2015 A G-quadruplex-binding macrodomain within the _SARS-unique domain_ is essential for the activity of the SARS-corona

2015·1.57 MB·English

Virology484(2015)313–322
ContentslistsavailableatScienceDirect
Virology
journal homepage: www.elsevier.com/locate/yviro
Brief Communication
A G-quadruplex-binding macrodomain within the “SARS-unique
domain” is essential for the activity of the SARS-coronavirus
replication–transcription complex
Yuri Kusova,b, Jinzhi Tana, Enrique Alvarez1,c, Luis Enjuanesc, Rolf Hilgenfelda,b,n
aInstituteofBiochemistry,CenterforStructuralandCellBiologyinMedicine,UniversityofLübeck,Lübeck,Germany
bGermanCenterforInfectionResearch(DZIF),Hamburg–Lübeck–BorstelSite,UniversityofLübeck,Germany
cDepartmentofMolecularandCellBiology,CentroNacionaldeBiotecnología(CNB-CSIC),CampusUniversidadAutónoma,Madrid,Spain
a r t i c l e i n f o a b s t r a c t
Articlehistory: Themulti-domainnon-structuralprotein3ofSARS-coronavirusisacomponentoftheviralreplication/
Received1March2015 transcription complex (RTC). Among other domains, it contains three sequentially arranged macro-
Returnedtoauthorforrevisions domains:theXdomainandsubdomainsSUD-NaswellasSUD-Mwithinthe“SARS-uniquedomain”.
19March2015 TheXdomainwasproposedtobeanADP-ribose-1”-phosphataseorapoly(ADP-ribose)-bindingprotein,
Accepted12June2015
whereasSUD-NMbindsoligo(G)-nucleotidescapableofformingG-quadruplexes.Here,wedescribethe
application of a reverse genetic approach to assess the importance of these macrodomains for the
Keywords: activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with
SARS-CoVreplicon selectivelydeletedmacrodomainswereconstructedandtheirabilitytomodulatetheRTCactivitywas
SARS-uniquedomain
examined.WhiletheSUD-NandtheXdomainswerefoundtobedispensable,theSUD-Mdomainwas
Macrodomain
crucialforviralgenomereplication/transcription.Moreover,alaninereplacementofchargedamino-acid
X-domain
residuesoftheSUD-Mdomain,whicharelikelyinvolvedinG-quadruplex-binding,causedabrogationof
Reversegenetics
G-quadruplex RTCactivity.
MERS-CoV &2015ElsevierInc.Allrightsreserved.
Introduction by the finding that SARS-CoV replicons are able to replicate
autonomously in transfected host cells (Almazán et al., 2006; Ge
TheSevereAcuteRespiratorySyndromecoronavirus(SARS-CoV) etal.,2007;Erikssonetal.,2008;Panetal.,2008;Wangetal.,2008;
belongstolineagebofthegenusBetacoronavirus.DuringtheSARS reviewed in Almazán et al., 2014). Replicons, in particular those
outbreak of 2003 (see Hilgenfeld and Peiris (2013), for a recent encoding a reporter gene, considerably facilitate the functional
review), the genome of SARS-CoV was sequenced within three analysis of molecular determinants that control the replication
weeksofthediscoveryofthevirus(Marraetal.,2003;Rotaetal., and transcription of the SARS-CoV genome. SARS-CoV replicons
2003) and subjected to detailed annotation shortly thereafter consistofthegenomic50 and30 untranslatedregionsandanopen
(Snijder et al., 2003). At the same time and in subsequent years, readingframe(ORF)encodingthetwopolyproteins(pp1aand,viaa
leadsforantiviraltherapyweredescribed(Anandetal.,2003;Yang (-1) frame-shift, pp1ab) that are processed into 16 non-structural
et al., 2005; see Hilgenfeld (2014), for a recent review). However, proteins (Nsp1 to Nsp16) to form the replication/transcription
thedetailedmolecularcharacterizationofviralgenomereplication complex (RTC, Fig.1A). Among the Nsps, Nsp3 is a multi-domain
and the assembly of viral particles was initially restricted to polypeptide comprising the following structurally organized
specialized high-safety laboratories. This limitation was overcome domains(Serranoetal.,2009;Johnsonetal.,2010):theubiquitin-
likedomain1(UB1),thepartiallydisorderedacidicdomain(Ac),the
Xdomain,andtheSARS-uniquedomain(SUD).Thisisfollowedby
the second ubiquitin-like domain (UB2), which is usually consid-
nCorrespondingauthor.
E-mailaddresses:[email protected](Y.Kusov), ered part of the next domain, the papain-like protease (PLpro). C-
[email protected](J.Tan),[email protected](E.Alvarez), terminaltothese,there isthe nucleic-acid-bindingdomain(NAB),
[email protected](L.Enjuanes), followed by a longer stretch of amino-acid residues apparently
[email protected](R.Hilgenfeld). lackingsecondarystructure(occasionallycalled“coronavirusgroup
iord1ePIrnevseensttigaadcdiorensess:CCeiennttríoficdaes.BUionloivgeíarsMidoadlecAuulatórnSoemvearodeOcMhoaad.riCdon(CseSjIoC-SUuApMer)-. 2 marker (G2M)”; Neuman et al., 2014) and the first transmem-
NicolásCabrera1,28049Madrid,Spain. braneregion(TM1).Thezinc-finger(ZF)istheonlydomainonthe
http://dx.doi.org/10.1016/j.virol.2015.06.016
0042-6822/&2015ElsevierInc.Allrightsreserved.
314 Y.Kusovetal./Virology484(2015)313–322
introducedinsubdomainspresumablyinvolvedintheinteraction
withG-quadruplexes(Tanetal.,2009).
