Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.
Identifieur interne : 001F17 ( Main/Exploration ); précédent : 001F16; suivant : 001F18Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus.
Auteurs : Trevor Scobey [États-Unis] ; Boyd L. Yount ; Amy C. Sims ; Eric F. Donaldson ; Sudhakar S. Agnihothram ; Vineet D. Menachery ; Rachel L. Graham ; Jesica Swanstrom ; Peter F. Bove ; Jeeho D. Kim ; Sonia Grego ; Scott H. Randell ; Ralph S. BaricSource :
- Proceedings of the National Academy of Sciences of the United States of America [ 1091-6490 ] ; 2013.
Descripteurs français
- KwdFr :
- ADN complémentaire (génétique), Amorces ADN (génétique), Attachement viral, Cellules cultivées, Coronavirus (génétique), Dipeptidyl peptidase 4 (métabolisme), Glycoprotéine de spicule des coronavirus (génétique), Glycoprotéine de spicule des coronavirus (physiologie), Humains, Maladies transmissibles émergentes (virologie), Moyen Orient, Polymorphisme de restriction, Protéines luminescentes, Réaction de polymérisation en chaine en temps réel, Régulation de l'expression des gènes viraux (génétique), Régulation de l'expression des gènes viraux (physiologie), Réplication virale (physiologie), Syndrome respiratoire aigu sévère (virologie), Technique de Northern, Technique de Western.
- MESH :
- génétique : ADN complémentaire, Amorces ADN, Coronavirus, Glycoprotéine de spicule des coronavirus, Régulation de l'expression des gènes viraux.
- métabolisme : Dipeptidyl peptidase 4.
- physiologie : Glycoprotéine de spicule des coronavirus, Régulation de l'expression des gènes viraux, Réplication virale.
- virologie : Maladies transmissibles émergentes, Syndrome respiratoire aigu sévère.
- Attachement viral, Cellules cultivées, Humains, Moyen Orient, Polymorphisme de restriction, Protéines luminescentes, Réaction de polymérisation en chaine en temps réel, Technique de Northern, Technique de Western.
English descriptors
- KwdEn :
- Blotting, Northern, Blotting, Western, Cells, Cultured, Communicable Diseases, Emerging (virology), Coronavirus (genetics), DNA Primers (genetics), DNA, Complementary (genetics), Dipeptidyl Peptidase 4 (metabolism), Gene Expression Regulation, Viral (genetics), Gene Expression Regulation, Viral (physiology), Humans, Luminescent Proteins, Middle East, Polymorphism, Restriction Fragment Length, Real-Time Polymerase Chain Reaction, Severe Acute Respiratory Syndrome (virology), Spike Glycoprotein, Coronavirus (genetics), Spike Glycoprotein, Coronavirus (physiology), Virus Attachment, Virus Replication (physiology).
- MESH :
- chemical , genetics : DNA Primers, DNA, Complementary, Spike Glycoprotein, Coronavirus.
- chemical , physiology : Spike Glycoprotein, Coronavirus.
- geographic : Middle East.
- genetics : Coronavirus, Gene Expression Regulation, Viral.
- chemical , metabolism : Dipeptidyl Peptidase 4.
- physiology : Gene Expression Regulation, Viral, Virus Replication.
- virology : Communicable Diseases, Emerging, Severe Acute Respiratory Syndrome.
- Blotting, Northern, Blotting, Western, Cells, Cultured, Humans, Luminescent Proteins, Polymorphism, Restriction Fragment Length, Real-Time Polymerase Chain Reaction, Virus Attachment.
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.
DOI: 10.1073/pnas.1311542110
PubMed: 24043791
Affiliations:
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Le document en format XML
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Yount</name></author><author><name sortKey="Sims, Amy C" sort="Sims, Amy C" uniqKey="Sims A" first="Amy C" last="Sims">Amy C. Sims</name></author><author><name sortKey="Donaldson, Eric F" sort="Donaldson, Eric F" uniqKey="Donaldson E" first="Eric F" last="Donaldson">Eric F. Donaldson</name></author><author><name sortKey="Agnihothram, Sudhakar S" sort="Agnihothram, Sudhakar S" uniqKey="Agnihothram S" first="Sudhakar S" last="Agnihothram">Sudhakar S. Agnihothram</name></author><author><name sortKey="Menachery, Vineet D" sort="Menachery, Vineet D" uniqKey="Menachery V" first="Vineet D" last="Menachery">Vineet D. Menachery</name></author><author><name sortKey="Graham, Rachel L" sort="Graham, Rachel L" uniqKey="Graham R" first="Rachel L" last="Graham">Rachel L. 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Baric</name></author></analytic><series><title level="j">Proceedings of the National Academy of Sciences of the United States of America</title><idno type="eISSN">1091-6490</idno><imprint><date when="2013" type="published">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Blotting, Northern</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Communicable Diseases, Emerging (virology)</term><term>Coronavirus (genetics)</term><term>DNA Primers (genetics)</term><term>DNA, Complementary (genetics)</term><term>Dipeptidyl Peptidase 4 (metabolism)</term><term>Gene Expression Regulation, Viral (genetics)</term><term>Gene Expression Regulation, Viral (physiology)</term><term>Humans</term><term>Luminescent Proteins</term><term>Middle East</term><term>Polymorphism, Restriction Fragment Length</term><term>Real-Time Polymerase Chain Reaction</term><term>Severe Acute Respiratory Syndrome (virology)</term><term>Spike Glycoprotein, Coronavirus (genetics)</term><term>Spike Glycoprotein, Coronavirus (physiology)</term><term>Virus Attachment</term><term>Virus Replication (physiology)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN complémentaire (génétique)</term><term>Amorces ADN (génétique)</term><term>Attachement viral</term><term>Cellules cultivées</term><term>Coronavirus (génétique)</term><term>Dipeptidyl peptidase 4 (métabolisme)</term><term>Glycoprotéine de spicule des coronavirus (génétique)</term><term>Glycoprotéine de spicule des coronavirus (physiologie)</term><term>Humains</term><term>Maladies transmissibles émergentes (virologie)</term><term>Moyen Orient</term><term>Polymorphisme de restriction</term><term>Protéines luminescentes</term><term>Réaction de polymérisation en chaine en temps réel</term><term>Régulation de l'expression des gènes viraux (génétique)</term><term>Régulation de l'expression des gènes viraux (physiologie)</term><term>Réplication virale (physiologie)</term><term>Syndrome respiratoire aigu sévère (virologie)</term><term>Technique de Northern</term><term>Technique de Western</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA Primers</term><term>DNA, Complementary</term><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="chemical" qualifier="physiology" xml:lang="en"><term>Spike Glycoprotein, Coronavirus</term></keywords><keywords scheme="MESH" type="geographic" xml:lang="en"><term>Middle East</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Coronavirus</term><term>Gene Expression Regulation, Viral</term></keywords><keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>ADN complémentaire</term><term>Amorces ADN</term><term>Coronavirus</term><term>Glycoprotéine de spicule des coronavirus</term><term>Régulation de l'expression des gènes viraux</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Dipeptidyl Peptidase 4</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Dipeptidyl peptidase 4</term></keywords><keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Glycoprotéine de spicule des coronavirus</term><term>Régulation de l'expression des gènes viraux</term><term>Réplication virale</term></keywords><keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>Gene Expression Regulation, Viral</term><term>Virus Replication</term></keywords><keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Maladies transmissibles émergentes</term><term>Syndrome respiratoire aigu sévère</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Communicable Diseases, Emerging</term><term>Severe Acute Respiratory Syndrome</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Blotting, Northern</term><term>Blotting, Western</term><term>Cells, Cultured</term><term>Humans</term><term>Luminescent Proteins</term><term>Polymorphism, Restriction Fragment Length</term><term>Real-Time Polymerase Chain Reaction</term><term>Virus Attachment</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>Attachement viral</term><term>Cellules cultivées</term><term>Humains</term><term>Moyen Orient</term><term>Polymorphisme de restriction</term><term>Protéines luminescentes</term><term>Réaction de polymérisation en chaine en temps réel</term><term>Technique de Northern</term><term>Technique de Western</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">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.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country><region><li>Caroline du Nord</li></region></list><tree><noCountry><name sortKey="Agnihothram, Sudhakar S" sort="Agnihothram, Sudhakar S" uniqKey="Agnihothram S" first="Sudhakar S" last="Agnihothram">Sudhakar S. Agnihothram</name><name sortKey="Baric, Ralph S" sort="Baric, Ralph S" uniqKey="Baric R" first="Ralph S" last="Baric">Ralph S. Baric</name><name sortKey="Bove, Peter F" sort="Bove, Peter F" uniqKey="Bove P" first="Peter F" last="Bove">Peter F. Bove</name><name sortKey="Donaldson, Eric F" sort="Donaldson, Eric F" uniqKey="Donaldson E" first="Eric F" last="Donaldson">Eric F. Donaldson</name><name sortKey="Graham, Rachel L" sort="Graham, Rachel L" uniqKey="Graham R" first="Rachel L" last="Graham">Rachel L. Graham</name><name sortKey="Grego, Sonia" sort="Grego, Sonia" uniqKey="Grego S" first="Sonia" last="Grego">Sonia Grego</name><name sortKey="Kim, Jeeho D" sort="Kim, Jeeho D" uniqKey="Kim J" first="Jeeho D" last="Kim">Jeeho D. Kim</name><name sortKey="Menachery, Vineet D" sort="Menachery, Vineet D" uniqKey="Menachery V" first="Vineet D" last="Menachery">Vineet D. Menachery</name><name sortKey="Randell, Scott H" sort="Randell, Scott H" uniqKey="Randell S" first="Scott H" last="Randell">Scott H. Randell</name><name sortKey="Sims, Amy C" sort="Sims, Amy C" uniqKey="Sims A" first="Amy C" last="Sims">Amy C. Sims</name><name sortKey="Swanstrom, Jesica" sort="Swanstrom, Jesica" uniqKey="Swanstrom J" first="Jesica" last="Swanstrom">Jesica Swanstrom</name><name sortKey="Yount, Boyd L" sort="Yount, Boyd L" uniqKey="Yount B" first="Boyd L" last="Yount">Boyd L. Yount</name></noCountry><country name="États-Unis"><region name="Caroline du Nord"><name sortKey="Scobey, Trevor" sort="Scobey, Trevor" uniqKey="Scobey T" first="Trevor" last="Scobey">Trevor Scobey</name></region></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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