Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning.
Identifieur interne : 000B38 ( Main/Exploration ); précédent : 000B37; suivant : 000B39Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning.
Auteurs : Doreen Muth [Allemagne] ; Benjamin Meyer [Allemagne] ; Daniela Niemeyer [Allemagne] ; Simon Schroeder [Allemagne] ; Nikolaus Osterrieder [Allemagne] ; Marcel Alexander Müller [Allemagne] ; Christian Drosten [Allemagne]Source :
- The Journal of general virology [ 1465-2099 ] ; 2017.
Abstract
Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×105 p.f.u. ml-1 and 3×105 p.f.u. ml-1, respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.
DOI: 10.1099/jgv.0.000919
PubMed: 28984231
Affiliations:
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Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin</wicri:regionArea><placeName><region type="land" nuts="3">Berlin</region><settlement type="city">Berlin</settlement></placeName></affiliation></author><author><name sortKey="Drosten, Christian" sort="Drosten, Christian" uniqKey="Drosten C" first="Christian" last="Drosten">Christian Drosten</name><affiliation wicri:level="3"><nlm:affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn</wicri:regionArea><placeName><region type="land" nuts="1">Rhénanie-du-Nord-Westphalie</region><region type="district" nuts="2">District de Cologne</region><settlement type="city">Bonn</settlement></placeName></affiliation></author></analytic><series><title level="j">The Journal of general virology</title><idno type="eISSN">1465-2099</idno><imprint><date when="2017" type="published">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×10<sup>5</sup> p.f.u. ml<sup>-1</sup> and 3×10<sup>5</sup> p.f.u. ml<sup>-1</sup>, respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.</div></front></TEI><affiliations><list><country><li>Allemagne</li></country><region><li>Berlin</li><li>District de Cologne</li><li>Rhénanie-du-Nord-Westphalie</li></region><settlement><li>Berlin</li><li>Bonn</li></settlement></list><tree><country name="Allemagne"><region name="Berlin"><name sortKey="Muth, Doreen" sort="Muth, Doreen" uniqKey="Muth D" first="Doreen" last="Muth">Doreen Muth</name></region><name sortKey="Drosten, Christian" sort="Drosten, Christian" uniqKey="Drosten C" first="Christian" last="Drosten">Christian Drosten</name><name sortKey="Meyer, Benjamin" sort="Meyer, Benjamin" uniqKey="Meyer B" first="Benjamin" last="Meyer">Benjamin Meyer</name><name sortKey="Muller, Marcel Alexander" sort="Muller, Marcel Alexander" uniqKey="Muller M" first="Marcel Alexander" last="Müller">Marcel Alexander Müller</name><name sortKey="Niemeyer, Daniela" sort="Niemeyer, Daniela" uniqKey="Niemeyer D" first="Daniela" last="Niemeyer">Daniela Niemeyer</name><name sortKey="Osterrieder, Nikolaus" sort="Osterrieder, Nikolaus" uniqKey="Osterrieder N" first="Nikolaus" last="Osterrieder">Nikolaus Osterrieder</name><name sortKey="Schroeder, Simon" sort="Schroeder, Simon" uniqKey="Schroeder S" first="Simon" last="Schroeder">Simon Schroeder</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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