2015 Coronavirus nonstructural protein 1_ Common and distinct functions in the regulation of host and viral gene express

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Virus Research

journal homepage: www.elsevier.com/locate/virusres

Coronavirus nonstructural protein 1: Common and distinct functions

in the regulation of host and viral gene expression

KrishnaNarayanana,∗,1,SydneyI.Ramirezb,1,2,KumariG.Lokugamagea,2,
ShinjiM akinoa,∗∗

aDepartmentofMicrobiologyandImmunology,TheUniversityofTexasMedicalBranchatGalveston,Galveston,TX77555-1019,UnitedStates
bDepartment of Pathology,Th eUn iversityofTex asM edicalBra nc hatG alveston, Galvest on ,TX77555 -1019,Unit ed States

a r t i c l e i n f o a b s t r a c t

Articlehistory: Therecentemergenceoftwohighlypathogenichumancoronaviruses(CoVs),severeacuterespiratory
Availab leonlinexxx synd rome CoVandMi dd leEa strespi ratorysynd romeCo V,hasignited astron gintere stint heidentifi-

cationofviralfactorsthatdeterminethevirulenceandpathogenesisofCoVs.Thenonstructuralprotein

Keywords: 1(nsp1)ofCoVshasattractedconsiderableattentioninthisregardasapotentialvirulencefactoranda
RNAviruses ta rgetfo rC oVva ccin edevelop mentbecause ofaccum ula ting eviden ce th atpointt oitsrolei nthed own -
Coro navirus regula tion of hostinn ateimmune response s toCoVinfecti on.Studie sha vere vea led bo th fun ctional
Nsp1
conservationandmechanisticdivergenceamongthensp1ofdifferentmammalianCoVsinperturbing
Translationinhibition
hostgeneexpressionandantiviralresponses.Thisreviewsummarizesthecurrentknowledgeaboutthe
mRNAcleavage
SARS biological functions of CoV nsp1 that provides an insight into the novel strategies utilized by this viral
proteintomodulatehostandviralgeneexpressionduringCoVinfection.

©2014ElsevierB.V.Allrightsreserved.

1. Introduction Netland,2009;Rotaetal.,2003;vanBoheemenetal.,2012;Zaki

etal.,2012).

Coronaviruses (CoVs) are found in a large variety of animal CoVsbelongtotheorderNidoviralesinthefamilyCoronaviri-

species, including humans, and primarily cause respiratory and dae,andarecurrentlyclassifiedintofourgenera,Alphacoronavirus,
entericd iseases(W eissand Nava s-Martin,2 005).I nhumans,c oro- Beta coro nav irus, Gam macoron avir us a nd Del tacoronavirus ((cid:2)-
navirus esusuall ycause ami ldrespiratoryd isease, lik ethecom mon CoV,(cid:3)-CoV,(cid:4)-Co Vand(cid:5)-CoV)inthe subfa milyCoronavirinae (de

cold(Falseyetal.,1997;vanderHoeketal.,2006).However,the Grootetal.,2011;Gorbalenyaetal.,2004;Snijderetal.,2003;Woo
iden tificatio n o f s evere acut e re spirat ory sy ndrom e CoV (SA RS- etal.,2 01 0,2 012). The(cid:2)-CoVs an d(cid:3) -CoVsa repred om in antlyf ound

CoV)astheetiologicalagentoftheSARSepidemicin2003andthe inmammalsandincludeseveralpathogenichumanCoVssuchas

recentdiscoveryofMiddleEastrespiratorysyndromeCoV(MERS- HCoV-229E,HCoV-HKU1,HCoV-OC43,HCoV-NL63,SARS-CoVand

CoV)asthecausativeagentofMERS,aviralrespiratorydiseasefirst MERS-CoV(Drexleretal.,2010;Drostenetal.,2003;Isaacsetal.,

reportedinSaudiArabiain2012,havehighlightedthepotentialfor 1983;Ksiazeketal.,2003;Larsonetal.,1980;Vabretetal.,2003,
zoonotic tra nsmis sionof h ighlyp atho genicCoVst oh umansca us- 2008; Werthe im et al., 20 13; Zak i e t al ., 2012 ). The (cid:4)- CoV s and
ingsever ediseasesint he huma npopulation (Dro ste netal., 2003; (cid:5)-CoV sareprima rily de tected inbir ds .Bat sappea rto bethen atu-

Ksiazek et al., 2003; Perlman and Dandekar, 2005; Perlman and ralreservoirinvolvedintheevolutionanddisseminationofmany

mammalianCoVs(Carringtonetal.,2008;Chanetal.,2013;Chu

etal.,2008;Gloza-Rauschetal.,2008;Poonetal.,2005;Reusken

etal.,2010;Tangetal.,2006).

CoVs possess a large, single-stranded, positive-sense RNA

∗ genomethatrangeinlengthfrom27to32kb,thelargestamong
Correspondingauthor.Tel.:[phone];fax:[phone].
∗∗ Corresponding author. Tel.: [phone]; fax: [phone]. anyofth eRN Aviru ses (Leee tal.,1 99 1;L om nic zi,1 977;Lo mniczi
E-mailaddresse s:krnar aya@ utm b.e du( K.Nara yan an) ,syi rami [email protected] and Ke nne dy,1 977).Th e5(cid:3)- m ost geneo ftheCoVg enom e,gene1,
(S.I. Ramire z), kgloku [email protected] (K.G. L oku gamage), shm [email protected] occu piesabou ttwo-t hird softheg enom e and con sistsoftw olarg e
(S.Makino).
12 TThele.:se+ 1au4t0h9or7s7 c2o8n1tr7i2b.uted equally to this study. aovreibrloaspopminagl ofrpaemne rsehaidftiinngg frsaigmneasl a(OtRthFse),j uOnRcFt i1oan aonfdt hOeRFtw 1ob, OwRitFhs

http://dx.doi.org/10.1016/j.virusres.2014.11.019
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Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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Fig.1. GenomeorganizationandproteolyticprocessingofORF1apolyproteinofselectedmembersinthe(cid:2)-CoVand(cid:3)-CoVgeneraofCoronaviridaefamily.Theopenreading

frames(ORFs)1aand1bconstitutegene1.Onememberofeachgenusisshownasarepresentativeexample.ThesitesinORF1apolyproteinprocessedbytheviralproteinases,
PLproan d3CLp ro, are ind icated.In theu pp erp anel,the do tted linesi nd icatet he p utativeproces singatth ens p1–n sp 2andn sp3–nsp4c leavagesi tes by TGEV PL1pro.The
predicted activity of TGEVPL1pr oa tth esecle avages ites remain stob edeterm ined .RFS:rib osomalfra me shif t;UTR:untr ansl atedregion. Thefigur eisn otd rawn toscale .

(Fig.1)(Bredenbeeketal.,1990;BrianandBaric,2005;Gorbalenya, (cid:2)-CoVssharenosignificantsequencesimilaritywith(cid:3)-CoVnsp1

2001;Leeetal.,1991;Ziebuhr,2005).Uponentryintohostcells, andtheirsizesarealsodifferent(ConnorandRoper,2007;Jansson,

theincomingviralgenomeistranslatedtoproducetwolargepre- 2013).Basedonthecomparativesequenceanalysisofthegenomes

cursorpolyproteins1a(pp1a)and1ab(pp1ab)thatareprocessed ofdifferentCoVs,nsp1couldbeconsideredasoneofthegenus-
byORF 1a-encodedv ira lprotei nase s,pa pain-like pro tein ase(PLpro) sp ecificmar kers(S nijde retal. ,20 03).Further mo re,b io info rmatics
and 3C -likeprotein ase( 3CLpro),into 16maturen onstructur alpro- analysis of the primary am in o acid sequence of nsp1 does not

teins (nsp1–nsp16, numbered according to their order from the revealanyknowncellularorviralhomologs,otherthaninCoVs,

N-terminustotheC-terminusoftheORF1polyproteins)(Ziebuhr, andalsorulesoutthepresenceofanyobviousfunctionalprotein

