Genome structure and transcriptional regulation of human ...

We described previously that the newly identified HCoV-NL63 virus has a typical coronavirus genome structure and gene order [6]. The nucleotide ... Skiptomaincontent Advertisement SearchallBMCarticles Search DownloadPDF Research OpenAccess Published:17November2004 GenomestructureandtranscriptionalregulationofhumancoronavirusNL63 KrzysztofPyrc1,MaartenFJebbink1,BenBerkhout1&LiavanderHoek1  VirologyJournal volume 1,Article number: 7(2004) Citethisarticle 21kAccesses 77Citations Metricsdetails AbstractBackgroundTwohumancoronavirusesareknownsincethe1960s:HCoV-229EandHCoV-OC43.SARS-CoVwasdiscoveredintheearlyspringof2003,followedbytheidentificationofHCoV-NL63,thefourthmemberofthecoronaviridaefamilythatinfectshumans.Inthisstudy,wedescribethegenomestructureandthetranscriptionstrategyofHCoV-NL63byexperimentalanalysisoftheviralsubgenomicmRNAs.ResultsThegenomeofHCoV-NL63hasthefollowinggeneorder:1a-1b-S-ORF3-E-M-N.TheGCcontentoftheHCoV-NL63genomeisextremelylow(34%)comparedtoothercoronaviruses,andwethereforeperformedadditionalanalysisofthenucleotidecomposition.Overall,theRNAgenomeisverylowinCandhighinU,andthisisalsoreflectedinthecodonusage.InspectionofthenucleotidecompositionalongthegenomeindicatesthattheC-countincreasessignificantlyinthelastone-thirdofthegenomeattheexpenseofUandG.Wedocumenttheproductionofsubgenomic(sg)mRNAscodingfortheS,ORF3,E,MandNproteins.WedidnotdetectanyadditionalsgmRNA.Furthermore,wesequencedthe5'endofallsgmRNAs,confirmingthepresenceofanidenticalleadersequenceineachsgmRNA.NorthernblotanalysisindicatedthattheexpressionlevelamongthesgmRNAsdifferssignificantly,withthesgmRNAencodingnucleocapsid(N)beingthemostabundant.ConclusionsThepresenteddatagiveinsightintotheviralevolutionandmutationalpatternsincoronaviralgenome.FurthermoreourdatashowthatHCoV-NL63employsthediscontinuousreplicationstrategywithgenerationofsubgenomicmRNAsduringthe(-)strandsynthesis.BecauseHCoV-NL63hasalowpathogenicityandisabletogroweasilyincellculture,thisviruscanbeapowerfultooltostudySARScoronaviruspathogenesis. BackgroundUntilrecentlyonlytwohumancoronaviruseswereknown–humancoronavirus(HCoV)229EandHCoV-OC43,representativesofthegroup1and2coronaviruses,respectively.Bothwereidentifiedin1960sandaregenerallyconsideredascommoncoldviruses.Anoutbreakofsevereacuterespiratorysyndrome(SARS)inthespringof2003ledtotherapididentificationofSARS-CoV[1,2],whichisconsideredtobeadistinctmemberofthegroup2coronaviruses[3]orthefirstmemberofgroup4coronaviruses[4,5].Weidentifiedearlierthisyearanotherhumanpathogenfromthisfamily:HCoV-NL63[6],avariantthatbelongstogroup1togetherwithHCoV-229EandPEDV.Theserecentfindingsmaybestriking,assincethe1960'snotasinglenewHCoVhadbeendescribed.ThegenomefeaturesofSARS-CoVanditstranscriptionstrategyhavebeendescribedindetail[1,5,7].Here,wepresentsuchananalysisforHCoV-NL63.HCoV-NL63isamemberofthecoronaviridaefamilythatclusterstogetherwitharteri-,toro-andronivirusesintheorderofthenidovirales.Coronavirusesareenvelopedviruseswithapositive,singlestrandedRNAgenomeofapproximately