A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

Suzannah J. Rihn; Andres Merits; Siddharth Bakshi; Matthew L. Turnbull; Arthur Wickenhagen; Akira J.T. Alexander; Carla Baillie; Benjamin Brennan; Fiona Brown; Kirstyn Brunker; Steven R. Bryden; Kerry A. Burness; Stephen Carmichael; Sarah J. Cole; Vanessa M. Cowton; Paul Davies; Chris Davis; Giuditta de Lorenzo; Claire L. Donald; Mark Dorward; James I. Dunlop; Matthew Elliott; Mazigh Fares; Ana da Silva Filipe; Joseph R. Freitas; Wilhelm Furnon; Rommel J. Gestuveo; Anna Geyer; Daniel Giesel; Daniel M. Goldfarb; Nicola Goodman; Rory Gunson; C. James Hastie; Vanessa Herder; Joseph Hughes; Clare Johnson; Natasha Johnson; Alain Kohl; Karen Kerr; Hannah Leech; Laura Sandra Lello; Kathy Li; Gauthier Lieber; Xiang Liu; Rajendra Lingala; Colin Loney; Daniel Mair; Marion J. McElwee; Steven McFarlane; Jenna Nichols; Kyriaki Nomikou; Anne Orr; Richard J. Orton; Massimo Palmarini; Yasmin A. Parr; Rute Maria Pinto; Samantha Raggett; Elaine Reid; David L. Robertson; Jamie Royle; Natalia Cameron-Ruiz; James G. Shepherd; Katherine Smollett; Douglas G. Stewart; Meredith Stewart; Elena Sugrue; Agnieszka M. Szemiel; Aislynn Taggart; Emma C. Thomson; Lily Tong; Leah S. Torrie; Rachel Toth; Margus Varjak; Sainan Wang; Stuart G. Wilkinson; Paul G. Wyatt; Eva Zusinaite; Dario R. Alessi; Arvind H. Patel; Ali Zaid; Sam J. Wilson; Suresh Mahalingam

doi:10.1371/journal.pbio.3001091

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

Suzannah J. Rihn, Andres Merits, Siddharth Bakshi, Matthew L. Turnbull, Arthur Wickenhagen, Akira J.T. Alexander, Carla Baillie, Benjamin Brennan, Fiona Brown, Kirstyn Brunker, Steven R. Bryden, Kerry A. Burness, Stephen Carmichael, Sarah J. Cole, Vanessa M. Cowton, Paul Davies, Chris Davis, Giuditta de Lorenzo, Claire L. Donald, Mark DorwardJames I. Dunlop, Matthew Elliott, Mazigh Fares, Ana da Silva Filipe, Joseph R. Freitas, Wilhelm Furnon, Rommel J. Gestuveo, Anna Geyer, Daniel Giesel, Daniel M. Goldfarb, Nicola Goodman, Rory Gunson, C. James Hastie, Vanessa Herder, Joseph Hughes, Clare Johnson, Natasha Johnson, Alain Kohl, Karen Kerr, Hannah Leech, Laura Sandra Lello, Kathy Li, Gauthier Lieber, Xiang Liu, Rajendra Lingala, Colin Loney, Daniel Mair, Marion J. McElwee, Steven McFarlane, Jenna Nichols, Kyriaki Nomikou, Anne Orr, Richard J. Orton, Massimo Palmarini, Yasmin A. Parr, Rute Maria Pinto, Samantha Raggett, Elaine Reid, David L. Robertson, Jamie Royle, Natalia Cameron-Ruiz, James G. Shepherd, Katherine Smollett, Douglas G. Stewart, Meredith Stewart, Elena Sugrue, Agnieszka M. Szemiel, Aislynn Taggart, Emma C. Thomson, Lily Tong, Leah S. Torrie, Rachel Toth, Margus Varjak, Sainan Wang, Stuart G. Wilkinson, Paul G. Wyatt, Eva Zusinaite, Dario R. Alessi, Arvind H. Patel, Ali Zaid, Sam J. Wilson, Suresh Mahalingam

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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.