2005).ManyofthenspsperformessentialfunctionsinviralRNA motifsinnsp1(ConnorandRoper,2007).Theseintriguingfeatures

replicationandtranscription(Bhardwajetal.,2004;Chengetal., of the primary amino acid sequence of nsp1 combined with the

2005;Fanetal.,2004;Imbertetal.,2006;Ivanovetal.,2004a,b; factthatalltheknownmammalianCoVs,includingthepathogenic

Minskaiaetal.,2006;Saikatenduetal.,2005;Snijderetal.,2003). humanCoVs,encodensp1hasputaspotlightonthisprotein,espe-

Besides the RNA-dependent RNA polymerase, helicase and pro- ciallysincetheSARSepidemicin2003.Indeed,nsp1hasgarnered

teases,someofthenspsareRNA-processingenzymessuchaspoly considerableattention,asevidencedbythegrowingbodyofliter-
(U)-spe cific e nd ori bonu clea se, 3(cid:3)-5(cid:3) exoribo nuclease, ribo se 2(cid:3)-O ature aimed at delinea tin g its struct ure , bi ological f uncti on s and
methyltrans ferase,adenosined iphosphate-ribose-1(cid:3)(cid:3)-p hospha tase impor tancei nC oVreplicati on andpatho genesis.Nu merouss tud-

andcyclicnucleotidephosphodiesterase(Leeetal.,1991;Snijder ieshaverevealedinterestingbiologicalpropertiesofnsp1andalso

etal.,2003;Thieletal.,2003;Ziebuhr,2005).Theenzymaticactivi- highlightednovelmechanismsofregulationofhostandviralgene

tiesandthefunctionaldomainsofmanyoftheseessentialnspsare expressionbynsp1(Table1).Anemergingthemefromthesestud-
pred icte dto beconser vedbetw ee nthe di fferen tgenera ofCo Vs, iesisthatth en sp1o f(cid:2)-CoV sa nd (cid:3)-CoVsex hibitre mark ablysi milar

indicatingtheirimportanceinviralreplication(Snijderetal.,2003; biologicalfunctions,despitethelackofoverallsequencesimilar-

Thieletal.,2003).Inadditiontothesenspswithdefinedfunctions, ityandknownproteinmotifs,suggestingitsimportanceinthelife

thereareseveralnspswhosebiologicalfunctionsandrolesinCoV cycleofthesedifferentlineagesofCoVs.

lifecyclestillremaintobecharacterized. Inthisreview,wewillsummarizethecurrentknowledgeabout

While nsp3–nsp16 from different CoV genera share several thepropertiesofCoVnsp1anddescribestudiesthathighlightthe
conserved functional d omain s, the N- term inal regi on of the ORF com monaswe lla sdis tinctb iolo gicalfunc tionsof (cid:2)-Co Vand(cid:3)-C oV

1 polyprotein, especially the nsp1 sequence, is highly divergent nsp1intheregulationofhostandviralgeneexpression.

amongCoVs(ConnorandRoper,2007;Snijderetal.,2003;Thiel

etal.,2003).Nsp1isthemostN-terminalcleavageproductreleased
fro m theOR F1ap ol ypro tein bytheactio nofPLpro (Fig.1)( Ziebuhr, 2. CoV nsp1: general features and biological functions
2n0sp015 )(.F Aigm .1o)n,g w thh eer feoausr (cid:4)C-oCVo gVe snae nrda,(cid:5) o -nCloyV (cid:2)s- Claoc Vksn asnpd1 (cid:3)a-nC dotV hsu esn,tchoedier 2.1. ˛-CoV nsp1

gene 1 encodes only 15 nsps (nsp2–nsp16) (Snijder et al., 2003; Thensp1of(cid:2)-CoVsisabout110aminoacidsinlength(Almeida
Wooetal.,2010;Ziebuhr,2005;Ziebuhretal.,2007).Thensp1of
et al., 2007; Jansson, 2013). In HCoV-229E, the processing of

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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Table1

Summaryofthebiologicalfunctionsofcoronavirusnsp1.

the ORF1a polyprotein by PL1pro releases nsp1 as an amino- thesequenceanalysisofnsp1fromdifferent(cid:2)-CoVsrevealedany

terminal9-kDaproteinalongwithnsp2,an87-kDaprotein(Herold conservedproteinmotifsordomainsthatcouldprovidesignificant
et al., 19 98; Zi ebuhr e t al., 2001 ). Bas ed on sequ ence co mpari- cluesabou tthefun ctions of (cid:2)-CoVns p1.A nalysi softhes ubcellular

son, this pattern of processing, resulting in the release of nsp1 localizationoftransientlyexpressedTGEVnsp1inhumanembry-

(alsoknownasp9),wasalsopredictedforanothercloselyrelated onic kidney (HEK) 293 cells using confocal microscopy showed
(cid:2)-Co V, trans m issib le ga stro enteritis v irus (TGEV) (Galan et al., the d istribut ion of TGEV nsp 1 in b oth the n ucleus and t he cyto-

2005). Comparative amino acid sequence analysis of the nsp1 plasm(Fig.2).TheprimarysequenceanalysisofTGEVnsp1didnot
of diffe rent (cid:2)-CoVs reveale d tha t HCoV-22 9E nsp1 sha res mod- reveal any can onic alnucle arlocaliza tionsign al .TGEV nsp 1co uld

erate sequence identities of 60% and 52% with HCoV-NL63 and diffusefreelyintothenucleusbecauseofitssmallmolecularweight
porci neepidem icdiarrhea v irusn sp1, resp ective ly(Huange tal., (∼9kDa ),whi chis bel owthes izeexclu sio nl imito fthenucle arpore

2011a). However, TGEV nsp1 has only 32% amino acid sequence complex(Gorlich,1998;Silver,1991).

similarity with HCoV-229E nsp1 but shares high sequence iden- Alimitednumberofstudieshaveexploredthebiologicalfunc-
tities of 97% and 93% w ith th e n sp1 of porc ine respir atory tions of (cid:2)-C oV nsp1 , p rimarily usin g transie nt g ene expre ssion

coronavirus and feline infectious peritonitis virus, respectively and cell-free in vitro translation systems. In mammalian cells,

(Huangetal.,2011a).Ahigh-resolutioncrystalstructureofTGEV both HCoV-229E and HCoV-NL63 nsp1 inhibit the expression of

nsp1combinedwithalignmentofnsp1sequencesfromdifferent reporter genes, under the control of constitutive promoters, like
(cid:2)-Co Vsprovide dadd itionalinfo rm ation aboutthes patia llocation SV40,HS V-TKa ndCM V,a swellas in ducibleprom otersofin nate
of the e volutiona rily conser ved regions of (cid:2)- CoV nsp1 ( Jansson, immu nerespo nseg enes, like inte rfe ron(IFN)-(cid:3) andIFN-st im ulated

2013). The TGEV nsp1 structure is defined by an irregular six- gene(ISG)15,carryingtheIFN-stimulatedresponseelement(ISRE)
strand ed(cid:3)- barrel flanke dbyan(cid:2)- he lixandm an yof theconse rved (Wan g et al., 2010; Zu st et al., 2007). Si milarly, T GEV nsp 1 also
residues form the hydrop ho bic core o f the (cid:3)-ba rr el fo ld, which inhibits th ee xpressi onof SV 40- promo ter-driven report ergen ein

wassuggestedtobemoreimportantforthestructuralstabilityof HEK293cellsaswellasinswinetestis(ST)cells,whichsupport

nsp1ratherthanforitsfunction(Jansson,2013).Inaddition,two TGEV replication (Huang et al., 2011a). Furthermore, TGEV nsp1

highlyconservedareasmaptothesurfaceofTGEVnsp1structure, stronglyinhibitshostproteinsynthesiswithoutaffectingthesta-

whicharethoughttobepotentiallyimportantforthefunctional bilityofhostmRNAsandalsosuppressesthetranslationofseveral

interactionofTGEVnsp1withotherproteins(Jansson,2013).How- different reporter mRNAs in a cell-free in vitro translation sys-

ever, neither the three-dimensional structure of TGEV nsp1 nor temlikeHeLaS10extract(Huangetal.,2011a).Intriguingly,TGEV

