2014 One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonucle

2014·1.23 MB·English

One severe acute respiratory syndrome coronavirus
protein complex integrates processive RNA
polymerase and exonuclease activities
LorenzoSubissia,ClaraC.Posthumab,AxelleColleta,JessikaC.Zevenhoven-Dobbeb,AlexanderE.Gorbalenyab,c,
EtienneDecrolya,EricJ.Snijderb,Bruno Canarda,1,andIsabelleImberta,1
aArchitectureetFonctiondesMacromoléculesBiologiques,CentreNationaldelaRechercheScientifique,UnitéMixtedeRecherche7257,Aix-Marseille
Université,13288Marseille,France;bMolecularVirologyLaboratory,DepartmentofMedicalMicrobiology,LeidenUniversityMedicalCenter,2300RC,Leiden,
TheNetherlands;andcFacultyofBioengineeringandBioinformatics,LomonosovMoscowStateUniversity,Moscow119899,Russia
EditedbyPaulAhlquist,UniversityofWisconsin,Madison,WI,andapprovedAugust5,2014(receivedforreviewDecember19,2013)
Inadditiontomemberscausingmilderhumaninfections,theCoro- respiratory tract infections, SARS-CoV caused a severe form of
naviridaefamilyincludespotentiallylethalzoonoticagentscaus- pneumoniawithacasefatalityrateofabout10%.Twoyearsafter
ing severe acute respiratory syndrome (SARS) and the recently theoutbreak,batswereidentifiedasthelikelyreservoirfrom
emergedMiddleEastrespiratorysyndrome.The∼30-kbpositive- which SARS-CoV had jumped to humans, probably using other
stranded RNA genome of coronaviruses encodes a replication/ animals as intermediate hosts (8, 9). Since April 2012, history
seems to repeat itself with the emergence of another zoonotic
transcriptionmachinerythatisunusuallycomplexandcomposed
CoV, Middle East respiratory syndrome coronavirus (MERS-
of16nonstructuralproteins(nsps).SARS-CoVnsp12,thecanonical
CoV)(10,11),whichhasthusfarinfectedover800peoplewith
RNA-dependent RNA polymerase (RdRp), exhibits poorly proces-
acasefatalityrateofabout35%.MERS-CoVismostcloselyrelated
siveRNAsynthesisinvitro,atoddswiththeefficientreplication
to several bat CoVs (12) and to a newly discovered coronavirus
ofaverylargeRNAgenomeinvivo.Here,wereportthatSARS-
(EriCoV)foundinhedgehogs(13).Thisnewepisodeemphasizes
CoV nsp7 and nsp8 activate and confer processivity to the RNA-
the ability of CoVs to cross species barriers and threaten public
synthesizing activity of nsp12. Using biochemical assays and re-
healthworldwide.
verse genetics, the importance of conserved nsp7 and nsp8 resi-
TheCoVRNAgenomecanbeupto32kblong(14),carries
dueswasprobed. Whereasseveralnsp7mutationsaffected virus a 5′-terminal cap structure, and is polyadenylated at its 3′ end
replicationtoalimitedextent,thereplacementoftwonsp8resi- (15).Thegenomeispolycistronicwiththe5′-proximaltwo-thirds
dues (P183 and R190) essential for interaction with nsp12 and being occupied by two large overlapping open reading frames
athird(K58)criticalfortheinteractionofthepolymerasecomplex (ORFs) (ORFs 1a and 1b) (16). These are translated to yield
withRNAwerealllethaltothevirus.Withoutalossofprocessivity, replicase polyproteins pp1a and pp1ab, with synthesis of the
thensp7/nsp8/nsp12complexcanassociatewithnsp14,abifunc- latter requiring −1 ribosomal frameshifting near the 3′ end of
tionalenzymebearing3′-5′exoribonucleaseandRNAcapN7-gua- ORF1a. Subsequently, the two polyproteins are cleaved by two
nine methyltransferase activities involved in replication fidelity or three ORF1a-encoded proteases to release a total of 16
and5′-RNAcapping,respectively.Theidentificationofthistripar- nonstructural proteins (nsp1 to nsp16). Next, these nsps assem-
tite polymerase complex that in turn associates with the nsp14 ble into a replication and transcription complex (RTC) that is
proofreadingenzymeshedslightonhowcoronavirusesassemble associated with modified intracellular membranes (17) and
anRNA-synthesizingmachinerytoreplicatethelargestknown directs a complex mechanism of viral RNA synthesis. This not
RNA genomes. This protein complex is a fascinating example of
thefunctionalintegrationofRNApolymerase,capping,andproof- Significance
readingactivities.
| The 2003 severe acute respiratory syndrome (SARS) epidemic
replicativecomplexreconstitution processivityfactor and recent emergence of Middle East respiratory syndrome
highlightthepotentiallethalityofzoonoticcoronavirusinfec-
A virus-encoded RNA-dependent RNA polymerase (RdRp) tions in humans. No specific antiviral treatment options are
isthecentralenzymeinthereplicativecycleofRNAviruses available.CoronavirusespossessthelargestknownRNAvirus
(1). In the case of mammalian positive-strand RNA (+RNA) genomesandencodeacomplexreplicationmachineryconsist-
viruses, the enzyme is generated by the translation of the in- ingof16viralnonstructuralproteins(nsps).Ourstudyreveals
comingviralgenome,whichyieldsapolyproteinprecursorfrom that the SARS-coronavirus RNA polymerase (nsp12) needs to
which the RdRp-containing subunit is released by proteolytic associatewithnsp7andnsp8toactivateitscapabilitytorepli-
cleavage.Subsequently,theRdRpisintegratedintoamembrane-
catelongRNA.Moreover,thiscomplexassociateswithnsp14,
associatedviralenzymecomplexthatdrivestheproductionofneg-
the proofreading subunit required to safeguard coronavirus
ative-strandRNA(−RNA),newgenomemolecules,andinmany
replicationfidelity.OurstudythusdefinesthecoreofanRNA-
virus groups also subgenomic (sg) messenger RNAs (mRNAs)
synthesizingmachinerythatisuniqueintheRNAvirusworld
(2–4). Compared with the replication of either viral or cellular
andincludesseveralkeytargetsforantiviraldrugdevelopment.
DNAsequences,RNAvirusgenomesarecopiedwithrelativelylow
fidelity,becausetheproductsofreplicationarebelievedtobenei-
Authorcontributions:L.S.,C.C.P.,A.E.G.,E.D.,E.J.S.,B.C.,andI.I.designedresearch;L.S.,
therproofreadnoredited(5).Thispropertyisamajorfactorinthe
C.C.P.,A.C.,J.C.Z.-D.,E.J.S.,B.C.,andI.I.performedresearch;A.E.G.contributednew
evolution,adaptation,andepidemiologyofRNAviruses. reagents/analytictools;L.S.,C.C.P.,A.E.G.,E.D.,E.J.S.,B.C.,andI.I.analyzeddata;and
Among+RNAviruses,coronaviruses(CoVs)(orderNidovirales) L.S.,C.C.P.,E.D.,E.J.S.,B.C.,andI.I.wrotethepaper.
stand out for having the largest single-stranded RNA genomes Theauthorsdeclarenoconflictofinterest.
knowntodate(6,7).Researchintothemolecularandstructural
ThisarticleisaPNASDirectSubmission.
biology of CoVs was boosted significantly by the emergence, in
1Towhomcorrespondencemaybeaddressed.Email:[email protected]
2003, of a previously undiscovered CoV that caused the severe [email protected].
acuterespiratorysyndrome(SARS)epidemic(6).Whereasestab-
Thisarticlecontainssupportinginformationonlineatwww.pnas.org/lookup/suppl/doi:10.
lished human CoVs are commonly associated with mild upper 1073/pnas.1323705111/-/DCSupplemental.
E3900–E3909 | PNAS | PublishedonlineSeptember2,2014 www.pnas.org/cgi/doi/10.1073/pnas.1323705111
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only entails the synthesis of new genomes from a full-length RNApolymerization,proofreading,andcappingactivitiesneeded P
negative-stranded template, but also a process of discontinuous toreplicatetheunusuallylongCoVRNAgenome.