Results
IntroductionofdeletionsandpointmutationsintotheSARS-CoV
repliconencodingRenillaluciferase
Comparing genome transcription with genome replication, it
was previously shown that the replicon pBAC-REP, which was
lackingareportergene,wasabletoreplicateinmammaliancells
(Almazán et al., 2006). These data encouraged us to introduce a
luciferasereportergeneintotherepliconpBAC-REPtoenablethe
quantitative detection of viral genome replication simply by
measuringtheluciferaseactivity.TheRenillaluciferasewaschosen
asareporterproteinbecauseofitslongerhalf-lifeascomparedto
thatofthefireflyluciferase(seeMaterialsandmethodsandTanaka
et al., 2012). However, the constructed full-length SARS-CoV
Fig.1. Schematicpresentations(nottoscale)ofthegeneticallyengineeredSARS-
CoVrepliconencodingRenillaluciferase(A),ofthedomainorganizationofNsp3 replicon DNA, pBAC-REP-RLuc, was too large (approximately
(B),andofconstructswithdeletedfragments(ΔX,SUD-ΔN,SUD-ΔM,SUD-ΔNM, 33kb) to ensure the correctness of desired deletions and/or
andSUD-ΔC)withindomainsX–SUDofNsp3(C).Nsp–nonstructuralprotein1–16, mutations. Therefore, the engineered mutants lacking different
Mpro – main (or 3C-like, 3CL) protease, prim/pol – non-canonical polymerase
genomefragments(completeSUD,orsubdomainsSUD-N,SUD-M,
activity(Xiaoetal.,2012;teVelthuisetal.,2012),ssRBP–single-strandedRNA-
binding protein, RdRp – RNA-dependent RNA polymerase, Hel – superfamily-1 SUD-NM,and,finally,SUD-C,Fig.1C)werefirstintroducedbysite-
helicase, ExoN/(7)N-GMtase – 30-to-50 exonuclease/7N-guanylmethyltransferase, directed mutagenesis into the shorter plasmid pBAC-SfoI-MluI
NendoU–U-specificendoribonuclease,20O-Mtase–20O-methyltransferase,TRS- (approx.15kb, Supplementary Fig.1B, Almazán et al., 2006). The
M–transcription-regulatorysequenceoftheMprotein,RLuc–Renillaluciferase,N fragmentscontainingthedeletionswerethenexcisedfromthese
– nucleocapsid protein, pA – a synthetic poly(A) tail, Rz – hepatitis deltavirus
ribozyme, BGH – bovine growth hormone termination and polyadenylation pBAC-SfoI-MluI-derived plasmids and transferred back into plas-
sequence,UB1–ubiquitin-likedomain1,Ac–acidicdomain,X–Xdomain,SUD midpBAC-REP-RLuc.Torecoverthereplication/transcriptionactiv-
–SARS-uniquedomain,UB2–ubiquitin-likedomain2precedingthepapain-like ity, the correctly oriented MluI-MluI fragment was inserted into
cysteineprotease(PLpro),NAB–nucleicacid-bindingdomain,G2M–coronavirus thefinalplasmids.SupplementaryFig.1depictsourmainstrategy
group2 marker, TM1 and TM2 – transmembrane regions, ZF – zinc-finger, Y – exemplified by the construction of the SUD-deleted SARS-CoV
uncharacterizeddomain.AsshownschematicallyinpanelC,Δindicatesadeletion
replicon encoding Renilla luciferase as reporter protein (pBAC-
oftheXdomainorofdomainsN,M,C,NM,orthecompleteSUD-codingsequences.
Δ
SUD-REP-RLuc).
To examine whether SUD function within the RTC might be
luminalsideof themembrane;thesecondtransmembraneregion connected toits binding to oligo(G)-stretches capable of forming
(TM2)and,finally,theuncharacterizedYdomainarelocalizednear G-quadruplexes(Tanetal.,2009),aSARS-CoVrepliconcontaining
theC-terminusofNsp3(Fig.1B,seeNeumanetal.(2014)). the mutations K565A, K568A, and E571A within the SUD-M
The X domain was shown to have a macrodomain fold and domain(referredtoasthemut4setofmutations)wasconstructed
proposedtobeanADP-ribose-1″-phosphatephosphatase(ADRP)or followingasimilarapproach.Wehadshownpreviouslythatthese
apoly(ADP-ribose)-bindingmodule(Saikatenduetal.,2005;Egloff mutations abrogate G-quadruplex binding to SUD-M (Tan et al.
etal.,2006).WehaveshownpreviouslythattheSUDcontainstwo 2009). The replicon containing the mutations K476A and K477A
consecutive macrodomains, called SUD-N and SUD-M (Tan et al., (the mut2 set of mutations) located near the C-terminus of the
2009). Although their functional role in the viral replication cycle SUD-N domain was also prepared for comparison, since these
remainsunknown,wehaveshownthatthesemacrodomainsbind mutationshadaweakereffectonSUD-oligo(G)bindingaccording
nucleicacidsthatcontainlongguaninestretchescapableofforming to zone-interference gel electrophoresis (Tan et al., 2009). To
G-quadruplexes(Tanetal.,2007,2009). further test our hypothesis, the mutation sets mut2 and mut4
Inthepresentstudy,weinvestigatedtheX,SUD-N,andSUD-M were also introduced into the replication-competent replicon
Δ
macrodomainstodeterminewhethertheyplayimportantrolesin pBAC- X-REP-RLuc lackingthe Xdomain (seebelowand Fig.4A
viralgenomereplication/transcriptionandwhethertheyactincis andB).
orintrans.Tothisend,wehaveintroducedin-framedeletionsinto
the X and SUD regions of the SARS-CoV replicon, into which a Replication-competenceofthereportergene-containingSARS-CoV
reportergene(Renillaluciferase,RLuc)wasintroducedunderthe repliconpBAC-REP-RLuc
control of the transcription regulatory sequence (TRS) for SARS-
CoVstructuralproteinM(Fig.1A).Inaddition,thefunctionalrole Toensurethatthereportergeneexpressiondirectlycorrelated
oftheSUDC-terminalregion(SUD-C,afrataxin-likedomainwith withviralgenomesynthesis,thelevelofviralRNAsynthesiswas
asyetunknownfunction(Johnsonetal.,2010)),wasassessedina measured in parallel to Renilla luciferase activity. This was
similar way. The reporter gene-containing replicons were tested achievedbyquantitativeRT-PCR(qRT-PCR)usingprimerscovering
fortheirabilitytosupporttheactivityoftheRTCinthesynthesis the non-structural protein 1 (Nsp1) region (see Materials and
of subgenomic replicon RNA and to assess whether the deleted methods). There was a direct correlation of Renilla luciferase
functionscouldberescuedintrans.Thedatapresentedindicatea expressionwiththeamountofviralRNAsynthesisinmammalian
crucialrolefortheSUD-MmacrodomainforviralRTCactivity,thus cells transfected with the SARS-CoV replicon pBAC-REP-RLuc
lending support to the significance of the previously observed (Fig. 2A and B, columns “REP”). Due to the specificity of the
binding of SUD to oligo(G)-containing nucleic acids (Tan et al., forward and reverse primers, the qRT-PCR system gave a non-
2007, 2009). This observation was additionally reinforced by the significant background (Fig. 2B, column “mock”). A replication-
analysis of replicons in which amino-acid mutations have been defective construct, pBAC-REP(NR)-RLuc, with a reverse
Y.Kusovetal./Virology484(2015)313–322 315
Fig.2. ComparisonofthelevelofRenillaluciferaseexpression(A)andviralRNAsynthesis(B)byreplication-competent(REP)andreplication-deficient(NR)SARS-CoV
replicons;time-courseofRLucexpressionbytheSARS-CoVreplicon(C);theeffectofSUDdeletion(twoclones,clon1andclon2)onRenillaluciferaseexpression(D),effectof
deletionoftheXdomain(ΔX),SUDmacrodomains(ΔNandΔM),andoftheSUD-Csubdomain(ΔC)ontheexpressionofRenillaluciferase(E)andonviralRNAsynthesis(F).