Original language	English
Article number	e3001091
Journal	PLoS Biology
Volume	19
Issue number	2
DOIs	https://doi.org/10.1371/journal.pbio.3001091
Publication status	Published - 25 Feb 2021
Externally published	Yes

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This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1371/journal.pbio.3001091Licence: CC BY

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Rihn, S. J., Merits, A., Bakshi, S., Turnbull, M. L., Wickenhagen, A., Alexander, A. J. T., Baillie, C., Brennan, B., Brown, F., Brunker, K., Bryden, S. R., Burness, K. A., Carmichael, S., Cole, S. J., Cowton, V. M., Davies, P., Davis, C., de Lorenzo, G., Donald, C. L., ... Mahalingam, S. (2021). A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biology, 19(2), Article e3001091. https://doi.org/10.1371/journal.pbio.3001091

@article{7f362512ef0948a7a6e6a6a117106b21,

title = "A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research",

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.",

author = "Rihn, \{Suzannah J.\} and Andres Merits and Siddharth Bakshi and Turnbull, \{Matthew L.\} and Arthur Wickenhagen and Alexander, \{Akira J.T.\} and Carla Baillie and Benjamin Brennan and Fiona Brown and Kirstyn Brunker and Bryden, \{Steven R.\} and Burness, \{Kerry A.\} and Stephen Carmichael and Cole, \{Sarah J.\} and Cowton, \{Vanessa M.\} and Paul Davies and Chris Davis and \{de Lorenzo\}, Giuditta and Donald, \{Claire L.\} and Mark Dorward and Dunlop, \{James I.\} and Matthew Elliott and Mazigh Fares and \{da Silva Filipe\}, Ana and Freitas, \{Joseph R.\} and Wilhelm Furnon and Gestuveo, \{Rommel J.\} and Anna Geyer and Daniel Giesel and Goldfarb, \{Daniel M.\} and Nicola Goodman and Rory Gunson and \{James Hastie\}, C. and Vanessa Herder and Joseph Hughes and Clare Johnson and Natasha Johnson and Alain Kohl and Karen Kerr and Hannah Leech and Lello, \{Laura Sandra\} and Kathy Li and Gauthier Lieber and Xiang Liu and Rajendra Lingala and Colin Loney and Daniel Mair and McElwee, \{Marion J.\} and Steven McFarlane and Jenna Nichols and Kyriaki Nomikou and Anne Orr and Orton, \{Richard J.\} and Massimo Palmarini and Parr, \{Yasmin A.\} and Pinto, \{Rute Maria\} and Samantha Raggett and Elaine Reid and Robertson, \{David L.\} and Jamie Royle and Natalia Cameron-Ruiz and Shepherd, \{James G.\} and Katherine Smollett and Stewart, \{Douglas G.\} and Meredith Stewart and Elena Sugrue and Szemiel, \{Agnieszka M.\} and Aislynn Taggart and Thomson, \{Emma C.\} and Lily Tong and Torrie, \{Leah S.\} and Rachel Toth and Margus Varjak and Sainan Wang and Wilkinson, \{Stuart G.\} and Wyatt, \{Paul G.\} and Eva Zusinaite and Alessi, \{Dario R.\} and Patel, \{Arvind H.\} and Ali Zaid and Wilson, \{Sam J.\} and Suresh Mahalingam",