RNA synthesis to produce subgenome-length negative-stranded
RNAs(18,19).Thelatterserveastemplatetoproduceanested Results
set of sg mRNAs required to express the viral structural and Nsp7andNsp8CooperateinActivatingthePrimer-DependentActivity
accessoryproteinsfromgenesnotaccessibletoribosomestrans- oftheNsp12RdRp.Thensp12subunitistheessentialRdRpofthe
latingtheviralgenomeRNA. coronavirusreplicativemachinery.Purifiedrecombinantnsp12is
The CoV replicase harbors awide variety ofenzymatic activ- able to extend a homopolymeric primer-template substrate by
ities(7,16,20).Severalnspshavealreadybeencharacterizedas a few dozen nucleotides in vitro, but in a nonprocessive (dis-
multifunctionalsubunits.WhereastheRdRpandhelicaseenzymes tributive)manner(21).Moreover,nsp12waspreviouslyreported
formtheC-terminaldomainsofnsp12andnsp13,respectively,and tointeractwithnsp8inGSTpull-downexperiments(39),which
aretypicallyfoundin+RNAviruses(21,22),lesscommonoreven displayedaprimase-likeRdRpactivity(30–32).Becauseoneof
uniqueRNA-processingactivitiesareassociatedwithothernsps. thetwodescribedstructuresofthensp7/nsp8complexresembles
For example, the N-terminal domain of the bifunctional nsp14 that of DNA processivity factors such as proliferating cell nu-
includesa3′to5′exoribonuclease(ExoN)(23)thathasbeenim- clear antigen (PCNA), it was hypothesized also that nsp7/nsp8
plicatedinauniquebutpoorlycharacterizedformofRNAviral maymodulatensp12RdRpactivity (29,40).
proofreading(24–26)andnsp15carriesanendoribonucleaseac- Recombinant SARS-CoV proteins carrying affinity tags were
tivity(NendoU)ofunknownfunction(27,28).Inaddition,SARS- expressed in Escherichia coli using a T7 RNA polymerase-free
CoVnsp8wascocrystallizedwithanotherviralprotein[nsp7(29)] systemandwerepurifiedbyaffinitychromatography(Fig.S1A).
asauniquehexadecamericringstructure,whichwasreportedto Pull-downexperimentsconfirmedtheinteractionbetweennsp12
exhibita“noncanonical,”denovo-initiatingRdRpactivity(30–32). andnsp8(Fig.S1B),ofwhichthelatterinturninteractswithnsp7
Moreover, evidence from genetics studies previously implicated (Fig. S1C), suggesting that a tripartite complex may be formed.
nsp8inaspecificinteractionwithRNAstructuresnearthe3′end However,such adirectinteractionbetweenthesethreeproteins
of the CoV genome (33). The nsp7/nsp8 protein complex was remainedundetected(Fig.S1D).
therefore proposed to act as a cofactor for the canonical nsp12 Nsp12alonewasunabletoextendtheprimerstrandofahet- Y
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RdRp.Indeed,bioinformaticsanalysesofthensp12RdRpdomain eropolymeric primer/template (P/T), of which the template O
identifiedtheso-called“Gmotif,”whichhasbeenimplicatedinthe mimicked the 3′-terminal 40 nt of the SARS-CoV genome [ex- BIOL
recognitionoftheprimerspredictedtoberequiredfortheinitia- cludingthepoly(A)tail],hereaftercalledLS2/LS1(Fig.1AandB). RO
tionofRNAsynthesisbynsp12(34). In the same manner, nsp8 alone was inactive on the LS2/LS1 MIC
Insights into how the RTC is assembled and operates are RNA primer/template, as previously described (30). Pairwise
criticalfortheadvancementofourbasicunderstandingofCoV combinations of nsp7, nsp8, and nsp12 displayed no significant
replication and for the development of efficient means to con- polymerase activity either (Fig. 1B). However, RNA synthesis
trolCoVinfections.Overthepast10years,theSARS-CoVRTC activitywasfoundwhennsp7,nsp8,andnsp12weresimultaneously
has been studied by using different approaches including bio- presentinthereactionmixture(Fig.1B).Toevaluatetherequire-
informatics,(reverse)genetics,aswellasbiochemicalandstruc- mentsforRdRpactivityinmoredetail,wepreincubatedallpos-
turalcharacterizationofrecombinantnsps.Clearly,theremustbe siblepairwisecombinationsofnsp7,nsp8,andnsp12forvarious
anetworkofinteractingreplicationproteinsthatcontrolstheCoV periodsoftime,beforeaddingthethirdproteinandassayingRNA
replicationcycle.Duringthepastyears,weandothershaveelu- synthesis.ThehighestRdRpactivitywasobtaineduponaddition
cidatedhowtheinvitroactivityofvariousCoVenzymescanbe ofanequimolaramountofnsp12toapreincubated1:1mixtureof
modulated by interactions with other viral nsps (24, 35). More- nsp7andnsp8,suggestingthatthelevelofassociationofnsp7and
over, the SARS-CoV RTC was partially purified from infected nsp8 may constitute a limiting factor for obtaining a high RNA
cellsandshowntoproducethefullspectrumofviralmRNAs(36). polymeraseactivity.
Exogenous RNA templates are, however, not replicated by this To overcome this limitation, we designed a set of gene con-
largemembrane-boundcomplex,whosesubunitcompositionwas structstoexpressfusionproteinsinwhichnsp7waseitherdirectly
notfullyresolved. fusedtonsp8(imitatingtheuncleavednsp7–nsp8,designated7–8)
Severalattemptshavebeenmadetocharacterizensp12RdRp or through a peptide linker of different lengths (7L8). Upon
activity in vitro. A weak polymerase activity was described for coexpressionwithnsp12inE.coli,allfiveengineeredfusionpro-
recombinantSARS-CoVnsp12expressedinfusionwithaGST- teinsboundtoandcopurifiedwithnsp12(Fig.S2A).Interestingly,
tag. The uncharacterized N-terminal domain of nsp12 was con- theproteincomplexconsistingofnsp12andafusion protein,in
cludedtoberequiredforactivity(37).Furtherenzymaticstudies whichthensp7Cterminusislinkedtothensp8Nterminusbyan
showedthat a full-length, C-terminally His -tagged nsp12 could engineered peptide of 6 (or 12) amino acids residues, is able to
6
initiate homopolymeric RNA synthesis in a primer-dependent efficientlypolymerizeRNA(Fig.S2B).Incontrast,whennsp12is
manner(21).However,thepolymeraseactivitydescribedinthese complexedwithanuncleavednsp7–nsp8polyprotein(either7–8/
studies appeared generally weak and nonprocessive. The proc- 12or7–8HC/12),theseproteincomplexesfailtoshowpolymerase
essivityofRNAsynthesis,definedasthenumberofnucleotides activity(Fig.S2B).Combined,theseresultsindicatethatinserting
polymerized during a single encounter of the RNA polymerase a linker between nsp7 and nsp8 may besufficient toimitate the
withitstemplate,bearsimportantbiologicalsignificance:ahighly effect of the nsp7–nsp8 cleavage and associated structural rear-
processiveRNApolymerasemaysynthesizeafull-lengthcopyof rangementsonthefunctioningofnsp7andnsp8intheactivationof
aviralRNAgenomewithoutfallingoffthetemplate. nsp12RNApolymeraseactivity.Thesedataarealsoinagreement
Here,wereportthattheprocessivityoftheSARS-CoVnsp12 withthoseofDemingetal.(41),whoshowedthatmurinehepatitis
polymerase in primer extension mode can be dramatically in- viruscouldnotberecoveredwhenthecleavagebetweennsp7–nsp8
creaseduponformationofacomplexwithnsp7andnsp8.Thus, wasprevented.BeingthemostefficientintermsofitsRNAprimer
this tripartite complex forms—uniquely among RNA viruses— conversionrate,the7L8/12complex,withthelinkerLconsistingof
aholoenzyme,whichisalsocapableofinitiatingRNAsynthesis sixhistidines,wasselectedforfurtherexperiments.Thisaffinity-
de novo, i.e., in the absence of an exogenous RNA primer. We purifiedcomplex(Fig.1C)isatleastthreefoldmoreactivethan
further established that nsp14, which harbors a N7-guanine cap a preincubated mixture of thethree separately purified subunits
methyltransferaseactivity(38)downstreamoftheExoNdomain (7+8+12; Fig. 1D), confirming that, in our experimental con-
(23),canjointhensp7/nsp8/nsp12complex,withoutsignificantly ditions, the nsp7/nsp8 association is a limiting factor to obtain
affecting either its polymerase activity or its processivity. The ahighlyactiveRNApolymerasecomplex.TheCterminusofnsp7
formation of this multifunctional nsp7/nsp8/nsp12/nsp14 com- andtheNterminusofnsp8arefarapartinthepublishedSARS-
plexdemonstratesthephysicalassociationofsubunitspossessing CoVnsp7–nsp8structure,indicatingthatthe7L8fusionproteinis
Subissietal. PNAS | PublishedonlineSeptember2,2014 | E3901
Two radiolabeled products are detected (Fig. 2), one with a
length similar to that of the RNA template and the other ap-
proximately twice as long. The size of the shorter product sug-
gestsittocorrespondtothecomplementarystrandof3R,implying
thatdenovoinitiationofRNAsynthesishasoccurred.Thelonger
productisinterpretedastheresultof“backpriming,”i.e.,thefor-
mationofahairpinstructureatthe3′endofthetemplate,which
canserveasprimertobeextendedintoaproductofabouttwicethe
templatelength(42,43).Noteworthy,nodenovopolymeraseac-
tivityisdetectedusingamixtureofthethreeproteinspurifiedin-
dependentlyfromE.coli.Previously,nsp8wasproposedtobean
RNA primase, possibly mediating the synthesis of small primers
(30–32)thatcouldbeelongatedbynsp12.Torevisitthishypothesis,
weperformeddenovoRNAsynthesisassaysusingtemplate3Rand
aproteincomplexconsistingof7L8copurifiedwiththeactivesite
knockoutmutantofnsp12(D760A).Undertheseconditions,nsp8-
mediatedsynthesisofshortprimersisnotdetected(Fig.S3).