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

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Fig.2. AnalysisofthesubcellularlocalizationofTGEVnsp1usingconfocalmicroscopy.HEK293cells,grownon4-wellLab-TekIIchamberslides(NalgeneNuncInternational),

weretransfectedwithaTGEVnsp1expressingplasmid,pCAGGS-nsp1(lowerrow)oranemptypCAGGSplasmid(mock;upperrow).At24hpost-transfection,thecells

werefixedin4%paraformaldehydefor15minandpermeabilizedwith0.1%TritonX-100.Subsequently,thecellsweresubjectedtoimmunofluorescenceanalysisusinga

TGEV-nsp1specificprimaryantibody,whichwasraisedbyimmunizingrabbitswithpurifiedfull-lengthC-terminallymyc-taggedTGEVnsp1protein,followedbyAlexaFluor

594-conjugatedsecondaryantibody(MolecularProbes)andDAPIcounterstaining(CellSignalingTechnology).ImageswerecollectedusingaZeissLSM-510METAconfocal

laser-scanningmicroscopewitha100XoilimmersionlensandprocessedwiththeLSMimagebrowser(Zeiss)andImageJ(NIH)softwareprogram.Nosignalwasdetected

inthenegativecontrol(mock;upperrow),confirmingthespecificityoftheanti-TGEVnsp1antibody.

nsp1lackstheabilitytoinhibitthetranslationofreportermRNAs andHKU5-5,thensp1isabout195aminoacidsinlengthwhereas
inth erabb itr eticuloc yt elysate (RR L)invitro tra nslation system in li neage D (cid:3)-C oVs ( (cid:3)- CoV ) tha t includ es th e b at CoV strains,
D

butregainsitstranslationinhibitionactivityinRRLsupplemented HKU9-1,HKU9-2,HKU9-3andHKU9-4,itisabout175aminoacids

with HeLa S10 extract or HeLa S100 postribosomal supernatant, long (Almeida et al., 2007; Tohya et al., 2009). There is only a

derivedfromHeLaS10extractaftercentrifugation(Huangetal., limiteddegreeofaminoacidsequencehomologyamongthensp1
2011a). Collec tively ,the sedata indica tethepresence ofaho stf ac- ofdiffer ent(cid:3)-C oV sbelo nging tothese fourlineag es.For exam ple,

tor(s)inHeLacellextractsandculturedcellsthatisutilizedbyTGEV SARS-CoVnsp1hasanaminoacidsequencesimilarityofonly20.6%

nsp1toexertitsinhibitoryeffectontranslation.Themechanisms and17.3%withMHVnsp1andBCoVnsp1,respectively,whereasit

of inhibition of reporter gene expression, host protein synthesis sharesahighsequenceidentityof92.2%withRm1,whichbelongs
an dmRNAtr ans lationby (cid:2)-Co Vnsp1have notb eenelu cidatedas tothes a meli neageasS ARS-CoV (T ohya etal., 2009) .Simil arly,the

yet.SomecluesareprovidedbytheobservationthatbothHCoV- nsp1ofthebatCoVstrains,133andHKU9-1,sharelowsequence

229E and HCoV-NL63 nsp1 associate with the ribosomal protein identities of 19.7% and 30.9% with SARS-CoV nsp1 (Tohya et al.,

S6,whichislocatedinthemRNAbindingsiteofthe40Sribosomal 2009).InMHVandBCoV,nsp1isreleasedasanN-terminal28-kDa

subunit,acentralcomponentofthecellulartranslationapparatus protein(p28)alongwithnsp2,a65-kDaprotein(p65)afterthepro-
(Wange ta l.,2010 ;Williamse ta l.,20 03).Int erestingly,T GEVnsp1 teolytic proce ssing of th e ORF 1 a polyp rotein b y PL1 pro ( Den ison
doesno tb ind tothe 40Sribo som al subun it,highlighting apos sible etal.,19 95,2004). Sim ilar ly,inS ARS-CoV,PL pro- mediatedcleav-
mech ani sticd iffe ren ceb etweenthe nsp1ofT GEVandoth e r(cid:2)-CoVs ag eat theco nsensu scleavage si teLXGGin theORF1apolyp rotein

(Huangetal.,2011a).Thecontributionofthesebiologicalactivities liberatestheanalogousnsp1andnsp2as20-kDaand70-kDapro-
of(cid:2)-CoV n sp1 toward the regulationof ho stgen eexpressio nduring teins,res pect ively(Pren ticee tal. ,2004 ).B othMH Van dSARS -CoV

CoVinfectionremainstobedetermined. nsp1 are localized exclusively in the cytoplasm of virus-infected

cells(Brockwayetal.,2004;Kamitanietal.,2006).
2.2. ˇ-CoVnsp1 Se veralstudi es hav einve stigatedt he bio logicalfunctionsof(cid:3)-

CoVnsp1.MHVnsp1expressioninmammaliancells,includingthe

The(cid:3)-CoVgenuscontainsfourdifferentlineages,A–D.Thesize murine 17Cl-1 and NIH 3T3 cells as well as the human embry-
of(cid:3)-Co Vnsp1 varies amongth evir usesofdi fferentlin eage swi thin oniclun gfibrob last LUce lls,i nhibi tsc ellpr oli ferat ionand induces
th is genu s. Th e nsp1 of vir uses in line ag e A of (cid:3) -CoV ((cid:3)- CoVA), cell cycle arrest in G0/ G1 p hase (Ch en et al., 2004). In t ransient
which include mouse hepatitis virus (MHV), bovine coronavirus expression studies, like HCoV-229E nsp1, MHV nsp1 also inhibit

(BCoV) and the human coronaviruses, HCoV-OC43 and HCoV- theexpressionofreportergenesunderthecontrolofconstitutive
HKU1, hasa bout 245am inoacidsresid uesandisals okn ownas and induciblep rom oterssu chasS V40,IF N-(cid:3) andISR E, suggestinga

p28(Almeidaetal.,2007;Jansson,2013).Thensp1ofSARS-CoVand functionalsimilaritybetweenthensp1ofCoVsbelongingtodiffer-
Rm1 ,aSARS- lik eb atCoV ,which belong tot helin ea geBof(cid:3)- CoV entphylog eneticline ages(Zus tet al.,2 00 7).Ad ditionally ,de letion
((cid:3)-Co V ),contain s18 0am inoac idresid ue s(A lmeidae t al. ,2007; ofth eC-terminal regionof MHV ns p1a bolish esitsactivityt oinhibit
JanssonB,2 013;Toh yae tal.,20 09). Inlineag eC(cid:3)-CoV s ((cid:3)- CoV ), re port ergeneex pressio n, revea ling theimpor tan ceofth is region
C
whichincludeMERS-CoVandseveralbatCoVs,suchasHKU4-1,133 foritsfunction(Zustetal.,2007).Mostimportantly,thisseminal

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

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studyusedarecombinantMHVencodingatruncatednsp1,carrying 2006).Subsequently,functionallysimilaractivitiesweredemon-
adele tion of 99nucleotid es(n ts)inthe n sp1-coding sequ ence,to strated forother(cid:2)-Co Vand(cid:3)-CoVn sp1.Ad etailedch aracte rization

examinetheroleofnsp1inthevirulenceandpathogenesisofMHV ofthemechanismofSARS-CoVnsp1-mediatedinhibitionofgene

inamurinemodelofCoVinfection(Zustetal.,2007).Thereplica- expressionrevealedanovelmodeofactionandalsoidentifiedspe-
tio n ofthism utant vi rus,M HV-nsp1 (cid:2)99, w ass imilar tow ild-type cificamino acidresid u esinn sp1th at areim port antf oritsfunct ions

MHV in cultured cells, including primary professional antigen- (Huangetal.,2011b;Kamitanietal.,2009;Lokugamageetal.,2012).

