title: Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.
abstract: Severe acute respiratory syndrome with high mortality 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 mutations 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 efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus 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 fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.
authors.affiliation: Departments of Epidemiology, Cell Biology and Physiology, and Microbiology and Immunology and Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7435.
authors.forename: Trevor
authors.initials: T
authors.lastname: Scobey
authors.forename: Boyd L
authors.initials: BL
authors.lastname: Yount
authors.forename: Amy C
authors.initials: AC
authors.lastname: Sims
authors.forename: Eric F
authors.initials: EF
authors.lastname: Donaldson
authors.forename: Sudhakar S
authors.initials: SS
authors.lastname: Agnihothram
authors.forename: Vineet D
authors.initials: VD
authors.lastname: Menachery
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Graham
authors.forename: Jesica
authors.initials: J
authors.lastname: Swanstrom
authors.forename: Peter F
authors.initials: PF
authors.lastname: Bove
authors.forename: Jeeho D
authors.initials: JD
authors.lastname: Kim
authors.forename: Sonia
authors.initials: S
authors.lastname: Grego
authors.forename: Scott H
authors.initials: SH
authors.lastname: Randell
authors.forename: Ralph S
authors.initials: RS
authors.lastname: Baric
mesh_terms: D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D021821:Communicable Diseases, Emerging / Q000821:virology*; D017934:Coronavirus / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D015967:Gene Expression Regulation, Viral / Q000235:genetics / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins; D008877:Middle East; D012150:Polymorphism, Restriction Fragment Length; D060888:Real-Time Polymerase Chain Reaction; D045169:Severe Acute Respiratory Syndrome / Q000821:virology*; D064370:Spike Glycoprotein, Coronavirus / Q000235:genetics / Q000502:physiology; D053585:Virus Attachment; D014779:Virus Replication / Q000502:physiology; D000097573:Red Fluorescent Protein
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D018819:Dipeptidyl Peptidase 4; D008164:Luminescent Proteins; D064370:Spike Glycoprotein, Coronavirus; D000097573:Red Fluorescent Protein
keywords: emerging pathogen; synthetic genome; zoonosis
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