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Volume 98, Issue 10

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

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 (1×10 p.f.u. ml and 3×10 p.f.u. ml, 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): MERS-CoV , recombination , reporter virus and reverse genetics
© 2017 The Authors
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2017-10-01
2024-04-19
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Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning
J. Gen. Virol. 98, 2461 (2017); https://doi.org/10.1099/jgv.0.000919
/content/journal/jgv/10.1099/jgv.0.000919
/content/journal/jgv/10.1099/jgv.0.000919
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