presentingcells,suchasdendriticcellsandmacrophages,butits SARS-CoVnsp1employsatwo-prongedstrategytoinhibithost

growthwasseverelyattenuatedinvivo,implyingthatMHVnsp1is gene expression by targeting the translation and stability of cel-

amajorvirulencefactor.Strikingly,comparedtotheseverelyatten- lularmRNAs;throughitstightassociationwiththe40Sribosomal
u atedgr owthofM HV-n sp1(cid:2)99in wild-type m ice, therepli cation subu nit, a ke y compo nen t of the cellular trans lati on m achinery,
ofMH V-nsp1(cid:2) 9 9wasrestored alm osttoth eleve lso fwild-type nsp1 inh ib its m RNA transl ati on a nd also induces an endonucle-
M HV in type I IFN rec eptor-de ficient m ic e. T hese d at a strongly olytic RNAcle avagein the5(cid:3)-UTR ofce llula rmRNAs (Fi g.3)(Huang

indicated the role of nsp1 in facilitating the efficient replication etal.,2011b;Kamitanietal.,2009).Theoutcomeofthiscleavageis

ofMHVinwild-typemicebycounteractingthetypeIIFNsystem. theacceleratedturnoverofcellularmRNAsastheinternallycleaved
In terest ing ly,inocula tionw ith MHV-nsp1(cid:2)9 9a lsopr o tect edmice mR NAsaresub sequently d egraded bythe ce llul arXrn1-m ediated
againstchalle ngewithwi ld-typ eMHV,highligh ting itspotent ialto 5(cid:3)-3(cid:3) ex onu cleolytic mRN A decay p ath wa y (Fig. 3 ) (Gaglia et al.,

bedevelopedasanoveltypeoflive-attenuatedCoVvaccine(Zust 2012). The binding of SARS-CoV nsp1 to the 40S ribosomal sub-

et al., 2007). More evidence for MHV nsp1 as a major virulence unit serves two important roles: it allows nsp1 to inactivate the

factorwasprovidedinastudythatexaminedthefunctionofarel- translationfunctionoftheribosomeandalsogainaccesstoactively

ativelyconservedaminoacidsequence,LLRKxGxKG,inMHVnsp1 translatinghostmRNAs.Abiologicallyinactivensp1,nsp1-mt,car-

thatisalsofoundinSARS-CoVnsp1(Leietal.,2013).AmutantMHV, ryingthemutationsK164AandH165AintheC-terminalregionof

MHV-nsp1-27D,carryingadeletionofthissequenceinnsp1,has SARS-CoVnsp1,isunabletobindthe40Ssubunit,demonstrating

similargrowthkineticsin17Cl-1cells,butwashighlyattenuatedin therequirementoftheseaminoacidresiduesandtheassociationof

mice(Leietal.,2013).ThemechanismbywhichMHVnsp1inhibits SARS-CoVnsp1withthe40Ssubunitforitsfunctions(Narayanan

hostgeneexpression,includingthetypeIIFNsystem,remainsto etal.,2008).

beelucidated. Interestingly, SARS-CoV nsp1 does not possess any intrinsic

SARS-CoV nsp1 has been the focus of many research efforts, nuclease activity and possibly, recruits a cellular endonucle-

includingourgroup,thathaverevealedsomenovelandinterest- ase for inducing mRNA cleavage (Huang et al., 2011b; Kamitani

ingbiologicalproperties.SARS-CoVnsp1isoneofthemostwell etal.,2009).Theidentityofthisputativecellularendonucleaseis

characterizednsp1ofCoVs,bothintermsofitsbiologicalfunctions stillunknown.Furthermore,SARS-CoVnsp1inducesatemplate-

andmodeofaction.Usingtransientgeneexpressioninmammalian dependentendonucleolyticcleavageofmRNAsbutdoesnottarget

cells,SARS-CoVnsp1wasthefirstCoVnsp1thatwasshowntoblock any specific nucleotide sequence in the mRNA substrate (Huang

theexpressionofreportergeneunderthecontrolofconstitutive et al., 2011b). In cell-free in vitro translation systems, SARS-CoV
pro motersasw ell astheind ucibl eIFN-(cid:3) pro moter( Ka mitanietal., ns p1 induces an endonuc leo lytic RNA cleava ge in the 5(cid:3)-UTR of

Fig.3. Two-prongedstrategyofSARS-CoVnsp1toinhibithostgeneexpression.SARS-CoVnsp1gainsaccesstohostmRNAsthroughitsassociationwiththe40Sribosomal

subunit.Consequently,nsp1inhibitsthetranslationofcappedcellularmRNAsbyprimarilyblockingthestepsinvolvedintheformationofelongation-competent80Sinitiation
complex .Nsp1alsorec ruits acellula re ndonucleas et oinduc eanen donucle oly ticRNAc leavagei nth e5(cid:3)-U TRofho st mR NAsthats ub sequentlyresultsinth eac celerated
degradati onof the5 (cid:3)-trunca te dinterm ediatebythe cel lularXr n1- mediated5(cid:3)–3(cid:3)e xonu cleolytic mR NA decayp at hway .

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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6 K.Narayananetal./VirusResearchxxx(2014)xxx–xxx

cappedmRNAsandwithintheribosomeloadingregionofmRNAs replicationofamutantSARS-CoV,encodingnsp1withthemuta-

carryingpicornavirustypeIandtypeIIinternalribosomeentrysites tionsR124SandK125E,wasstronglyattenuatedincellswithan

(IRESes),whereasmRNAscarryingtheIRESesofcricketparalysis intactIFNresponse(Watheletetal.,2007).Collectively,thesestud-

virus, hepatitis C virus or classical swine fever virus are resis- ies highlight the role of SARS-CoV nsp1 in regulating the innate

tant to nsp1-induced RNA cleavage (Huang et al., 2011b). It has immuneresponseduringvirusinfectionandalsolendfurthersup-

beenproposedthatthetemplate-dependentnatureofSARS-CoV porttothenotionthatSARS-CoVnsp1isapotentialvirulencefactor

nsp1-induced mRNA cleavage could be due to differences in the thatcontributestoviralpathogenesis.

requirement of translation initiation factors and mechanism of Itisworthnotingthatthensp1ofbatCoVsbelongingtodifferent
translation in iti ation among capped c ellular mRN As and mRN As (cid:3)-Co V lineag esalso exhi bit funct ion als imila ritieswith S ARS-CoV

with different IRESes (Huang et al., 2011b). However, the exact nsp1 (Tohya et al., 2009). Nsp1 of the bat CoV strains, Rm1, 133
mech anismun derlyin gthetem pla te- depende ntmRNAc leava geby andH KU9-1, be lon gingto (cid:3)-Co V ,(cid:3)-C oV and (cid:3)-CoV linea ges,
B C D

SARS-CoVnsp1stillremainstobeclarified. respectively,alsodisplayedanabilitytoinhibithostproteinsyn-

A nuclear magnetic resonance (NMR) structure of the N- thesis and promote host mRNA degradation in mammalian cells

terminalregionofSARS-CoVnsp1revealedsomeuniquestructural (Tohyaetal.,2009).Inaddition,expressionofthesebatCoVnsp1
features, includi ng acomplex irreg ular(cid:3)-ba rrelfo ld(Alm eidaetal., intrans in hibi tsthein du ctionoft ypeIIFNand IF N-stim ula tedg enes

2007). This study also alluded to the potential role of positively incellsinfectedwiththeIFN-inducingSCoV-mt(Tohyaetal.,2009).

charged residues exposed on the surface of SARS-CoV nsp1 in However,thesebatCoVnsp1haddifferentialinhibitoryactivities,

themRNAdegradationactivityofSARS-CoVnsp1(Almeidaetal., indicatingpossibledifferencesintheirmechanismofaction(Tohya

2007).Inlinewiththispossibility,amutatedSARS-CoVnsp1,car- etal.,2009).Nevertheless,theevidenceofaconservedbiological
ryinga lan ine subst ituti onofthech a rgedresi duesR124 andK 125 fu ncti onamo ngthensp1o fSA RS-CoVa nd b atCoVsin the(cid:3)-CoV

thatareexposedonthesurfaceofnsp1,lacksthemRNAcleavage genuscouldhavepotentiallysignificantimplications,considering
func tion but reta ins the transla tio n inh ibitio n ac tivity, implying theide ntifica tion ofbatsasth enaturalre servoirofsev eral(cid:2)-CoVs
the impo rtan ce of th ese residues fo r the mRN A cleava ge prop- and (cid:3)-CoVs,inclu di ngth os ecl oselyre latedtoS AR S-CoV, andthe

ertyofSARS-CoVnsp1(Lokugamageetal.,2012).Theisolationof potentialoftheirviromeasthesourceofemerginghumanCoVs

thiscleavage-defective(CD)mutantofSARS-CoVnsp1,nsp1-CD, (SmithandWang,2013).