SARS-CoVrepliconswithdeleteddomainsweretransfectedinVeroE6cellsandafter24hofincubation,RenillaluciferaseactivityandtheamountofviralRNAwere
measured(seeMaterialsandmethods).RenillaluciferaseactivityandviralRNAsynthesiswerealsomeasuredaftertransfectionofareplication-deficientSARS-CoVreplicon
(NR)andinmock-transfectedcells(Cells).Datashownarefromquadruplicateexperiments,expressedasthemeanvalueofRNAcopies7standarddeviation(SD).The
differenceinexpressionofthefull-lengthSARS-CoVrepliconanditsvariousmutantswasfoundtobesignificant,implyingthatvaluesamongthemaregreaterthanwould
beexpectedbychance(0.014p40.001).Notethatinthereplication-deficientSARS-CoVreplicon(NR),thesequenceoftheMluI-MluIfragmentisreversed,thuspreventing
theformationofthereplicasecomplex.RLU–relativelightunits.
orientationoftheMluI-MluIfragmentusedasanegativecontrol, correlated with viralgenomesynthesis, Renilla luciferase activity
neither expressed Renilla luciferase nor was able to synthesize expressed by constructs lacking individual domains (in this case
viral RNA (Fig. 2, A and B, columns “NR”). These results clearly theXdomain)wasdirectlycomparedwithRNAsynthesis.Ahigh
indicate that Renilla luciferase activity is a good reporter for level of viral RNA synthesis was observed for the X-domain-
analyzingviralgenomereplication. deleted construct (Fig. 2F, column “ΔX”). Thus, not only is the
Tofindouttheoptimumtimeforthereporter-geneexpression ADRP activity of the X domain not required for coronavirus
by the engineered SARS-CoV replicon, the Renilla luciferase replication in tissue culture, as shown for HCoV 229E (Putics
activity was analyzed at different time-points after transfection et al., 2005), but the entire X domain is dispensable in case of
of pBAC-REP-RLuc into Vero E6 cells. As shown in Fig. 2C, the SARS-CoV(ourdata).
reportergeneexpressionshowedalinearincreasebetween18and
53h posttransfection (hpt), with the highest value of Renilla SUDisindispensableforSARS-CoVgenometranscription/replication
luciferase activity detected around 53hpt. Therefore, the expres-
sionbetween24and48hptwasrecordedinourstudy. IncaseoftheSUD,wehaveshownthatitpreferentiallybinds
oligo(G) stretches (G-quadruplexes) (Tan et al., 2007, 2009). To
TheXmacrodomainofSARS-CoVisdispensableforRTCactivity assesstheimportanceofSUDfortheactivityoftheRTC,theentire
SUD-encodingsequencewasdeletedfromtheSARS-CoVreplicon,
Afewregionsnearthe50endofthecoronavirusgenome(nsp1 as described above. The ability of the deleted construct pBAC-
Δ
of murine hepatitis virus, MHV (Denison et al., 2004; Brockway SUD-REP-RLuctoreplicateitsgenomewascomparedtothatof
and Denison, 2005, Tanaka et al., 2012), and nsp2 of MHV and the parental replicon pBAC-REP-RLuc. In contrast to the latter
SARS-CoV (Graham et al., 2005)) have been showntobe dispen- (Fig. 2D, column “REP”), two independently prepared clones of
Δ
sable for virus replication (reviewed in Neuman et al., 2014). In pBAC- SUD-REP-RLuc,transfectedinVeroE6cells,expressedthe
addition, the ADRP of Nsp3b, i.e. the X domain of human Renilla luciferase gene only at a level similar to background
coronavirus 229E (HCoV 229E) can be inactivated by mutation activity, suggesting that the SUD is indispensable for SARS-CoV
without significantly affecting viral replication in cell culture RTCactivity(Fig.2D,cf.columns“ΔSUD“withcolumn“mock”).
(Putics et al., 2005). To find out whether the X domain may
display another, as yet uncharacterized activity involved in viral Lackoftrans-complementationofthereplication-deficientSUD-
replicationortranscription,weremovedthecodingsequencefor lackingSARS-CoVrepliconbythefull-lengthSUDandSUD-NM
thecompletedomainfromtheSARS-CoVrepliconandfoundthat
Δ
the resulting replicon, pBAC- X-REP-RLuc, was replication- To answer the questionwhether the SUD function is required
competent. Approximately 70–75% of the parental replicon exclusively in cis or can be provided in trans, the full-length
(pBAC-REP-RLuc) activity (Fig. 2E, column “REP”) was observed SUD or its more stable SUD-NM fragment was co-transfected
Δ
for the expression of the X domain-deleted replicon (Fig. 2E, togetherwiththeSUD-deletedrepliconpBAC- SUD-REP-RLucin
column “ΔX”). To ensure that the reporter-gene expression Huh-T7 cells susceptible to SARS-CoV (Gillim-Ross et al., 2004;
316 Y.Kusovetal./Virology484(2015)313–322
Hattermannetal.,2005).However,thereportergeneactivitydid expression using vaccinia virus (VV) MVA-T7 as a helper virus.
not exceed background level (Fig. 3, panel A). None of the Previously, we have successfully used MVA-T7 to efficiently
constructsexpressingSUDorSUD-NM(columns2and3,respec- express hepatitis A virus genes (Kusov et al., 2002). Indeed, the
tively) was able to considerably enhance the extremely low transfectionofconstructsexpressingSUDorSUD-NMfollowedby
reporter gene activity of the SUD-lacking replicon co-transfected infection with the helper virus MVA-T7 (a procedure known as
with vector alone (column 1). Intriguingly, the co-expression of transinfection, Kusov et al., 2002) allowed immunological detec-
SUD or SUD-NM with the replication-competent SARS-CoV repli- tion of SUD and SUD-NM using either polyclonal SARS-CoV anti-
con (REP-RLuc, columns 5 and 6, respectively) slightly inhibited Nsp3(Rockland;resultnotshown)ormonoclonalanti-His (Qia-
4
the Renilla luciferase activity expressed by the replicon co- gen) (Fig. 3C). Note that the level of Renilla luciferase expression
transfected with the vector pIVEX used as a control (column 4). bytheSUD-deletedrepliconwasalsoincreased,probablybecause
Assuming that the inability to complement the SUD-deleted of more efficient mRNA transcription from a cryptic promoter or
replicon by providing SUD or SUD-NM in trans was due to low duetoahelpereffectofVVforrepliconRNAsynthesis,asweand
levels of protein production, we increased the amount of others have noticed previously (Sutter et al., 1995; Kusov et al.,
Fig.3. ImpactofSUDandSUD-NMproteins,providedintrans,ontheactivityofthefull-lengthandSUD-deletedSARS-CoVreplicons.IndicatedDNAswereco-transfectedin
Huh-T7cells(A)oradditionallyinfectedwithvacciniavirusMVA-T7asahelpervirus(seeMaterialsandmethods)(B).Theinfectionand/orco-transfectionmixtureswere
replacedwithgrowthmediumandincubatedforanadditional48h.ThecelllysateswereanalyzedforRenillaluciferase(AandB,po0.01)andanti-Hisimmunological
activityinmixtures2and3showninpanelB(C).Allexperimentswererunintriplicateandtheaveragewasemployedforanalysis.Errorbarsrepresentstandarddeviations
ofthemeanvalues.Thedifferenceinreporterproteinactivityofthefull-lengthrepliconandtheSUD-lackingrepliconwasfoundtobestatisticallysignificant(panelA,
p¼0.005;panelB,p¼0.01).