To exclude that the products detected in the de novo assay
weredueto3′-terminalnucleotidetransferaseactivityof7L8/12,
the3′endoftemplate3Rwasblockedusingperiodateoxidation.
This chemical modification results in cleavage of the 2′C—3′C
bondintheriboseofthe3′-terminalnucleotide,thuspreventing
anyfurthernucleotideadditiontothe3′endoftheRNAtemplate.
WhenusingthismodifiedRNAtemplate,theputativebackpriming
productisnolongerobserved,whereasafull-lengthproductcom-
plementarytothetemplateisstillproduced,albeitwithloweref-
ficiency(Fig.S4A,lane1).ThisreductioninRNAsynthesislikely
reflects reduced binding of the polymerase complex to the peri-
odate-treated 3′ end of the template, as previously described for
Fig.1. SARS-CoVnsp12polymeraseactivityisactivatedbynsp7andnsp8. denguevirusRdRp(44).
(A)SequenceoftheRNAprimer/templateusedinthisstudy;the20-ntprimer Tobettermimictheinvivocontextfordenovoinitiation,we
LS2was5′-radiolabeled(markedby*)andannealedtothe40-nttemplateLS1. alsotestedavariantofthe3Rtemplatecarryinga3′-terminal
(B) Primer extension polymerase assays were performed using LS2*/LS1 as 20-residue poly(A) tail (named3RA). The efficiency of 7L8/12-
substrateanddifferentcombinationsofseparatelypurifiednsp12,nsp8,and mediatedRNAsynthesisisunchangedonthistemplatecompared
nsp7.RNAproductswereseparatedbydenaturinggelelectrophoresis(20% withthatofthetemplatewithoutpoly(A)tail(Fig.S4B).Taken
polyacrylamide/7Murea)andanalyzedbyautoradiography.Thepositionsof together,theseresultsshowthatthe7L8/12polymerasecomplex
the primer (20-mer) and the full-length extension product(40-mer) are in- iscapable ofdenovoinitiationofRNAsynthesisona template
dicated.(C)WTormutant(D760A)nsp12werecoexpressedinE.coliwiththe with the same sequence as the 3′-proximal domain of the viral
nsp7-L-nsp8fusionprotein(7L8).Afterpurificationofthe7L8/nsp12complex
RNA genome, and of subsequent elongation of RNA products
on a Strep-Tactin column, analysis by 12% SDS/PAGE and Coomassie blue to>300nt.
stainingoftheproteinsconstitutingthecomplex(nsp12WTorD760Amutant)
wasdone.#indicatesthepositionofE.coliproteincontaminants(definedby
The Nsp7/Nsp8 Complex Confers Processivity to Nsp12 RdRp. The
MALDI-TOFanalysis).(D)Comparisonofprimerextensionpolymeraseactivities
activation of nsp12 RdRp activity in the presence of nsp7 and
ofWTandmutant(D760A)nsp12inthepresenceofnsp7andnsp8.Nsp7,nsp8,
andnsp12wereeitherpurifiedandaddedseparately(laneslabeled7+8+12)or nsp8 suggests that the nsp7/nsp8 complex enhances processivity
copurifiedfromE.coliasdescribedabove(laneslabeled7L8/12).Thereactions of nsp12-mediated RNA synthesis. We next analyzed the proc-
wereperformedonRNAtemplateLS2*/LS1(seeB).Primerconversionrates essivityof7L8/12onthe3Rtemplate.Toavoidrecyclingofthe
(at60min):40%for7+8+12;67%for7L8/12;and0%for7+8+12(D760A)and polymerase once it had terminated a series of nucleotide addi-
7L8/12(D760A). tions, we made use of a 200-fold excess of an exogenous RNA
highlyunlikelytoassembleintoasimilarhexadecamericstructure,
due to topological constraints. Another assembly type than the
SARS-CoVhexadecamerwasreportedforfelinecoronavirus,in
which the nsp7/nsp8 complex forms a heterotrimer (32), clearly
pointingtotheplasticityofthisCoVproteincomplexthatcouldbe
relevantfor7L8.
To investigate whether the observed primer extension was
indeed catalyzed by the nsp12-RdRp domain, we mutated the
conservedmotifCoftheRdRp(SDDtoSAD,orD760A).This
nsp12(D760A)mutantisstillabletobindto7L8(Fig.1C)butis
incapableofRNAsynthesisinthepresenceofeitherseparately
purifiednsp7andnsp8orcopurified7L8(Fig.1D).
The 7L8/12 Polymerase Complex Catalyzes de Novo RNA Synthesis.
An assay to investigate de novo initiation of RNA synthesis by
Fig.2. The7L8/12polymerase complexcatalyzesdenovoRNA synthesis.
7L8/12 was designed using a 339-nt-long heteropolymeric RNA
DenovoRNApolymeraseassayswereperformedusing500nM7L8/12with
template,correspondingtothe3′-nontranslatedregion(3′-NTR) 500nMRNAtemplate(3R),representingthe3′-terminal339ntoftheSARS-CoV
of the SARS-CoV genome (named 3R). The protein complex
genome. Reaction products, collected at the indicated time points, were
andtemplate3Rwereusedforatimecourseexperimentinthe analyzedbydenaturingagarosegelelectrophoresisandautoradiography.
presence of [α-32P]ATP and reaction products were analyzed ReactionproductsareidentifiedontheRightofthegel,andradiolabeled
usingdenaturingagarosegelelectrophoresisandautoradiography. RNAsizemarker(M)isshowntotheLeftofthepanel.
E3902 | www.pnas.org/cgi/doi/10.1073/pnas.1323705111 Subissietal.
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substrate,thepreviouslyusedLS2/LS1primer/template(Fig.1A) LS1trapwereintroducedsimultaneously(setup#2),thesynthesis P
thatservedasa“polymerase trap.”Thelatterwasaddedeither of long products derived from template 3R is barely detectable
atthesametimeasthe3Rtemplate,orafter7L8/12/3Rtemplate (Fig. 3B, compare #1 and #2) indicating that LS2/LS1 out-
complex formation had been allowed to occur (Fig. 3A, setups competes 3R. However, when the same excess of LS2/LS1 was
#2and#3,respectively).Inthelattersetup,exogenousLS2/LS1 added after 7L8/12/3R complex formation had occurred (setup
shouldtrapallfreepolymerasecomplexesassoonastheywould #3),thetwolongproducts(fromdenovoandbackpriminginiti-
falloffthe3Rtemplate.Thus,thislastconditionwouldallowvi- ationmodes)aretheonlyproducts>40ntdetected(Fig.3B,setup
sualizationofRNAproductsresultingfromasingledenovo(or #3).Theabsenceofanyshorterproductresultingfromabortive
backpriming)initiationevent.Allpolymeraseassayswerestarted RNA synthesis shows that RNA synthesis proceeds with a very
bytheadditionoftheNTPmixture,including[α-32P]ATP(NTP*). low dissociation rate once a productive polymerase/3R complex
As a control (setup #1), a reaction was performed without the isformed.