demonstratesthatthetranslationinhibitionfunctionofSARS-CoV

nsp1isindependentandseparablefromitsmRNAcleavageactivity

(Lokugamageetal.,2012).Furthermore,itwasshownthatSARS- 3. CoVnsp1:rolesintheregulationofviralgeneexpression

CoVnsp1inhibitsthetranslationofmRNAsattheinitiationstepby

targetingmultiplestages,dependingonthedifferentmechanisms MultiplelinesofevidencehavesuggestedtheroleofCoVnsp1

ofinitiationoperatingoncappedandIRES-drivenmRNAtemplates inregulatingviralreplicationandgeneexpression.Apointmuta-

(Lokugamageetal.,2012). tion,introducedintotheTGEVgenomebyreversegenetics,atthe
SARS-CoV nsp 1a lsoblockstheactivationofIFN-induciblegenes pred ictedPL1pro cleav age sitet hatsever ely affected theproc ess ing
byinhibitingt hevi rus- andIFN -de pendenta nti viralsignaling path- of the OR F1a polyprotein and the release of the P L1pr o cleavage

ways(Kamitanietal.,2006;Watheletetal.,2007).Theintroduction products,includingnsp1,alsocausedadrasticreductionintheeffi-

of mutations R124S and K125E in SARS-CoV nsp1 significantly ciencyofinfectiousTGEVrescuefromcDNA(Galanetal.,2005).In

reducedtheabilityofnsp1toinhibittheantiviralsignalingpath- addition,therecoveredviruseshadasmall-plaque-sizephenotype

ways,furtherhighlightingtheimportanceoftheseresiduesforthe andalsoshowedarapidreversionoftheintroducedmutationto

inhibitoryactivityofSARS-CoV(Watheletetal.,2007).Anexhaus- the original wild type TGEV sequence (Galan et al., 2005). These
tive mutat ional an al ysis of SAR S-CoV nsp 1, spe cifical ly t argeting data suggest edth eimp ortan ceofthep roteins lib erat edbyP L1pro-

thesolventexposedresiduesofnsp1,suggestedthattheinhibition mediatedcleavage,whichincludesnsp1,inTGEVreplication.

ofhostgeneexpressionandantiviralsignalingpathwayscouldbe Mutagenesis of the coding region of MHV nsp1 in the viral

mediatedbydistinctbutoverlappingregionsofnsp1interacting genome, using reverse genetics, identified two domains in nsp1

withdifferenthostfactors(Jaureguietal.,2013).Inagenome-wide that are important for virus replication (Brockway and Denison,

yeast two-hybrid screen, several members of the immunophilin 2005). Deletions in the N-terminal half of nsp1 were lethal

andcalcipressinfamilieswereidentifiedasinteractingpartnersof and infectious viruses could not be recovered (Brockway and

SARS-CoVnsp1(Pfefferleetal.,2011).Thisstudyalsoshowedthat Denison, 2005). Furthermore, introduction of point mutations in

nsp1expressionaswellasSARS-CoVinfectionstronglyenhanced the N-terminal region of nsp1 resulted in the recovery of viable

signalingthroughtheCalcineurin/NFATpathway,whichismodu- MHV mutants that exhibited replication defects in cell culture,

lated by immunophilins and plays an important role in immune suggestingthepresenceofcriticalreplicationdeterminantswithin

cellactivation(Pfefferleetal.,2011).Alongsimilarlines,SARS-CoV the N-terminal half of MHV nsp1 that are required for optimal

nsp1expressioninducedthesecretionofchemokines,suchasCCL5, virusreplication(BrockwayandDenison,2005).Itshouldbenoted

CXCL10andCCL3,inhumanlungepithelialcells(Lawetal.,2007). thatthesereplicationelementscouldbeanRNAstructureformed

However,theinvolvementofnsp1intheinductionofchemokines bysequenceswithinthisregionofMHVnsp1and/orafunctional

inthecontextofSARS-CoVinfectionremainstobevalidated.Nev- moiety in the nsp1 protein. Mutations in the C-terminal half of
er thel ess,thes es tudiessug gestapos siblerole fo rn sp1inimm une MHVn sp1 ,ha rborin gthePL 1pro cleava ge site, thatabolish edt he

dysregulation,asobservedinlaterstagesofSARS. releaseofnsp1fromtheORF1apolyproteinshowedthatthecleav-

Most importantly, studies have also investigated the role of age between nsp1 and nsp2 is not required for virus replication

SARS-CoV nsp1 in the context of virus replication in cell cul- (BrockwayandDenison,2005;Denisonetal.,2004).However,the

ture (Narayanan et al., 2008; Wathelet et al., 2007). Notably, rescuedMHVmutantsformedsmallplaquesandexhibiteddelayed

experiments using a mutant SARS-CoV (SCoV-mt), encoding the growthkineticswithreducedviralRNAsynthesisandpeakviral
biologicallyi nactive n sp1(nsp 1-mt),sho wedthatns p1suppre sses titers, in dicating the importan ce of the PL1pro-rele ased prot eins,

theexpressionofhostgenes,includingtheinnateimmuneresponse includingnsp1,inMHVreplication(BrockwayandDenison,2005;

genes like type I IFN, ISG15 and ISG56, by inhibiting host pro- Denisonetal.,2004).Thepotentialroleofnsp1inMHVreplication

tein synthesis and promoting the degradation of host mRNAs in isalsosuggestedbythelocalizationofMHVnsp1toviralreplica-

SARS-CoV-infectedcells(Narayananetal.,2008).Furthermore,the tioncomplexesduringvirusinfectionanditsinteractionwithnsp7

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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Fig.4. ViralmRNAsarenotspatiallyseparatedfromnsp1inSARS-CoV-infectedcells.RNAfluorescentinsituhybridization(RNA-FISH)andconfocalmicroscopyanalyses

ofviralmRNAsandnsp1inSARS-CoV-infectedcells.VeroE6cells,grownon4-wellLab-TekIIchamberslides(NalgeneNuncInternational),wereeithermock-infected
(M ock) orinfec tedw ithw t SARS-CoV(SCoV-W T)at amu ltip licity ofinfec tio n(MOI )of5.At 1 2hp.i.,th ecell swerefix edin 4%paraformald ehyd efor1 6hat4◦Cand
permea bil izedwith 0.5% Trit onX-100f or5minatro om temperature .Su bsequent ly,the cel ls we rep re cipi tate dwith 70% ethan ola t4 ◦Covernightandsu bjec ted t oR NA-F ISH
andimmunoflu ores cenc eanaly ses.A dig o xigen in (DIG )-labeledanti senseriboprob e,c orres pond ingtothenu cleo tides (nt)29 ,08 4–2 9,608att he3 (cid:3)-endofth e SARS-CoV

genome,wasusedtodetectviralmRNAs,includingmRNAs1–9.ViralmRNAs(green)werevisualizedwithaprimarysheepanti-DIGantibodyandAlexa488-conjugatedanti-

sheepsecondaryantibody(Invitrogen).Nsp1(red)wasvisualizedusinganaffinity-purifiedrabbitanti-nsp1polyclonalantibody,whichwasraisedbyimmunizingrabbits

withpurifiedfull-lengthnsp1protein,followedbyAlexa594-conjugatedsecondaryantibody(Invitrogen).ImageswerecollectedusingaZeissLSM-510METAconfocal
micro scopew itha63×,1 .40nu mericala pertureo ilim mersionlensandpro cessedwith theLSM imagebrowse randM etamo rphsoftw are(M o lecula rDevices ,Down ingtown,

PA).Inthelowerpanel(SCoV-WT),theinsettotherightofthemergedimagerepresentstheindicatedregion(smallsquarepanel)inthemergedimage.Thehistogram

displaysthefluorescencesignalintensitiesalongthewhitearrowintheinset,whichisarepresentativelineforasinglelinescanshowingthefluorescencesignalintensity

