietal.,2007;

Solaetal.,2011).In mostnidovirus families,includingAVsand CoVs,thesgmRNAsconsistofsequencesthatarenon-contiguous intheviralgenome:acommon5^‘leader’sequenceisattachedto different‘body’segmentsrepresentinga variablepartofthe3^- proximalregionofthegenome (Fig.1A).The joiningofleader- andbody-encodingsequencesoccursduringdiscontinuousminus strandRNAsynthesis(Fig.5).Followinginitiationofminusstrand

RNAsynthesis,whichinvariablyoccursatthegenomic3^ end,a uniquemechanism of‘polymerasejumping’ formsthebasis for leader-to-bodyjoining.Thisstepappearstobeprimarilydirected byTRSs,theshortconservedsequencemotifsthatarepresentin thegenomictemplatebothatthe3^endoftheleadersequenceand atthe5^endofeachofthesgmRNAbodies.Whenminusstrand RNAsynthesishasbeenattenuatedatabodyTRS,thebodyTRS complement,whichformsthe3^endofthenascentminusstrand, canbasepairwiththegenomicleaderTRStodirectre-initiation of RNA synthesis. Subsequently, the subgenome-length minus strandiscompletedbyadditionofthecomplementofthegenomic leadersequence.Theimportanceof(−)TRS-(+)TRSbasepairingwas probedand confirmedextensively bysite-directedmutagenesis andreversegeneticsforbothAVsandCoVs(reviewedin(Pasternak etal.,2006;Sawickietal.,2007;Solaetal.,2011).Inadditionto TRSbasepairing,higher-orderRNAstructurelikelyco-determines therelativeefficiencyofdifferentTRSsinnidovirustranscription, whichmechanisticallyresemblestheprocessofcopy-choiceRNA recombination,asitisthoughttocommonlyoccuramong+RNA viruses(Pasternaketal.,2001;Solaetal.,2015;Yountetal.,2006).

The above strongly suggests that nidovirus replication and transcription are competing for common factors, like the RNA polymerase,and that thebalancebetweencontinuous and dis- continuous minus strandRNA synthesis must be regulated. In additiontoregulatoryRNAsignalsliketheTRSs,specificreplicase subunitsmayinteractwiththeRNApolymerasetoinfluenceits behaviouronthegenomictemplate.Solidsupportforthishypoth- esis wasobtainedin theEAV model, inparticular for thensp1

“transcriptionfactor”andthensp10helicase.Specificmutations inthesetwosubunitscan(nearly)completelyinactivatetranscrip- tion (Nedialkova et al., 2010; Seybert et al.,2005; Tijms etal., 2001; vanDintenet al., 1997), withsignificantupregulationof genomereplicationbeingaprominentandstrikingside-effect.EAV nsp1wasconcludedtocontrolthelevelsatwhichthegenome- lengthand differentsubgenome-lengthminusstrandtemplates accumulateintheinfected cell(Nedialkovaetal.,2010).TheN- terminalzincfingerdomainofnsp1isimportantforthisfunction, butalsomutationsinothernsp1domainscanstronglyinfluence transcription (Nedialkovaet al.,2010; Tijmset al.,2001).Nsp1 mutagenesisandpseudorevertantanalysisprovidedgeneticevi- dencethatabalancedratiobetweenreplicationandtranscription, andalsobetweenindividualsgmRNAspecies,isvitaltothevirus (Nedialkovaetal.,2010).However,itremainsunclearhowAVnsp1 interactswithe.g.thenetworkofTRSsignals,theviralRNApoly- merase(AVnsp9),orthehelicase(AVnsp10),whichhasalsobeen implicatedintranscriptionalcontrol(Dengetal.,2014;Lehmann etal.,2015b).Theproteincoulde.g.modulateRTCstallingatbody TRSs,nascentminusstrandtransfertotheleaderTRSorreinitia- tionofRNAsynthesisfollowing(−)TRS-(+)TRSbasepairing.Studies aimingtodetectAVnsp1-nsp9ornsp1-nsp10interactionshavenot beensuccessfulsofar,butitisconceivablethatsuchcomplexes areshort-lived.Clearly,arobustinvitroAVRdRpassaycouldbea ground-breakingtooltoexploreattenuationduringminus-strand RNAsynthesis,TRSbasepairingandtheroleofregulatoryprotein factorslikeAVnsp1andnsp10.

ThecompositionoftheRNA-synthesizingcomplexesthatother nidovirusesemployforreplicationandtranscriptionhasnotyet beenstudiedingreatdetail.Theirmuchlargergenomesizethan theAVgenomeclearlyprovidesamplegroundstospeculatethat theymayencodemechanismsandfactorsthatarelackinginthe AVsystem.Still,thusfaronlyafew studieshave suggestedthe existenceofCoV“transcriptionfactors”withanimpactresembling thatofAVnsp1.Apointmutationinthehelicase(nsp13)oftheCoV infectiousbronchitisviruswasreportedtocauseaspecificblockin transcription(Fangetal.,2007),butthisobservationhasnotbeen followedupinmoredetailforanyCoVthusfar.Morerecently,N