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 : 000B29 ( PubMed/Corpus ); précédent : 000B28; suivant : 000B30Transgene 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 ; Benjamin Meyer ; Daniela Niemeyer ; Simon Schroeder ; Nikolaus Osterrieder ; Marcel Alexander Müller ; Christian DrostenSource :
- 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
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Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Meyer, Benjamin" sort="Meyer, Benjamin" uniqKey="Meyer B" first="Benjamin" last="Meyer">Benjamin Meyer</name><affiliation><nlm:affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Niemeyer, Daniela" sort="Niemeyer, Daniela" uniqKey="Niemeyer D" first="Daniela" last="Niemeyer">Daniela Niemeyer</name><affiliation><nlm:affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Schroeder, Simon" sort="Schroeder, Simon" uniqKey="Schroeder S" first="Simon" last="Schroeder">Simon Schroeder</name><affiliation><nlm:affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Osterrieder, Nikolaus" sort="Osterrieder, Nikolaus" uniqKey="Osterrieder N" first="Nikolaus" last="Osterrieder">Nikolaus Osterrieder</name><affiliation><nlm:affiliation>Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Muller, Marcel Alexander" sort="Muller, Marcel Alexander" uniqKey="Muller M" first="Marcel Alexander" last="Müller">Marcel Alexander Müller</name><affiliation><nlm:affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</nlm:affiliation></affiliation></author><author><name sortKey="Drosten, Christian" sort="Drosten, Christian" uniqKey="Drosten C" first="Christian" last="Drosten">Christian Drosten</name><affiliation><nlm:affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</nlm:affiliation></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><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">28984231</PMID><DateRevised><Year>2020</Year><Month>01</Month><Day>08</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Electronic">1465-2099</ISSN><JournalIssue CitedMedium="Internet"><Volume>98</Volume><Issue>10</Issue><PubDate><Year>2017</Year><Month>Oct</Month></PubDate></JournalIssue><Title>The Journal of general virology</Title><ISOAbbreviation>J. Gen. Virol.</ISOAbbreviation></Journal><ArticleTitle>Transgene expression in the genome of Middle East respiratory syndrome coronavirus based on a novel reverse genetics system utilizing Red-mediated recombination cloning.</ArticleTitle><Pagination><MedlinePgn>2461-2469</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1099/jgv.0.000919</ELocationID><Abstract><AbstractText>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.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Muth</LastName><ForeName>Doreen</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>German Centre for Infection Research (DZIF), Inhoffenstraße 7, 38124 Braunschweig, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Meyer</LastName><ForeName>Benjamin</ForeName><Initials>B</Initials><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Niemeyer</LastName><ForeName>Daniela</ForeName><Initials>D</Initials><AffiliationInfo><Affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Schroeder</LastName><ForeName>Simon</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Osterrieder</LastName><ForeName>Nikolaus</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Müller</LastName><ForeName>Marcel Alexander</ForeName><Initials>MA</Initials><AffiliationInfo><Affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Drosten</LastName><ForeName>Christian</ForeName><Initials>C</Initials><AffiliationInfo><Affiliation>Institute of Virology, University of Bonn Medical Centre, Sigmund-Freud-Str. 25, 53127 Bonn, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>German Centre for Infection Research (DZIF), Inhoffenstraße 7, 38124 Braunschweig, Germany.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Virology, Helmut-Ruska-Haus, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>J Gen Virol</MedlineTA><NlmUniqueID>0077340</NlmUniqueID><ISSNLinking>0022-1317</ISSNLinking></MedlineJournalInfo><CitationSubset>IM</CitationSubset><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">MERS-CoV</Keyword><Keyword MajorTopicYN="N">recombination</Keyword><Keyword MajorTopicYN="N">reporter virus</Keyword><Keyword MajorTopicYN="N">reverse 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