Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Trevor Scobey, Boyd L. Yount, Amy C. Sims, Eric F. Donaldson, Sudhakar S. Agnihothram, Vineet Menachery, Rachel L. Graham, Jesica Swanstrom, Peter F. Bove, Jeeho D. Kim, Sonia Grego, Scott H. Randell, Ralph S. Baric

Research output: Contribution to journalArticle

101 Citations (Scopus)

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 rMERSCoV, 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.

Original languageEnglish (US)
Pages (from-to)16157-16162
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number40
DOIs
StatePublished - Oct 1 2013
Externally publishedYes

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Reverse Genetics
Complementary DNA
Viruses
Severe Acute Respiratory Syndrome
Clone Cells
Genome
Dipeptidyl Peptidase 4
Alveolar Epithelial Cells
Lung
Coronavirus
Mutation
Proteins
Tropism
Consensus Sequence
Lycopersicon esculentum
Northern Blotting
Sequence Analysis
Glycoproteins
Vaccines
Endothelial Cells

Keywords

  • Emerging pathogen
  • Synthetic genome
  • Zoonosis

ASJC Scopus subject areas

  • General

Cite this

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. / Scobey, Trevor; Yount, Boyd L.; Sims, Amy C.; Donaldson, Eric F.; Agnihothram, Sudhakar S.; Menachery, Vineet; Graham, Rachel L.; Swanstrom, Jesica; Bove, Peter F.; Kim, Jeeho D.; Grego, Sonia; Randell, Scott H.; Baric, Ralph S.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 110, No. 40, 01.10.2013, p. 16157-16162.

Research output: Contribution to journalArticle

Scobey, T, Yount, BL, Sims, AC, Donaldson, EF, Agnihothram, SS, Menachery, V, Graham, RL, Swanstrom, J, Bove, PF, Kim, JD, Grego, S, Randell, SH & Baric, RS 2013, 'Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus', Proceedings of the National Academy of Sciences of the United States of America, vol. 110, no. 40, pp. 16157-16162. https://doi.org/10.1073/pnas.1311542110
Scobey, Trevor ; Yount, Boyd L. ; Sims, Amy C. ; Donaldson, Eric F. ; Agnihothram, Sudhakar S. ; Menachery, Vineet ; Graham, Rachel L. ; Swanstrom, Jesica ; Bove, Peter F. ; Kim, Jeeho D. ; Grego, Sonia ; Randell, Scott H. ; Baric, Ralph S. / Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus. In: Proceedings of the National Academy of Sciences of the United States of America. 2013 ; Vol. 110, No. 40. pp. 16157-16162.
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