LS2/LS1 trap. Fig. 3B (left lanes of the gel) shows that it yields TogaininsightintotheassociationkineticsofRNAtemplate
somesmaller“abortiveproducts,”presumablyderivingfrompoly- and7L8/12,weperformedtrappingexperimentssimilarlytothose
merase complexesfallingoff the 3R template, inaddition to the presentedinFig.3.Theobtainedk value(Fig.S5;7.9·10−3s−1)
off
two expected full products resulting from de novo initiation and indeedreflectsaverylowdissociationrate.Together,theseresults
backpriming.TheproductsfromabortiveRNAsynthesisareonly clearly demonstrate that the 7L8 cofactor activates and confers
detectedafterlongerincubationtimes(>5min),inlinewiththe processivitytotheSARS-CoVnsp12RdRp.
polymeraserecyclinghypothesis.Whenthe3RtemplateandLS2/
Nsp7andNsp8ConferRNABindingCapacitytoNsp12.Howdoesthe
nsp7/nsp8 complex stimulate nsp12 RdRp activity? We first
compared the RNA binding properties of nsp12, 7L8, and the
7L8/12 complex. Electrophoretic mobility shift assays (EMSAs)
indicate that nsp12 itself does not bind significantly to RNA
(LS2/LS1), that 7L8 interacts weakly with it, whereas 7L8/12
strongly binds to RNA (Fig. 4A). Moreover, the 7L8/12 RNA Y
G
binding affinity is higher in elongation than in initiation mode, O
L
bshaisfetd(Foing.p4rAo)t.eAinltcoogmetphleerx,tqhueasnetditaietas suusgegdestottohbatse7rLve8manedRiaNteAs OBIO
R
affinityofthecomplex forRNA. MIC
Next, we sought to identify nsp7 and nsp8 residues that may
confer RNA template-binding ability to the nsp7/nsp8/nsp12
complex.BasedonsequenceconservationacrossCoronaviridae,
theirexposureonthesurfaceofthepublishednsp7/nsp8crystal
structures (29, 32), and amino acid properties, 5 nsp7 residues
and16nsp8residueswereselectedforsubstitutionwithalanine
(Fig.S6).Primerextensionassayswerefirstperformedusingthe
five nsp7 mutants in combination with separately purified WT
nsp8andWTnsp12.Nsp7residuesK7andN37arefoundtobe
essentialforRdRpactivity,whereasreplacementofH36confers
a moderate decrease in primer-dependent polymerase activity
(38%residualactivity;Table1).Next,toaddresstheimpacton
de novo polymerase activity, 7L8 proteins carrying these three
mutations were expressed in E. coli and copurified with nsp12.
Complexes containing the nsp7 H36A and N37A mutants show
strongly reduced de novo polymerase activity (less than 7% of
the WT control), whereas the complex containing K7A exhibits
33% oftheactivity oftheWTcontrol.Then, EMSAs were per-
formedwithcomplexescontainingthesesamensp7mutants(K7A,
H36A,andN37A). Replacementofeach ofthesensp7 residues
severelyreducestheRNAbindingcapacityofthensp7/nsp8/nsp12
complexduringelongation,indicatingthatnsp7playsacrucialrole
inRNAbinding(Fig.4BandTable1).
Thesameapproachwasusedtoanalyzemutantsinwhichone
of 16 conserved nsp8 residues had been mutated. Polymerase
activities of these nsp8 mutants were assayed either in the con-
textofseparatelypurified(nsp7plusnsp8plusnsp12)forprimer
extension assaysor aspartofthe copurified 7L8/12polymerase
complex for de novo polymerase assays. Of note, primer exten-
sion activities of nsp8 WT and a set of nsp8 mutants were also
assayedbothinthecontextofseparatelypurifiednsp7,nsp8,and
nsp12 and in the copurified 7L8/12 polymerase complex. Com-
parable relative values of primer extension activities are ob-
Fig.3. The7L8/12polymerasecomplexprocessivelyreplicatesthe3RRNA servedinbothsetups,supportingthebiologicalrelevanceofdata
template.(A)Schematicrepresentationofthethreeexperimentalsetupsused,
obtained with the 7L8 fusion protein (Table S1). Five nsp8
allincluding500nMof7L8/12polymerasecomplexand100nMofthe339-nt
mutations (K58A, D99A, P116A, P183A, and R190A) are asso-
template3R;theLS2/LS1template(Fig.1A)wasusedasatrapat20μM.NTPs*
representsamixofthefourNTPs(500μMGTP,UTP,CTP,and50μMATP)and ciated with a strong decrease of both primer-dependent and de
0.17μM[α-32P]ATP(0.5μCi/μL).Nucleotideincorporationwasfollowedintime novo polymerase activities (Table 2). The 7L8/12 complex con-
taining the nsp8 K58A mutant shows strongly reduced RNA
asindicatedinthethreeflowcharts.(B)Reactionswerestartedbytheaddi-
tionofNTPs*.RNAproductswereseparatedbydenaturingacrylamide/urea binding(Fig.4B),consistentwiththeobservedlossofpolymerase
gels and visualized by autoradiography. RNA products and residual radio- activity, but without providing an explanation for the difference
labeledATP*areindicatedontheRightoftheautoradiograph. in activity observed between primer extension and de novo
Subissietal. PNAS | PublishedonlineSeptember2,2014 | E3903
The Nsp8/Nsp12 Interaction Is Critical for SARS-CoV RdRp Activity.
To further unravel the involvement of nsp8 residues in nsp12
RdRpstimulation,wecharacterizedthefourothernsp8mutants
exhibitingadecreaseinbothprimerextensionanddenovopoly-
meraseactivities(D99A,P116A,P183A,R190A;Table2).These
mutants, in the context of 7L8, were analyzed for their ability
tointeractwithnsp12inaprotein–proteininteractionstudyalso
including the D50A, K82A, and S85A nsp8 mutants (displaying
unchanged primer-dependent polymerase activities) and nsp8
mutant K58A, which wasassociated with a critical decrease of
the interaction between polymerase complex and RNA. Using
theE.colicoexpressionsystem,WTormutant7L8proteinswere
copurified with nsp12. Proteins were separated by SDS/PAGE,
and peaks corresponding to nsp12 and 7L8 were quantified.
Table2presentsthelevelofcopurificationbetweennsp12and7L8
(mutants)relativeto7L8(WT),whichwastakentodefine100%of
nsp12/nsp8 protein interaction. Nsp8 mutants D99A, P116A,
P183A,andR190Alosetheirabilitytointeractwithnsp12,likely
explainingthelackofpolymeraseactivity,whereastheinteraction
betweennsp12andnsp8D50A,K58A,K82A,andS85Amutants
isunaffected(Table2).
Ourstudythusidentifiesfournsp8residues(D99,P116,P183,
andR190)thatarecriticalfortheinteractionofnsp8withnsp12
in vitro, and thereby essential for processive RNA polymeriza-
Fig.4. Nsp7 andnsp8 confer RNA-bindingcapacity to the nsp12 RdRp.
tiontooccur.
EMSAs were performed with 100 nM radiolabeled RNA template (LS2*/
LS1) and increasing concentrations of the indicated proteins. With the
exception of 7L8/12 in initiation mode, all of the other reactions were ReverseGeneticsConfirmstheCriticalRoleofNsp7andNsp8inSARS-
donewithNTPs(500μMCTP,UTP,GTPand50μM3′-dATP)tofacilitate CoVReplication.Theimportanceofthecoronavirusnsp7andnsp8
formationofastalledRNA/enzymecomplex,andwith3′-dATPpreventing subunits for virus replication in vivo has barely been addressed
synthesisoffull-lengthRNAproduct.Reactionswereincubatedfor60min thusfar.Basedonourinvitrostudies,mutationsofnsp7andnsp8
at30°Candproductswereanalyzedbynative6%PAGE.(A)Noprotein residuesthatareimportantforpolymeraseactivity(i.e.,nsp7K7,
(np)controlandincreasingconcentrationsofnsp12,7L8,and7L8/12,as H36, and N37; nsp8 K58, D99, P116, P183, and R190; Tables 1
indicated above each panels. (B) No protein (np) control and increasing and2)werereverse-engineeredintotheSARS-CoVgenomeus-
concentrations(0.5,0.75,and1μM)of7L8/12polymerasecomplexescon- ingaBAC-basedfull-lengthcDNAclone(45).Also,nsp8residues
tainingdifferentnsp7andnsp8mutants,asindicatedaboveeachpanel. S11 (with constrained Ser/Thr variability in coronaviruses) and
7L8/12inelongationmodegave42.5%±7.5%ofshiftedprobe;7L8,9.0%± N43, one of the few invariant nsp8 residues among CoVs (Fig.
