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 : 001C04 ( Ncbi/Checkpoint ); précédent : 001C03; suivant : 001C05Transgene 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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<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>
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