year = "2021",

month = feb,

day = "25",

doi = "10.1371/journal.pbio.3001091",

language = "English",

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Rihn, SJ, Merits, A, Bakshi, S, Turnbull, ML, Wickenhagen, A, Alexander, AJT, Baillie, C, Brennan, B, Brown, F, Brunker, K, Bryden, SR, Burness, KA, Carmichael, S, Cole, SJ, Cowton, VM, Davies, P, Davis, C, de Lorenzo, G, Donald, CL, Dorward, M, Dunlop, JI, Elliott, M, Fares, M, da Silva Filipe, A, Freitas, JR, Furnon, W, Gestuveo, RJ, Geyer, A, Giesel, D, Goldfarb, DM, Goodman, N, Gunson, R, James Hastie, C, Herder, V, Hughes, J, Johnson, C, Johnson, N, Kohl, A, Kerr, K, Leech, H, Lello, LS, Li, K, Lieber, G, Liu, X, Lingala, R, Loney, C, Mair, D, McElwee, MJ, McFarlane, S, Nichols, J, Nomikou, K, Orr, A, Orton, RJ, Palmarini, M, Parr, YA, Pinto, RM, Raggett, S, Reid, E, Robertson, DL, Royle, J, Cameron-Ruiz, N, Shepherd, JG, Smollett, K, Stewart, DG, Stewart, M, Sugrue, E, Szemiel, AM, Taggart, A, Thomson, EC, Tong, L, Torrie, LS, Toth, R, Varjak, M, Wang, S, Wilkinson, SG, Wyatt, PG, Zusinaite, E, Alessi, DR, Patel, AH, Zaid, A, Wilson, SJ & Mahalingam, S 2021, 'A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research', PLoS Biology, vol. 19, no. 2, e3001091. https://doi.org/10.1371/journal.pbio.3001091

TY - JOUR

T1 - A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

AU - Rihn, Suzannah J.

AU - Merits, Andres

AU - Bakshi, Siddharth

AU - Turnbull, Matthew L.

AU - Wickenhagen, Arthur

AU - Alexander, Akira J.T.

AU - Baillie, Carla

AU - Brennan, Benjamin

AU - Brown, Fiona

AU - Brunker, Kirstyn

AU - Bryden, Steven R.

AU - Burness, Kerry A.

AU - Carmichael, Stephen

AU - Cole, Sarah J.

AU - Cowton, Vanessa M.

AU - Davies, Paul

AU - Davis, Chris

AU - de Lorenzo, Giuditta

AU - Donald, Claire L.

AU - Dorward, Mark

AU - Dunlop, James I.

AU - Elliott, Matthew

AU - Fares, Mazigh

AU - da Silva Filipe, Ana

AU - Freitas, Joseph R.

AU - Furnon, Wilhelm

AU - Gestuveo, Rommel J.

AU - Geyer, Anna

AU - Giesel, Daniel

AU - Goldfarb, Daniel M.

AU - Goodman, Nicola

AU - Gunson, Rory

AU - James Hastie, C.

AU - Herder, Vanessa

AU - Hughes, Joseph

AU - Johnson, Clare

AU - Johnson, Natasha

AU - Kohl, Alain

AU - Kerr, Karen

AU - Leech, Hannah

AU - Lello, Laura Sandra

AU - Li, Kathy

AU - Lieber, Gauthier

AU - Liu, Xiang

AU - Lingala, Rajendra

AU - Loney, Colin

AU - Mair, Daniel

AU - McElwee, Marion J.

AU - McFarlane, Steven

AU - Nichols, Jenna

AU - Nomikou, Kyriaki

AU - Orr, Anne

AU - Orton, Richard J.

AU - Palmarini, Massimo

AU - Parr, Yasmin A.

AU - Pinto, Rute Maria

AU - Raggett, Samantha

AU - Reid, Elaine

AU - Robertson, David L.

AU - Royle, Jamie

AU - Cameron-Ruiz, Natalia

AU - Shepherd, James G.

AU - Smollett, Katherine

AU - Stewart, Douglas G.

AU - Stewart, Meredith

AU - Sugrue, Elena

AU - Szemiel, Agnieszka M.

AU - Taggart, Aislynn

AU - Thomson, Emma C.

AU - Tong, Lily

AU - Torrie, Leah S.

AU - Toth, Rachel

AU - Varjak, Margus

AU - Wang, Sainan

AU - Wilkinson, Stuart G.

AU - Wyatt, Paul G.

AU - Zusinaite, Eva

AU - Alessi, Dario R.

AU - Patel, Arvind H.

AU - Zaid, Ali

AU - Wilson, Sam J.

AU - Mahalingam, Suresh

PY - 2021/2/25

Y1 - 2021/2/25

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pbio.3001091

DO - 10.1371/journal.pbio.3001091

M3 - Article

SN - 1544-9173

VL - 19

JO - PLoS Biology

JF - PLoS Biology

IS - 2

M1 - e3001091

ER -

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research

Abstract

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