3.5%;7L8(K58A)/12, 10.0% ± 6.5%,whereas nsp12 alone andthe other S6),weretargetedinthisreverse-geneticsstudy,aswellasthree
mutantspresentedheredidnotshowanyabilitytobindtheprobe(0%). residues previously identified as important for in vitro nsp8
These values are averages from quantification of three independent
function (i.e., D52, K82, and S85) (30, 31), which have pheno-
experiments.
typescomparabletothatoftheWTcontrolinourbiochemical
studies (Table 2).
Mutant viruses were launched by electroporation of in vitro-
initiationassays(70%versus99%lossofactivity,respectively;
generated RNA transcripts into BHK-Tet-SARS-N cells, which
Table 2).
express the viral nucleocapsid (N) protein (46). These cells can
Inconclusion,ourstudyidentifiesthreensp7residues(K7,H36,
be transfected efficiently and release progeny virus, but do not
andN37)andonensp8residue(K58)thatappeartobeimportant
support further viral spread because they lack the ACE2 recep-
fortheinteractionofthepolymerasecomplexwithRNA,thusalso torusedbySARS-CoV.Forthisreason,transfectedcellswere
providingthefirstevidence(toourknowledge)foraroleofnsp7 mixed with (susceptible) Vero-E6 cells and the replication of
inCoVRNAsynthesis. mutant viruses was characterized at different time points using
Table1. BiochemicalpropertiesofSARS-CoVnsp7mutants
Summaryofquantificationfornsp7mutantsinprimerextensionanddenovopolymeraseactivities,aswellas
RNAbinding.Quantificationsofpolymerizationactivitiesweredoneafter60minofincubationandbyusing
ImageGaugesoftware.ND,notdetermined.WTabsolutevaluescorrespondingto100%activityareasfollows:
42%±4%ofprimerconversion(at60min)forprimer-dependentexperiments;42.5%±7.2%ofshiftedprobe
for EMSAs.Forthedenovoassays,polymeraseactivities ofthedifferentmutantswere always analyzed on
denaturatinggelinparallelwiththeWTpolymeraseproteincomplex,whosebandintensitiesweresetat100%.
SDsarecalculatedfromthreeindependentexperiments.
*Nsp7WTplusnsp8WTplusnsp12WTprimerextensionactivitywassetat100%.
†7L8/12WTcomplexfordenovopolymeraseactivitywassetat100%.
‡RNAbindingof7L8/12WTcomplexwassetat100%.Mutantshighlightedindarkgrayindicateimportant
residuesforinvitropolymeraseactivities.
E3904 | www.pnas.org/cgi/doi/10.1073/pnas.1323705111 Subissietal.
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Table2. BiochemicalpropertiesofSARS-CoVnsp8mutants P
Summaryofquantificationfornsp8mutantsinprimerextensionanddenovopolymeraseactivities,aswellas
innsp8/nsp12interactionassays.Quantificationsofpolymerizationactivitiesweredoneafter60minofincuba-
tion and by using Image Gauge software. ND, not determined. WT absolute values corresponding to 100%
activity are as follows: 42% ± 4% of primer conversion (at 60 min) for primer-dependent experiments and GY
42% ± 8.2% of 7L8 band intensity (compared with nsp12 band intensity) for nsp8/nsp12 interaction assays. LO
O
Forthedenovoassays,polymeraseactivitiesofthedifferentmutantswerealwaysanalyzedondenaturating BI
gelinparallel with theWT polymerase protein complex,whoseband intensities weresetat100%. SDsare RO
C
calculatedfromthreeindependentexperiments. MI
*Nsp7WTplusnsp8WTplusnsp12WTprimerextensionactivitywassetat100%.
†7L8/12WTcomplexfordenovopolymeraseactivitywassetat100%.
‡Levelofcopurificationof7L8(WT)relativetonsp12wastakentorepresent100%ofnsp12/nsp8proteinin-
teraction.Mutantshighlightedindarkgrayindicateimportantresiduesforinvitropolymeraseactivities.
immunofluorescencemicroscopyandprogenyvirustitration(plaque testedbyreversegenetics.MutantD52Adisplayedareducedpla-
assays). Each mutant was tested twice, by transfection of RNA quesize,althoughitsgrowthkineticswasquitesimilartowhatwas
derived from two independently generated cDNA clones. For foundfortheWTvirus(Fig.5).ForK82A,noevidenceofrepli-
mutants producing infectious progeny, the 42-h posttransfection cation was observed at the earliest time point after transfection
(p.t.)harvestwasusedtoinfect freshVeroE6,fromwhich RNA analyzed (18 h p.t.; Table S2), but this changed later on. This
wasisolatedtoamplifythensp7-,nsp8-,andnsp12-codingregions transition coincided with the discovery of second-site mutations,
by RT-PCR. This material was used to confirm the presence of specifically a D78N substitution in one experiment and a mix of
the engineered mutation(s) and investigate the presence of pos- E77T andD78N inthesecondexperiment. The results obtained
siblesecond-sitereversions.Thepropertiesofthevariousmutants forthesetwomutantsonceagainhighlighthowtheuseofdiverse
aresummarizedinTableS2. assaystoanalyzetheeffectofmutationsinviralproteinsisacritical
Substitution of the invariant nsp7 residues H36 and N37 by partoftheirfunctionalcharacterization.Interestingly,withregard
alanine supportsthe importance of nsp7for virus replication in totheonlythreeinvariantresiduesidentifieduponthecomparative
vivo. Although both mutants were viable, their replication was analysis of CoV nsp8 sequences (N43, K58, and P183; Fig. S6),
impairedcomparedwiththatoftheWTvirusaswasinitiallyev- replacements of K58 and P183 impair polymerase activity and
identfromtheirreducedplaquesize(Fig.5).Similarobservations resulted in a nonviable phenotype in reverse genetics, whereas
were madefor mutant nsp7K7A.The phenotype ofthese three mutantN43AsurprisinglydisplayedaWT-likephenotypebothin
nsp7mutantswasfurthercomparedbydeterminingtheirgrowth- vitroandinvivo.
curves, for which fresh Vero-E6 cells were infected [multiplicity Althoughthe lethal effectofan nsp8 full-lengthdeletion pre-
ofinfection(M.O.I.)of5]withsupernatantharvestedat42hp.t. viouslysuggestedtheoverallimportanceofnsp8(41),thisstudyis
from cells transfected with mutant RNA (Fig. 5). This more de- thefirst(toourknowledge)toestablishthedirectfunctionalim-
tailedcomparisonconfirmsthatallthreensp7mutantsare(about portance of specific nsp8 residues (K58, P183, and R190) for
equally) crippled compared with the WT control, reaching final virusreplication.
progeny titers[24 h postinfection(p.i.)] that are about 0.5–1 log
reduced(Fig.5). TheNsp7/Nsp8/Nsp12PolymeraseComplexIsAbletoAssociatewith
The10nsp8mutantstesteddisplayedavarietyofphenotypes anActiveBifunctionalNsp14.Inagreementwiththeproposedrole
(TableS2),rangingfromnonviable(K58A,P183A,andR190A), of the nsp14 ExoN domain in coronavirus proofreading during
viacrippled (D52A,K82A,D99A,andP116A),toquite similar replication (24, 25, 47), the nsp14 replicase subunit binds to
totheWTcontrol(S11A,N43A,andS85A).Forthemutantsin nsp12(Fig.S1Bandref.39). Wetherefore addressed theques-
thefirstandthirdgroups,theinvivophenotypesareingeneral tionwhethernsp14isstillabletobindtonsp12inthecontextof
agreementwiththeresultsfromthebiochemicalassays(Table2). the 7L8/12complex andif such an interaction could potentially
This is less clearcut for the second group, which for example affect any of the three-enzymatic activities (RdRp, 3′-5′ ExoN,
containsnsp8mutants(D99A,P116A)withmajordefectsinthe andN7-MTase).
biochemicalassaysbutaviable,althoughcrippledinvivopheno- Pull-down experiments were performed as described in the
type,asreflectedintheirreducedplaquesizeandsomewhatlower legendofFig.S1.Interactionof7L8,nsp12,andnsp14isreadily
progenytiters(Fig.5).Conversely,twomutants(D52A,K82A)not detected(Fig.6A),andtheproteincomplex(hereafter7L8/12/14)
showingmajordefectsinbiochemicalassayswereimpairedwhen can be purified (Fig. S7). The polymerase activities of the
Subissietal. PNAS | PublishedonlineSeptember2,2014 | E3905
number of nucleotides polymerized during a single encounter
with its template, is considered critical for efficient virus repli-
cation(54–58).
CoronavirusescarryRNAgenomesatleasttwicelarger(∼30kb)
thanthatofthemajorityofanimalRNAviruses(∼3to∼15kb).