(yaxis)inbothchannels(redandgreen)inanarbitraryscaleversusdistance(xaxis)(inpixel)overthatline.

andnsp10,twoORF1a-derivedproteinsthathavebeenshownto defectiveinterfering(DI)RNAreplication(Suetal.,2014).Taken

playcriticalrolesinregulatingviralRNAsynthesisandpolyprotein together,thesestudiespointtowardafunctionallinkbetweennsp1

processing (Brockway et al., 2004; Deming et al., 2007). MHV andCoVreplication.

nsp1 is associated with intracellular membranes and displays a The effect of SARS-CoV nsp1 on viral gene expression has

temporalpatternofsubcellularlocalizationduringvirusinfection beenstudiedprimarilyusingcell-freeinvitrotranslationsystems

(Brockway et al., 2004). At early times postinfection, MHV nsp1 (Huang et al., 2011b; Lokugamage et al., 2012). Strikingly, nsp1

colocalizes with viral proteins that are components of the repli- efficientlyinhibitsthetranslationofSARS-CoVmRNAsinRRLand

cationcomplexthatincludensp7,nsp10,nsp12(RNA-dependent HeLa extracts but unlike host mRNAs, nsp1 does not induce the

RNA polymerase), nsp13 (helicase), and the viral nucleocapsid endonucleolyticcleavageofviralmRNAsintheRRLsystem(Huang
prote in, N (Brockw ay et al., 2004). At la te t imes postinfection, etal.,2011b);th epresen ce ofth e5(cid:3)-end le ade rse quence, acom-

MHVnsp1localizestositesofMproteinaccumulation,hintingat monfeatureofalltheviralmRNAs,protectstheviralmRNAsfrom

itspossibleroleinvirionassembly(Brockwayetal.,2004). nsp1-inducedRNAcleavage(Huangetal.,2011b).Nsp1inhibitsthe

BCoVnsp1hasbeenshowntobeanRNA-bindingproteinthat translationofSARS-CoVmRNAsattheinitiationstagebyprimarily
interacts with cis-a cting replica tio nel em entsinthe5(cid:3)u ntransl ated blocking th e steps invo lved in th e co nversion of th e 4 8S initia-

region(UTR)oftheBCoVgenome,implyingitspotentialroleinthe tioncomplexintotheelongation-competent80Sinitiationcomplex

regulationofviraltranslationorreplication(Gustinetal.,2009). (Lokugamageetal.,2012).

SARS-CoV nsp1 also binds to a stem-loop structure, SL1, in the Toclarifytheeffectofnsp1onviralmRNAtranslationinSARS-
5(cid:3)-UTR of SARS- CoV genom e a n d it has be en suggest ed th at this CoV-in fected cel lsand ga inabe tte rund erstan dingofthei nt erplay

interactionenhancesvirusreplication(Tanakaetal.,2012).Inboth betweenviralandhostgeneexpressioninSARS-CoVinfection,we

BCoVandMHV,thereisevidenceofalong-rangeRNA-RNAinterac- examinedtheeffectofnsp1onviralgeneexpressioninSARS-CoV-
tionb etw eenth e5(cid:3)-U TR andthe cod i ngregiono fnsp1(Gu anetal., infectedce lls.

2012).Furthermore,synthesisofanascentBCoVnsp1proteincar- To investigate the possibility that viral mRNAs are spatially

ryingtheN-terminalaminoacidsequenceWAPEFPWM,whichis separated from nsp1 in infected cells that would facilitate
conse rved amongthe nsp1 of(cid:3)- CoV ,isre quiredincis forBCo V the escap e of v iral m RNA s from t he ns p1-in duced t ranslation
A

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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8 K.Narayananetal./VirusResearchxxx(2014)xxx–xxx

inhibition,weexaminedthelocalizationofviralmRNAsandnsp1

inSARS-CoV-infectedcellsbycombiningRNAfluorescentinsitu

hybridization (RNA-FISH) and immunofluorescence analyses to

detectviralmRNAsandnsp1,respectively(Fig.4).Nosignalswere

detected in mock-infected cells (Fig. 4, upper panel), confirming

thespecificityoftheriboprobeandanti-nsp1antibody.InSARS-

CoV-infected cells, we observed areas of colocalization of nsp1

with viral mRNAs, as demonstrated by the similar fluorescence

histogrampatternsofviralmRNAsandnsp1(Fig.4,lowerpanel).

This data implied that nsp1 was not excluded from the sites of

viralmRNAtranslationinSARS-CoV-infectedcells.

TheabovedataledustohypothesizethatviralmRNAtransla-

tionisalsosusceptibletoinhibitionbynsp1inSARS-CoV-infected

cells.ThishypothesispredictsthatthetranslationofviralmRNAs

would be less efficient in SARS-CoV-infected cells than in SARS-

CoV-mt-infectedcells,becauseoftheabortivetranslationofviral

mRNAs,causedbythetranslationally-inactive48S-nsp1complex,

andthereducedpoolofbiologicallyactive40SsubunitsinSARS-

CoV-infected cells, due to nsp1-induced inactivation of the 40S

subunit.BothSARS-CoVandSARS-CoV-mtdisplayedsimilarrepli-

cationkineticswithsimilarvirusyieldsandlevelsofaccumulation

of viral mRNAs (Fig. 5A and B). While the level of nsp1-mt was

marginally higher than nsp1 at 10h postinfection (p.i.), the lev-

els of nsp1 and nsp1-mt were similar at 12h p.i. (Fig. 5C). As

describedpreviously(Narayananetal.,2008),nsp1-mtmigrated

slightlyslowerthannsp1(Fig.5C).

Next,weperformedmetabolicpulse-radiolabelingexperiments

toteaseouttheeffectofnsp1onviralmRNAtranslationininfected

cells. SARS-CoV replication induced a more prominent inhibi-

tion of host protein synthesis than SARS-CoV-mt replication in

infected cells, as reported previously (Fig. 6A) (Narayanan et al.,

2008).Thesynthesisofthemajorviralstructuralprotein,N,was

markedlylowerinSARS-CoV-infectedcellsthanthatinSARS-CoV-

mt-infected cells at both 10h and 12h p.i. (Fig. 6A), despite the

similar levels of accumulation of mRNA 9, encoding N protein,

inSARS-CoVandSARS-CoV-mt-infectedcells(Fig.5B).Thesedata

stronglyindicatedthatnsp1,butnotnsp1-mt,inhibitedthetrans-

lation of mRNA 9. Radiolabeled M protein, another major viral

structural protein, was not detected due to M protein aggrega-
tion, caus ed by th e inc uba tion of sa mple s a t 1 00◦C fo r 15min

in SDS-sample buffer for the complete inactivation of SARS-CoV

infectivity (Lee et al., 2005; Sturman et al., 1980). Western blot

analysisofthecellextractsclearlyshowedthatthelevelsofthe

viralstructuralproteins,NandS,andtheaccessoryproteins,3a,6
Fig.5. ViralgrowthkineticsandmRNAaccumulationaresimilarinSARS-CoVand
and7a,werelowerinSARS-CoV-infectedcellsthaninSARS-CoV-
SARS-CoV-mt-infectedcells.VeroE6cellswereeithermock-infected(Mock)or
mt-infectedcells(Fig.6B).Insummary,despitethesimilarlevels
ofaccumula tiono fvir alm RN Asencodin gthese prot einsin SARS- Kin1f6ec4tAeda nwdiHth1 6w5tA SiAnRnSs-pC1o,Va t(aWnTM) OorI oSfA5R.S(-AC)oCVu-lmtutr e(msutp),e crnarartyainntgs wtheer emcuotlaletciotends

tChoeV aacncdu SmAuRlSa-tCioonV-omfvt-irinaflescttreudc tcuerllasl, tahned syacnctehsessoisr yofp Nro ptreointesinw aenrde iant Vtheer oi nEd6i ccaetlelds. tTihmee sr epsu.i.l,t sa nredp vreirsuesn tt ittheers awveerraeg ed eotfe rtmhrienee din bdye pTeCnIdDe5n0t aenxaplyesriis-
ments.(B)TotalRNAswereextractedat10hand12hp.i.TheviralmRNAswere
markedly lower in SARS-CoV-infected cells than in SARS-CoV-mt- detecte dby Nort hernb lotana lysisusing a ribo p robe tha t bind sto the3 (cid:3)-endofS ARS-
infectedcells.Thesedatasupportourhypothesisthatnsp1inhibits CoVgeno m e,asdesc ribed inthel egend forFig.1. Repr esenta ti ved atafro m three
thetrans lation ofvir alm RNAsin SAR S-CoV-infec ted cells. inde pendent exp eriments are sho wn.(C )At 10h a nd12hp.i.,tot alpro teins were
O ur seemin gly cou nter-int uit ive finding that SA RS-CoV nsp1 extracted,an dWesternblo tan alysisw as per for m edto de t ectN sp1p roteinus inga
rabbitanti -nsp 1polyclo nal antibody .Rep resentative d atafrom thre eindep enden t
inhibited the translation of viral mRNAs in SARS-CoV-infected
experimentsareshown.
cells is nevertheless consistent with the following observations.