Y.Kusovetal./Virology484(2015)313–322 317
α
2002).However,noneoftheproteins,SUD(Fig.3B,column2)or are located in the loop connecting the second -helix with the
β
SUD-NM (column 3), was able to increase the reporter-gene third -strand of the SUD-M domain, which was found to be
Δ
expression of the SUD-deleted replicon pBAC- SUD-REP-RLuc essentialforreplicationoftheSARS-CoVreplicon(Fig.2EandF).In
(column 1), indicating that the function(s) of SUD cannot be contrast, the mutations K476A and K477A (the mut2 set of
complemented in trans. To double-check these results, we have mutations) located in the dispensable SUD-N domain (Fig. 2E
tried to supplement the parental SARS-CoV replicon pBAC-REP- and F) had only a minor effect on oligo(G)-binding in vitro (Tan
RLuc.InlinewiththedatapresentedinFig.3A,theco-expression etal.,2009).
ofSUDorSUD-NMratherpartiallyinhibitedtheRenillaluciferase Tocomparetheeffectofmutationsonviralgenomereplication/
activity of the replication-competent replicon (Fig. 3, cf. columns transcriptioninvivo,the twosets ofmutations (mut2 and mut4)
4and5inpanelsAandB),implyingthatpossiblyafinebalanceof wereintroducedseparatelyintotwoSARS-CoVrepliconscontain-
Δ
the proteins is crucial for the formation of the active SARS-CoV ing the full-length SUD sequence, pBAC-REP-RLuc and pBAC- X-
replicasecomplex.Takentogether,thefunctionalactivityofSUDin REP-RLuc,whichwereabletoefficientlyreplicatetheirgenomeas
ciscouldnotbesupplementedintrans,neitherbythefull-length judged by Renilla luciferase expression and viral RNA synthesis
Δ
SUDnorbyitsmorestableversionSUD-NMconsistingofthetwo (Fig. 2). A replicon lacking the SUD-C subdomain, pBAC-SUD C-
macrodomains,SUD-NandSUD-M. REP-RLuc, which contains both the SUD-N and SUD-M domains,
was not considered for site-directed mutagenesis because of its
TheSUD-Mmacrodomainiscrucialfortheactivityofthereplication/ lowactivity.
transcriptioncomplex Theimpactofthemut2andthemut4setofmutationsonthe
activityoftheRTCwasexaminedbyreportergeneexpressionand
ToassesstheroleofthesubdomainsofSUDfortheactivityof viralRNAsynthesisasmentionedabove.Comparedtotheoriginal
theRTC,theSUD-NandSUD-MmacrodomainsandtheC-terminal constructs,thenon-mutatedrepliconpBAC-REP-RLuc(Fig.4A)and
Δ
subdomain (SUD-C) were deleted in separate experiments from itsXdomain-deleted derivativepBAC- X-REP-RLuc (Fig.4B),the
thesequenceoftheSARS-CoVreplicon(Fig.1).Wecomparedthe corresponding replicons containing the mut2 set of mutations
activities of the replicons lacking individual SUD subdomains to (K476A and K477A) expressed Renilla luciferase activity three
thatoftheparentalSARS-CoVreplicon,pBAC-REP-RLuc.TheSARS- times more weakly (Fig. 4A, -SUDmut2-), or even at the same
Δ
CoVrepliconlackingtheSUD-Ndomainexpressed30to35%ofthe level (Fig. 4B, - X-SUDmut2-). Probably, this difference is con-
Renillaluciferaseactivityoftheparentalreplicon(Fig.2E,column nectedwiththereducedactivityoftheSARS-CoVrepliconlacking
“ΔN”).Accordingly,itsRNAlevelwaswelldetectableincontrastto theXdomain(seeFig.2EandF).Incontrasttotheeffectofmut2
other SUD subdomain-deleted replicons (Fig. 2F, compare the variants,theRenillaluciferaseactivityofrepliconscontainingthe
column “ΔN” with those for ΔM and ΔC). These data indicate mut4 set of mutations was negligible, thereby emphasizing the
thattheSUD-Nmacrodomainmayalsobeconsidereddispensable crucialroleoftheresiduesalteredinthemut4setofmutationsfor
Δ
for SARS-CoV replication, similar to the X domain (see above), genomereplication(Fig.4AandB,-SUDmut4-and- X-SUDmut4-
althoughtheireffectsonreplicationmaybeconsidered“minor”(X ). These results were in close agreement with the levels of
domain) and “moderate” (SUD-N domain), respectively. In sharp quantified viral RNA (not shown). Taken together, the effect of
contrast, the Renilla luciferase activity expressed by the replicon chargedamino-acidresiduesonthein-vitrobindingofSUD-NMto
with the SUD-M domain deleted (Fig. 2E, column “ΔM”) did not G-quadruplexesstrictlycorrelatedwiththeactivityofthemutated
exceed the level of the activity expressed by the replication- SARS-CoVrepliconsinvivo.
deficientrepliconpBAC-REP(NR)-RLuc(Fig.2E,column“NR”).This
activity was similar to the background level detected in mock-
transfectedcells(Fig.2E,column“Cells”).Thelackofreplicationof Discussion
theSUD-Mdomain-deletedreplicon,deducedfromthenegligible
levelofRenillaluciferaseactivity,wasconfirmedbyquantification The highly infectious and virulent Severe Acute Respiratory
of the viral genome (Fig. 2F, column “ΔM”). The pBAC-SUDΔC- Syndromecoronavirus(SARS-CoV),classifiedasabiosafetylevel-3
REP-RLuc replicon was able to replicate only to a significantly agent (BSL3), can only be handled in specially equipped labora-
lowerextentthantheconstructlackingtheSUD-Ndomain(Fig.2, tories. To overcome this limitation and to avoid the use of
E and F, cf. columns “ΔC” and “ΔN”). Nevertheless, its Renilla dangerous live virus, SARS-CoV replicons have been engineered
luciferaseexpressionandthesynthesisofviralRNAwerealwaysat (Almazánetal.2006,2014;Geetal.,2007;Erikssonetal.,2008;
detectablelevels(Fig.2,EandF,columns“ΔC”),incontrasttothe Pan etal.,2008; Wangetal.,2008).Here,wehaveconstructeda
activityoftheSUD-M-lackingreplicon(column“ΔM”)ortothatof SARS-CoV replicon containing Renilla luciferase as reporter gene,
therepliconwiththeSUD-NMdomainsdeleted(notshown).The thusallowingnotonlytheeasyscreeningofchemicallibrariesfor
RTCactivityofthelatterrepliconswaseitheratbackgroundlevel antivirals interfering with replication of viral RNA and the char-
(columns “Cells”) or at the level of the replication-deficient acterization of antiviral lead compounds, but also studies of the
construct (columns “NR”). In summary, among all tested SARS- function of various viral proteins and regulatory sequence ele-
CoVrepliconswiththeabove-mentioneddeletions,thereplication mentsbyreversegenetics.
