tetano
Editor, Senior Moderator
PLoS Biol
. 2021 Feb 25;19(2):e3001091.
doi: 10.1371/journal.pbio.3001091. eCollection 2021 Feb.
A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research
Suzannah J Rihn[SUP] 1 [/SUP], Andres Merits[SUP] 2 [/SUP], Siddharth Bakshi[SUP] 1 [/SUP], Matthew L Turnbull[SUP] 1 [/SUP], Arthur Wickenhagen[SUP] 1 [/SUP], Akira J T Alexander[SUP] 1 [/SUP], Carla Baillie[SUP] 3 [/SUP], Benjamin Brennan[SUP] 1 [/SUP], Fiona Brown[SUP] 3 [/SUP], Kirstyn Brunker[SUP] 4 [/SUP], Steven R Bryden[SUP] 1 [/SUP], Kerry A Burness[SUP] 3 [/SUP], Stephen Carmichael[SUP] 1 [/SUP], Sarah J Cole[SUP] 1 [/SUP], Vanessa M Cowton[SUP] 1 [/SUP], Paul Davies[SUP] 3 [/SUP], Chris Davis[SUP] 1 [/SUP], Giuditta De Lorenzo[SUP] 1 [/SUP], Claire L Donald[SUP] 1 [/SUP], Mark Dorward[SUP] 3 [/SUP], James I Dunlop[SUP] 1 [/SUP], Matthew Elliott[SUP] 3 [/SUP], Mazigh Fares[SUP] 1 [/SUP], Ana da Silva Filipe[SUP] 1 [/SUP], Joseph R Freitas[SUP] 5 [/SUP], Wilhelm Furnon[SUP] 1 [/SUP], Rommel J Gestuveo[SUP] 1 6 [/SUP], Anna Geyer[SUP] 3 [/SUP], Daniel Giesel[SUP] 3 [/SUP], Daniel M Goldfarb[SUP] 1 [/SUP], Nicola Goodman[SUP] 3 [/SUP], Rory Gunson[SUP] 7 [/SUP], C James Hastie[SUP] 3 [/SUP], Vanessa Herder[SUP] 1 [/SUP], Joseph Hughes[SUP] 1 [/SUP], Clare Johnson[SUP] 3 [/SUP], Natasha Johnson[SUP] 1 [/SUP], Alain Kohl[SUP] 1 [/SUP], Karen Kerr[SUP] 1 [/SUP], Hannah Leech[SUP] 3 [/SUP], Laura Sandra Lello[SUP] 2 [/SUP], Kathy Li[SUP] 1 [/SUP], Gauthier Lieber[SUP] 1 [/SUP], Xiang Liu[SUP] 5 [/SUP], Rajendra Lingala[SUP] 8 [/SUP], Colin Loney[SUP] 1 [/SUP], Daniel Mair[SUP] 1 [/SUP], Marion J McElwee[SUP] 1 [/SUP], Steven McFarlane[SUP] 1 [/SUP], Jenna Nichols[SUP] 1 [/SUP], Kyriaki Nomikou[SUP] 1 [/SUP], Anne Orr[SUP] 1 [/SUP], Richard J Orton[SUP] 1 [/SUP], Massimo Palmarini[SUP] 1 [/SUP], Yasmin A Parr[SUP] 1 [/SUP], Rute Maria Pinto[SUP] 1 [/SUP], Samantha Raggett[SUP] 3 [/SUP], Elaine Reid[SUP] 3 [/SUP], David L Robertson[SUP] 1 [/SUP], Jamie Royle[SUP] 1 [/SUP], Natalia Cameron-Ruiz[SUP] 1 [/SUP], James G Shepherd[SUP] 1 [/SUP], Katherine Smollett[SUP] 1 [/SUP], Douglas G Stewart[SUP] 1 [/SUP], Meredith Stewart[SUP] 1 [/SUP], Elena Sugrue[SUP] 1 [/SUP], Agnieszka M Szemiel[SUP] 1 [/SUP], Aislynn Taggart[SUP] 1 [/SUP], Emma C Thomson[SUP] 1 [/SUP], Lily Tong[SUP] 1 [/SUP], Leah S Torrie[SUP] 9 [/SUP], Rachel Toth[SUP] 3 [/SUP], Margus Varjak[SUP] 1 [/SUP], Sainan Wang[SUP] 2 [/SUP], Stuart G Wilkinson[SUP] 3 [/SUP], Paul G Wyatt[SUP] 9 [/SUP], Eva Zusinaite[SUP] 2 [/SUP], Dario R Alessi[SUP] 3 [/SUP], Arvind H Patel[SUP] 1 [/SUP], Ali Zaid[SUP] 5 10 [/SUP], Sam J Wilson[SUP] 1 [/SUP], Suresh Mahalingam[SUP] 5 [/SUP]
Affiliations
Abstract
The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.