Nsp1wasnotspatiallyseparatedfromviralmRNAsinSARS-CoV-

infected cells (Fig. 4). Nsp1 inhibits the translation of SARS-CoV

mRNA9inRRLprimarilybyblockingthestepsinvolvedinthecon- subunitbynsp1couldleadtoareductioninthepoolofbiologically

versionofthe48Scomplexintothe80Scomplex,whichsuggests active 40S subunits in infected cells. Based on these data, we

that the nsp1-40S complex can load onto viral mRNAs to form proposethatthenon-productiveinteractionofnsp1-40Scomplex

the48Scomplexininfectedcells(Lokugamageetal.,2012).Nsp1 withviralmRNAsininfectedcellsthatleadstoabortivetranslation

associatestightlywiththe40Ssubunit,indicatedbytheresistance andthensp1-inducedreductioninthepoolofbiologicallyactive

ofthisinteractiontostringenthigh-salttreatmentconditionsthat 40SsubunitsbothcontributetotheinhibitionofviralmRNAtrans-

isknowntocausethedissociationoftranslationinitiationfactors lation by nsp1 in SARS-CoV-infected cells. Importantly, because

andthe60Ssubunitfromthe40Ssubunit(Kamitanietal.,2009; viralmRNAsareresistanttonsp1-inducedmRNAcleavage(Huang

Merrick, 1979); this data suggests that the inactivation of 40S et al., 2011b), the translationally-competent intact viral mRNAs

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
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K.Narayananetal./VirusResearchxxx(2014)xxx–xxx 9

dataimplythateventhoughnsp1inhibitedviralmRNAtranslation,

thereducedlevelsofviralstructuralproteinsinSARS-CoV-infected

cells are still sufficient and above the threshold of viral proteins

requiredforoptimalvirusreplicationandassembly.Incontrastto

thestructuralandaccessoryproteins,thelevelsofaccumulation

of nsp1 and viral mRNAs were similar in SARS-CoV- and SARS-

CoV-mt-infectedcells.Wespeculatethattheefficientsynthesisof

nsp1,whichistranslatedfrommRNA1,theviralgenomicRNA,as

partofthereplicasepolyproteins,occurspriortothetranslationof

viralsubgenomicmRNAsencodingtheviralstructuralandacces-

soryproteins,andduringtheearlystagesofinfectionwhenmRNA

1isundergoingtranslation,thelevelsofnsp1arenotsufficientto

inhibitthesynthesisofreplicaseproteins,includingnsp1,resulting

inthesimilaraccumulationofnsp1andviralmRNAsinSARS-CoV

andSARS-CoV-mt-infectedcells.

A study by Tanaka et al. suggested that a specific interaction
ofns p1wit hth e5(cid:3)un tra nsla tedregion (UTR ) ofSARS- CoVmRNA

protects viral mRNAs from nsp1-mediated translational shutoff

in SARS-CoV-infected cells (Tanaka et al., 2012). In addition, the

authorsalsospeculatedthatnsp1promotesviralproteinsynthe-

sisandviralRNAreplicationthroughthisinteractionbecausethis

effect was not observed with a mutated nsp1 protein, carrying
R124A mu tatio n that ab olishe d its intera ction with th e 5(cid:3) UTR

of viral mRNA (Tanaka et al., 2012). Our data that viral protein

synthesisandaccumulationarelowerinSARS-CoV-infectedcells

than in SARS-CoV-mt-infected cells do not support the possibil-

ity that the interaction of nsp1 with viral mRNA augments viral

mRNAtranslation,becausetheputativensp1-mediatedenhance-

ment of viral protein synthesis would have resulted in a more

efficient production of viral proteins in SARS-CoV-infected cells

than in SARS-CoV-mt-infected cells. Based on our studies with

thecleavage-defectivensp1mutant,nsp1-CD,carryingR124Aand

K125A mutations, the nsp1R124A mutant most probably lacked

thehostmRNAcleavagefunctionbutretainedtheabilitytobind

andinactivatethe40Ssubunit(Lokugamageetal.,2012).Accord-

ingly,inthestudybyTanakaetal.,thensp1-mediatedenhancement

of viral protein synthesis could be an indirect consequence of

the degradation of host mRNAs, induced by nsp1 but not by the

nsp1R124Amutant,thatwouldeliminatethecompetitionbetween

viralandhostmRNAsforthelimitingamountsoftranslationally-

competent40Ssubunits,therebytiltingthebalanceinfavorofviral

mRNAtranslation.Furthermore,thensp1-induceddegradationof

host mRNAs could also liberate the translation initiation factors

fromhostmRNAsthatcanbeutilizedbytheintactviralmRNAs

Fig.6. SARS-CoVnsp1inhibitedthetranslationofviralmRNAsininfectedcells. in SARS-CoV-infected cells. Therefore, it is conceivable that the

VeroE6cellswereeithermock-infected(Mock)orinfectedwithwtSARS-CoV(WT) cleavageofhostmRNAsbynsp1andtheresistanceofviralmRNAs
oofr S5A. R(AS )-CCoeVll -smwt (emr et)r, acdair orlyaibnegl ethdef moru1ta5 tmioinns Kw1 it6h4 A5 0a0nd(cid:6) CH i1o6f5AT r iann n[3s5pS1], laatb aenl M(MOPI to nsp1-i nd uced RNA cle av age a re st rate gies that S AR S-Co V could
haveevolvedtocompensatefortheinhibitionofviralmRNAtrans-
Biomedicals)at10hand12hp.i.Equivalentamountsoftheintracellularproteins
wereanalyze do na 1 2%S DS -P AG Eandvisua lizedbya ut orad iography.Ar rowhead lationbynsp1therebyfacilitatingtheproductionofviralproteins
indica testhep osi ti ono fNprotein .Re presentativ ed atafromthreein dependent inSAR S-C oV-in fectedc ells.

experimentsareshown.(B)Totalproteins,extractedat10hand12hp.i.in(A),

weresubjectedtoWesternblotanalysistodetecttheviralstructuralandaccessory

proteins,S,N,3a,6and7a.Anti-SARS-CoVSantibodies(IMG-541;Imgenexand
4. Concludingremarks
AP6000a; Abgent) and anti-SARS-CoV N antibody (IMG-548; Imgenex) were used to
detectSandNproteins,respectively.Rabbitanti-SCoV3a,6and7aantibodieswere

usedtodetect3a,6and7aproteins,respectively,asdescribedpreviously(Huang The accumulated knowledge of CoV nsp1 has revealed con-
etal. ,20 06a,b, 200 7 ).An ti-a ctinantib ody(SantaC ru zBiotechn ology)was usedto served functionsand divergentm ech anis msam ong different CoVs
de tec t(cid:3)-actin andth edetection ofsimila ramou ntso f(cid:3)-actininbot hSA RS-C oV
toblockhostgeneexpressionandantagonizehostinnateimmune
andSARS-CoV-mt-infectedcellsconfirmedtheloadingofsimilaramountsofcell
extr actsineachlane.Repre senta tivedatafro mt hreeind ep endent experime nts are responses that provide an insight into the expanding repertoire
shown. of novel v iral s trategie s of immun e ev asio n. In additi on, it also

highlights functionally significant correlations between the nsp1

aremostprobablytranslatedbythebiologicallyactivensp1-free ofCoVsbelongingtodifferentgenera,despitethelackofobvious