ability was mostly affected by the deletion of the SUD-M Our aim was to elucidate the functional role of the three
macrodomain. sequential macrodomains within Nsp3 for the activity of the
SARS-CoV RTC. To this end, we have created various SARS-CoV
Chargedamino-acidresiduesoftheSUD-Mmacrodomain repliconswithdeletedand/ormutatedmacrodomains.First,theX
presumablyinvolvedinG-quadruplexbindingareessentialforSARS- domain-encoding sequence was deleted from the Renilla
CoVRTCfunction luciferase-containingSARS-CoVrepliconandtheresultingreplicon
was tested for its ability to express the reporter gene. The X
Among all previously tested sets of amino-acid replacements domainhasbeenshowntoexhibit aweakADRPactivity inmost
thatwereabletoaffectthebindingofSUDtoG-quadruplexes,the coronavirusesexamined, butwehave previouslyshownthatthis
mut4 setof mutations, comprising alanine substitutions of K565, activityisnotcompletelyconservedacrossthefamily;thus,theX
K568, and E571 of Nsp3, was most efficient in preventing the domainoftheBeaudettestrainofInfectiousBronchitisVirus(IBV)
binding of oligo(G) (Tan et al., 2009). These amino-acid residues isunabletobindADP-riboseduetoreplacementofaGly-Gly-Gly
318 Y.Kusovetal./Virology484(2015)313–322
Fig.4. Effectofamino-acidreplacementsintheSUDmacrodomainsonviralgenometranscription/replication.IndicatedmutationsinSUD-NandSUD-M(designated–
SUDmut2-and-SUDmut4-,respectively)wereintroducedintotheSARS-CoVrepliconpBAC-REP-RLuc(A)anditsderivativelackingtheXdomain,pBAC-ΔX-REP-RLuc(B)
(seediagramsattherightsideoftheconstructs).TheabilityoftheconstructstoexhibitRenillaluciferaseactivityispresentedasmeanvaluesoftriplicateexperiments7SD
(po0.05).AputativemodelofG-quadruplexbindingtoSUD-NM,obtainedbyautomateddockingintothecrystalstructure(C,modifiedfromTanetal.,2009).TheSUD-N
andSUD-Mmacrodomainsareinvioletandcyan,respectively.TheG-quadruplexasfoundintheBCL2promoterregion(PDBcode:2F8U,Daietal.,2006)isinorange.The
mut2setofmutations(K476AþK477A),locatedattheC-terminusoftheSUD-Ndomain,isindicatedbyyellowsticks.Themut4setofmutations(K565AþK568AþE571A),of
SUD-M,isindicatedbygreensticks.Theseresiduesbelongtoaclusterofchargedamino-acidresidueslocatedinthesecondα-helixofSUD-Mandintheloopconnectingit
withthethirdβ-strand(R562,K563,K565,K568,andE571,green,red,blue,magenta,andorangelabel,respectively)(D).
tripletinthebindingsitebyGly-Ser-Gly(Piotrowskietal.,2009). positioned macrodomains, i.e. X, SUD-N, and SUD-M, may have
Furthermore,Puticsetal.(2005)reportedthattheADRPactivityof (an) as yet unidentified function(s) enhancing SARS-CoV genome
HCoV229Eisnotessentialforgenomereplicationofthisvirusin replication.Sincefull-lengthSUDwasfoundtobecrucialforSARS-
cellculture.Inthisstudy,wefindthattherepliconpBAC-ΔX-REP- CoV genome replication (Fig. 2D) and not able tocomplement in
RLuc expressed a high level of Renilla luciferase and was able to trans (Fig. 3), we decided to gain further insight into the role of
synthesizeviralRNAaftertransfectionintomammaliancells(Vero each SUD macrodomain for RTC activity. In addition, the C-
E6 or Huh-T7, a derivative of SARS-CoV-susceptible Huh-7 cells terminal SUD subdomain (SUD-C) was also investigated by
(Gillim-Rossetal.,2004;Hattermannetal.,2005)).Thisindicates removalof theSUD-C-coding sequencefromtheSARS-CoVrepli-
that the X domain does apparently not carry another, as yet con.ThedeletionoftheSUD-Mdomaincompletelyabolishedboth
unidentified, activity that would be essential for SARS-CoV repli- replicon activities, thus indicating that the SUD-M domain is
cation/transcription.InsharpcontrasttoresultswiththedeletedX indispensable for SARS-CoV genome replication. In contrast, the
Δ
domain, the SUD-lacking replicon, pBAC- SUD-REP-RLuc, abro- SUD-N domainwas revealed to be non-essential for RTC activity
gated reporter gene expression and the synthesis of viral RNA since its removal abolished neither Renilla luciferase expression
(Fig.2Dandnotshown,respectively).Toourknowledge,thisisthe norviralRNAsynthesis(Fig.2,EandF,columns“ΔN”).Thesame
first description of the indispensability of SUD for SARS-CoV istruefortheSUD-Csubdomain,althoughtheRTCactivityofthe
genome replication. The most plausible explanation for this SUD-C-lackingrepliconwasalwaysatalowlevel(Fig.2,EandF,
observation is an essential role of the SUD-M macrodomain for columns “ΔC”). Intriguingly, three amino-acid residues (K565,
α
viralRNAsynthesis(seebelow). K568, and E571) located in the loop following the second -
Interestingly,allourattemptstocomplementintranstheSUD helix of SUD-M and responsible for the interaction of SUD with
function by co-expression of the full-length SUD or SUD-NM oligo(G)-nucleotides have previously been identified (Tan et al.,
failed, albeit the expression was evidenced by immunological 2009). We propose that the SUD-oligo(G) interaction is required
detection of the proteins (see Fig. 3). Also, the replication of the forSARS-CoVgenomereplication.TheSARS-CoVgenomecontains
completeSARS-CoVrepliconwasnotenhancedwhenafull-length a number of conserved G stretches (Johnson et al., 2010) that
4
SUD or SUD-NM were provided in trans (Fig. 3). The inability to couldbebinding-partnersforSUD.Oligo(G)sequencesarecapable
supply SUD activity in trans rules out a hypothetical enzymatic of forming G-quadruplexes, even if they comprise a few non-
activity of SUD. Taken together, these results indicate that the guanines;thus,theNMRstructureoftheprototypeG-quadruplex
functionally active SUD is required only in cis or, alternatively, a thatweusedfordockingintotheSUD-NMcavitycontains8non-
fine balance of the proteins is essential for the formation of the guaninesamong23nucleotides(Daietal.,2006).Therefore,oligo
activeSARS-CoV replicasecomplex. These observations maysub- (G) regions such as, e.g., 50-GGGAGGUAGG-30, which is found
stantiatethepreviouslypublisheddataonenhancementofSARS- conservedintheNsp2-andNsp12-codingregionsinthegenomes
CoV reporter activity by the co-expression of Nsp3.1, which ofdifferentSARS-CoVstrains(Johnsonetal.,2010),arecandidates
comprises domains X and SUD (Pan et al., 2008). Now we can forinteractionwithSUD.
assumethatthisenhancementwasmainlyduetothepresenceof WhiletheseobservationssupportanessentialroleoftheSUD-
the X domainwithin Nsp3.1. Alternatively, the three sequentially M domain in replication/transcription of the SARS-CoV genome,
Y.Kusovetal./Virology484(2015)313–322 319
probably via interaction with G-rich stretches forming G-quad- butnegativelycharged(Fig.4D,orangelabel,andSupplementary
ruplexes, we have to consider a potential negative effect of SUD Fig. 3) needs to be further investigated. In summary, we have
domain deletions on the activity of the papain-like protease identified amino-acid residues essential for SARS-CoV genome
(PLpro),locatedimmediatelydownstreamofSUD.Suchamodula- replication,whichrequiresSUD-oligo(G)interaction.Interestingly,
tion of PLpro activity by flanking regions has been reported for thisclusterofchargedamino-acidresidueslocatedinthesecond
SARS-CoV PLpro (Harcourt et al., 2004; Han et al., 2005) and α-helixoftheSUD-Mdomain,orintheloopfollowingit(Fig.4D;
alphacoronavirus PL2pro (Ziebuhr et al., 2001, 2007). Therefore, R562, K565, K568, and E571, green, blue, magenta, and orange
to demonstrate a direct correlation between the G-quadruplex labels,respectively),isconservedamongestablishedhumanSARS-
interactionandthereplicationabilityofSARS-CoVrepliconsandto CoV strains (Urbani, Frankfurt, Tor2, GZ02, and BJ01) and SARS-
exclude a possible modulating effect of domains preceding the CoV-relatedbatandcivetisolates(RsSHC014,RS3367,Rs672/2006,
PLpro,wereasonedtoleavetheSUDintactbutmutatetheamino- HKU3-1, Rf1, Rm1, Rp3, SZ3, SZ16, Bat273, Bat 279, and BM48)
acid residues putatively involved in SUD-binding to G- (SupplementaryFig.3).