Our present results suggest that to ensure processivity of their
nsp12-polymerase, coronaviruses have evolved a mechanism of
RNA synthesis that is unique among RNA viruses. Based on in
vitroassays,SARS-CoVnsp12RdRpwaspreviouslyreportedtobe
anonprocessiveprimer-dependentRNApolymerase(21,37).We
nowdemonstratethatthesimultaneousadditionofnsp7andnsp8
activatesandconfersprocessivitytothensp12RdRp.Bydisplaying
averylowdissociationratefromRNA,thensp7/nsp8/nsp12com-
plexcansynthesizeanRNAof,atleast,359ntwhensynthesisis
startedwithoutaprimer(denovo).Clearly,associationwithother
nspsmayberequiredtocopythe30-kbfull-lengthgenome.
Thethreeaminoacidsinnsp7(K7,H36,andN37)thatconfer
RNA-binding properties to the polymerase complex (Fig. 4B)
arealsoimportantforvirusreplicationinvivo(Fig.5).Indeed,in
A nsp12 bound to B
Fig.5. Reverse-geneticsanalysisoftheimpactofselectedSARS-CoVnsp7 StrepTactin StrepTactin
andnsp8mutations.Plaquephenotypesofviablebutcrippledmutantswith beads + beads +
ewnegrienepeerrefodrnmsepd7awnidthnespa8rlympurtoagtieonnysavrireuisl,luhsatrravteesdteodnftrhoemLceeftll.sPalatq1u8ehaspsaoysts nsp14 + 7L8 7L8 nsp14 nsp14 + 7L8 nsp14 + 7L8 7L8/12 7L8/12/14
full-lengthRNAtransfection(MaterialsandMethods).OntheRight,growth
curvesareshownforcripplednsp7andnsp8mutantsandtheWTparental 40-mer
virus.Fortheseexperiments,mutantviruswasharvestedfromtransfected nsp14
cellsat42hp.t.Aftertitrationofthesevirusstocks,freshVero-E6cellswere
infected(M.O.I.,5),andvirusproductionatthegiventimepointswasmea-
suredbyplaqueassay.GraphsdisplaymeantitersandSDsderivedfromthree
independentexperiments.Nonviablemutantsandmutantswithaphenotype LS2*/LS1
similartoWTvirusarelistedattheBottom. 7L8 0 15 60 0 15 60 time (min)
7L8/12 and 7L8/12/14 complexes are comparable (Fig. 6B). In C D
thesepolymeraseassays,RNAdegradationisnotdetectedinthe
presence of nsp14, indicating that nsp14 ExoN activity is either
7000
very weak due to the stimulating-factor absence (24) or sup-
LS3*/LS1
pressedwhenthe7L8/12/14complexisin“polymerizationmode.” 6000
The3′-5′ExoNactivityofthe7L8/12/14complexwasthenassayed
5000
using the optimal experimental conditions previously described
for ExoN (24). Under these conditions, ExoN activity is barely m4000
p
detected(Fig.6C).Whennsp10isaddedtothe7L8/12/14com- c
3000
plex,theRNAexonucleaseactivityissignificantlyenhanced(Fig.
6C),indicatingthatnsp10isabletoeitherenhancensp14-ExoN 2000
activityinsituaspreviouslyreported(24),ortopartiallydisplace 1000
nsp14 from 7L8/12/14 and activate nsp14-ExoN activity sepa-
0
rately.Moreover,nsp14N7-MTaseactivityisalsofoundasso- 0 10 30 0 10 30 0 10 30 time (min) nsp14 7L8/12/14 7L8/12
ciated with the 7L8/12/14 complex (Fig. 6D). We therefore
concludethatthensp7/nsp8/nsp12/nsp14complexrepresents Fig. 6. The SARS-CoV 7L8/12/14 complex possesses RdRp, ExoN, and N7-
a unique coronavirus nsp assembly that incorporates RdRp, MTase activities. (A) Strep-tagged SARS-CoV nsp12 was bound to Strep-
Tactinbeadsandincubatedwith7L8,nsp14,orbothsimultaneously.After
exoribonuclease,andN7-MTaseactivities.
SDS/PAGEandWesternblotting,his-taggedproteins(7L8andnsp14)were
Discussion revealedusingananti-His5-HRPantibody.(B)Timecourseprimerextension
polymeraseassayswereperformedusingeitherthe7L8/12(500nM)orthe
In the infected cell, viral RNA replication is challenged at sev- 7L8/12/14(500nM)complexeswithLS2*/LS1asprimer*/templatewhereLS2
erallevels. RNAsynthesismustberegulatedtominimizeexpo- was 5′-radiolabeled (markedby *).RNAproductswere separatedina de-
suretothehostcell’sdefensemechanisms.Moreover,compared naturingpolyacrylamide/ureagelandvisualizedbyautoradiography.(C)Time
withDNA-basedorganisms,RNAvirusesaregenerallybelieved courseexoribonucleaseassayswereperformedusingthe7L8/12/14(500nM)
toreplicatetheirgeneticmaterialwithlowaccuracy,astheyare complexintheabsenceorpresenceof100nMnsp10,andascontrolwith7L8/
believedtolackproofreadingandcorrectionmechanisms(48,49). 12(500nM)plusnsp10(100nM).TheRNAsubstratewasa40-ntRNA(LS1)
Hence, RNA replication has evolved toward an equilibrium at annealedwith5′-radiolabeledLS3primercarryingonenoncomplementary
whichaheterogeneouspopulationofviralRNAs,commonlyre- baseatits3′end(LS3*)andnamedLS3*/LS1.Digestionproductsweresepa-
ratedbydenaturingpolyacrylamide/ureagelelectrophoresisandvisualizedby
ferredtoasaquasispecies,isreproducedwithhighefficiency(50,
autoradiography(Fuji).The“α”symbolindicatesRNAcleavageproducts.(D)
51). The viral RdRp, fine-tuned during evolution, is the central
AdoMet-dependentN7-MTaseactivityofthe7L8/12/14complex.Thedifferent
enzymeinthisprocess.Itisthoughttopossesstheoptimumcom-
purifiedproteinsorproteincomplexes(nsp14,300nM;7L8/12/14,300nM;and
binationofRNAsynthesisefficiencyandnucleotideincorporation
7L8/12,300nM)wereincubatedwithsubstrateGpppAC4RNAoligonucleotide
fidelity(52,53).Attheenzymelevel,speedandaccuracycanbe inthepresenceof[3H]AdoMet.ThemethyltransfertothecappedRNAsub-
measured and evaluated using a number of parameters. Among stratewasdeterminedbyusingafilter-bindingassay(asdescribedinref.73).
them, the processivity of the RNA polymerase, reflected in the Allexperimentsweredoneintriplicate(SDsarepresented).
E3906 | www.pnas.org/cgi/doi/10.1073/pnas.1323705111 Subissietal.
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reverse-genetics studies, these mutations delayed virus growth fromanRNAduplex(24).Thensp7/nsp8/nsp12complexbindsto P
comparedwiththeWTcontrol,althoughtheirimpact islessse- thebifunctionalexoribonuclease/N7-guaninecapmethyltransfer-
verethanonemighthaveanticipatedgiventheirphenotypeinthe ase nsp14, with no apparent modulation of polymerase, ExoN,
invitroassays.Thelackofcorrelationforsomemutantsbetween orN7-MTaseactivities.Thereconstitutionofannsp7/nsp8/nsp12/
invitroandinvivostudiessuggeststhattheinvitrobiochemical nsp14complexmakesitnowpossibletodissectinvitrothecoro-
assays reflect only a part of the complexity of coronavirusRNA navirus proofreading mechanism discovered through prior re-
synthesisinvivo.Itisconceivablethattheeffectsofsingle-point search(25,26,47).
mutationsonpropertieslikeRNAbindingaremoredramaticin Inanycase,thiscomplexprovidesafascinatingexampleofthe
thecontextofthetrimericcomplexinvitrothaninthecontextofa integrationofRNApolymerization,proofreading,andcap-mod-
16-nsp complex in vivo, where, e.g., the presence of other repli- ifying activities into a multifunctional protein assembly. This
case subunits may provide some form of compensation. None- macromolecularassembly,possiblyincombinationwiththeheli-
theless,thesedataconstitutethefirstevidence(toourknowledge) case/RNAtriphosphatase nsp13and the 2′O-MTase nsp16 may
fora role ofnsp7incoronavirusreplication.Moreover, our bio- constitute the core of the coronavirus replication/transcription
chemical(Tables1and2)andreverse-genetics(Fig.5andTable machinery,whichisabletoengageincoordinatedRNAsynthesis
S2) data strengthen the case for a role for nsp8 in viral RNA andprocessingactivities.
