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Reverse genetics with a full length infectious cDNA of the Middle East respiratory syndrome coronavirus Trevor Scobey a Boyd L Yount a Amy C Sims a Eric F Donaldson a Sudhakar S Agnihothram a Vineet D Menachery a Rachel L Graham a Jesica Swanstrom a Peter F Bove b Jeeho D Kim c Sonia Grego d Scott H Randell c and Ralph S Baric a e 1 Departments of a Epidemiology c Cell Biology and Physiology and e Microbiology and Immunology and b Cystic Fibrosis Pulmonary Research and Treatment Center University of North Carolina at Chapel Hill Chapel Hill NC 27599 7435 and d Center for Materials and Electronic Technologies Research Triangle International Durham NC 27709 Edited by Peter Palese Mount Sinai School of Medicine New York NY and approved August 13 2013 received for review June 18 2013 Severe acute respiratory syndrome with highmortality rates 50 is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus MERS CoV We synthesized a panel of contiguous cDNAs that spanned the entire genome Following contig assembly into genome length cDNA transfected full length transcripts recovered several recombinant viruses rMERS CoV that contained the expected marker muta tions inserted into the component clones Because the wild type MERS CoV contains a tissue culture adapted T1015N mutation in the S glycoprotein rMERS CoV replicated 0 5 log less ef ciently than wild type virus In addition we ablated expression of the accessory protein ORF5 rMERS ORF5 and replaced it with tomato red uorescent protein rMERS RFP or deleted the entire ORF3 4 and 5 accessory cluster rMERS ORF3 5 Recombinant rMERS CoV rMERS CoV ORF5 and MERS CoV RFP replicated to high titers whereas MERS ORF3 5showed1 1 5 logs reduced titer compared with rMERS CoV Northern blot analyses con rmed the associated molecular changes in the recombinant viruses and sequence analy sis demonstrated that RFP was expressed from the appropriate con sensus sequence AACGAA We further show dipeptidyl peptidase 4 expression MERS CoV replication and RNA and protein synthesis in human airway epithelial cell cultures primary lung broblasts primary lung microvascular endothelial cells and primary alveolar typeII pneumocytes demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus The avail ability of a MERS CoV molecular clone as well as recombinant virusesexpressingindicatorproteins willallowforhigh throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines emerging pathogen zoonosis synthetic genome E merging respiratory virus infections can cause considerable morbidity and mortality in human populations Some in cluding in uenza A viruses and human coronaviruses CoVs can spread rapidly and cause acute lung injury and its more se vere form acute respiratory distress syndrome ARDS These devastating end stage lung diseases are often associated with high mortality rates 30 50 and can progress to chronic conditions such as pulmonary brosis 1 CoVs frequently transmit between species 2 4 Divided into three main groups the betacor onaviruses represent a large collection of phylogenetically dis tant strains that include several human coronaviruses HCoVs such as HCoV OC43 HCoV HKU1 group 2a and the severe acute respiratory syndrome coronavirus SARS CoV group 2b 5 In 2003 SARS CoV emerged from zoonotic sources in China and caused atypical pneumonia and ARDS with 10 mortality rates 5 In 2012 a new group 2c betacoronavirus was detected in the Middle East 3 Designated MERS CoV Middle East re spiratory syndrome coronavirus it has caused at least 114 con rmed cases with 54 deaths as of September 10 2013 The virus replicates ef ciently in human airway epithelial cells HAEs and uses human and bat dipeptidyl peptidase 4 DPP4 as recep tors for entry 6 7 Human to human transmission coupled with the unusual severity and high mortality rates dictates a need for key resources for the testing of MERS CoV therapeutics and vac cines 8 The 30 119 nt MERS CoV genome encodes a typical portfolio of ORFs expressed from both genome length ORF1a 1b 9 and subgenomic length mRNAs sgRNAs which are arranged in a nested set from the 3 end of the genome The MERS CoV transcription regulatory sequence TRS is predicted to be AACGAA but remains poorly de ned in culture In addition to the classic structural proteins a 180 90 kDa spike glycoprotein S an 23 kDa membrane glycoprotein M a small envelope protein E and an 50 kDa nucleocapsid protein N MERS CoV also encodes at least four group speci c ORFs designated ORF3 ORF4a ORF4b and ORF5 Previously we constructed or synthesized full length infectious clones from several CoVs including SARS CoV HCoV NL63 and HKU3 BAT SRBD 10 12 In this manuscript we describe the synthesis and assembly of a full length molecular clone for MERS CoV and show that wild type MERS CoV EMC2012 substrain has evolved a unique tissue culture adapted mutation in the S glycoprotein that is not present in the recombinant virus