quadruplexes. The replacement of charged amino-acid residues Despite strong evidence for SUD-M domain - G-quadruplex
byalanine(K565A,K568A,andE571A,mut4setofmutations)on interactiondemonstratedinvitro(Tanetal.,2007,2009)aswellas
the surface of the SUD-M domain that is oriented towards the invivo(thiswork),wedonotruleoutaroleinreplicationofother
SUD-N domain and remote from the PLpro (Tan et al., 2009), possibleSUDfunctions,suchastheSUD-SUD,SUD-UB1,andSUD-
completely abrogated the Renilla luciferase expression (Fig. 4) X domain-domain interactions described previously (Neuman et
andthesynthesisofviralRNA(notshown),bothinthecontextof al., 2008). Nevertheless, supporting a crucial role for oligo(G)-
the SARS-CoV repliconand its X-lacking version. In contrast, and binding in viral genome replication, a similar, but not identical,
consistent with the previously described marginal effect of the stretch of charged amino-acid residues was also found to be
lysineresiduesatpositions476and477oftheSUD-Ndomainon conservedin thegenomeof the newlyemerging human Middle-
G-quadruplex-binding invitro(Tan et al., 2009), the introduction East Respiratory Syndrome coronavirus (MERS-CoV) and in the
of themut2setofmutations(K476AandK477A)intotheabove- genomesofthecloselyrelatedbatCoVs,HKU4andHKU5,aswell
mentioned replicons had only a minor effect on expression of as in the genome of CoVs isolated from dromedars, which are
Renillaluciferase(Fig.4)andviralRNAsynthesis(notshown).In supposedtobeaprimaryanimalreservoirofMERS-CoV(Memish
agreementwiththis,therepliconwiththeSUD-Ndomaindeleted etal.,2014)(seeSupplementaryFig.3).Intriguingly,asseenforthe
wasabletoreplicate(Fig.3EandF,columns“ΔN”). SUD-Mmacrodomain,wefoundtheputativeMdomainofMERS-
These results were consistent with our hypothesis that SARS- CoVtobindexclusivelyoligo(G)(andnotoligo(A),oligo(C),oroligo
CoVgenomereplicationrequirestheinteractionofSUDwitholigo (U)) nucleotides (Lei et al., personal communication). It is highly
(G)-containing nucleic acids (Tan et al., 2007, 2009). Amino-acid probable that this property is attributable to the stretch of
replacements(mut4),whichinin-vitroexperimentsabrogatedthe conserved charged residues on the surface of the M domain of
interaction of mutated SUD-NM with oligo(G) (Tan et al., 2009), MERS-CoV (see Supplementary Fig. 3). Further studies will be
resultedinareplication-defectiveconstruct.Incontrast,themut2 required to elucidate the impact of the deletions and mutations
setofmutationswithnegligibleeffectonoligo(G)-bindingtoSUD- described here at the level of the full-length SARS-CoV genome
NMinvitro(Tanetal.,2009)resultedinaviablereplicon. and,inparticular,toanswerthequestionwhetheranysecond-site
Taken together, these data suggest SUD amino-acid residues mutation(s)canrescuethemutatedvirus.
thatarestrictlyrequiredforSARS-CoVgenomereplication.These
residues(lysineresidues565,568,andglutamate571)arelocated
α β
intheloopconnectingthesecond -helixwiththethird -strand Conclusion
oftheSUD-Mmacrodomain;interestingly,thetwolysineresidues
belongtoaregionshowntobeinvolvedinG -bindingbyNMR- In this contribution, we demonstrate, for the first time, the
10
shiftperturbationanalysis(Johnsonetal.,2010)andareinvolved functional role of the SUD subdomains within the replication–
in interactions with a G-quadruplex according to our docking transcription activity of the Severe Acute Respiratory Syndrome
model(Fig.4CandD).Consideringtheirelectrostaticpotential,the Coronavirus (SARS-CoV). Contrary to the dispensable SUD-C and
replacementoftheseamino-acidresiduesbyalaninewillaffectthe SUD-N subdomains aswellas totheX domainpreceding SUD in
chargedistributionwithinthebindingsiteforG-quadruplexesor thegenome,theSUD-Mmacrodomainwasfoundtobecrucialfor
othernucleicacids(SupplementaryFig.2andSupplementarytext the activity. The indispensability of this subdomain might be
B).Suchamodificationofthebindingsite'selectrostaticpotential connected with its ability to bind oligo(G) stretches/G-quadru-
results in abrogation of the SUD – nucleic-acid interaction (Tan plexesasconcludedfromtheresultsofsite-directedmutagenesis
etal.,2009)thatisrequiredforSARS-CoVgenomereplicationina ofchargedamino-acidresiduesintheloopconnectingthesecond
α β
cell-based assay (Supplementary Fig. 2). On the other hand, the -helix of SUD-M with the third -strand. Intriguingly, a similar,
replacementoflysinebyalanineatpositions476and477didnot butnotidentical,clusterofresiduesisobservedinthegenomesof
significantlyaffecteithertheelectrostaticpotentialofthebinding the newly emerging human Middle-East Respiratory Syndrome
site (Supplementary Fig. 2) or the direct interaction with the coronavirus (MERS-CoV), MERS-related dromedary camel CoV,
G-quadruplex(Fig.4C).Althoughwedidnotconstructthereplicon andbatCoVsHKU4andHKU5.
containing arginine to alanine replacement at position 562, we
speculate that this highly conserved arginine residue is also
involved in G-quadruplex binding, since it is located in close Materialsandmethods
proximity to the binding cavity (Supplementary Fig. 3 and
Fig. 4D; R562, green label) and has also been identified as part Cellsandviruses
of the nucleic-acid binding site in the NMR-shift perturbation
experiments reported by Johnson et al. (2010). In contrast, the Africangreenmonkeykidneycells(VeroE6)andHuh-T7cells,
lysineresidueatposition563seemstobeorientedawayfromthe a derivative of human hepatocellular carcinoma Huh-7 cells
binding pocket (Fig. 4D; K563, red label), thereby preventing its (Nakabayashi et al., 1984) that constitutively expresses the T7
participation in oligo(G) interaction. The participation of the RNA polymerase (Shultz et al., 1996), were grown in Dulbecco's
strictlyconservedE571,whichislocatedclosetothebindingsite modifiedminimalessentialmedium(DMEM)supplementedwith
320 Y.Kusovetal./Virology484(2015)313–322
2mM glutamine, 100U/ml penicillin, 100mg/ml streptomycin plasmid (ΔSUD), which encoded the Renilla luciferase reporter
sulfate, and fetal calf serum (10% v/v). Huh-T7 cells were addi- gene,wasstillreplication-deficientbecauseoftheabsenceofthe
tionally supplemented with geneticin (G-418 sulfate, 400mg/ml). MluI-MluI fragment. To restore all replicase components, this
Therecombinant,non-cytopathicvacciniavirus(VV)MVA-T7was fragmentwasre-introducedattheMluIrecognitionsiteaccording
usedtoproduceSUDoritssubdomainsNþM(SUD-NM)inorder totheprocedureforcloninglongDNAfragments(seeabove).The
to complement in trans the activity of the SARS-CoV replicon correctorientationoftheMluI-MluIfragmentwasprovenbyStuI
lacking SUD. MVA-T7 was propagated in BHK-21 baby hamster digestion prior to sequencing. The plasmid with reverse orienta-
kidney cells and titrated as described previously (Kusov et al., tion of the MluI-MluI fragment was used as negative, non-
2002). Other cell and culture conditions have been described in replicating(NR)control.