synthesis, with three nsp8 residues(K58, P183, and R190) being
identified as critical for SARS-CoV genome replication. Our MaterialsandMethods
biochemical assays further indicate that nsp8 residues P183 and Cloning, Expression, and Purification of SARS-CoV Proteins. All SARS-CoV
R190areinvolvedinnsp8/nsp12interaction,whereasresidueK58 proteins used in this study were expressed in a bacteriophage T7 RNA
modulatestheinteractionofthepolymerasecomplexwithRNA. polymerase-freesystem,withtheexceptionofnsp10andnsp14.Allplasmids
Nsp8 was previously suggested to be a second, noncanonical wereexpressedinE.coliC2523cells(NEBiolabs),whereasE.coliC41(DE3;
coronavirus RdRp, which would be able to synthesize small pLysS)(Novagen)wasusedfornsp10andnsp14expressions.SARS-CoVnsp7
oligomers(<6nt)alone(30)orincomplexwithnsp7(31,32).In fusedtoaC-terminalhexahistidinetag(namednsp7)wasclonedintothe
anefforttounderstandhowdenovoRNAsynthesisisachieved vectorpASK3-Kana,amodifiedpASK3vector(IBA)inwhichtheampicillin
resistancecassetteisreplacedbythatforkanamycin.Nsp7expressionwas
and possibly demonstrate the role of nsp8 as primase in this done overnight at 17 °C after induction with 200 μg/L anydrotetracyclin. GY
complex, we tested nsp12 catalytic mutant in de novo assays, in O
the presence of the nsp7/nsp8 complex. Full-length products PSAroRtSe-iCnopVunrisfpic8atwioitnhwaaCs-tpeerrmfoinrmalehdeaxashdisetsicdriinbeedtapgre(nvaiomuesldynfospr8n)swp9as(6p9u)-. BIOL
wereabsentasexpected.However,wewerealsounabletodetect O
the nsp8-driven formation of short RNAs (Fig.S3), even inthe rinifsieerdtiansgdaeshcerxibaehdistpidreinvieoutasglyl(in31k)e.rTbheetwfueseionnnpspro7t-eainnd7Ln8spw8a-csogdeinngersaetqeudebny- MICR
presence of Mn2+, an element described as essential for nsp8- ces.ThisgenewasclonedintothepASK3-Kanabackboneandproteinpuri-
RdRpactivity(30).Onepossibleexplanationofthisdiscrepancy ficationwasperformedasdescribedfornsp7.SARS-CoVnsp12wasexpressed
maybethatnsp8incomplexwithnsp12andnsp7adoptsastruc- fromasyntheticgeneoptimizedforexpressioninE.coliandequippedwith
turalconformationthatpreventsthesynthesisofshortRNAs.The aC-terminalStrep-Tag,whichwasclonedintoplasmidpJ404(purchasedfrom
abilityofthensp7/nsp8complextoadaptdifferentconformations DNA2.0).Nsp12expressionwasinducedbyadditionof500μMisopropylβ-D-1-
(refs.29and32;seebelow)mayreflectitsinvolvementindistinct thiogalactopyranosidewhentheOD600nmvalueoftheculturereached0.5,
functions, including primer synthesis. It is thus plausible that andwasallowedtoproceedfor16hat17°C.Lysisbuffer[50mMHepes
aparticularfunctionofcoronavirusnsp7andnsp8mayinfact (pH7.5);150mMKCl;5mMMgCl2;5%(vol/vol)glycerol]supplementedwith
be triggered by the nature of their partnership and/or quater- 10 μg/mL DNase I, 1 mM PMSF, and 0.25 mg/mL lysozyme were used to
narystructure. resuspendpellets.AStrep-Tactin(IBA)purificationstepwasperformedand
Nsp7 and nsp8 are shown to interact with each other, and nsp12 wasrecoveredusing lysis buffer supplemented with 2.5 mMD-des-
thiobiotin. A second chromatography step was then performed. The salt
nsp7/nsp8 complexes were crystallized and solved for different
concentrationofnsp12proteinsolutionwasloweredto25mMKCl,andnsp12
coronaviruses. The presence of nsp7 seems to be required to
wasthenincubatedduring1hwithheparinbeadsandfinallyelutedwith
crystallize nsp8. The structure of the SARS-CoV nsp7/nsp8 alineargradientofKCl(25–500mM).
complex(29)revealedahexadecamericcomplexwithapositively
The 7L8/12 complex was obtained after cotransformation of plasmids
charged central tunnel supposedly accommodating RNA. This
encoding7L8andnsp12,andwaspurifiedasdescribedabovefornsp12,with
structure is reminiscent of processivity-promoting factors such
anadditionalsizeexclusionchromatographystepperformedonaKW804
as, e.g., the eukaryotic PCNA sliding clamp (59–61), or the
column(Shodex).
β-subunitboundtoE.coliDNApolymeraseIII(62).Incontrast,
Site-directedmutagenesistogenerateplasmidsexpressingmutantpro-
feline coronavirus nsp7 and nsp8were shownto form ahetero- teinswasperformedusingtheQuikChangeSite-DirectedMutagenesisKit
trimerbyassociationoftwonsp7moleculeswithonemoleculeof (Stratagene)accordingtothemanufacturer’sinstructions.Forrelevantnsp7
nsp8withouttheformationofahollowstructure(32).Itisthus (K7A,H36A,andN37A)andnsp8(K58A,D99A,P116A,P183A,andR190A)
likely that coronavirus nsp7 and nsp8 may in fact be endowed mutantsaswellasfornsp7WTandnsp8WT,correctfoldingwasconfirmed
withseveralfunctionstriggeredbythenatureoftheirpartnership bycirculardichroism(CD)analysis.TheCDspectrawererecordedonaJasco
and/or quaternary structure. They would thus constitute addi- 810dichrographusing1-mm-thickquartzcells.
tional examples of RNA virus proteins that have evolved a re-
markableplasticitytoaccommodatemultiplefunctions.Another RNA Templates. Synthetic RNAs (LS1, LS2, and LS3) were purchased from
recent example of this kind of functional flexibility is the Ebola Biomers(HPLCgrade).Templates3RAand3RRNAtemplates,corresponding
virus matrix protein VP40, which was shown to be subject to tothelast339ntoftheSARS-CoV(strainFrankfurt;GenBankaccessionno.
structural rearrangements giving rise to at least three distinct AY291315)genomic3′-NTRwithorwithout20adenines,respectively,were
structures, each executing a distinct function during the viral producedusinganinvitroT7transcriptionkit,andpurifiedasdescribedby
replication cycle(63). themanufacturer(Ambion).RNAmarker(Promega;catalogno.G3191)was
radiolabeledwiththeT4polynucleotidekinase(PNK)(NEB)and[γ-32P]ATP.
PolymeraseprocessivityfactorshavebeenidentifiedforDNA
viruses such as the gp45 protein of bacteriophage T4 (64), the
UL42 protein of herpes simplex virus 1 (65, 66), or E. coli thi- Pull-Down,WesternBlot,andNsp8/Nsp12InteractionAssays.Nsp12wasbound
toStrep-Tactinbeadsandincubatedwith1mg/mLBSAfor15minatroom
oredoxinforbacteriophageT7(67,68).Toourknowledge,coro-
temperature. Then, the Strep-Tactin beads with or without bound nsp12
navirusesnowarethefirstRNAvirusesknowntouseanRdRp wereincubatedfor2hinthepresenceof15μgofhis-taggedpartnerprotein
processivity factor, to expedite replication of their ∼30-kb RNA
tobetested.Next,unboundproteinswereextensivelywashedawaywith
genome.Remarkably,thefunctionofthecoronavirusRNApoly-
washingbuffer(50mMTris,pH8;150mMNaCl;0.05%Triton).Thebeads
meraseprocessivityfactorappearstobecompatiblewiththeca- weredilutedin2×SDS/PAGEloadingbuffer[100mMTris,pH6.8;200mM
pabilityofthensp7/nsp8/nsp12complextoassociatewithanactive DTT;4%(vol/vol)SDS;0.2%bromophenolblue;20%(vol/vol)glycerol]and
nsp14-exonuclease that is able to remove terminal mismatches loaded on an SDS/PAGE gel. A Western blot was then performed using
Subissietal. PNAS | PublishedonlineSeptember2,2014 | E3907
asingleHRP-conjugateantibody(anti-His5HRPconjugate;Qiagen),and ElectroporationsweredoneusingtheAmaxaCellLineNucleofectorKitTand
bandswerevisualizedwithaKodakImageStation400MMPro(CareStream programT-020ofanAmaxaNucleofector(Lonza),accordingtothemanu-
Health). facturer’sinstructions.BHK-Tet-SARS-Ncellsweremixedina1:1ratiowith
Tostudytheinteractionbetweennsp8andnsp12,WTormutant7L8were Vero-E6cells,andseededoncoverslipsinsix-wellclustersforimmunofluo-
coexpressedwithnsp12inE.coli.AfterpurificationonaStrep-Tactincol- rescencemicroscopyanalysisandanalysisofvirusproduction.Eachmutant
umn,theelutedproteinswereanalyzedusingSDS/PAGE,andthepeakin- waslaunchedtwicefromtwoindependentlygeneratedBACclones.Allwork
tensitiescorrespondingtothe7L8andnsp12proteinswerequantified.
withliveSARS-CoVwasperformedinsidebiosafetycabinetsinabiosafety
level3facilityatLeidenUniversityMedicalCenter.