derived from the molecular clone Recombinant viruses were also isolated that express a red uorescent indicator tomato red red uorescent protein RFP from sgRNA In addition we also generate mutants lacking one or more of the MERS CoV group speci c ORFs The availability Signi cance The identi cation of a novel emerging human coronavirus with 50 mortality designated Middle East respiratory syn drome coronavirus MERS CoV emphasizes the importance of the rapid development of reagents that can be used to i characterize the replication and pathogenesis of emerging pathogens and ii develop therapeutics for treatment In this report we describe the development of a cassette based in fectious cDNA clone of MERS CoV and verify that it functions similarly to the wild type isolate in terms of replication protein and RNA expression and spike attachment protein processing We also show that the virus replicates preferentially in differ entiated primary lung cells Author contributions T S B L Y and R S B designed research T S B L Y A C S E F D S S A V D M R L G J S and J D K performed research P F B S G and S H R contrib uted new reagents analytic tools T S B L Y A C S E F D S S A V D M R L G J S and R S B analyzed data and T S A C S E F D R L G J D K and R S B wrote the paper The authors declare no con ict of interest This article is a PNAS Direct Submission Freely available online through the PNAS open access option 1 To whom correspondence should be addressed E mail rbaric ad unc edu This article contains supporting information online at www pnas org lookup suppl doi 10 1073 pnas 1311542110 DCSupplemental www pnas org cgi doi 10 1073 pnas 1311542110 PNAS October 1 2013 vol 110 no 40 16157 16162 MICROBIO L OGY of a molecular clone for MERS CoV along with marker and deletion viruses provides opportunities for understanding novel gene functions tools for high throughput screening of antiviral drugs and essential reagents for the rational design of candidate live attenuated virus vaccines Results Design of MERS CoV Full Length cDNAs Rapid response and control of emerging respiratory viruses require high throughput strate gies for the manipulation and recovery of recombinant viruses by reverse genetic strategies The MERS CoV genome was initially synthesized as a panel of six contiguous cDNAs A F that span the genome and are linked by class II restriction endonuclease sites BglI GCCNNNN NGGC which cleave at symmetrical pal indromic sequences but leave different asymmetric 3 nt overhangs Due to instability in the plasmid the D fragment was split into two separate fragments resulting in seven total cDNA cassettes Fig 1 Thus rational cDNA design allows for the systematic directional and ef cient assembly of the individual MERS A F cDNAs into a genome length cDNA by in vitro ligation 12 In addition naturally occurring BglI sites were removed by G C and C T changes at positions 494 and 17713 respectively and an A G change was introduced at position 2393 to ablate a poten tial T7 pause site in the derived synthetic molecular clone 12 The MERS D fragment was unstable in microbial vectors and was thus bisected into two pieces designated MERS D1 and MERS D2 which were very stable BglI sites were engineered by introducing silent mutations between the A B B C C D1 D1 D2 D2 E and E F junctions Fig 1 The MERS A fragment contains a T7 start site whereas the F subclone terminates in 25 T residues allowing for in vitro transcription of capped polyadenylated transcripts Characterization of Recombinant Viruses To assemble the MERS CoV molecular clone each subclone was digested with BglI puri ed and ligated and the resulting product was used as a template for in vitro transcription Because N gene transcripts enhance the infectivity of CoV full length transcripts 12 re combinant rMERS CoV transcripts were mixed with capped N transcripts and electroporated into Vero cells Within 48 72 h posttransfection cytopathic effects and leader containing tran scripts were detected within transfected cultures but not in controls rMERS CoV replicated to titers that approach 10 7 to 10 8 pla que forming units mL pfu mL and formed small plaques in Vero 81 cells Figs 2 and 3 rMERS CoV but not wild type virus should encode several unique marker mutations at the junctions associated with the assembly of the component clones Therefore RT PCR was used to generate cDNA amplicons which were puri ed and then subjected to restriction fragment length polymorphism RFLP analysis with BglI MERS CoV contains a BglI site at position 17717 that was ablated in rMERS CoV Fig 2B In contrast rMERS CoV contains a BglI site inserted between the component E F junctions Fig 2C a genotype that was subsequently veri ed by genome length sequence analysis Interestingly wild type MERS CoV appeared to replicate 0 5 log more ef ciently and formed larger plaques than rMERS CoV Figs 2 and 3 Genome length sequence analysis of our passage 9 wild type MERS CoV stock