40SsubunitstoproduceviralproteinsinSARS-CoV-infectedcells. primarysequencehomologywitheachother,suggestingtheirevo-

BothSARS-CoVandSARS-CoV-mtexhibitedsimilarreplication lutionaryrelatednessandroleintheadaptationofCoVstodifferent

kinetics with similar virus yields, despite the reduced level of hostspecies.Thefactthatnsp1ofdifferentCoVsshareacommon

accumulation of viral structural and accessory proteins in SARS- biologicalfunctiontoinhibithostgeneexpression,butusediffer-

CoV-infectedcellscomparedtoSARS-CoV-mt-infectedcells.These ent modes of action to exert this function, has also raised some

Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019
G Model
ARTICLE IN PRESS
VIRUS-96459; No.ofPages12

10 K.Narayananetal./VirusResearchxxx(2014)xxx–xxx

importantquestionsabouttheimpactofthesefunctionsanddiver- CommitteeonTaxonomyofViruses.AcademicPress,Ltd.,London,UnitedKing-
gentmech anismson thev irul encean d patho genesisof em erging dom,pp.80 6– 828.
Deming,D .J., Graham,R.L.,Denison,M.R.,Baric,R.S.,2007.Processingofopenreading
hdeugmraadna CtiooVns.a Fctoirv ietxyaomfpSlAe,R wS-hCaotV isn tshpe1 ctoontthriebuvtiriounle onfc ethoef mSARRNSA- frreapmli cea 1tiao rne.pJ.liVciarsoe l .p8r1o t(e1i9n)s, 1n0sp2 78 0t–o1 n0s2p9110. in m urine hepatitis v ir us str ain A59
CoV?Studie stoexam in etherole ofnsp 1 asa potential vir ulence Denison,M.R.,H u ghes,S .A. ,Wei ss,S.R.,1995.Identificationandcharacterizationofa
65-kD apro teinpro cesse dfrom the gene1 polyproteino fthe murinecoronav iru s
factor in vivo are now feasible with the availability of SARS-CoV MHV-A 59.Viro logy207(1 ),31 6–3 20.
nsp1mutantsandsuitablemousemodelsystems,whichwillyield Denison,M.R., Yount,B. ,Bro ckw ay,S.M.,Graham,R.L.,Sims,A.C.,Lu,X.,Baric,R.S.,
valua bleinform ati onforth eration aldesi gnoflive -attenu ated vac- 2004 .Cleav agebe twe enreplicas epro teinsp28 and p65of mou seh ep atitisv irus
cines aga inst CoVs (M cCr ay e t al., 200 7; Rob er ts et al., 2007; T seng Drexisle nro,tJ .rFe.,qGuilroezda -fRoar uvsircuhs, rFe.,pGlicleantido en,. JJ.., VCiororm l. 7a8n ,(1V1.M) , .5,9M5 7u –th5,9D65.,. Goettsche ,M.,
et al., 2007). Also, does the nsp1 of MERS-CoV, another highly Seeb ens, A.,Niedrig,M .,P fefferle, S., Yordanov ,S.,Z helyaz kov ,L.,Herman ns,
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(cid:3)-CoVC, po ssess sim ilar functions, inc lu ding the mRNA cleavage c2o0r1 o0n. aGveirn uosm iinc cEhuarroapcetae nrizba atitosna onfd s ecvlaesrsei fiaccauttieo nr esopf ircaotroornya svy irnudsr eosmbea-sreelda toend
property, as SARS-CoV nsp1? A recent study comparing the reg- partial RNA -de pendent R NA p olym erase gene s equ ences. J. Virol . 84 (2 1),
ulation of gl obal ISG re sponse s by high ly pat hogenic res pira tory 11336– 11349.
viruses ,in cluding SAR S-CoVand M ERS-Co V,hassugge stedapos- Drosten,C.,Gunther,S.,Preiser,W.,vanderWerf,S.,Brodt,H.R.,Becker,S.,Rabenau,
H.,Pa nn ing,M.,K ole snikova ,L., Fou chie r,R.A. ,Be rger,A .,Bur guiere, A.M .,Cinatl,
tshibeleh mosetcIhFaNnrisetsipc odnivseer(gMenecnea cahmeorynge tthael. ,tw20o1 C4o).VIst iwn iallnbtaegoofngizrienagt JR.,i cE kicekrmts,aVn.n, S, tMu.r ,m Esecrr,iMou.,,V Nie., tGh r,yS w.,Knlae,n Kk.,, KH r.Dam.,Om sete, Srh., aMu as,nA u.gDu.,eSrcrha,m J.iC t.z, ,MHu., lDleore, rSr.,,
inte rest toe xplorethe roleofMER S- CoV nsp1i n mod ula tin gthe H.W.,20 03. Identific atio nofa no velcor onav irusinpati ents withsev ere acute
respir atory syndrome.N.E ng l. J.Med .348(20),1 96 7–1976,P MID :12690 091.
hnospst1 ,imwmithunites irnetsrpigounisnegs pdruorpinegrt iMesEaRnSd-CcohVa riancfteecrtiisotnic. sO,ivsearanlle, xCcoitV- FalseKyo,l aAs.sRa.,, J.ME. c,C1a9n9n7,. TRh.Me c.,o m Hamllo, n Wc .oJ.l,d Cinr idfrdali el,o lMde. Mrp., eFrsoormnsi:ciam , pMa.cAt.,o fWrhyicnoofvf,i rDu.s,
ing av enue fo r future re search tha t co uld potentially lea d to the andcoro nav irusin ase niordayc arec en ter. J.Am. Geriatr.S oc.45( 6) ,706–711.
Fan,K.,W ei,P.,Feng, Q. ,C hen,S. ,Huang, C.,Ma,L ., Lai,B .,Pei,J., Liu, Y.,C hen ,J.,Lai,L.,
discovery of novel players and pathways of host gene regulation. 2 00 4.Bio sy nthes is,p urific ati on,and sub stra te spe cifi city of sev ere acute re spir a-
torysy ndromecoro navirus3C-li kep roteinase .J.Biol.Che m .279(3 ),163 7–1642,
PMID :1456174 8.
Acknowledgments Gaglia,M.M .,Covarrubias,S.,Wong,W.,Glaunsinger,B.A.,2012.Acommonstrategy
for hostR NAdegradat ion bydiv erg entviruses.J. Viro l.86(1 7 ),9527–9 530.
We thank Adriana Paulucci-Holthauzen (Optical Microscopy Galagne,n Ce.,d Eifn fejureann teisa,l lLy.,a Afflemctasz caon r, oFn.,a v2i0r0u5s.g Aen poominet v me rusutastim onin iwgeitnho inm tehree prelipcalitciaosne.
Core, University of Texas Medical Branch) for support with the J.Viro l.79(24),15 016–15 026.
confo cal microsc op y analy sis. The work in Ma kino labo rator y is Gloza -Raus ch, F., Ip sen, A., Seebens, A., Gottsche, M., Panning, M., Drexler, J.F.,
supporte d by Public Health Se rvic e gran ts AI72493 and AI9910 7 Petersen,N .,A nnan, A., Grywna,K .,M uller,M., Pfef ferle,S.,D ros ten,C.,2 008.
Detection an dpreva len cepatter ns ofgroup Ic oronaviru ses inbats, nor thern
from the National Institute of Health. GorbGaelremnyaan,yA. .EEm.,e2 r0g0. 1In.fBeicgt.n Did iso. v1i4ru (s4)g, e6n2o 6m–6e3.1W .h encountand ord erof domains
matter.A dv.E xp.M ed.B iol.494,1 –17.
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Pleasecitethisarticleinpressas:Narayanan,K.,etal.,Coronavirusnonstructuralprotein1:Commonanddistinctfunctionsinthe

regulationofhostandviralgeneexpression.VirusRes.(2014),http://dx.doi.org/10.1016/j.virusres.2014.11.019