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Proc Natl Acad Sci USA. Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus
[Source: Proceedings of the National Academy of Sciences of the United States of America, full page: (LINK). Abstract, edited.]
Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus
Trevor Scobey<SUP>a</SUP>, Boyd L. Yount<SUP>a</SUP>, Amy C. Sims<SUP>a</SUP>, Eric F. Donaldson<SUP>a</SUP>, Sudhakar S. Agnihothram<SUP>a</SUP>, Vineet D. Menachery<SUP>a</SUP>, Rachel L. Graham<SUP>a</SUP>, Jesica Swanstrom<SUP>a</SUP>, Peter F. Bove<SUP>b</SUP>, Jeeho D. Kim<SUP>c</SUP>, Sonia Grego<SUP>d</SUP>, Scott H. Randell<SUP>c</SUP>, and Ralph S. Baric<SUP>a</SUP>,<SUP>e</SUP>,<SUP>1</SUP>
<SUP></SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Epidemiology, <SUP>c</SUP>Cell Biology and Physiology, and <SUP>e</SUP>Microbiology and Immunology and <SUP>b</SUP>Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7435; and <SUP>d</SUP>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)
Significance
The identification of a novel, emerging human coronavirus with ∼50% mortality, designated Middle East respiratory syndrome 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 infectious 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 differentiated primary lung cells.
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.
emerging pathogen ? zoonosis - synthetic genome
Footnotes
<SUP>1</SUP>To whom correspondence should be addressed. E-mail: rbaric@ad.unc.edu.
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. contributed 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 conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311542110/-/DCSupplemental.
Freely available online through the PNAS open access option.
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Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus
Trevor Scobey<SUP>a</SUP>, Boyd L. Yount<SUP>a</SUP>, Amy C. Sims<SUP>a</SUP>, Eric F. Donaldson<SUP>a</SUP>, Sudhakar S. Agnihothram<SUP>a</SUP>, Vineet D. Menachery<SUP>a</SUP>, Rachel L. Graham<SUP>a</SUP>, Jesica Swanstrom<SUP>a</SUP>, Peter F. Bove<SUP>b</SUP>, Jeeho D. Kim<SUP>c</SUP>, Sonia Grego<SUP>d</SUP>, Scott H. Randell<SUP>c</SUP>, and Ralph S. Baric<SUP>a</SUP>,<SUP>e</SUP>,<SUP>1</SUP>
<SUP></SUP>
<SUP></SUP>
Author Affiliations: Departments of <SUP>a</SUP>Epidemiology, <SUP>c</SUP>Cell Biology and Physiology, and <SUP>e</SUP>Microbiology and Immunology and <SUP>b</SUP>Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7435; and <SUP>d</SUP>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)
Significance
The identification of a novel, emerging human coronavirus with ∼50% mortality, designated Middle East respiratory syndrome 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 infectious 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 differentiated primary lung cells.
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.
emerging pathogen ? zoonosis - synthetic genome
Footnotes
<SUP>1</SUP>To whom correspondence should be addressed. E-mail: rbaric@ad.unc.edu.
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. contributed 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 conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1311542110/-/DCSupplemental.
Freely available online through the PNAS open access option.
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