JOURNALOFVIROLOGY,Dec.2008,p.11948–11957 Vol.82,No.23 0022-538X/08/$08.00(cid:1)0 doi:10.1128/JVI.01804-08 Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved. Systematic Assembly of a Full-Length Infectious Clone of Human (cid:1) Coronavirus NL63 † Eric F. Donaldson,1,2§ Boyd Yount,2§ Amy C. Sims,2 Susan Burkett,3 Raymond J. Pickles,1,3 and Ralph S. Baric1,2* DepartmentsofMicrobiologyandImmunology1andEpidemiology2andCysticFibrosis/PulmonaryResearchand TreatmentCenter,3UniversityofNorthCarolina,ChapelHill,NorthCarolina D o w Received27August2008/Accepted19September2008 n lo Historically,coronaviruseswerepredominantlyassociatedwithmildupperrespiratorydiseaseinhumans. a d More recently, three novel coronaviruses associated with severe human respiratory disease were found, e d including (i) the severe acute respiratory syndrome coronavirus, associated with a significant atypical pneu- f moniaand10%mortality;(ii)HKU-1,associatedwithchronicpulmonarydisease;and(iii)NL63,associated ro with both upper and lower respiratory tract disease in children and adults worldwide. These discoveries m establish coronaviruses as important human pathogens and underscore the need for continued research h t toward the development of platforms that will enable genetic manipulation of the viral genome, allowing for tp rapid and rational development and testing of candidate vaccines, vaccine vectors, and therapeutics. In this :/ / report, we describe a reverse genetics system for NL63, whereby five contiguous cDNAs that span the entire jv genome were used to generate a full-length cDNA. Recombinant NL63 viruses which contained the expected i.a markermutationsreplicatedasefficientlyasthewild-typeNL63virus.Inaddition,weengineeredtheheter- s m ologousgreenfluorescentproteingeneinplaceofopenreadingframe3(ORF3)oftheNL63clone,simulta- . o neously creating a unique marker for NL63 infection and demonstrating that the ORF3 protein product is r g nonessentialforthereplicationofNL63incellculture.TheavailabilityoftheNL63andNL63gfpclonesand / recombinantvirusesprovidespowerfultoolsthatwillhelpadvanceourunderstandingofthisimportanthuman o n pathogen. M a r c h Coronaviruses(CoVs)arethelargestknownsingle-stranded Croup is a disease caused by many different viruses which is 1 positive-sense RNA viruses; they encode 5(cid:2)-capped, polyade- characterizedbythesuddenonsetofadistinctivebarkycough, 6 , nylated genomes ranging in size from 27 to 32 kb. Until re- stridor, hoarse voice, and respiratory distress resulting from 2 0 cently, CoVs were predominantly associated with severe dis- upper-airway obstruction (6). Croup accounts for roughly 1 5 ease in domestic animals, including bovines (bovine CoV), 250,000 hospitalizations each year in the United States, and b swine(porcineepidemicdiarrheavirusandtransmissiblegas- cases severe enough to require hospitalization can be fatal y T troenteritis virus [TGEV]), avians (infectious bronchitis virus (24). In addition, although understudied, hCoV infection can h [IBV])(2,8,30,36),andmice(mousehepatitisvirus[MHV]) result in a particularly severe pneumonia in the elderly, as e U (42),whileinfectionsinhumanswereprimarilyassociatedwith evidencedbyanoutbreakofhCoV-OC43inaretirementcom- n mild upper respiratory tract diseases caused by human CoVs munitythatwasassociatedwithan(cid:3)10%mortalityrate(41). iv e (hCoVs) hCoV-229E and hCoV-OC43 (30). However, the Taxonomically,CoVsareclassifiedasmembersoftheorder r s identification of a novel CoV as the etiological agent respon- Nidovirales, family Coronaviridae, genus Coronavirus (14, 30, it y sible for severe acute respiratory syndrome (SARS), an atyp- 37). Currently, the Coronavirus genus is further divided into o icalpneumoniawitha10%mortalityrate(53),indicatedthat threeprimarygroupsbaseduponserologicalandphylogenetic f I hCoVs are capable of causing severe disease in humans and data. Among the hCoVs, group 1 contains NL63 and hCoV- ow that unidentified hCoVs continue to exist in nature. More- 229E,whilegroup2strainsincludehCoV-OC43,HKU-1,and a recent discoveries have led to the identification of two addi- SARS-CoV (14). The CoVs are roughly 100 nm in diameter, Lib tional hCoVs: (i) HKU-1, which has been associated with areenveloped,andcontainthreecorestructuralspikes,includ- r a chronic pulmonary disease in humans (32), and (ii) NL63, inga180-to190-kDaspikeglycoprotein(S),a26-kDamem- rie whichhasbeenassociatedwithbothupperandlowerrespira- brane glycoprotein (M), and an envelope protein (E) of (cid:3)9 s torytractdiseaseinchildrenandadultsworldwide(1,5,9–11, kDa.ThegenomicRNAissurroundedbyahelicalnucleocap- 13, 23, 27, 28, 57, 62, 63). In addition, NL63 has been associ- sidcomposedofthe(cid:3)50-to60-kDanucleocapsidprotein(N) ated with croup in infants and young children (45, 60, 61). (46). Interestingly, despite large differences in S glycoprotein se- *Correspondingauthor.Mailingaddress:2107McGavran-Greenberg, quences (less than 50% identity at the nucleotide level) be- CB# 7435, Chapel Hill, NC 27599-7435. Phone: (919) 966-3895. Fax: tween SARS-CoV and NL63, both viral S glycoproteins have (919)966-0584.E-mail:[email protected]. been reported to interact with human angiotensin-converting †Supplemental material for this article may be found at http://jvi enzyme-2(ACE2)asareceptorfordockingandentryintocells .asm.org/. §Theseauthorscontributedequallytothiswork. (25, 34, 44, 52). Upon entry into the host cell, the genomic (cid:1)Publishedaheadofprinton25September2008. RNA is uncoated and immediately translated into two large 11948 VOL.82,2008 FULL-LENGTH INFECTIOUS CLONE OF NL63 11949 polyproteins(30,36).Thefirsttwo-thirdsoftheCoVgenome TABLE 1. Primersusedtogenerateinfectiousclonefragments encodes nonstructural replicase proteins in two overlapping andforPCR open reading frames (ORFs). The final one-third of the ge- Primer Nucleotideposition Comment nomeconsistsofthestructuralproteinsS,E,M,andN,aswell 5(cid:2)T7NL63(cid:1) 5(cid:2)endofgenome Creates5(cid:2)T7RNA as accessory proteins specific to different strains which are polymerasepromoter translated from a nested set of 3(cid:2) coterminal subgenomic NL63:A(cid:5) 6907–6928 CreatesBsmBIjunction mRNAs (30, 36). For NL63, there are six genes with a gene betweenAandB order of 5(cid:2)-replicase-S-ORF3-E-M-N-3(cid:2), wherein gene 1 en- NL63:B(cid:1) 6922–6948 CreatesBsmBIjunction codes the nonstructural