revealed a unique consensus T1015N change in the S glycoprotein that was not present in the published sequence To evaluate whether this con sensus mutation altered virus growth kinetics or plaque morphol ogy in vitro we introduced this mutation into the molecular clone and isolated a recombinant virus rMERS CoV T1015N rMERS CoV T1015N more closely replicated wild type virus replication kinetics and plaque formation Figs 2D and 3E These data suggest that T1015N is a tissue culture adapted mutation that arose during serial in vitro passage rMERS CoV Derivative Virus Phenotypes The MERS CoV genome encodes several accessory ORFs of unknown function To eval uate the roles of these accessory ORFs in MERS CoV replica tion we introduced a stop codon into ORF5 at nucleotide A 27162 G rMERS CoV ORF5 which truncated ORF5 replaced ORF5 with the RFP gene rMERS CoV RFP or deleted the down stream subgenomic ORFs 3 4a 4b and 5 rMERS CoV ORF3 5 Following electroporation of full length transcripts into Vero cells recombinant viruses were isolated Compared with rMERS CoV rMERS CoV ORF5 and rMERS CoV RFP replicated to titers from 10 7 to 10 8 pfu mL In contrast the replication of rMERS CoV ORF3 5 was signi cantly reduced by 1 1 5 logs in Vero cells Fig 2A Consonant with the presence of sgRNA transcripts encoding RFP uorescent microscopy revealed RFP uorescence at different times postinfection in rMERS CoV RFP but not in rMERS CoV or wild type control infected cultures Fig 3 A D To evaluate virus RNA synthesis Northern blot analyses revealed the expected set of appropriately sized RNAs in wild type MERS CoV rMERS CoV rMERS CoV RFP and rMERS CoV ORF5 infected cells which included genome length RNA and seven sgRNAs Fig 4A As RFP and ORF5 are of similar size we did not note any visible alterations in the size of sgRNA5 between these two viruses In contrast rMERS CoV ORF3 5 removed several ORFs and TRSs from the ge nome resulting in a loss of sgRNAs 3 4 and 5 and increased expression and mobility of sgRNA2 S glycoprotein Fig 4A To evaluate recombinant virus structural protein expression we cloned and expressed the MERS CoV S and N proteins in Venezuelan equine encephalitis virus strain 3526 replicon con structs virus replicon particle VRP S VRP N inoculated mice and isolated antisera against these viral proteins Using Western blot analysis we con rmed robust S glycoprotein and N protein expression in wild type MERS CoV rMERS CoV rMERS CoV ORF5 rMERS CoV RFP and rMERS CoV ORF3 5 infected cells Fig 4B Interestingly increased S glycoprotein expression was noted in rMERS CoV 3 5 infected cells a phenotype coincident with increased sgRNA2 expression Fig 4B The MERS CoV S glycoproteins are cleaved more ef ciently at later times and were heavily glycosylated as evidenced by increased electrophoretic mobility following peptide N glycosidase F PNGase F treatment Fig S1 As downstream Fig 1 Organization of the MERS CoV molecular clone A The organiza tion of the MERS CoV genome B The full length MERS CoV genome was ultimately divided into seven contiguous cDNAs designated MERS A F and anked by unique BglI sites that allow for directed assembly of a full length cDNA MERS A nucleotides 1 4692 MERS B 4693 8811 MERS C 8812 12258 MERS D1 12259 15470 MERS D2 15471 18806 MERS E 18807 24397 and MERS F 24398 30119 16158 www pnas org cgi doi 10 1073 pnas 1311542110 Scobey et al TRSs are thought to in uence the abundance of upstream tran scripts it seems likely that the deletion of the regulatory domains ofsgRNAs3 4 and5enhancedtheexpressionofsgRNA2 resulting in increased S glycoprotein expression and fusion phenotypes An increasedfusionphenotypewasnotedinrMERS CoV ORF3 5 that was rarely evident in wild type or recombinant control virus infected cells Fig 4 C and D Recombinant Virus Growth in Primary Lung Cultures MERS CoV has been reported to replicate in nonciliated HAEs and alveolar type II pneumocytes in ex vivo cultures 7 13 To evaluate MERS CoV tissue speci city in other lung cells we obtained primary human lung broblasts microvascular endothelial cells alveolar type II pneumocytes and ciliated HAEs and infected these cultures with MERS CoV rMERS CoV RFP or rSARS CoV Wild type MERS CoV titers increased during infection in alveolar type II pneumocytes microvascular endothelial cells HAEs and broblasts approaching titers of 10 6 to 10 7 pfu mL within 30 h postinfection Fig 5 RFP expression was clearly visible in broblast and ciliated HAE cultures but not in endo thelial cells or alveolar type II pneumocytes most likely due to high auto uorescence levels in these cultures Fig 6A MERS CoV replicated most ef ciently in HAE and lung broblast cultures slightly less ef ciently in type II pneumocytes and least ef ciently in microvascular endothelial cells Under identical conditions SARS CoV only replicated ef ciently in the HAE cultures To con rm robust MERS CoV replication we per formed Northern blot analysis and clearly