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1、Dependence of Coronavirus RNA Replication on an NH2-Terminal Partial Nonstructural Protein 1 in cis Yu-Pin Su, Yi-Hsin Fan, David A. Brian Department of Biomedical and Diagnostic Sciences, University of Tennessee College of Veterinary Medicine, Knoxville, Tennessee, USA ABSTRACT Genomesofpositive(?)
2、-strandRNAvirusesuse cis-actingsignalstodirectbothtranslationandreplication.Hereweexamine two5=-proximalcis-replicationsignalsofdifferentcharacterinadefectiveinterfering(DI)RNAofthebovinecoronavirus (BCoV)thatmapwithina322-nucleotide(nt)sequence(136ntfromthegenomic5=untranslatedregionand186ntfromthe
3、 nonstructuralprotein1nsp1-codingregion)notfoundintheotherwise-identicalnonreplicatingsubgenomicmRNA7 (sgmRNA7).ThenaturalDIRNAisstructurallyafusionofthetwoendsoftheBCoVgenomethatresultsinasingleopenread- ingframebetweenapartialnsp1-codingregionandtheentireNgene.(i)Inthefi rstexamination,mutationana
4、lysesofare- centlydiscoveredlong-rangeRNA-RNAbase-pairedstructurebetweenthe5=untranslatedregionandthepartialnsp1-coding regionshowedthatit,possiblyinconcertwithadjacentstem-loops,isa cis-actingreplicationsignalinthe(?)strand.Wepostu- latethatthehigher-orderstructurepromotes(?)-strandsynthesis.(ii)In
5、thesecondexamination,analysesofmultipleframe shifts,truncations,andpointmutationswithinthepartialnsp1-codingregionshowedthatsynthesisofaPEFPcoreaminoacid sequencewithinagroupAlineagebetacoronavirus-conservedNH2-proximalWAPEFPWMdomainisrequiredin cisforDIRNA replication.Wepostulatethatthenascentprote
6、in,aspartofanRNA-associatedtranslatingcomplex,actstodirecttheDIRNA toacriticalsite,enablingRNAreplication.Wesuggestthattheseresultshaveimplicationsforviralgenomereplicationandex- plain,inpart,whycoronavirussgmRNAsfailtoreplicate. IMPORTANCE cis-ActingRNAandproteinstructuresthatregulate(?)-strandRNAv
7、irusgenomesynthesisarepotentialsitesforblockingvirus replication.Herewedescribetwo:apreviouslysuspected5=-proximallong-rangehigher-orderRNAstructureandanovelnas- centNH2-terminalproteincomponentofnsp1thatarecommonamongbetacoronavirusesofgroupAlineage. W hat constitutes the cis-acting requirements fo
8、r coronavirus RNA replication has remained an intriguing question since it was discovered that the subgenomic mRNAs (sgmRNAs) of coronaviruses (used primarily to synthesize viral structural pro- teins)areboth(i)5=and3=coterminalwiththegenomeforatleast ?70 and 1,670 nucleotides (nt), respectively, le
9、ngths greater than thoseofmanyviralRNApolymerasepromoters(13),and(ii)are present in sgmRNA-length replication-intermediate-like double- stranded RNA structures that are involved in sgmRNA synthesis (46) yet fail to replicate when transfected, as synthetic tran- scripts, into virus-infected cells (Fi
10、g. 1) (7). If replication of the coronavirussgmRNAsnormallyoccursduringinfection,itmight be expected that they would replicate following their transfection into virus-infected cells, since all trans-acting factors required for viral RNA replication are present. In coronaviruses, the 5= two- thirdsof
11、thesingle-strandedpositive(?)-strand?30-kbcorona- virus genome is used as mRNA for synthesis of overlapping poly- proteins1a(?4,000aminoacidsaa)and1ab(?7,000aa),which are proteolytically processed into the 16 replicase proteins that make up the replication/transcription complex, whereas the 3= one-t
12、hird of the genome is transcribed into a 3= nested set of sgmRNAs that are coterminal with the genome but are translated separately (3, 8, 9). One model widely used to explain the origin of the sgmRNA-length replication-intermediate-like double- strandedRNAswasproposedbySawickietal.(4,6,10,11).Inthi
13、s model, (i) the genome is envisioned as the only template for neg- ative(?)-strandRNAsynthesis,and(ii)anRNA-dependentRNA polymerase (RdRp) template-switching event takes place during (?)-strandsynthesisfromtheviralgenometemplateatintergenic donor core sequence (also termed transcription-regulating
14、se- quence) sites (UCUAAAC in bovine coronavirus BCoV and mouse hepatitis virus MHV) to the 5=-proximal leader acceptor core sequence (UCUAAAC) on the genome (i.e., a discontinuous transcription step) to create a sgmRNA-length (?)-strand RNA. In this model, the sgmRNA-length (?)-strand RNA (5, 12, 1
15、3) then functions as a template for synthesis of new sgmRNA. The termproposedforthesgmRNA-length,partiallydouble-stranded structure hence became “transcriptive intermediate” (4, 11) rather than “replicative intermediate,” as was initially used (5, 6, 12),tomoreclearlyidentifytheviralgenomeastheonlyt
16、emplate for sgmRNA (?)-strand synthesis. This model for sgmRNA syn- thesis from the genome was tested fi rst by reverse genetics in an arterivirus (14), a fellow member of the Nidovirales order with a very similar pattern of sgmRNA generation, and second in the coronavirus (15), and the results with both viruses are consistent withtheSawickimodel.Morerecently,ithasbeenlearnedthatthe Received 13 March 2014 Accepted 21 May 2014 Published ahead of print 28 May 2014 Editor: S. Perlman Address corres