replicase proteins, gene 2 encodes S, betweenAandB NL63:B(cid:5) 13537–13562 CreatesBsmBIjunction gene 3 encodes an accessory protein of unknown function betweenBandC D knownasORF3,gene4encodesE,gene5encodesM,gene6 NL63:C(cid:1) 13556–13579 CreatesBsmBIjunction o w encodesN,andanoverlappingORF6bhasbeenpredictedto betweenBandC n encode an additional accessory protein of unknown function NL63:C(cid:5) 19988–20011 CreatesBsmBIjunction lo betweenCandD a (47,59).AllCoVgenomescontaingroup-specificgenesinthe NL63:D(cid:1) 19991–20014 CreatesBsmBIjunction d finalone-thirdofthegenome,andmanyofthesegenesencode e betweenDandD d group-specific accessory proteins of undetermined function NL63:D(cid:5) 23845–23875 CreatesBstAPIjunction f r that are dispensable for replication (17, 68). Interestingly, betweenDandE o ORF3 of NL63 encodes a 225-amino-acid protein that is ho- NL63:E(cid:1) 23854–23882 CreatesBstAPIjunction m betweenDandE h mologous to ORF4 of hCoV-229E (53% similarity) and to NL63:E(cid:5) 3(cid:2)endofgenome Creates3(cid:2)poly(A)tailat ttp ORF3AofSARS-CoV(23%similarity)(39),andbothofthese endofgenome :/ / proteinshaveunknownfunctions. NL63-N1s Leadersequence Real-timePCRprimer jv Full-lengthcDNAconstructsofCoVgenomeshaverevolu- NL63-N1a 69–47antisenseofN Real-timePCRprimer i.a tionizedreversegeneticapplicationsincoronavirology(7,66– NL63-NR 255–236antisenseofN RTfo-PrC1R16-pnrtimamerp(liwciotnh sm 68). The strategy employed by our laboratory has been to NL63-N1s)for302-nt .o divide the genome into stable cDNA fragments flanked by amplicon rg native or engineered type IIS restriction endonuclease sites NL63-7(cid:1)3002 23582–23599genomic (cid:3)350nt5(cid:2)ofBstAPIsite / that form unique junctions at the ends of each fragment. In NL63-7R 24490–24471antisense (cid:3)650nt3(cid:2)ofBstAPIsite on addition,aT7promotersiteisaddedtothefirstfragment(at genomic M the5(cid:2)endofthegenome)toenableinvitrotranscriptionofthe ar c full-lengthcDNAfragmentafterligation,andapoly(A)tailis h included at the end of the last fragment (at the 3(cid:2) end). For 1 specimensresectedfrompatientsundergoingelectivesurgeryunderUNCInsti- 6 assembly, the fragments are cleaved by restriction digestion, tutionalReviewBoard-approvedprotocolsbytheUNCCysticFibrosisCenter , 2 whichremovesthenonnativeportionoftherestrictionsiteand TissueCultureCore.Briefly,primarycellswereexpandedonplastictogenerate 0 1 sequence, leaving unique ends that allow for a seamless, uni- passage1cellsandplatedatadensityof250,000cellsperwellonpermeable 5 directional ligation of the full-length cDNA clone. Transcrip- Transwell-Col(12-mmdiameter)supports(18,43).HAEculturesweregener- b atedbytheprovisionofanair-liquidinterfacefor4to6weekstoformwell- y tionofthefull-lengthcDNAisdrivenbytheT7promoter,and differentiated,polarizedculturesthatresembleinvivopseudostratifiedciliated T the full-length infectious RNA is transfected into cells. The epithelium(43). he individual fragments can be easily stored and amplified, and DesignoftheicNL63andicNL63gfpclones.Initialattemptsatgeneratinga U the smaller cDNA sizes are more manageable for targeted syntheticNL63clonebaseduponthegenomicNL63sequenceoriginallydepos- n mutagenesis studies. This infectious clone strategy has been itedinGenBankinJune2004withaccessionnumberNC_005831wereunsuc- iv cessful. However, this sequence was later updated with several corrections e successfullyemployedforTGEV(65),MHVstrainA59(67), (NC_005831.2);thesecorrectionswereengineeredintothesyntheticclone,but rs hCoVSARS-CoVstrainUrbani(66),andIBV(64). wewerestillunabletosuccessfullyrescuerecombinantvirus.Wethenacquired ity Inthisstudy,wereportandcharacterizethefirstfull-length thevirus(asakindgiftfromLiavanderHoek),sequencedit,andattemptedto o infectious clone of NL63 (icNL63). In addition, we replaced generatetheclonefromthissequence,butyetagainwereunsuccessfulatres- f I cuingrecombinantvirus.ThisviralsequencewasdifferentfromNC_005831.2atsix o ORF3,whichencodesaproteinofunknownfunction,withthe w positions,andthisviralstockwaslaterdeterminedtobeproblematic.Asecond a heterologous green fluorescent protein gene (GFP), simulta- shipmentofviruswasrequestedandusedtosuccessfullygeneratetheclonede- L neouslydevelopinganewmarkerforNL63infectionanddem- scribedhere(FJ211861).ItisimportanttonotethattheNL63genomeisATrich ib onstratingthattheproteinproductofORF3isnonessentialfor (66%),whichlikelycontributedtoproblemswithcloningandsequencing. ra Onceareliablevirussampleandsequencewereestablished,icNL63was r efficient viral replication in LLC-MK2 cells and primary cul- amplifiedfromviralcDNA(FJ211861)andclonedasasetoffivefragments ie turesofhumanciliatedairwayepithelium(HAE). s (Table1).Thefirstfragment,NL63-A,wasPCRamplifiedusingprimerset 5(cid:2)T7NL63(cid:1)(5(cid:2)-GGTACCTAATACGACTCACTATAGCTTAAAGAATTT TTCTATCTATAG-3(cid:2))andNL63:A(cid:5)(5(cid:2)-GCGGCCGCGTCTCCAGGAGC MATERIALSANDMETHODS TGTGGGTTGAACAG-3(cid:2)).TheseprimerscreatedaT7RNApromoterat Virusandcells.TheNL63virusandLLC-MK2cellsweregenerouslyprovided the 5(cid:2) end of the fragment and a BsmBI restriction site at the 3(cid:2) end, byLiavanderHoek.TheLLC-MK2celllineisanepitheliallineestablishedin respectively. The PCR product was gel isolated and then cloned into the the1950sfromapooledsuspensionpreparedfromkidneytissueofsixadult pCR-XLTOPOcloningvector(Invitrogen).Thesecondfragment,NL63-B, rhesusmonkeys(Macacamulatta)(26).TheLLC-MK2cellsweremaintainedat wasamplifiedusingprimersNL63:B(cid:1)(5(cid:2)-GCGGCCGCGTCTCCTCCTGC 37°Cwith5%CO inminimalessentialmediumsupplementedwith10%fetal ATATGTTATTATTGATAAG-3(cid:2))andNL63:B(cid:5)(5(cid:2)-GCGGCCGCGTCTC 2 cloneII(Gibco),10%tryptosephosphatebroth,andgentamicin(0.05(cid:4)g/ml)– TGCTGGGGAAGAAGCTATTATCAAG-3(cid:2)). Fragment NL63-C was am- kanamycin(0.25(cid:4)g/ml).NL63waspropagatedonthesecells,andtheinfections plifiedwithprimersNL63:C(cid:1)(5(cid:2)-GCGGCCGCGTCTCCCAGCACTCGTT weremaintainedat32°Cinincubatorsmaintainedat5%CO. GATCAACGCAC-3(cid:2))andNL63:C(cid:5)(5(cid:2)-GCGGCCGCGTCTCTCTTTAGA 2 Humannasalandtracheobronchialepithelialcellswereobtainedfromairway GACATTTTCACCATC-3(cid:2)).Bothofthesefragments,whichareflankedwith 11950 DONALDSON ET AL. J.VIROL. mega)overnightat4°Cinatotalreactionmixturevolumeof(cid:3)200(cid:4)ltogenerate thewild-type(wt)icNL63.FortheNL63cloneexpressingGFP(icNL63gfp),the NL63-EgfpfragmentwasusedinsteadoftheNL63-Efragment. Transfection of full-length transcripts. The full-length cDNAs were then furtherpurifiedbychloroformextractionandisopropanolprecipitation,tran- scribedusingaT7transcriptionkit(Ambion/AppliedBiosystems),cotransfected into 8 (cid:6) 106 LLC-MK2 cells in parallel with the N gene driven by an SP6 promoter,andtranscribedwithanSP6transcriptionkit(Ambion/AppliedBio- systems).LLC-MK2cellswereefficientlytransfectedwithonepulseat200Vand 950(cid:4)ferradsusingaBio-Rad(Hercules,CA)GenePulserXcellelectroporator. ElectroporatedLLC-MK2swereplatedinT25flasksandincubatedat32°Cfor FIG. 1. ThegeneorderofNL63andthestrategiesusedtogenerate upto7days. D theinfectiousclones.