27to32kb.The5'two-thirdofacoronavirusgenomeencodesapolyproteinthatcontainsallenzymesnecessaryforRNAreplication.Theexpressionofthecompletepolyproteinrequiresa-1ribosomalframeshiftduringtranslationthatistriggeredbyapseudoknotRNAstructure[8,9].Thepolyproteinundergoesautocatalyticcleavagebytheviralpapain-likeproteinaseandachymotrypsin-likeproteinase.The3'one-thirdofacoronavirusgenomeencodesseveralstructuralproteinssuchasspike(S),envelope(E),membrane(M)andnucleocapsid(N)that,amongotherfunctions,participateinthebuddingprocessandthatareincorporatedintothevirusparticle.Someofthegroup2virusesencodeahemagglutininesterase(HE)[10,11].Non-structuralproteingenesarelocatedbetweenthestructuralgenes.Theseaccessorygenesvarysignificantlyinnumberandsequenceamongcoronavirusspecies.Theirprecisefunctionisunknown,butseveralreportsindicatethattheycanmodulateviralpathogenicity[12].Coronavirusreplicationisacomplex,notyetfullyunderstoodmechanism[13,14].The5'endofthegenomicRNAcontainstheuntranslatedleader(L)sequencewiththeTranscriptionRegulationSequence(TRS)inthedownstreampart.TheLTRSisverysimilartosequencesthatcanbefoundinfrontofeachopenreadingframe(bodyTRSs).TheRNA-dependentRNA-polymerasehasbeenproposedtopauseafterabodyTRSofaparticulargeneiscopiedduring(-)strandsynthesis,subsequentlyswitchingtotheLTRSandthusaddingacommonLsequencetoeachsgmRNA[15].Thisdiscontinuoustranscriptionmechanismisbasedonbase-pairingofthenascent(-)strandcopyRNAwiththe(+)strandLTRS.Thenestedsetof(-)strandsgmRNAsaresubsequentlycopiedintoasetof(+)strandsgmRNA.OtherfactorsbesidesthesequencesimilaritybetweenbodyandLTRSinfluencetheefficiencyoftranscription.TheleveloftranscriptionofaparticulargenehasbeenreportedtobeinverselyrelatedtothedistanceofaparticularTRStothe3'endofthegenome[16–19].Inthisstudy,weanalyzedthegenomestructureofHCoV-NL63.First,wefocusontheunusualnucleotidecompositionoftheRNAgenome.Wedescribeindetailthebiasinthenucleotidecompositionanditsinfluenceonthecodonusageofthisvirus.Weprovideapossiblemechanisticexplanationforashiftinnucleotidebiasattwo-thirdoftheHCoV-NL63genomethatisbasedontheRNAreplicationmechanism.Second,wedescribeindetailthedifferentsgmRNAsgeneratedduringHCoV-NL63replicationandtheirrelativeabundance.ResultsNucleotidecontentoftheHCoV-NL63genomeWedescribedpreviouslythatthenewlyidentifiedHCoV-NL63virushasatypicalcoronavirusgenomestructureandgeneorder[6].Thenucleotidecompositionofthegenomic(+)strandRNAofseveralcoronaviridaemembersispresentedinFigure1,demonstratingacommonpatternwithUasthemostabundantnucleotideandGandinparticularCasunderrepresentednucleotides.HCoV-NL63hasthemostextremenucleotidebiasamongthecoronaviridae,with39%Uandonly14%C.Asageneraltrend,UandCseemtocompetedirectly,becausethegenomeswiththelowestC-count(HCoV-NL63,HCoV-OC43andBCoV)havethehighestU-countandviceversa(Figure1).Figure1NucleotidecontentofcoronaviridaeRNAgenomes.WearrangedthevirusesbasedontheirC-count,whichrangesfrom14%(HCoV-NL63)to20%(SARS-CoV).FullsizeimageToinvestigateifallcodingregionsofHCoV-NL63displayasimilarlystrongpreferenceforUandagainstC,wealsoplottedthenucleotidecountfortheindividualgenesand5'and3'non-codingregions(Figure2).Thetypicalnucleotidebiasisobservedinallgenomesegments.ThehighestU-countisfoundintheORF3andEgenes(43%)andthelowestC-countinthe1a/1bgenesandthe3'UTR(13%,14%and14%,respectively).TheNgeneismostmoderateinitsnucleotidebias,with21%Cand32%U,confirmingthe"competition"ideathatwasalreadysuggestedbyinspectionofFigure1.Figure2NucleotidecontentofindividualHCoV-NL63genesandthe5'/3'untranslatedregions(UTR).FullsizeimageWeplottedthenucleotidedistributionalongthegenome(Figure3)todeterminewhetherthereisanysignificantvariation.WeobservedthatlocalchangesinA-countareinverselylinkedtochangesinG-count.Thisismoststrikinginthe20400–26000ntregion,wherethreeApeaksaremirroredbythreeGanti-peaks.Althoughthetypicalbiasismaintainedalongthegenome,themostnotablevariationoccursinthelastone-thirdofthegenome,whereanincreaseinCandadecreaseinGcontentisapparent.Thisregionencodesthestructuralproteins.Figure3NucleotidedistributionalongtheHCoV-NL63genome.ThechangeintheC-andG-countattwo-thirdofthegenomeisstatisticallysignificantforalltestedcoronaviruses(HCoV-NL63,HCoV-229E,SARS-CoV,HCoV-OC43)withp<0.01forC-countandp<0.05forG-countinMann-WhitneyUtestfortwoindependentsamples.FullsizeimageRecently,GrigorievreportedaninterestingfeaturewithincoronaviralgenomesthatisvisiblewhenthecumulativeGC-skewisplotted[20,21].CumulativeGCskewgraphisawaytovisualizethelocalG:Cratioalongthegenome,discardingthelocalfluctuations.Abiphasicpatternwasdescribedthatseparatesthe1a/1bpolyproteingenesandthestructuralgenes.ThecumulativeGC-skewsforHCoV-NL63andfourothercoronaviruses:HCoV-OC43,HCoV-229E,PEDVandSARS-CoVarepresentedinFigure4.Inthe1a/1bgenes,theG:Cratioreacheshighlevels,whereasforallcoronaviruses,includingHCoV-NL63,the3'endofthegenomedisplaysaflatteningofthecurve,astheG:Cratioreachesvalue~1orless.Grigorievproposedthatthisbiphasicpatternisduetothediscontinuoustranscriptionprocess[20].Hesuggestedthatthefrequentdeaminationofcytosineonthe(-)strandRNAresultsinadecreaseofGonthe(+)strandintheregionencodingthestructuralgenes.Inthediscussionsectionwewillpresentanalternativemechanisticexplanation.Figure4CumulativeGC-skewdiagramsforseveralcoronaviralRNAgenomes.Theverticalbarindicatestheborderbetweenthe1a/1bandthestructuralgenes.FullsizeimageHCoV-NL63codonusageThebiasinthenucleotidecountledustocomparethecodonusageofHCoV-NL63withthatofhumanmRNA(Table1).ThecodonusageofHCoV-NL63differsmarkedlyfromthatofhumanmRNAs.Third-basechoicesinthefour-codonfamilies(Thr,Pro,Ala,Gly,Val)provideaconvenientexampleofthiscontrastingcodonusage.Forinstance,theGlycodonsinhumanmRNAspreferC(34%)overG(25%),A(25%)andU(16%).Incontrast,HCoV-NL63prefersU(83%)overA(7%),C(8%)andG(2%).Thisresultstronglysuggeststhatthecodonusageisshapeddirectlybytheunusualnucleotidecompositionoftheviralgenome,thatisahighU-countandalowG/C-count.AllHCoV-NL63genes,exceptfortheEgene,followthistrend(Table1).ThecoronaviraladdictiontotheUnucleotideismostprominentinthe"free"thirdpositionofdegeneratecodons.Forthecompletegenome,theU-countatthethirdpositionisupto58%whereastheA-countis20%,G-countis13%andC-countisonly9%(Figure5).ThisillustratesthattheU-pressuremainlyaffectsthe%Cand%G.Figure5Nucleotidecompositionofthefirst,secondandthirdcodonpositionsintheHCoV-NL63genome.FullsizeimageTable1CodonusageofHCoV-NL63comparedwiththatofhumangenesFullsizetableIdentificationoftheHCoV-NL63TRSelementsThe5'endofHCoV-NL63genomeRNAcontainstheLsequenceof72nucleotidesthatendswiththeLTRSelement.ThisTRShasahighsimilaritytoshortsequencesthatarelocatedinfrontofeachopenreadingframe(S-ORF3-E-M-N)[22].WepreviouslyidentifiedtheLTRSandbodyTRSoftheNgeneusingacDNAbank[6],whichallowedustopredictthebodyTRSoftheothergenes.Toconfirmthesepredictions,weamplifiedandsequencedallsgmRNAfragmentswithageneralLprimerandgene-specific3'primersinanRT-PCRprotocol.InspectionofsgmRNAjunctionsindicatedthattheyareindeedcomposedofthepartoftheHCoV-NL63genomethatisdirectlydownstreamofaparticularbodyTRS,withits5'endderivedfromtheleadersequence.Apparently,strandtransferoccurredonthe5'endofthebodyTRS,asindicatedinFigure6.ThemostconservedTRSregionwasdefinedbymultiplesequencealignmentasAACUAAA(graybox).ThiscoresequenceisconservedinallsgmRNA,exceptfortheEgenethatcontainsthesub-optimalTRScoreAACUAUA(Figure6).Interestingly,theEgenecontainsa13-nucleotidesequenceupstreamofthecoresequencewithperfecthomologytotheLsequence.PerhapstheupstreamsequencecompensatesfortheabsenceofanoptimalTRScoreduringdiscontinuous(-)strandsynthesis.Thiswouldsuggestthatthesesequencesarecopiedduring(-)strandsynthesis,andthattheactualstrandtransferwithintheEsequencesoccurredaftercopyingofthecoreTRSandthenext13nucleotides.Evidenceforsucha"delayed"strandtransferisprovidedbythejunctionanalysisoftheMandNsgmRNAs,whichclearlydemonstratesthatthenucleotidesdirectlyupstreamofthecoreTRSarederivedfromthebodyTRSelementandnotfromtheleader.Figure6Body-leaderjunctionsofallHCoV-NL63sgmRNAs.Shownontopistheleader(L)sequenceandbelowthespecificsequencesupstreamofthestructuralgenes.Thefusionof5'Lsequencesto3'sgRNAisindicatedbytheboxes.Sequencehomologybetweenthestrandsnearthejunctionismarkedbyasterisks,theconservedAACUAAATRScoreishighlightedingray.FullsizeimageAnalysisofthesubgenomicmRNAsofHCoV-NL63TodeterminewhetherthepredictedsgmRNAsencodingtheS-ORF3-E-M-Nproteinsareproducedinvirus-infectedcells,weperformedNorthernblotanalysisontotalcellularRNA(Figure7).Weuseda(-)strandNgeneprobethatannealstobothgenomicRNAandallsg(+)strandmRNAs.WeincludedRNAfromMHV-infectedcellstoobtaindiscretesizemarkers.SixdistinctmRNAsareproducedinHCoV-NL63infectedcells.ThesizesoftheRNAfragmentswereestimatedandthesevaluesnicelyfitthesizeofthegenomicRNAandthefivepredictedsgmRNAs.AllHCoV-NL63ORFsthathavethepotentialtoencodeviralproteinsareindeedtranscribedintosgmRNAs(Figure7).Figure7TheleftpanelshowstheNorthernblotanalysisofHCoV-NL63RNAininfectedLLC-MK2cells.RNAofHCoV-NL63(NL63lane)wascomparedwithRNAofMHVstrainA59(MHVlane).Non-infectedLLC-MK2cellsareincludedasanegativecontrol(controllane).MHVRNAbandsrepresentthecompletegenome(1)andsgmRNAs2a(2),S(3),17.8(4),13.1andE(5),M(6),N(7).HCoV-NL63RNAincludesthecompletegenome(1)andsgmRNAsforS(2),ORF3(