. 2021 Feb 25;19(2):e3001091.
doi: 10.1371/journal.pbio.3001091. eCollection 2021 Feb.
A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research
Suzannah J Rihn[SUP] 1 [/SUP], Andres Merits[SUP] 2 [/SUP], Siddharth Bakshi[SUP] 1 [/SUP], Matthew L Turnbull[SUP] 1 [/SUP], Arthur Wickenhagen[SUP] 1 [/SUP], Akira J T Alexander[SUP] 1 [/SUP], Carla Baillie[SUP] 3 [/SUP], Benjamin Brennan[SUP] 1 [/SUP], Fiona Brown[SUP] 3 [/SUP], Kirstyn Brunker[SUP] 4 [/SUP], Steven R Bryden[SUP] 1 [/SUP], Kerry A Burness[SUP] 3 [/SUP], Stephen Carmichael[SUP] 1 [/SUP], Sarah J Cole[SUP] 1 [/SUP], Vanessa M Cowton[SUP] 1 [/SUP], Paul Davies[SUP] 3 [/SUP], Chris Davis[SUP] 1 [/SUP], Giuditta De Lorenzo[SUP] 1 [/SUP], Claire L Donald[SUP] 1 [/SUP], Mark Dorward[SUP] 3 [/SUP], James I Dunlop[SUP] 1 [/SUP], Matthew Elliott[SUP] 3 [/SUP], Mazigh Fares[SUP] 1 [/SUP], Ana da Silva Filipe[SUP] 1 [/SUP], Joseph R Freitas[SUP] 5 [/SUP], Wilhelm Furnon[SUP] 1 [/SUP], Rommel J Gestuveo[SUP] 1 6 [/SUP], Anna Geyer[SUP] 3 [/SUP], Daniel Giesel[SUP] 3 [/SUP], Daniel M Goldfarb[SUP] 1 [/SUP], Nicola Goodman[SUP] 3 [/SUP], Rory Gunson[SUP] 7 [/SUP], C James Hastie[SUP] 3 [/SUP], Vanessa Herder[SUP] 1 [/SUP], Joseph Hughes[SUP] 1 [/SUP], Clare Johnson[SUP] 3 [/SUP], Natasha Johnson[SUP] 1 [/SUP], Alain Kohl[SUP] 1 [/SUP], Karen Kerr[SUP] 1 [/SUP], Hannah Leech[SUP] 3 [/SUP], Laura Sandra Lello[SUP] 2 [/SUP], Kathy Li[SUP] 1 [/SUP], Gauthier Lieber[SUP] 1 [/SUP], Xiang Liu[SUP] 5 [/SUP], Rajendra Lingala[SUP] 8 [/SUP], Colin Loney[SUP] 1 [/SUP], Daniel Mair[SUP] 1 [/SUP], Marion J McElwee[SUP] 1 [/SUP], Steven McFarlane[SUP] 1 [/SUP], Jenna Nichols[SUP] 1 [/SUP], Kyriaki Nomikou[SUP] 1 [/SUP], Anne Orr[SUP] 1 [/SUP], Richard J Orton[SUP] 1 [/SUP], Massimo Palmarini[SUP] 1 [/SUP], Yasmin A Parr[SUP] 1 [/SUP], Rute Maria Pinto[SUP] 1 [/SUP], Samantha Raggett[SUP] 3 [/SUP], Elaine Reid[SUP] 3 [/SUP], David L Robertson[SUP] 1 [/SUP], Jamie Royle[SUP] 1 [/SUP], Natalia Cameron-Ruiz[SUP] 1 [/SUP], James G Shepherd[SUP] 1 [/SUP], Katherine Smollett[SUP] 1 [/SUP], Douglas G Stewart[SUP] 1 [/SUP], Meredith Stewart[SUP] 1 [/SUP], Elena Sugrue[SUP] 1 [/SUP], Agnieszka M Szemiel[SUP] 1 [/SUP], Aislynn Taggart[SUP] 1 [/SUP], Emma C Thomson[SUP] 1 [/SUP], Lily Tong[SUP] 1 [/SUP], Leah S Torrie[SUP] 9 [/SUP], Rachel Toth[SUP] 3 [/SUP], Margus Varjak[SUP] 1 [/SUP], Sainan Wang[SUP] 2 [/SUP], Stuart G Wilkinson[SUP] 3 [/SUP], Paul G Wyatt[SUP] 9 [/SUP], Eva Zusinaite[SUP] 2 [/SUP], Dario R Alessi[SUP] 3 [/SUP], Arvind H Patel[SUP] 1 [/SUP], Ali Zaid[SUP] 5 10 [/SUP], Sam J Wilson[SUP] 1 [/SUP], Suresh Mahalingam[SUP] 5 [/SUP]
Affiliations
- PMID: 33630831
- DOI: 10.1371/journal.pbio.3001091
Abstract
The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.