(A)TheNL63genomecontainssixgenes,with Detection of recombinant NL63 and NL63gfp replication. To determine if o ORF1aand-bencodingtheviralnonstructuralproteinsinvolvedpri- replicationoccurredintheicNL63-transfectedcultures,cellswereexaminedat w mpraortielyinisn,wreipthlicOaRtioFn3.(Ggeennees3)2etnoc6odeinncgodaeprthoetesintruocftuunraklnaonwdnafcucnecstsioorny. r7edgauylasrpionsttetrrvaanlssffeocrticoynt,ospoathhailcfeofffetchte(CcePllEs)a.nHdoswuepveernr,aCtaPnEtswwaesrenoptadsseafigneidtivweitaht nloa (B)TheNL63genomewasdividedintofivecDNAfragments(desig- freshcellsandmedia,andculturesobservedforanadditional7days,priortoa d nated NL63-A to NL63-E) flanked by unique type IIS restriction thirdpassage.Ateachpassage,infectedcellswereharvestedinTrizolreagent, ed endonucleasesitesthatenableaseamless,unidirectionalassemblyof total RNA was isolated, and reverse transcriptase PCR (RT-PCR) targeting f the entire genome. Fragment NL63-A contains a T7 promoter se- subgenomicRNAwasconductedusingprimersspecifictotheleadersequence ro quence and NL63-E a poly(A) tail [A(n)]. In addition, ORF3 was andthe5(cid:2)endoftheNgene(Table1).Briefly,viralRNAwasreversetran- m deletedandaheterologousgfpgeneinsertedinitsplaceintheNL63-E scribedtocDNAbyusingSuperScriptIII(Invitrogen)withmodificationtothe h fragmenttoformfragmentNL63-Egfp,whichwasusedtoengineeran protocol as follows. Random hexamers (300 ng) and total RNA (5 (cid:4)g) were ttp NL63clonewithGFPasamarkerofinfection. incubatedfor10minat70°C.Theremainingreagentswerethenaddedaccording : / tothemanufacturer’srecommendation,andthereactionmixturewasincubated /jv at55°Cfor1hfollowedby20minat70°CtodeactivatetheRT.ForRT-PCR, i. aforwardprimerintheleadersequence(NL63-N1s,GATAGAGAATTTTCT a s BsmBIsites,weregelisolatedandclonedintotheBigEasyv2.0linearcloning TATTTAGACTTTGTG)andareverseprimer(cid:3)250nucleotides(nt)intotheN m vector (Lucigen). Fragment NL63-D was amplified using primer NL63:D(cid:1) gene (NL63-NR, AGGTCCAGTACCTAGGTAAT) were used to generate a .o (5(cid:2)-GGTGAAAACGTCTCTAAAGATGG-3(cid:2))andprimerNL63:D(cid:5)(5(cid:2)-CA 302-bpproductbyPCR(Table1). r g GCAGCACAGTATGCAGAAAAAGCAAACC-3(cid:2)).Thisprimersetcreated Real-timeRT-PCRwasalsoconductedwiththesamecDNAtemplatesby / aBsmBIsiteatthe5(cid:2)endandaBstAPIrestrictionsiteatthe3(cid:2)end.Thelast usingaSmartCyclerII(Cepheid)withSybrgreen(dilutedto0.25(cid:6);Cepheid)to o n fragment,NL63-E,wasPCRamplifiedusingprimersNL63:E(cid:1)(5(cid:2)-TTTCT detect subgenomic cDNA with primers (7.5 pM) optimized to detect 116 nt M GCATACTGTGCTGCTGCCAACTG-3(cid:2)) and NL63: E(cid:5) (5(cid:2)-TTTTTTTTT spanningfromtheleadersequence(NL63-N1s;GATAGAGAATTTTCTTAT TTTTTTTTTTTTTTTGTGTATCCATATCAAAAACAATATCATTAACA TTAGACTTTGTG)tothe5(cid:2)endoftheNgene(NL63-N1a;CATGTAAAAT ar AGTACC-3(cid:2))andcontainedaBstAPIsiteatits5(cid:2)end.TheBstAPIsiteat GAAGGAGGAGGAA)(Table1).ThecDNAfromtheRTreactionofeach ch theNL63-DandNL63-Ejunctionwasengineeredbysilentmutagenesisinto viruswasusedatavolumeof2(cid:4)lforeachreactionmixture,withatotalreaction 1 thegenomicsequencesuchthatitwouldberetainedafterligationofthetwo mixture volume of 25 (cid:4)l. Omnimix beads (Cepheid) containing all reagents 6 , fragments, providing a unique marker for confirming that recombinant vi- exceptSybrgreen,primers,andtemplatewereusedtostandardizethereaction 2 ruseswerederivedfromtheclonedcDNA.Thelasttwofragments,NL63-D conditions.Inaddition,allproductswereverifiedbymeltingcurveanalysis. 0 1 and NL63-E, were gel purified and subsequently cloned into the pCR-XL For icNL63gfp, replication was confirmed by observing GFP fluorescence. 5 TOPOvector.The5(cid:2)approximately630bpoftheNL63-EfragmentwasPCR Infectionswerepassagedasdescribedaboveuntilnearly100%ofcellswereGFP b amplifiedusingtheprimersetNL63:E(cid:1)andNgfp2(cid:5)(5(cid:2)-CCATTATTGAA positive,atwhichtimethesupernatantsandcellswereharvested.Replication y CGTGGACCTTTTC-3(cid:2)).ThegeneencodingGFPwasamplifiedwithprimer wasfurtherverifiedbyRT-PCR,usingprimersspecifictosubgenomicNtran- T Ngfp1(cid:1) (5(cid:2)-GAAAAGGTCCACGTTCAATAATGGTGAGCAAGGGCGA scripts. he GG-3(cid:2))andprimerNgfp3(cid:5)(5(cid:2)-GGTCACCTTACTTGTACAGCTCGTCCA Plaque purification and titration of rescued virus. Supernatants harvested U TG-3(cid:2)).ThesetwoampliconswerejoinedinanoverlappingextensionPCR, frompassage3ofthetransfectionswerediluted1:10,and200(cid:4)lofdilutions n and the resulting product was cloned into the pCR-XL cloning vector. A from100to10(cid:5)5werepouredontoLLC-MK2cellsinsix-wellplates.Aftera1-h iv consensus clone was generated by using standard recombinant DNA tech- adsorptionperiod,5mlofoverlay(0.8%[wt/vol]LEagar[Lonza,Inc.],10%fetal e niques,andtheBstAPItoBstEIIfragmentfromthisclonewasinsertedinto cloneII,40%2(cid:6)minimalessentialmedium,1%gentamicin-kanamycin)was rs theNL63-Efragment,whichhadalsobeendigestedwithBstAPIandBstEII. addedtoeachculture,andtheinfectionsweremaintainedat32°Cfor7days.To ity TheresultingplasmidthencontainedgfpinplaceoftheNL63ORF3,andthis helpvisualizetheplaques,theplateswerestainedwithneutralredfor1hat o fragmentwasdesignatedNL63-Egfp(Fig.1). 