Almazánetal.(2006)andKusovetal.(2006). A similar cloning strategy was employed to introduce point
Δ
mutations into the replicon pBAC- X-REP-RLuc lacking the X
ConstructionoftheSARS-CoVrepliconcontainingreportergene domainandintothefull-lengthrepliconpBAC-REP-RLuc.Twosets
ofmutations–K476AandK477A(mut2)intheSUD-Ndomainand
TogenerateaSARS-CoVrepliconcontainingareportergene,we K565A, K568A, and E571A (mut4) in the SUD-M domain – were
havetakenadvantageofthestrategypreviouslydescribedforthe introduced into both replicons. Phosphorylated asymmetric for-
construction of a SARS-CoV replicon lacking a reporter gene ward and reverse primers overlapping only within a short
(Almazán et al., 2006). A Renilla luciferase as a reporter gene sequence (Supplementary Table 1) were employed for site-
(RLuc, Renilla reniformis, also known as sea pansy) was PCR- directed mutagenesis as described above. Mut2- and mut4-
amplified using pRL-SV40 DNA as a template (Promega; acc. containing clones were identified by BtsI and BstAPI digestion,
AF025645), forward and reverse primers (Supplementary respectively.TheORFofallconstructedSARS-CoVrepliconsbear-
Table 1), and proof-reading DNA polymerase (AkkuPrime Pfx ingdeletionsormutationswithinNsp3,schematicallydepictedin
Δ Δ Δ Δ Δ Δ
SuperMix, Invitrogen). The transcription regulatory sequence for Fig.1C ( SUD, X, SUD- N, SUD- M, SUD- NM, and SUD- C
Δ
theSARS-CoVMprotein(TRSM)andaKozaksequenceenhancing replicons)andFig.4(-SUDmut2-and-SUDmut4-,- X-SUDmut2-
expression in eukaryotic cells were included in the forward RLuc and-ΔX-SUDmut4-),wasverifiedbycompletesequencingofSfoI-
primer(Almazánetal.,2004;Kozak,1989).ThePCRampliconwas MluIfragments(LGCGenomics).Detailsofthecloningprocedures,
treated with AscI and BamHI and introduced between the same restriction analysis of constructed plasmids, their maps and
restrictionsitesoftheSARS-CoVrepliconpBAC-REP-URB(Almazán sequencescanbeprovideduponrequest.
etal.,2006)producingthereportergene(RLuc)-containingSARS-
CoV replicon, referred to as pBAC-REP-RLuc (Supplementary TransfectionofSARS-CoVrepliconsinVeroE6orHuh-T7cells
Fig. 1). To exactly conform to the Kozak sequence, the second
amino-acidresidueofRluc(tyrosine)wasreplacedbyalanine.This Grown in twelve-well plates to 95% confluence, Vero E6 or
replacement was successfully employed in the RLuc expression Huh-T7cells(1(cid:2)105cells/well)weretransfectedwithdeletedor
vectorpBS-35S-Rluc-Ala(acc.numberAY189983). mutatedSARS-CoV repliconsbyusingLipofectamin 2000accord-
ingtothemanufacturer'sspecifications(Invitrogen).Atindicated
IntroductionofdeletionsandpointmutationsintotheRenillagene- time-points (see figure legends), the cells were lysed and the
containingSARS-CoVreplicon Renillaluciferaseactivityand/orviralRNAgenomewasmeasured
in cell lysates (see below). All experiments were performed in
Alldesireddeletionsandpointmutationswereintroducedinto triplicate or quadruplicate and the mean values and standard
the pBAC-REP-RLuc plasmid encoding polyproteins pp1a and deviations(SD)arepresented.PlasmidpRL-SV40DNAs(Promega,
pp1ab as replicase and RLuc as reporter protein (see above). acc.AF025645)andthewild-typeSARS-CoVrepliconwereapplied
However,tosimplifythecloningprocedure,weusedastemplate ascontrolreportersinalltransfectionexperiments.
forsite-directedmutagenesistheshorterplasmidpBAC-SfoI-MluI
encodingonlytheN-terminalhalfofpolyprotein1a(Nsp1–Nsp3) AssayofRenillaluciferaseactivity
of SARS-CoV (Almazán et al., 2006). In brief, a Phusion Hot Start
DNA polymerase (Phusion Site-Directed Mutagenesis Kit, Finn- To lyse the cells,150ml/well of Passive lysis buffer (Promega)
zymes),which ensureshighfidelityfor the amplification oflarge wasaddedtothewashedcellmonolayerin12-wellplates(1ml/
plasmids, was employed to extend perfectly matched 50-phos- well,phosphate-bufferedsaline,PBS)andincubatedfor20minat
phorylatedforwardandreverseprimers(SupplementaryTable1) room temperature (RT) with rigorous shaking. The cells were
that border the deleted area as schematically exemplified in further lysed by a freeze ((cid:3)801C) / thaw procedure, vortexed,
SupplementaryFig.1BfordeletionofthecompleteSUDsequence. and centrifuged (18,400(cid:2)g, 1min). To a 20-ml aliquot of clear
The amplification mixture was treated with DpnI to destroy the supernatant, a mixture of Renilla luciferase assay and enhancer
original template DNA and the amplicon was circularized with solutions (50ml each, Biotium) was added and the luminescence
Quick T4 DNA ligase (Finnzymes). An aliquot of the ligation wasimmediatelymeasuredusinganAnthosLucy-3luminescence
mixture was transformed by electroporation (2.5kV, 200Ω, plate reader (Anthos Labtec Instruments). Data presented in
25mF) into electrocompetent E. coli cells (DH10B, NEB 10-beta, Figs. 2, 3, and 4 are from quadruplicate experiments and are
New England Biolabs) or HST02 (Takara) that are suitable for expressed as the mean value7standard deviation (SD). The
transformation of long-size plasmids. Positive clones were differences in Renilla luciferase expression of the full-length
initially identified by restriction analysis and then confirmed by SARS-CoV replicon and its various mutants were analyzed with
sequencing. An agarose gel-purified SfoI-ΔSUD-MluI fragment Sysstat (SigmaPlot Software Inc.) and found to be statistically
was transferred into dephosphorylated pBAC-REP-RLuc significant implying that values are greater than would be
(Supplementary Fig. 1A) that was restricted with SfoI and MluI. expectedbychance.