3),E(4),M(5)andN(6).TherightpanelshowstheMHVandHCoV-NL63genomeorganizationandtheHCoV-NL63sg-mRNAs.FullsizeimageTodeterminetheexpressionlevelofeachsubgenomicRNA,wemeasuredtheintensityofthesignals.Whenplottedasafunctionofthegenomeposition(Figure8),thereappearsacorrelationbetweentherelativedistanceofagenetothe3'terminusanditsRNAexpressionlevel,withtheexceptionoftheEgene.Figure8ExpressionlevelsoftheHCoV-NL63genomicandsgmRNAs.FullsizeimageDiscussionWeanalyzedthenucleotidecompositionoftheHCoV-NL63genomic(+)RNA,whichwasfoundtoexhibitatypicalcoronaviruspatternwithanabundanceofU(39%)andshortageofG(20%)andC(14%).Infact,HCoV-NL63hasthemostpronouncednucleotidebiasamongthecoronaviridae.ThereisasignificantfluctuationinthenucleotidecountamongtheHCoV-NL63genes.Forinstance,ORF3andMappearasextremeU-richandA-poorislands.Itispossiblethattheuniquenucleotidecompositionofsomestructuralgenesreflectstheirevolutionaryorigin,perhapssuggestingthatsomeofthesefunctionswereacquiredrecentlyfromanotherviralorcellularoriginbygenetransfer.Thesepropertiesmimicthepathogenicityislandsofprokaryoticgenomes[23].Consistentwiththisgenetransferhypothesisistheobservationthatthereisalotofvariationinthenumberandidentityofthe3'genesamongcoronaviridae.Inspectionofthenucleotidecompositionalongthegenomeindicatesabi-phasicpattern.The5'two-thirdofthegenomeencodingthe1abpolyproteinhasastablenucleotidecountwiththetypicalU>A>G>Corder,butratherstrikingdifferencesareobservedinthe3'one-thirdofthegenomethatencodesthestructuralproteins(Figure2).Mostnotably,theC-countincreasessignificantlyattheexpenseofGandU.Grigorievrecentlyreportedthetypicalnucleotidebiasofcoronaviralgenomesandtheswitchinnucleotidecountattwo-thirdsofthegenome[20].HeperformedananalysisbasedoncumulativeGC-skew,andsuggestedthatthedropinGC-ratioisinfactduetoadecreaseinG-count.However,inspectionoftheHCoV-NL63nucleotidecompositionindicatesthattheswitchisduetoasuddenincreaseinC-count,withaslightdropinG-count.InspectionofothercoronaviralgenomesconfirmsthatCgoesup(withhighestsignificanceingroup1coronaviruses)andGgoesdown(withhighestsignificanceingroup2coronaviruses)attwo-thirdoftheviralgenome(resultsnotshown).Grigorievpresentedapossiblemechanisticexplanation.Hesuggestedthatthe3'-terminalone-thirdoftheviralgenomic(-)strandismorelikelytobesinglestrandedbecause(-)sgmRNAsynthesisonthe(+)strandtemplatefrequentlydisruptstheprotectiveduplexinthatregion.Thiswouldmakethispartofthe(-)strandgenomemorevulnerabletoCtoUtransitions,whichwouldeventuallyleadtoadecreaseoftheG-countonthe(+)strand.ThisscenarioexplainstheGdecrease,butobviouslyisnotconsistentwiththelocalincreaseinC-count.Wethereforeproposeanalternativemechanismthatisalsodictatedbytheviraltranscriptionstrategy.Thecentral1a/1bportionoftheviral(+)strandgenomeislesslikelytobeannealedtocomplementary(-)strandduringviralreplicationbecausemost(-)strandRNAsaresub-genomic,whichlackthis1a/1bdomain.The1a/1bportionofthegenomethusbecomesmorevulnerabletoCtoUdeamination,whichcorrelateswiththehighU-countandthelowC-count.Obviously,theremaybemanyothercellularconditionsandviralpropertieslikehigheramountofsecondarystructuresonthe3'partofthegenomethatcouldhaveshapedthecoronavirusgenomeoveranevolutionarytimescale,butthisscenarioexplainstheswitchinnucleotidecountattwo-thirdsoftheviralgenome.WeshowthatU-countsreachthehighestvaluesandC-countsthelowestvaluesatthethirdpositionoftheHCoV-NL63codons(Figure5).AnalysisofthesynonymouscodonusageindicatesthatcodonswithahighUandAcontentarepreferredoverCandGrichcodons(Table1).Thus,thepeculiargenomecompositionhasadirecteffectonthecodonusageofHCoV-NL63,andpossiblyevenanindirecteffectontheaminoacidcompositionofcoronaviralproteinsbyaffectingthenon-synonymouscodonusage[24–26].ThesynonymouscodonusageofHCoV-NL63clearlydiffersfromthatinhumancells.Thus,thegenomemayhavebeenshapedbycytosinedeaminationoveranevolutionarytimescale,butitispossiblethatthetranslationalmachineryhasrestrictedthisgenomedriftbecauseoftheavailabilityoftRNAmolecules.Inspectionoftheviralgenomesequenceledustopredictthatthe1abpolyproteinisexpressedfromthegenomicRNAandthe3'structuralproteinsandORF3from5distinctsgmRNAs.Thiswasconfirmedexperimentally.WeobservedthatsgmRNAsaremoreabundantwhenthecorrespondingTRSislocatedclosertothe3'endofthegenome.TheexceptionisformedbytheEsgmRNA,whichisrelativelyunderexpressed.Thismaycorrelatewiththelowexpressionlevelofthisprotein.Thegeneraltrendofincreasedgeneexpressionalongthegenomehasbeenreportedpreviouslyforothercoronaviruses[19].ApossiblemechanisticexplanationisthattheviralpolymerasedensityisreducedalongthegenomeorthatthepolymerasebecomeslesssusceptibletoexecuteatransferfrombodyTRStoLTRSduringextended(-)strandsynthesis.Fine-tuningoftheefficiencyofthestrand-transferprocessesmaybemodulatedbymanyotherfeatures,includingthelocalsequenceandstructureofthecorebodyTRSanditsflankingregions.Itwasreportedpreviously[27]thatthecoreoftheLTRSofgroup1coronavirusesispresentedinthesinglestrandedloopofamini-hairpin.WefoundsimilarmotifsinHCoV-NL63(resultsnotshown).Althoughnotexcessivelystable,thisstructuralmotifsispredictedtofoldaspartofthecompleteleadersequence,anditmayparticipateinthestrandtransferprocess.ThecoresequenceAACUAAAisconservedintheLTRSandallbodyTRSs,exceptfortheEgenethathasasinglemismatchAACUAUA.Thepresenceofasub-optimalcoresequencemayinfactexplainthelowerthanexpectedexpressionlevelofthissgmRNA(Figure8).ButthereisanotherstrikingfeatureoftheEbodyTRS:ithas13additionalupstreamnucleotidesincommonwiththeleaderTRS.Ifoneassumesthatstrandtransferdoesnotoccuratthecoresequencebutupto13nucleotidesfurtherupstream,thissequencehomologywillresultinadditionalbasepairinginteractionsthatmaystimulatethestrandtransferprocess.Thus,theextendedTRShomologymaycompensateforthesub-optimalcoreelement.Aremarkablysimilarscenarioofsub-optimalcoreandextendedTRSisapparentintheEgenesequenceofPEDV(resultsnotshown).AfurtherindicationthattheviralpolymerasefrequentlycopiesbeyondthecoresequenceisprovidedbytheactualsequenceoftheMandNsgmRNAs,whichapparentlyhavecopiedtheTRSnucleotidethatflanksonthe5'sidethecoreelementofbodyTRS.MethodsGenomeAnalysisThenucleotidecontentofdifferentCoronaviridaefamilymemberswasassessedusingBioEditsoftware.ThenucleotidedistributionwasdeterminedusingaMicrosoftExceldatasheet(300nucleotide(nt)windowand10-ntstep).CodonusagewasassessedusingDNA2.0software.DatawasprocessedinMicrosoftExceldatasheetandallstatisticalanalysiswasperformedwithSPSS11.5.0software.Thelevelofsignificanceofthenucleotidebiaswasestablishedfor300-ntnon-overlappingwindowswiththenon-parametricMann-WhitneyUtestfortwoindependentsamples.CumulativeGC-skewgraphsweregeneratedasdescribedpreviously[20]withthevalueinstepndefinedasthesumof(G-C)/(G+C)fromstep0ton(200-ntslidingwindow,10-ntstep).ViralRNAisolationHCoV-NL63RNAwasobtainedfromvirus-infectedLLC-MK2cells(2×107)after6daysofculture(viruspassage7).MouseHepatitisVirus(MHV)RNAwasobtainedbyinfecting2×107LR7cellswithMHVstrainA59.Themediumwasremovedandthecellsweredissolvedin15mlTRIzol®andRNAwasisolatedaccordingtothestandardTRIzol®procedure.RNAwassubsequentlyprecipitatedwith0.8volumeofisopropanol,driedanddissolvedin50μlH2O.IntegrityoftheRNAwasanalyzedbyelectrophoresisonanon-denaturating0.8%agarosegel.RNAwasstoredat-150°C.RT-PCRThecDNAusedforsequencingandprobeconstructionwasmadebyMMLV-RTonviralRNAwith1μgofrandomhexamerDNAprimersin10mMTrispH8.3,50mMKCl,0.1%Triton-X100,6mMofMgCl2and50μMofeachdNTPsat37°Cfor1hour.ThesinglestrandedcDNAproductwasmadeintodouble-strandedDNAinastandardPCRreactionwith1.25UofTaqpolymerase(Perkin-Elmer)perreactionwithappropriateprimers(seebelow).NorthernBlotGelelectrophoresisofviralRNAwasperformedona1%agarosegelwith7%offormaldehydeat100Voltin1×MOPSbuffer(40mMMOPS,10mMsodiumacetate,pH7.0).Transferontoapositivelychargednylonmembrane(BoehringerMannheim)wasdoneovernightbymeansofcapillaryforce.RNAwaslinkedtothemembraneinaUVcrosslinker(Stratagene).ForgenerationoftheHCoV-NL63probe,theRT-PCRproductwasfurtheramplifiedwith5'primerN5PCR1(CTGTTACTTTGGCTTTAAAGAACTTAGG)and3'primerN3PCR1(CTCACTATCAAAGAATAACGCAGCCTG).Similarly,theMHVprobewasamplifiedwith5'primerMHV_UTR-B5'(GATGAAGTAGATAATGTAAGCGT)and3'primerMHV_UTR-B3'(TGCCACAACCTTCTCTATCTGTTAT).LabelingoftheprobeswasdoneinastandardPCRreactionwithspecific3'primers(N3PCR1andMHV_UTR-B3')inpresenceof[α-32P]dCTP.PrehybridizationandhybridizationwasdoneinULTRAhybbuffer(Ambion)at50°Cfor1and12hours,respectively.Themembranewasthenwashedatroomtemperaturewithlow-stringencybuffer(2×SSC,0.2%SDS)andat50°Cinhighstringencybuffer(0.1×SSC,0.2%SDS).ImageswereobtainedusingtheSTORM860phosphorimager(AmershamBiosciences)anddataanalysiswasperformedwiththeImageQuantsoftwarepackage.ThesizeofsgmRNAfragmentsofHCoV-NL63wereestimatedfromtheirmigrationontheNorthernblotusingthesgmRNAofMHVassizemarker.SequenceanalysisofTRSmotifsTheL/bodyTRSjunctionswerePCR-amplifiedfromanHCoV-NL63cDNAbank.Weperformed35cyclePCRwiththe5'Lprimer(L5–TAAAGAATTTTTCTATCTATAGATAG)andgenespecific3'primers(Sgene–SL3'–ACTACGGTGATTACCAACATCAATATA;ORF3–4L3'–CAAGCAACACGACCTCTAGCAGTAAG;Egene–EL3'–TATTTGCATATAATCTTGGTAAGC;Mgene–ML3'–GACCCAGTCCACATTAAAATTGACA;Ngene–3-163-F15–ATTACCTAGGTACTGGACCT).ThePCRproductswereanalyzedbyelectrophoresisona0.8%agarosegelandproductsofdiscretesizewereusedforsequencingusingtheBigDyeterminatorkit(ABI)andABIPrism377sequencer(PerkinElmer).SequenceanalysiswasperformedbySequenceNavigatorandAutoAssembler2.1software.SequencesThecompletegenomesequenceofHCoV-NL63[6]isdepositedinGenBank(accessionnumber:NC_005831).sgmRNAsequencesaredepositedinGenBankundertheaccessionnumbers:AY697419-AY697423.TheGenBankaccessionnumberofthesequencesusedinthisgenomeanalysisare:MHV(mousehepatitisvirus,strainMHV-A59):NC_001846;HCoV-229E:NC_002645;HCoV-OC43strainATCCVR-759:NC_005147;PEDV(porcineepidemicdiarrheavirus,strainCV777):AF353511;TGEV(transmissiblegastroenteritisvirus,strainPurdue):NC_002306;SARS-CoVisolateTor2:NC_004718;IBV(avianinfectiousbronchitisvirus,strainBeaudette):NC_001451;BCoV(bovinecoronavirus,isolateBCoV-ENT):NC_003045. 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PubMed  GoogleScholar  DownloadreferencesAcknowledgementsWethankBerendJanBoschandPeterRottierforprovidingMHVinfectedcellsandAlexanderNabatovandBarbaravanSchaikfortechnicalsupport.AuthorinformationAffiliationsDepartmentofHumanRetrovirology,UniversityofAmsterdam,1105,Meibergdreef15,CollegeStation,AZ,NetherlandsKrzysztofPyrc, MaartenFJebbink, BenBerkhout & LiavanderHoekAuthorsKrzysztofPyrcViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarMaartenFJebbinkViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarBenBerkhoutViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarLiavanderHoekViewauthorpublicationsYoucanalsosearchforthisauthorin PubMed GoogleScholarCorrespondingauthorCorrespondenceto LiavanderHoek.AdditionalinformationCompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Authors'contributionsKPcarriedouttheviralRNAisolation,RT-PCR,sequencingofsgmRNAs,Northernblotevaluationandallcomputeranalysisdoneinthisstudy;MFJcarriedoutthefullgenomesequencing;allauthorsparticipatedinwritingthemanuscript.Lv/dHandBBaretheprincipalinvestigatorsAuthors’originalsubmittedfilesforimagesBelowarethelinkstotheauthors’originalsubmittedfilesforimages.Authors’originalfileforfigure1Authors’originalfileforfigure2Authors’originalfileforfigure3Authors’originalfileforfigure4Authors’originalfileforfigure5Authors’originalfileforfigure6Authors’originalfileforfigure7Authors’originalfileforfigure8RightsandpermissionsReprintsandPermissionsAboutthisarticleCitethisarticlePyrc,K.,Jebbink,M.F.,Berkhout,B.etal.GenomestructureandtranscriptionalregulationofhumancoronavirusNL63. VirolJ1,7(2004).https://doi.org/10.1186/1743-422X-1-7DownloadcitationReceived:29October2004Accepted:17November2004Published:17November2004DOI:https://doi.org/10.1186/1743-422X-1-7SharethisarticleAnyoneyousharethefollowinglinkwithwillbeabletoreadthiscontent:GetshareablelinkSorry,ashareablelinkisnotcurrentlyavailableforthisarticle.Copytoclipboard ProvidedbytheSpringerNatureSharedItcontent-sharinginitiative KeywordsCodonUsageSevereAcuteRespiratorySyndromePorcineEpidemicDiarrheaVirusSevereAcuteRespiratorySyndromeSynonymousCodonUsage DownloadPDF Advertisement VirologyJournal ISSN:1743-422X Contactus Submissionenquiries:AccesshereandclickContactUs Generalenquiries:[email protected]