32°C,andfiveplaqueswerepickedforeachvirus.Eachplaquewasincubatedin f I Systematicassemblyoffull-lengthNL63cDNAsforicNL63andicNL63gfp. phosphate-bufferedsaline(PBS)at32°Cfor30minandthenpouredontofresh o w Forassemblingtheinfectiousclones,plasmidsincorporatingcDNAfragments LLC-MK2cellsandgrownat32°Cforupto9daystoallowforthepropagation a NL63-AthroughNL63-EweretransformedintochemicallycompetentTop10 ofpurifiedvirus.FortheNL63gfprecombinantvirus,plaqueswereclearlyvisible L cells (Invitrogen) by heat shock at 42°C for 2 min and then plated on Luria by fluorescent microscopy, and five plaques were picked and propagated as ib Bertani(LB)plateswithappropriateselection(kanamycin[25(cid:4)g/ml]orchlor- described above. The titers for both recombinant icNL63 and recombinant r a amphenicol [20 (cid:4)g/ml]). Colonies were picked and grown under appropriate icNL63gfpweredeterminedbyplaqueassayusingLLC-MK2cells.Briefly,LLC- r selectionconditionsin5mlofLBbrothmaintainedat28.5°Cfor16to24hand MK2cellswereinfectedinduplicatewith200(cid:4)lsofeachserialdilutionof100 ie s thenpurifiedandscreenedbyrestrictiondigestion.Larger20-mlstockswere to10(cid:5)5ofrecombinanticNL63orrecombinanticNL63gfpinsix-wellplateswith grownat28.5°Cfor24hto48hforeachofthecDNAs.Purifiedplasmidswere a1-hadsorptionperiod.Fivemillilitersofoverlay(0.8%[wt/vol]LEagar[Lonza, then digested as follows: NL63-A, NL63-B, and NL63-C were digested with Inc.],10%fetalcloneII,40%2(cid:6)minimalessentialmedium,1%gentamicin- BsmBI,andNL63-DandNL63-Eweredigestedundertheappropriatecondi- kanamycin)wasaddedtoeachinfection,andtheplatesweremaintainedat32°C tionswithBsmBIandBstAPI.NL63-EgfpwasdigestedwithBstAPIandBsmBI. untilplaqueswereobserved(between4and7days).Tovisualizeplaques,plates Ofnote,thefragmentboundarieswereestablishedbytrialanderror,astoxic werestainedwithneutralredfor2hat32°Candthenincubatedovernightprior regionsinthegenomepreventedthecloningofseveralpreliminaryfragments. tocounting. Afterdigestion,fragmentswereelectroporatedon0.8%(wt/vol)agarosegel, Detectionofmarkermutations.AuniqueBstAPIrestrictionendonucleasesite and appropriate bands were excised and gel purified by using a Qiaex II gel was engineered into both the icNL63 and icNL63gfp clone to facilitate the extractionkit(Qiagen)withmodifications(67).Briefly,allfragmentswerere- unidirectionalligationoftheNL63-DandNL63-Efragments.Thisengineering suspendedin620(cid:4)lofQXIbuffer,11(cid:4)lQiaexIIsilicagelparticles,and12.5(cid:4)l introducedauniquebutsilentBstAPIrestrictionendonucleasesitefromposi- 3Msodiumacetateandelutedin35(cid:4)lofelutionbufferheatedto70°C.Purified tion23916to23925ofbothclones.Thissitewasusedtoverifythattheplaque- fragmentsNL63-AthroughNL63-EwereligatedbyusingT4DNAligase(Pro- purifiedvirusesharvestedoriginatedfromtheinfectiousclones.Primersflanking VOL.82,2008 FULL-LENGTH INFECTIOUS CLONE OF NL63 11951 the marker mutation (NL63-7(cid:1)3002 [ATAAGATTCAGGATGTTG] and and collected after a 10-min incubation at 32°C. All samples were stored at NL63-7R[GCAACAACCACAACAACCTG])(Table1)wereusedtoamplify (cid:5)80°CuntilassayedforplaqueformationonLLC-MK2cells. thisregionofthegenomeofwtNL63,recombinanticNL63,andrecombinant icNL63gfpbyRT-PCR.Inallcases,an(cid:3)1,000-bpPCRproductwasdetectedby electroporationona0.8%agarosegel,andthebandforeachviruswasexcised RESULTS andgelpurifiedbyusingaQiaexIIgelextractionkit(Qiagen)withmodifications (67)asdescribedabove.Analysisofthegenotypewasconductedbyrestriction DesignandassemblyoficNL63andicNL63gfp.Afull-length digestionofthe1,000-bpDNAwiththeBstAPIrestrictionendonuclease.Briefly, consensus sequence for NL63 was not possible, as all of the 25(cid:4)lofDNAforeachviruswasincubatedwith1(cid:4)lBstAPI,3(cid:4)lNEB(New full-lengthsequencesavailableattheNationalCenterforBio- EnglandBiolabs)buffer3,and1(cid:4)ldouble-distilledwaterat60°Cfor2hand thenelectroporatedona0.8%agarosegel.Theremaining5(cid:4)lofDNAwasused technologyInformation(NCBI)differedsignificantly(seeFig. tosequencethefragmentforgenotypeverification. S1inthesupplementalmaterial).Therefore,wesequencedthe D GrowthkineticsandRNAanalysis.Forthegrowthcurveanalysis,LLC-MK2 virusfromanefficientlyreplicatingstockandbuiltthecDNA o cellswereinoculatedatamultiplicityofinfection(MOI)of0.003PFU/cellin w clone based upon this sequence (FJ211861). For icNL63, the 12-wellplateswitha1-hadsorptionperiod,followedbythreewasheswithPBS. n Twomillilitersofmediumwasaddedtoeachculture,andtheinfectionsmain- NL63genomewasdividedintofivecDNAfragments(NL63-A lo a tainedat32°C.Thesupernatantswereharvested,at300(cid:4)lpertimepointwith through NL63-E) with unique type IIS endonuclease restric- d 300(cid:4)lofmediumaddedback,at0,8,24,48,72,96,120,144,168,and192h tion sites flanking each junction (Fig. 1). For icNL63gfp, the e d postinoculation(p.i.).Thetiterforeachvirusateachtimepointwasdetermined samestrategywasused,althoughtheheterologousGFPgene f byplaquetitrationinLLC-MK2cellsmaintainedat32°C,asdescribedabove. was inserted in place of and under the control of the same ro ForNorthernblotanalysis,totalRNAwasharvestedinTrizolreagent(Invitro- m gen),followingthemanufacturer’sprotocol,fromcellsinfectedatanMOIof transcriptional regulatory sequence as the accessory ORF3 in h 0.003PFU/mlandharvestedat96hp.i.ThetotalRNAwasdiluted,and5(cid:4)gwas NL63-E,andthisconstructwasdesignatedNL63-Egfp(Fig.1). tt p usedforeachvirus,includingwtNL63,recombinanticNL63,andrecombinant Toassembletheclones,thefragmentswerecutbyrestriction : / icNL63gfp.TheRNAfromeachinfectionwasseparatedbygelelectrophoresis, digestion (BsmBI and/or BstAPI) to remove the nonnative /jv t(r3a(cid:2)n-CsfTeCrrTedTGtoAaAnCitAroTcTelCluCloAsAeTmAemAbCrCanAeA,aTnCdTpGrCobTeCdTw-5it(cid:2)h;Na3g1e-nnetpcDosNitAionpsro1b51e portion of the restriction site and sequence, leaving unique, i.a to 180, italicized residues were biotinylated) designed to detect genomic and asymmetrical sticky ends. The digested fragments were then sm subgenomicRNAsbyusingaNorthernMax-Glysystem(Ambion)followinga ligated to generate the full-length cDNA clones, with NL63- . o modifiedprotocol.Briefly,theexactprocedurewasfolloweduptoandincluding EgfpbeingusedinsteadofNL63-EforicNL63gfp(Fig.1).A rg theovernight42°ChybridizationoftheprobetoRNAcross-linkedtothemem- T7 promoter site engineered at the 5(cid:2) end of the genome in / brane.Thenextmorning,themembranewaswashedonetimeinlow-stringency o fragmentNL63-Awasusedtodriveinvitrotranscriptionofthe n wash solution for 10 min, followed by a second wash in low-stringency wash solutionat45°Cfor2min.Athirdandfinalwashwasconductedfor2minat full-lengthcDNAtoinfectiousRNA(Fig.1).LLC-MK2cells M a 45°Cina50/50mixtureofhigh-stringencyandlow-stringencywashsolutions. weretransfectedwiththefull-lengthRNAforeachclone,and r c DetectionofbandswasaccomplishedbyusingaBrightStarBioDetectsystem thecellsmonitoredforCPE. h (Ambion)followingthemanufacturer’sprotocol.Themembranewasthenex- DetectionofrecombinanticNL63andrecombinanticNL63gfp 1 posedtofilm,whichwaspreparedforpublicationbyusingAdobePhotoshopCS. 6 IFA.LLC-MK2cellsweregrownto70to80%confluenceonfour-wellcham- replication.TodetermineifreplicationoccurredintheicNL63 , 2 ber slides (Lab-Tek, NUNC) and inoculated with recombinant icNL63 at an transfection cultures, cells were examined at regular intervals 0 MOI of (cid:3)1 PFU/cell or mock inoculated (medium alone). At 48 h p.i., the for CPE, which in LLC-MK2 cells is discernible as rounded 15 medium was aspirated, and the cells were fixed and permeabilized in (cid:5)20°C cells that appear on top of the monolayer. In the case of b methanolovernight.ThecellswererehydratedinPBSfor30minandblockedin y recombinanticNL63,CPEwasnotconclusiveatanytime,but buffercomprisedofPBSwith5%bovineserumalbumin.Allsubsequentimmu- T nofluorescence assay (IFA) steps were conducted at 25°C in IFA assay wash recoveryofrecombinanticNL63wasdetectedinpassage3by h e buffercomprisedofPBScontaining1%bovineserumalbuminand0.05%Non- RT-PCR amplification of leader-containing transcripts and U idetP-40.Afterbeingblocked,thecellswereincubatedintheprimaryantibody, furtherverifiedbyanIFAwithanti-Nantibodyandbyplaque n anti-N(anti-NL63N;generouslyprovidedbyLiavanderHoek),diluted1:1,000, titration(Fig.2AtoD). iv for1h.ThecellswerethenwashedinIFAassaywashbufferthreetimesat10 e min/wash.Next,thecellswereincubatedinthesecondaryantibody(goatanti- For recombinant icNL63gfp, replication was confirmed by rs rabbitAlexa488,diluted1:1,000;MolecularProbes)for45min.Next,thecells observingGFPfluorescencefollowingtransfectionorinocula- ity werewashedthreetimesat10min/wash,followedbyafinalwashof30minin tion(Fig.3).Whilefluorescentfociwereobservedasearlyas o PBS.Thecellswerethenvisualizedbyfluorescentmicroscopy.Theimageswere 2 days posttransfection at 32°C, additional passages at 7-day f I preparedforpublicationbyusingAdobePhotoshopCS. o intervals were necessary to infect most of the cells in the w Westernblotting.LLC-MK2cellsweremockinoculated(mediumalone)or a inoculatedwithwtNL63,recombinanticNL63,orrecombinanticNL63gfpatan culture.By7daysp.i.ofpassage3,therewasobviousCPEin L MOIof0.003,andat144hp.i.,cellswerewashedin1(cid:6)PBS,lysedinbuffer therecombinanticNL63gfp-infectedcells(Fig.3A).Cellsand ib containing 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% deoxycholine, 1% supernatantswereharvestedwhennearly100%ofcellsshowed ra NonidetP-40,0.1%sodiumdodecylsulfate(SDS)andpostnuclearsupernatants r wereaddedtoanequalvolumeof5mMEDTA–0.9%SDS,resultinginafinal strong evidence of GFP fluorescence. Replication and the ie presenceofviralsubgenomicmRNAencodingviralstructural s SDSconcentrationof0.5%.Equivalentsamplevolumeswereloadedonto4to 20%Criteriongradientgels(Bio-Rad)andtransferredtopolyvinylidenediflu- proteins or GFP were further verified by RT-PCR (data not oridemembranes(Bio-Rad).Theblotswereprobedwithpolyclonalrabbitan- shown). tiseradirectedagainsttheNL63Nprotein(kindlyprovidedbyLiavanderHoek) Plaque purification and titration of rescued virus. Viruses diluted1:1,000orwithantiseradirectedagainstGFP(Clontech)diluted1:1,000 rescued from the icNL63 and icNL63gfp transfections were anddevelopedusingchemiluminescencereagents(AmershamBiosciences). InoculationofHAEcultures.Priortoapicalinoculation,theapicalsurfacesof plaque purified, and stocks were propagated on LLC-MK2 HAEwererinsedthreetimesover30minwithPBSat37°C,andinoculations cells. For recombinant icNL63, plaques were round and clear were performed at 32°C with 200 (cid:4)l of recombinant icNL63 or recombinant with an approximate diameter of 2.5 to 3 mm (Fig. 2). For icNL63gfpvirusstock((cid:3)104PFU/ml).Followinga2-hincubationat32°C,the recombinant icNL63gfp, plaques were clearly visible by fluo- inoculantwasremoved,andHAEwasmaintainedat32°Cfortheremainderof rescent microscopy (Fig. 3D) and similar to recombinant theexperiment.Togenerategrowthcurvesatspecifictimesafterviralinocula- tion,120(cid:4)loftissueculturemediumwasappliedtotheapicalsurfaceofHAE icNL63, with the main difference observed between recombi- 11952 DONALDSON ET AL. J.VIROL. D o w n lo a d e d f r o FIG. 3. Detection of replication in LLC-MK2 cells infected with m FIG. 2. Detection of replication in cells inoculated with icNL63 icNL63gfp.(A)CPEwasevidentincellstransfectedwithicNL63gfp h passage 3 supernatants. (A) Supernatants from the icNL63 transfec- afterpassage3,asindicatedbyroundedclumpsofcellsthatgrewon tt top of the monolayer, forming long continuous striations. (B) GFP p tionflaskwereusedtoinoculatefour-wellchamberslidesofLLC-MK2 : fluorescencewasdetectedasearlyas24hp.i.;however,thespreadof / cellstoassayforthepresenceofNL63NproteinbyIFA.Nucleocapsid / GFP fluorescence to nearly every cell required three passages. jv plorcoatleiiznatiwoanspdaettteecrnte.d(Bin) Ithnesmeoccekl-lisn,feacntdeditceelxlsh,ibNitepdroatepineriwnausclneaort (C) Cells infected with wt NL63 virus generated no detectable fluo- i.a rescencebeyondthenormalbackgroundlevel.(D)Cellsinfectedwith s detected.(C)WhileCPEwasnotdramatic,thesupernatantsfromthe m recombinant icNL63gfp and covered with overlay medium formed icNL63 transfection resulted in various sizes of plaques. (D) Sub- . plaquesdistinguishedbyfluorescentfociwithinthemonolayer. o genomictranscriptionwasverifiedbyreal-timeRT-PCRbyassaying r g forleader-containingtranscripts.AmplificationoftheviralcDNAfor / wtNL63(f)andrecombinanticNL63(Œ)occurredatnearlyidentical o n cyclethresholds,suggestingthatrecombinanticNL63generatedquan- M titiesofsubgenomicNgenethatweresimilartothequantitiesgener- atedbywtNL63.LLC-MK2cells(●)andthecontrolwithouttemplate sequenced to verify that the marker mutation was present in ar ((cid:6))showednoamplification. the two clones, and this was the case for both recombinant c h viruses(Fig.4). 