demonstrated the presence of both full length and subgenomic length MERS CoV transcripts during virus infection of the primary lung cell types tested with the exception of undifferentiated human bronchial cells Fig 6B Consonant with these data real time RT PCR and Western blot analysis demonstrated DPP4 mRNA and protein expression in all these cell types Fig 6 C and D Discussion Rapid response platforms are needed to control newly emerg ing virus pathogens MERS CoV likely emerged in the Middle East from exotic animals perhaps bats although the exact timing and origins of this pathogen in human populations remains un clear and is under study MERS CoV has caused 54 deaths at thetimeofwriting 50 mortality including documented instances of human to human transmission 3 8 Therefore the epidemic potential of MERS CoV is high and its biology life cycle gene function and pathogenic mechanisms clearly warrant additional study Developing full length infectious cDNAs of emerging RNA viruses is assisted by the availability of high throughput se quencing gene synthesis and the use of class II IIS restriction endonucleases that allow seamless assembly of consensus syn thetic cDNA fragments into full length infectious cDNAs In this instance we synthesized a molecular clone from the published sequence assembled full length genomes and isolated recombi nant viruses in culture Recently similar approaches have been applied to newly emerging in uenza viruses and can direct the rapid assembly of genome length microbial genomes exceeding millions of base pairs 10 12 14 It is noteworthy that rMERS CoV replicated less ef ciently than wild type MERS CoV a phe notype that was linked to a tissue culture adapted mutation in the S2 glycoprotein of the passage 9 stock of wild type virus S2 con tains the fusion peptide transmembrane domain and two heptad repeat regions HR1 and HR2 conserved features of class I fu sion proteins that form the classic six helix bundle believed to drive virion cell membrane fusion 15 As substitutions in S2 may alter properties ofS1and S includingstabilizingS1 S2association altering fusion behavior and changing tissue tropism 16 it is likely that the T1015N mutation enhances entry and or egress phenotypes in vitro Although the origin of the tissue culture ad aptation is unclear other studies have demonstrated the rapid emergence of tissue culture adaptations in the M and S glyco proteins of human and zoonotic strains of SARS CoV 17 18 As our stock was derived from an inoculum used for primate experiments 19 it is possible that this tissue culture adaptation altered MERS CoV pathogenic outcomes after in vivo infection MERS CoV like other CoVs expresses full length and sgRNAs arranged in the form of a nested set from the 3 end of the Fig 2 Recombinant virus growth A Cultures of Vero 81 cells were infected with MERS CoV rMERS CoV rMERS CoV ORF5 rMERS CoV RFP and rMERS CoV ORF3 5 at an MOI of 0 01 Virus samples were harvested at different times postinfection and titered by plaque assay MERS CoV rMERS CoV rMERS CoV ORF5 rMERS CoV RFP rMERS CoV ORF3 5 B RFLP analysis spanning a naturally occurring BglI site at 17 717 bp that is ablated in rMERS CoV uncut vs digested with Bgll C RFLP analysis across the engineered rMERS CoV E F junction at approximately nucleotide 24397 either uncut or cut with Bgll D Wild type MERS CoV rMERS CoV and rMERS CoV T1015N growth were compared in triplicate in Vero 81 cells MERS CoV rMERS CoV rMERS CoV T1015N Error bars represent SD from the mean Fig 3 rMERS CoV RFP expression and comparative plaque morphology A D Cultures of Vero cells were infected with rMERS CoV RFP A and C or rMERS CoV B and D At 12 h postinfection the cultures were visualized for tomato red uorescence A and B or by light microscopy C and D E Plaque size differences between wild type MERS CoV rMERS CoV and rMERS CoV T1015N Scobey et al PNAS October 1 2013 vol 110 no 40 16159 MICROBIO L OGY genome The expression of sgRNAs is regulated by coordinated interactions between TRSs located at the 5 end of the genome and just upstream of many downstream ORFs Sequence analysis of a subset of the 5 ends of sgRNAs encoding natural or in dicator transgenes indicates that the core sequence AACGAA functions as a site for discontinuous transcription during nega tive strand synthesis MERS CoV encodes several unique group speci c ORFs Deletion analysis clearly demonstrates that these group speci c ORFs are not essential for virus replication al though the absence of these genes as a group attenuates peak viral titers Fig 2A It is interesting that deletion of ORFs 3 5 resulted in enhanced expression of sgRNA2 which encodes the S glycoprotein These ndings are consistent with a model of dis continuous transcription during negative strand synthesis where downstream TRSs in uence the abundance of upstream tran scripts 20 Increased sgRNA2 transcript production resulted in enhanced S glycoprotein expression that was as