For this procedure, the DNA ligase 〈long〉 optimized for cloning
largeDNAfragmentswasusedasrecommendedbythemanufac- ViralRNAquantification
turer(Takara).Theefficiencyoftransformationwastremendously
increased after removing components of the ligation buffer by To isolate the viral RNA from Vero E6 cells transfected with
sodium acetate/ethanol precipitation. The resulting SUD-lacking SARS-CoV replicons containing deletions or mutations, the cells
Y.Kusovetal./Virology484(2015)313–322 321
were trypsinized as usual and spun down (1000(cid:2)g, RT, 5min). virus MVA-T7 (multiplicity of infection, moi, around 5 as pre-
ThecellpelletwaswashedwithPBSandcellsweresuspendedin viouslydescribed(Sutteretal.1995;Kusovetal.,2002)).Afterone
100mlPBSbeforedividingintotwoaliquots.A20-mlaliquotofcell hour, the infection mixture was replaced with growth medium
suspension was used for the evaluation of Renilla luciferase and incubation was continued for 48h. To measure Renilla
activityafter lysis of pelleted cells in 50ml Passive lysis buffer as luciferase activity, the cells were lysed using Passive Lysis Buffer
described(seeabove).ThetotalRNAwasisolatedfrom80mlofcell (Promega) as described above. Complementation experiments
suspensionusingtheRNeasyextractionkitandDNaseItreatment were run in triplicate and the mean values were employed for
asrecommendedbythesupplier(Qiagen).TraceamountsofDNA analysis. To detect viral proteins, aliquots of cell lysates were
wereremovedfromRNApreparations(20ml)byadditionaltreat- boiled with 1% sodium dodecyl sulfate (SDS) and proteins were
ment (371C, 30min) with 1 unitofRNase-free DNaseIin DNase separated by 12% denaturing polyacrylamide gel (SDS-PAGE)
buffercontainingMgCl .TheDNasewasinactivatedbyadding1ml before transfer to PVDF membranes (Immobilon-P, Millipore).
2
of25mMEDTAsolutionandheatingat651Cfor10min.Theyield His-taggedproteins (full-length SUD or SUD-NM) wereimmuno-
of total RNA was quantified by using a NanoDrop 1000 UV/Vis logically detected using either polyclonal SARS-CoV anti-Nsp3
spectrophotometer(ThermoFisher).Anequalamount(500ng)of (dilution 1:1000; Rockland, not shown) or monoclonal anti-His
4
RNAsamplesextractedfromcellstransfectedwithdeletedSARS- (1:5000,Qiagen)asprimaryantibody.Alkalinephosphatase(AP)-
CoV replicons was directly used as template for the first-strand conjugatedanti-rabbitoranti-mouseIgGwasusedasasecondary
cDNAsynthesis.Thereactionmixture(25ml)additionallycontain- antibody(1:10000,Sigma).
Δ
ingareverse Xprimer(200nM,seeSupplementaryTable1)and
allfourstandarddNTPs(800mMeach)waspre-incubatedat651C
DockingofaG-quadruplextoSUD-NM
for5min,chilledonicetodestroyanysecondarystructureofviral
RNAand,afteradditionofRNaseinhibitor(1ml,Ribolock,Thermo
Atomic coordinates for a typical G-quadruplex were obtained
Scientific)andreversetranscriptase(40units,ThermoScientific),
fromtheProteinDataBank(PDB;PDBcode2F8U,Daietal.,2006).
further incubated at 451C for 60min. The reverse transcriptase
The G-quadruplex was docked into the SUD-NM structure deter-
wasinactivatedbyincubationat701Cfor5min.Asimilarmixture
minedpreviously(Tanetal.,2009)usingtheprogramAUTODOCK
withoutreversetranscriptasewasusedasacontrol.A5-mlaliquot
(Morrisetal.,2009).
of the mixture was used for Real-Time PCR after addition of
forward and reverse primers (0.3mM each, see Supplementary
Table 1; note that these primers represent a sequence of Nsp1
Acknowledgments
allowingtodeterminegenomereplication,butnottranscription),
a fluorescence-quenching primer (6FAM-ACCATCAAGTATGGTGA-
CAGCTGCTCT-BBQ, 0.2mM; TIB MolBiol), and a Maxima Probe We thank Doris Mutschall for excellent technical assistance,
qPCR Master Mix (Fermentas, MBI) in a total volume of 20ml. A Liliya Grin and Luisa Stroeh for help with the preparation and
analysisofseveralconstructs,andDr.JianLeifordiscussion.This
calibrationcurvewaspreparedbylog dilutionofpBAC-REP-RLuc
10
workwassupportedbytheGermanCenterforInfectionResearch
in nuclease-free water. The measurements were performed in
(DZIF), the European Commission (through its projects SILVER
triplicate or quadruplicate; mean values of genome copies and
(Contract no. 260644) and EMPERIE (Contract no. 223498)), the
SD are presented. Data shown are from quadruplicate experi-
ments, expressed as the mean value of RNA copies7standard MinistryofScienceandInnovationofSpain(BIO2010-16705),the
U.S.NationalInstitutesofHealth(Grantnos.2P01AI060699-06A1
deviation (SD). The statistical analysis of RNA copies for the full-
andCRIP-HHSN266200700010C),andtheSino-GermanCenterfor
length SARS-CoV replicon and mutants was performed as
the Promotion of Research, Beijing, China (GZ 236 (202/9)). The
describedabove.
fundershadnorolein studydesign, datacollection andanalysis,
decisiontopublish,orpreparationofthemanuscript.
Generationofplasmidstoexpressfull-lengthSUDandSUD-NMin
mammaliancells
AppendixA. Supportinginformation
To express in Huh-T7 cells the full-length SUD and its more
stable derivative SUD-NM comprising the two macrodomains
(SUD-N and SUD-M) (Tan et al., 2007), their coding sequence Supplementarydataassociatedwiththisarticlecanbefoundin
theonlineversionathttp://dx.doi.org/10.1016/j.virol.2015.06.016.
wasplacedundercontroloftheT7promoterinthecontextofthe
pIVEXWGvector(Roche).AcompleteSUDsequencewasobtained
frompET-Blue2-SUD(Tanetal.,2007)bydigestionwithNcoIand References
XhoIrestrictionenzymes.Then,thepurifiedinsertwasclonedinto
NcoI-XhoI-treated and dephosphorylated vectors resulting in Almazán,F.,Galan,C.,Enjuanes,L.,2004.Thenucleoproteinisrequiredforefficient
plasmids named pIVEX-SUD. A SUD-NM sequence was PCR- coronavirus genome replication. J. Virol. 78, 12683–12688. http://dx.doi.org/
amplified using the proof-reading Pfu DNA polymerase, the 10.1128/JVI.78.22.12683-12688.2004.
Almazán,F.,DeDiego,M.L.,Galan,C.,Escors,D.,Alvarez,E.,Ortego,J.,Sola,I.,Zuniga,
template pQE30-Xa-SUD-NM, and the forward and reverse pri-
S., Alonso, S., Moreno, J.L., Nogales, A., Capisco, C., Enjuanes, L., 2006.
mers(SupplementaryTable1).Thepurifiedampliconwastreated ConstructionofaSevereAcuteRespiratorySyndromeCoronavirusinfectious
withNcoIandSmaIandinsertedintothedephosphorylatedvector cDNAandareplicontostudycoronavirusRNAsynthesis.J.Virol.80,10900–-
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ComplementationintransofSUDandSUD-NMandtheir virusres.2014.05.026.
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immunologicaldetection mainproteinase(3CLpro)structure:basisfordesignofanti-SARSdrugs.Science
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SUD-lacking or parental SARS-CoV replicon and 1mg pIVEX-SUD nRsNpA1-scyondtihnegsirse,gainodnvidireanltriefipelsicraetsioidnu.eVsiriomlopgoyrt3an40t,fo2r09p–ro2t2e3in.hpttrpoc:/e/sdsxin.dgo,i.voirrga/l
or pIVEX-SUD-NM plasmids were infected with helper vaccinia 10.1016/j.virol.2005.06.035.
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