1 Growth kinetics and RNA analysis. To determine if the 6, recombinant viruses rescued from the two clones generated 2 nanticNL63andrecombinanticNL63gfpplaquesbeingoneof 0 similarquantitiesofviralmRNAs,Northernblotanalysiswas 1 definition, as the recombinant icNL63 plaques were clearly 5 performed(Fig.5A).Theresultsofthisanalysisdemonstrated visible, while the recombinant icNL63gfp virus formed fuzzy b thatwhilerecombinanticNL63gfpdidnotgenerateamountsof y plaquesthatwereslightlysmaller.Interestingly,allplaquesfor T recombinant icNL63gfp were fluorescent. The viral titers de- viralmRNAsequivalenttothosegeneratedbywtNL63,itdid h rived from recombinant icNL63 plaques reached 2 (cid:6) 104 containaunique,appropriatelysizedmRNAindicativeofGFP e U PFU/ml in LLC-MK2 cells, while the titers for recombinant (Fig.5A).Todetermineiftherecombinantvirusesgenerated n icNL63gfp were slightly higher, reaching a titer of (cid:3)8 (cid:6) 104 fromthetwoclonesgrewwithgrowthkineticssimilartothose iv e PFU/ml.TherecombinanticNL63virustiterswereconsistent ofwtNL63,agrowthcurveanalysiswasconducted.Ingeneral, rs withthepeakvirustitersreportedpreviouslyforwtNL63virus viral growth was similar for wt NL63 and both recombinant ity (2(cid:6)105PFU/ml)(46,47,59). viruses, although recombinant icNL63 appeared to have a o Detection of marker mutations in the rescued viruses. As shorter lag phase than wt NL63 and recombinant icNL63gfp f I o part of the cloning strategy, a silent BstAPI restriction endo- (Fig.6). w nuclease site was engineered into both the icNL63 and Analysis of recombinant viruses by Western blotting. A a L icNL63gfp clones at the NL63-D and NL63-E or NL63-Egfp Westernblotanalysiswasconductedtocomparethelevelsof ib junctiontofacilitatetheunidirectionalligationoftheNL63-D viralproteinexpressionofwtNL63,recombinanticNL63,and ra and NL63-E fragments. To verify that each clone had this recombinanticNL63gfpbyusingantiseraagainstNL63Nand rie marker mutation, viral RNA was harvested from cultures in- GFP.WhileallthreevirusesgenerateddetectablelevelsofN s fected with plaque-purified stocks, and the (cid:3)1,000-nt region protein, there was an obvious reduction in the recombinant flankingtheBstAPIsitewasamplifiedbyRT-PCR(Fig.4).In icNL63gfplane,suggestingthatthisvirusdoesnotproducewt all cases, an (cid:3)1,000-nt PCR product was present following levelsofviralproteins(Fig.5C);onlyrecombinanticNL63gfp electroporation, and the band for each virus was excised and expressedthe28-kDaGFPprotein(Fig.5B).Theseresultsare gel purified (Fig. 4). The purified 1,000-nt DNA from each consistent with those of Northern blotting, which demon- viruswasthendigestedwiththeBstAPIrestrictionendonucle- strated that recombinant icNL63gfp is also deficient in RNA ase. Viral cDNA harvested from icNL63 and icNL63gfp re- synthesisbutgeneratesasubgenomicRNAconsistentwiththe combinant viruses was digested into two bands of 600 nt and GFP gene engineered into the clone (Fig. 5A). The recombi- 400nt,respectively(Fig.4),whilethewtNL63viralcDNAwas nant icNL63 virus mimics wt NL63 in RNA synthesis and not cleaved by this enzyme (Fig. 4). Further, this region was proteinexpression(Fig.5AtoC). VOL.82,2008 FULL-LENGTH INFECTIOUS CLONE OF NL63 11953 couldreplicateefficientlyintheseculturesofHAE.Theinfec- tionofHAEbyrecombinanticNL63gfpwasdetectedasfluo- rescentcellsonday1(24hp.i.),andthefluorescenceincreased in intensity at each time point of the experiment (Fig. 7), although its spread to additional cells appeared to be limited (Fig. 7). In HAE, recombinant icNL63 CPE was not evident, althoughviruswasisolatedanddeterminedtoreachpeaktiters of 5 (cid:6) 104 on day 4 (96 h p.i.). In contrast, recombinant icNL63gfp achieved peak titers of 7.5 (cid:6) 103 on day 5 (120 h p.i.)(Fig.7). D These results indicate that recombinant NL63 viruses de- o w rivedfromthecDNAclonesreplicatedasefficientlyasbiolog- n icallyderivedNL63andgrewinLLC-MK2cellsandHAEand lo a thatthereplacementofORF3withthegfptransgeneallowed d theexpressionofGFPininfectedcells.WhileORF3appears e d to be nonessential in cell culture, there were differences in f r RNA synthesis, protein expression, plaque morphology, and o m FIG. 4. Verificationofthemarkermutationinrescuedvirusfrom growthinHAEthatsuggestthatORF3mayplayanimportant h icNL63 and icNL63gfp. A silent BstAPI site introduced into both undeterminedroleduringinvivoinfection. t t clonesattheNL63-DandNL63-EorNL63-Egfpjunctionswasusedto p : verifythatthevirusesrescuedfromthetransfectionflasksweregen- / / eratedfromtheclonedcDNA.(A)A1,000-ntregionflankingthissite jv wasamplifiedbyPCR,digestedbyBstAPI,andanalyzedbygelelec- DISCUSSION i.a trophoresis.Lane1,marker;lane2,wtNL63wasnotcutbyBstAPI; s lane3,theDNAfromthisregionintheicNL63recombinantviruswas AreversegeneticssystemforNL63providesaplatformfor m . cleaved by BstAPI; and lane 4, the DNA from this region in the studyingthisvirusindepthandisanecessarycomponentinthe o r icNL63gfprecombinantviruswasalsocleavedbyBstAPI.Toverifythe developmentofvaccinecandidates,vaccinevectors,andther- g / genotype, this region was sequenced for icNL63, icNL63gfp and wt apeutics.Inthisstudy,wedevelopedareversegeneticssystem o NL63.Molecularsizesinnucleotidesareshownontheleft.(B)The n for NL63 and rescued recombinant NL63 viruses by utilizing chromatogramsoficNL63andicNL63gfpwereidenticalinthisregion M andareshownhere.(C)ThesequencechromatogramofwtNL63in the same cloning strategy employed to generated infectious a r thisregion.Thedifferencesbetweenthetwochromatogramsareindi- clones of TGEV (65), MHV (67), IBV (7), and SARS-CoV c catedbytheboxes.TheBstAPIrecognitionsiteisGCANNNNNTGC. (66). In general, plaque-purified wt NL63 and recombinant h 1 icNL63 viruses were indistinguishable in cell culture, as both 6 , generatednearlyroundplaquesof2.5to3mmindiameterin 2 0 Recombinant NL63 infections in HAE cultures. A primary LLC-MK2 cells (Fig. 2), exhibited similar levels of RNA syn- 1 5 target for infection by other hCoVs, like SARS-CoV and thesis and protein expression (Fig. 5), and replicated with b hCoV-229E, is ciliated cells (51) of the upper airways. As similargrowthkinetics(Fig.6).Interestingly,althoughrecom- y ciliated cells express robust levels of ACE2 (20), we next de- binant icNL63 appeared to have a shortened lag phase, this T h termined if the recombinant icNL63 and icNL63gfp viruses differencefellwithintherangeoferrorfortheexperimentand e U n iv e r s it y o f I o w a L ib r a r ie s FIG. 5. VerificationofreplicationbyNorthernandWesternblotting.(A)ANorthernblotanalysiswasconductedusingviralRNAharvestedfrom LLC-MK2cellsinfectedwithwtNL63,recombinanticNL63,orrecombinanticNL63gfp.Sixbandsweredetectedwhichcorrespondtothesixviralgenes. Theproteinproductthatarisesfromeachgeneisindicatedinparentheses.TherecombinanticNL63gfpvirusproducedanmRNAbandthatcorresponds totheGFPtransgeneandlackedthemRNAcorrespondingtogene3(ORF3),whereaswtNL63andrecombinanticNL63virusesproducedthesix expectedviralRNAs.