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

被引：137

作者：

Rihn, Suzannah J. ^{[1
]}

Merits, Andres ^{[2
]}

Bakshi, Siddharth ^{[1
]}

Turnbull, Matthew L. ^{[1
]}

Wickenhagen, Arthur ^{[1
]}

Alexander, Akira J. T. ^{[1
]}

Baillie, Carla ^{[3
]}

Brennan, Benjamin ^{[1
]}

Brown, Fiona ^{[3
]}

Brunker, Kirstyn ^{[4
]}

Bryden, Steven R. ^{[1
]}

Burness, Kerry A. ^{[3
]}

Carmichael, Stephen ^{[1
]}

Cole, Sarah J. ^{[1
]}

Cowton, Vanessa M. ^{[1
]}

Davies, Paul ^{[3
]}

Davis, Chris ^{[1
]}

De Lorenzo, Giuditta ^{[1
]}

Donald, Claire L. ^{[1
]}

Dorward, Mark ^{[3
]}

Dunlop, James I. ^{[1
]}

Elliott, Matthew ^{[3
]}

Fares, Mazigh ^{[1
]}

da Silva Filipe, Ana ^{[1
]}

Freitas, Joseph R. ^{[5
]}

Furnon, Wilhelm ^{[1
]}

Gestuveo, Rommel J. ^{[1
,6
]}

Geyer, Anna ^{[3
]}

Giesel, Daniel ^{[3
]}

Goldfarb, Daniel M. ^{[1
]}

Goodman, Nicola ^{[3
]}

Gunson, Rory ^{[7
]}

Hastie, C. James ^{[3
]}

Herder, Vanessa ^{[1
]}

Hughes, Joseph ^{[1
]}

Johnson, Clare ^{[3
]}

Johnson, Natasha ^{[1
]}

Kohl, Alain ^{[1
]}

Kerr, Karen ^{[1
]}

Leech, Hannah ^{[3
]}

Lello, Laura Sandra ^{[2
]}

Li, Kathy ^{[1
]}

Lieber, Gauthier ^{[1
]}

Liu, Xiang ^{[5
]}

Lingala, Rajendra ^{[8
]}

Loney, Colin ^{[1
]}

Mair, Daniel ^{[1
]}

McElwee, Marion J. ^{[1
]}

McFarlane, Steven ^{[1
]}

Nichols, Jenna ^{[1
]}

机构：

[1] Univ Glasgow, Ctr Virus Res CVR, MRC, Glasgow, Lanark, Scotland

[2] Univ Tartu, Inst Technol, Tartu, Estonia

[3] Univ Dundee, Sch Life Sci, MRC Prot Phosphorylat & Ubiquitylat Unit, Dundee, Scotland

[4] Univ Glasgow, Inst Biodivers Anim Hlth & Comparat Med, Glasgow, Lanark, Scotland

[5] Griffith Univ, Menzies Hlth Inst Queensland, Emerging Viruses Inflammat & Therapeut Grp, Gold Coast, Qld, Australia

[6] Univ Philippines Visayas, Coll Arts & Sci, Div Biol Sci, Iloilo, Philippines

[7] West Scotland Specialist Virol Ctr, Glasgow, Lanark, Scotland

[8] Indian Immunol Ltd IIL, Hyderabad, Telangana, India

[9] Univ Dundee, Sch Life Sci, Wellcome Ctr AntiInfect Res, Drug Discovery Unit DDU, Dundee, Scotland

[10] Griffith Univ, Sch Med Sci, Gold Coast, Qld, Australia

来源：

PLOS BIOLOGY | 2021年 / 19卷 / 02期

基金：

英国医学研究理事会; 澳大利亚国家健康与医学研究理事会; 英国惠康基金; 比尔及梅琳达.盖茨基金会;

关键词：

CLEAVAGE SITE; SPIKE; INFECTION; MEMBRANE; PROTEIN; ENTRY; SARS;

D O I：

10.1371/journal.pbio.3001091

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 , constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

引用

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