Polymerase Assays. All radioactive reagents were purchased from Perkin-
Elmer.Polymeraseassayswereperformedinpolymeraseassaybuffer(20mM
SequenceAnalysisoftheSARS-CoVNsp7-,Nsp8-,andNsp12-CodingRegions.
T(5r0is0,pnHM8);,1n0spm7M(1K.5Clμ;M1m),ManDdTnTs;p28m(1M.5MμgMC)l2o)rw5it0h0snepMar7aLt8e/l1y2pucorimfiepdlenxsapn1d2 FreshVero-E6cellswereinfectedwithharveststakenat42hp.t.andSARS-
CoVRNAwasisolated18hp.i.asdescribedaboveandamplifiedbyRT-PCR
250nMRNAtemplate,unlessspecifiedotherwise.Primers(LS2orLS3)were
radiolabeledattheir5′endsusing[γ-32P]ATPandPNK,andtermedLS2*and using random hexamers for the RT reaction, and primers 5′-CACCTTA-
LS3*,respectively.LS2*orLS3*werethenannealedtothecomplementary CAGTGTATCATGC-3′and5′-CTGGAACCACCTTGTAGGTTTG-3′forPCRamplifi-
templateLS1byheatingat70°Cfor10minandthencoolingdowntoroom cationofthensp7–8codingregion.Thensp12-codingregionwasamplifiedin
temperature(withaprimer/templateratioof1.2:1).Primerextensionassays twooverlappingfragments,byusingthefollowingprimersets:5′-TTGCCTAC-
werealwaysperformedwithLS2*/LS1astemplate,andreactionswerestar- TATAACAATTCG-3′togetherwith5′-CAGAACTTCCTTCCTTAAAGAAACCTT-3′;
tedbyadding500μMNTPmix.Denovoassays,usingeither3Ror3RAas 5′-GCAGCTTCTGGCAATTTATTGC-3′togetherwith5′-GGAATGGTCTCCTAATA-
template,werestartedbyadding500μMGTP,UTP,CTP,50μMATP,and CAGGC-3′.RT-PCRproductsweresequencedtoverifythepresenceofthein-
0.17 μM [α-32P]ATP (0.5 μCi/μL). After incubation at 30 °C, reactions were troducedmutationsandpossiblesecond-sitereversions.
quenchedbytheadditionofanequalvolumeofloadingbuffer(form-
amidewith10mMEDTA).RNApolymerizationproductsofprimerextension ImmunofluorescenceMicroscopy.Tomonitorthepresenceand/orprogression
assayswereanalyzedin20%(wt/vol)polyacrylamide/7Mureagels,whereas ofSARS-CoVinfection,transfectedcellswereseededoncoverslipsandfixed
productsfromdenovoassayswereanalyzedin1%agarose-formaldehyde atvarioustimepoints.Immunofluorescenceassaysweredonefollowingapre-
gels,unlessspecifiedotherwise.RNAproductswerevisualizedusingphoto-
viouslydescribedprotocol(71)usingarabbitantiserumagainstSARS-CoVnsp4
stimulatedplatesandaphosphorimager(Fuji).
(36)andamousemonoclonalantibodyagainsttheSARS-CoVNprotein(72).
EMSAs.SerialdilutionsofSARS-CoVproteinsinpolymeraseassaybufferwere
VirusTitration.Vero-E6cellsseededinsix-wellclusterswereinfectedwith
preincubatedwith100nMprimer*/template(LS2*/LS1)for30minat30°C.
Unlessotherwisestated,500μMCTP,UTP,GTP,and50μM3′-dATPwere serial 10-fold dilutions in PBS, 0.005% DEAE, and 2% (vol/vol) FCS of
addedtofacilitateRNA–enzymecomplexformation.Thereactionmixture supernatantsfromtransfectedcells,incubatedfor1hat37°C,afterwhich
theinoculumwasreplacedbyanoverlayof1.2%(wt/vol)Avicel(FMCBio-
wasincubatedfor60minat30°C,andsampleswereloadeddirectlyonto
6%(vol/vol)acrylamidegelsandwererunin0.5×TBEat100Vfor60minat Polymer)inDMEMsupplementedwith50mMHepes,2%(vol/vol)FCS,and
4°C.Bandswerevisualizedusingaphosphorimager(Fuji). incubatedat37°C.After3d,theoverlaywasremoved,andcellswerefixed
with7.4%(vol/vol)formaldehydeinPBSandstainedwithcrystalvioletto
Exonuclease Assay. Nuclease reactions were performed as previously de- visualizeplaques.Titerswereexpressedinplaque-formingunitspermilliliter.
scribed (24) with 500 nM radiolabeled LS3*/LS1 substrate. Primer LS3 has
thesamesequenceasLS2withtheadditionofanadenosineatits3′end, ACKNOWLEDGMENTS.WethankLaureGluais,FrançoisFerron,AliceBlouet,
causinganA:AmismatchwhenannealedwithtemplateLS1. JéromeDeval,BarbaraSelisko,AartjanteVelthuis,AlexanderKravchenko,
DmitrySamborskiy,IgorSidorov,IsabelleVarlet,andRenaudVincentellifor
helpfuldiscussionsand/ortechnicalassistance.WethankChristianDrosten
SARS-CoVReverseGenetics.Verocells(ATCC;CCL-81)wereculturedinEagle’s andSusannePfefferle(UniversityofBonn)forprovidingtheSARS-CoVre-
MEM(Lonza)with8%(vol/vol)FCS(PAA)andantibiotics.BHK-Tet-SARS-N versegeneticssystem,andYingFang(KansasStateUniversity)forproviding
cells(46) wereculturedinthesame mediumcontaining100μg/mL G418. the anti-N monoclonal antibody. We acknowledge Dr. Deborah Byrne for
MutationsintheSARS-CoVnsp7-ornsp8-codingregionwereengineeredin editorialassistance.ThisworkwassupportedbytheFrenchNationalResearch
prSCV,apBeloBac11derivativecontainingafull-lengthcDNAcopyofthe Agencyunderreference“ANR-08-MIEN-032”and“ANR-12-BSV3,”theNeth-
SARS-CoVFrankfurt-1sequence(45)asdescribedinref.70.Full-lengthBAC erlandsOrganizationforScientificResearch(TOP-GOGrant700.10.352),and
by the European Union Seventh Framework Programme (FP7/2007-2013)
clones were linearized with NotI, extracted with phenol-chloroform, and
throughtheEUVIRNAproject[EuropeanTrainingNetworkon(+)RNAVirus
transcribedwithT7RNAPolymerase(mMessage-mMachineT7kit;Ambion)
ReplicationandAntiviralDrugDevelopment;GrantAgreement264286],and
usinganinputof2μgofBACDNAper20-μLreaction.RNAtranscriptswere theSILVERproject(SmallInhibitorLeadsVersusEmergingandNeglectedRNA
precipitated with LiCl according to the manufacturer’s protocol. Sub- Viruses;GrantAgreement260644),andfinallybytheCollaborativeAgree-
sequently,6μgofRNAwaselectroporatedinto5×106BHK-Tet-SARS-Ncells, mentinBioinformaticsBetweenLeidenUniversityMedicalCenterandMos-
whichexpresstheSARS-CoVNproteinafterinductionwith2μMdoxycyclin. cowStateUniversity(MoBiLe),andLeidenUniversityFund.
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Subissietal. PNAS | PublishedonlineSeptember2,2014 | E3909