(B)AWesternblotwasconductedusingtheanti-GFPantibodytodetecttheGFPproteinfrominfected-celllysates,whichwas onlydetectablefortherecombinanticNL63gfpvirus.(C)AWesternblotwasconductedusingtheanti-NantibodytodetecttheNprotein.Nprotein expressionwasreducedfortherecombinanticNL63gfpvirus.Molecularmassesinkilodaltonsareshownbetweentheblots. 11954 DONALDSON ET AL. J.VIROL. D o w n lo a d e d f r o FIG. 6. GrowthkineticsofwtNL63andrecombinanticNL63and m icNL63gfpviruses.Allthreevirusesgrewwithsimilargrowthkinetics, althoughrecombinanticNL63virus((cid:1))appearedtohaveashorterlag ht phase. The growth kinetics of wt NL63 (●) and recombinant tp icNL63gfp (Œ) were nearly identical at every time point until day 7 :// (168 h p.i.), when wt NL63 reached peak titers of 5 (cid:6) 105 PFU/ml. jv RecombinanticNL63reachedapeaktiterof3(cid:6)105onday6(144h i.a p.i.),andrecombinanticNL63gfpreachedatiterof1.5(cid:6)105PFU/ml s m on the same day. All virus titers at each time point fall within 1 FIG. 7. TimecourseofinfectionofHAEandviraltitersproduced . standarddeviation,suggestingthatalltitersaresimilar. byrecombinanticNL63andicNL63gfp.HAEcultureswereinoculated o r withrecombinanticNL63andrecombinanticNL63gfpandmonitored g for6days.FluorescencewasdetectedinHAEinfectedwithrecombi- / o nanticNL63gfpby24hp.i.andcontinuedtobedetectedthroughout n waslikelyduetodifferencesincellcultureandnotdifferences thecourseoftheexperiment.(AtoE)Imagesshowingtherecombi- M nant icNL63gfp-infected HAE at 24-h intervals beginning with the a in the recombinant icNL63 virus (Fig. 6). In addition, recom- 24-hp.i.timepointandcontinuingthrough120hp.i.(F)SARS-CoV- rc binanticNL63viralRNAcontainedtheuniquemarkerintro- GFP-infectedHAEculturesat120hp.i.producedsmallerfluorescent h duced into the clone sequence to allow verification that the foci.(G)TitersweredeterminedforbothrecombinanticNL63(black) 1 6 virus was derived from the engineered clone (Fig. 4). To test andrecombinanticNL63gfp(hatched)ateachtimepoint. , 2 the utility of this reverse genetics system, we removed the 0 1 accessory ORF3 from the NL63 genome and replaced it with 5 the gene for GFP, creating a unique system for monitoring aminoacidlevelinallisolates,whilemostORF3genesvaried b y NL63 infection in real time. In addition, the results of this 1to2%atthenucleotidelevel.Whilethissuggestsanimpor- T experimentdemonstratedthattheORF3proteinisnonessen- tantrolefortheORF3proteinproductinvivo,ORF3deletion h e tialforthereplicationofNL63inLLC-MK2cells.Thisobser- from icNL63gfp was not deleterious to replication in LLC- U vationwasinagreementwiththeresultsofseveralotherstud- MK2 cells. This finding was not surprising given that the dis- n ieswhichhaveshownthatCoVaccessoryandluxuryORFsare tantly homologous proteins ORF4 in hCoV-229E (12) and iv e dispensableforinvitroreplication(17,66,68). ORF3a in SARS-CoV (17, 68) have also been shown to be rs ThereplacementofORF3withtheheterologousGFPgene nonessential in cell culture. Group-specific ORFs of several ity resulted in infected cells that were detectable by fluorescent different CoVs have been deleted, and while some deletions o f microscopy(Fig.3),andtherecombinanticNL63gfpvirusgen- attenuated pathogenesis or viral growth in vitro, the function I o eratedtitersandexhibitedgrowthkineticsthatwereessentially ofmostisunknown.TwoexceptionsaretheORF3bandORF6 w a identical to those of wt NL63 and recombinant icNL63 in products of SARS-CoV, which have been characterized as L LLC-MK2cells(Fig.6).Interestingly,recombinanticNL63gfp interferon antagonists (16, 29). Whether ORF3 of NL63 en- ib virusgeneratedplaquesthatwereslightlysmaller(2to2.5mm codes interferon antagonist activities remains to be deter- ra r in diameter versus 2.5 to 3 mm), with irregular borders, and mined. In preliminary studies, we have observed that GFP- ie were considerably less-well defined than wt NL63 plaques taggedORF3proteinlocalizestothenucleuswhentransfected s (datanotshown).Althoughthedifferentplaquephenotypedid intocells(datanotshown). notcorrelatetoareductioningrowthkinetics(Fig.6),recom- InadditiontothetransfectionofLLC-MK2cells,recombi- binant icNL63gfp had modestly reduced levels of RNA syn- nant icNL63 and recombinant icNL63gfp were used to infect thesis(Fig.5A)andproteinexpression(Fig.5C)comparedto primary HAE, which supports the infection and spread of those of wt NL63. The lack of an animal model for studying other respiratory pathogens, such as influenza virus, respira- NL63madeitimpossibletodetermineifORF3playsarolein tory syncytial virus (RSV), SARS-CoV, and paramyxoviruses viralpathogenesisinvivo. (4,49–51,56,69).SinceNL63infectsboththeupperandlower At the time of this study, 12 NL63 genomes containing a respiratory tracts and HAE cultures maintain the form and full-length ORF3 sequence were available at NCBI, and functionofhumanciliatedairways,theseculturesrepresenta among these, ORF3 was strictly (100%) conserved at the relevant and authentic model for studying this virus. Not sur- VOL.82,2008 FULL-LENGTH INFECTIOUS CLONE OF NL63 11955 prisingly, both recombinant viruses grew in HAE (Fig. 7G), optimalmucosalimmuneinduction;(ii)virusinductionofro- andrecombinanticNL63gfpwasdetectablebyfluorescenceby busthumoral,mucosal,andpossiblycellularimmunerespons- 24 h p.i. with increased fluorescent intensity over time, al- es; and (iii) a genome size, organization, and helical nucleo- thoughitsspreadfromcelltocellwassomewhatlimited(Fig. capsid assembly scheme that allow (a) coordinated gene 7AtoE).Incontrast,SARS-CoVexpressingGFPinanacces- expression;(b)thedeletionofluxurygenesthatarenonessen- sory ORF was used to infect HAE cultures, and spreading of tial for replication; and (c) stable incorporation of multiple, this virus was evident over the course of the infection (Fig. largegeneinserts(17,66,68).Asaproofofprinciple,inthis 7G).SpreadingofRSVinHAEhasalsobeenobserved(70). report we demonstrated that replacing the luxury ORF3 with Interestingly, the fluorescent foci detected with recombinant heterologous gfp allowed stable targeting of GFP to the cells icNL63gfp infection were smaller and generally more diffuse infectedbyNL63.Hypothetically,multipleheterologousanti- D than those observed in HAE infected with the recombinant genswithnoveltranscriptionalregulatorysequencescouldbe o w SARS-CoVexpressingGFP(Fig.7F)(50).Althoughthismay engineered into the intergenic space between a propagation- n