Fachbereich Veterinärmedizin



    Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning (2017)

    Zeitschriftenartikel / wissenschaftlicher Beitrag
    Muth, Doreen
    Meyer, Benjamin
    Niemeyer, Daniela
    Schroeder, Simon
    Osterrieder, Nikolaus (WE 5)
    Müller, Marcel Alexander
    Drosten, Christian
    The journal of general virology; 98(10) — S. 2461–2469
    ISSN: 0022-1317
    DOI: 10.1099/jgv.0.000919
    Pubmed: 28984231
    Institut für Virologie

    Robert-von-Ostertag-Str. 7-13
    Gebäude 35
    14163 Berlin
    +49 30 838 51833

    Abstract / Zusammenfassung

    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(5) p.f.u. ml(-1) and 3×10(5) 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.