beduetovariabilitybetweencultures,wecannotruleoutthe deficient set of structural genes, providing a multivalent, rep- lo a possibility that ORF3 is nonessential for replication in LLC- lication-competent, propagation-deficient virus vector vaccine d MK2cellsbutmayplayaroleinmore-relevanttissuesthatare approach capable of immunizing against multiple viruses si- ed relatedtoreplicationinnonimmortalizedcelllines.Theresults multaneously. The complementation of such a vector in cells fr of previous studies have shown that parainfluenza virus and o expressingthepropagation-deficientgenecouldbeutilizedto m RSV infection of HAE mimic their in vivo replication capac- assembleviablevirusesthatwouldactasone-hitvectors,gen- h ities, while in cell lines, attenuation is not seen (69, 70). We t eratingantigenatthetargetedcellwhilelackingthenecessary t p speculatethatORF3mightberequiredforefficientviralegress componentstogenerateaviableviralparticle.Asimilarstrat- :/ / in HAE, as spreading within cultures was reduced in the re- egywasreportedforTGEVwherebytheEgenewasexpressed jv combinanticNL63gfpvirus.Thisissupportedbythefactthat in a replicon cell system, which allowed the TGEV vaccine i.a recombinanticNL63gfpappearedtogrowlessefficientlythan vectortobepackagedasaviablevirusandgrowntohigh-titer sm recombinanticNL63inHAE(Fig.7G). . replicon stocks (40). An NL63-based vaccine vector would o Engineering GFP into icNL63 and rescuing recombinant potentially replicate extensively in the upper and, to a lesser rg viruses expressing this marker protein provides an important / extent,lowerrespiratorytractbytargetingcellpopulationson o reagent enabling the testing of drugs and therapeutic agents n mucosalsurfacesthatexpressACE2,suchasHAE,lungalve- againstinfectionsinrealtime.Severalotherviralsystemshave M olarepithelialcells,andoralandnasalmucosa(21). a utilized a similar approach to generate novel reagents which r Acurrentimpedimentinthefieldisthelackofeitherasmall c allowhigh-throughputtherapeuticscreening(3,15,19,22,31, h or large animal model of NL63 replication or pathogenesis. 33,35,38,54,58).InLLC-MK2cells,weobservedviralspread 1 WhilemiceexpressanACE2variant,virusreplicationhasnot 6 throughouttheculture,eventhoughtherewerenodetectable , been detected in mice infected with NL63. Moreover, the 2 differences in CPE. While only a few fluorescent foci were 0 SARS-CoV receptor binding domain required adaptations in 1 presentatearlytimesposttransfection,overtimeweobserved 5 the spike protein to accommodate the structural differences more and more fluorescence spreading to neighboring cells. b imposed by the variations between the human and mouse y Fluorescence was also detectable in the HAE, providing a T ACE2molecules(48).SinceNL63utilizesadifferentreceptor platformtomonitortheinfectionofprimaryHAEinrealtime. h binding domain and a different set of interactions, there may e Importantly,inallcasestheGFPtransgenewashighlystablein U beevenmorechangesnecessarytoadaptNL63toreplicatein theNL63genomeforover2monthsinculture,animportant n featureforthedevelopmentofhCoVvaccinevectors. mice. In addition, more-robust cell culture systems will be iv e AllhCoVs,withtheexceptionofSARS-CoV,growpoorlyin required for the propagation of NL63 as a vaccine vector rs cell culture, while some, including hCoV-OC43 and hCoV- system.Ingeneral,icNL63makesapowerfulvaccineplatform, ity 229E, do not generate plaques, making downstream assays asCPEcanbedetectedinLLC-MK2cells;itmayusethesame o difficult to perform. Moreover, a new hCoV associated with receptor as has been described for SARS-CoV, a homologue f I o pneumonia in adults, known as HKU-1, has never been suc- ofwhichispresentinmice,andthestableexpressionofGFP w cessfully cultured in vitro. Poor growth in culture makes it willallowreal-timemonitoringofinfections.Thesecharacter- a L extremely difficult to rescue recombinant viruses from full- istics are in contrast to the hCoV-229E clone, which grows ib length cDNA clones, which makes manipulating these virus poorlyandisdifficulttodetectbyCPE(55). ra genomesdifficult.NL63hasanintermediategrowthphenotype The infectious clones described in this report provide a re- rie in cell culture, where it grows at an optimal temperature of versegeneticsplatformthatcanbeusedtodevelopcandidate s 32°C, requiring 7 days to reach peak titer in LLC-MK2 cells, vaccinestrainsthatmightonedayreducetheimpactofNL63 whileSARS-CoVgrowsat37°Cwithadistinctgrowthadvan- asanimportantrespiratorypathogenthatinfectschildrenand tage,allowingittoreachpeaktitersin(cid:7)48hp.i.inVerocells. adults worldwide. The benefits of such a vaccine would be to These observations indicate that more-robust culture systems reduce the overall disease burden in children and perhaps are needed for the development of NL63 as a vaccine vector reduce cases of croup. The availability of icNL63 and forhumanuse. icNL63gfpprovidesresearchopportunitieswhichwilladvance There are several distinct features that suggest that NL63 our understanding of in vivo tropisms and assist in the devel- would be an efficacious vaccine vector, and these include (i) opment of small and large animal models of infection. More- natural targeting of respiratory pathogen antigens to the ap- over,detailedgeneticmanipulationofthegenomewillassistin propriatemucosalepithelialcellsliningtheupperairwaysfor understandingtheroleofviralgenesinreplicationandpatho- 11956 DONALDSON ET AL. J.VIROL. genesisandleadtothedevelopmentofhCoV-basedvectored A.D.Osterhaus,W.Timens,A.J.Turner,G.Navis,andH.vanGoor.2007. vaccines. TheemergingroleofACE2inphysiologyanddisease.J.Pathol.212:1–11. 21. Hamming,I.,W.Timens,M.L.Bulthuis,A.T.Lely,G.J.Navis,andH.van Goor.2004.TissuedistributionofACE2protein,thefunctionalreceptorfor SARS coronavirus. A first step in understanding SARS pathogenesis. ACKNOWLEDGMENTS J.Pathol.203:631–637. WegratefullyacknowledgeLiavanderHoekandKrzysztofPyrcfor 22. Hammoumi,S.,C.Cruciere,M.Guy,A.Boutrouille,S.Messiaen,S.Lecol- linet, and L. Bakkali-Kassimi. 2006. Characterization of a recombinant providing NL63 virus, LLC-MK2 cells, viral RNA, and sequence in- encephalomyocarditisvirusexpressingtheenhancedgreenfluorescentpro- formation. tein.Arch.Virol.151:1783–1796. This work was supported by research project grants AI023946-15 23. Han, T. H., J. Y. Chung, S. W. Kim, and E. S. Hwang. 2007. 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