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

10.1371/journal.pbio.3001091

Plos Biology

Contributing to research themes:

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 list:

Affiliations:

  1. MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom, 
  2. Institute of Technology, University of Tartu, Tartu, Estonia, 
  3. MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom, 
  4. Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow, United Kingdom, 
  5. Emerging Viruses, Inflammation and Therapeutics Group, Menzies Health Institute Queensland, Griffith University, Gold Coast, Queensland, Australia,
  6. Division of Biological Sciences, College of Arts and Sciences, University of the Philippines Visayas, Miagao, Iloilo, Philippines, 
  7. West of Scotland Specialist Virology Centre, Glasgow, United Kingdom, 
  8. Indian Immunologicals Ltd (IIL), Rakshapuram, Gachibowli Post, Hyderabad Telangana, India, 
  9. Drug Discovery Unit (DDU), Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dundee, United Kingdom, 
  10. School of Medical Sciences, Griffith University, Gold Coast, Queensland, Australia

Authors:

Suzannah J. Rihn1‡, Andres Merits2‡, Siddharth Bakshi1‡, Matthew L. Turnbull1‡, Arthur Wickenhagen1‡, Akira J. T. Alexander1, Carla Baillie3, Benjamin Brennan1, Fiona Brown3, Kirstyn Brunker4, Steven R. Bryden1, Kerry A. Burness3, Stephen Carmichael1, Sarah J. Cole1, Vanessa M. Cowton1, Paul Davies3, Chris Davis1, Giuditta De Lorenzo1, Claire L. Donald1, Mark Dorward3, James I. Dunlop1, Matthew Elliott3, Mazigh Fares1, Ana da Silva Filipe1, Joseph R. Freitas5, Wilhelm Furnon1, Rommel J. Gestuveo1,6, Anna Geyer3, Daniel Giesel3, Daniel M. Goldfarb1, Nicola Goodman3, Rory Gunson7, C. James Hastie3, Vanessa Herder1, Joseph Hughes1, Clare Johnson3, Natasha Johnson1, Alain Kohl1, Karen Kerr1, Hannah Leech3, Laura Sandra Lello2, Kathy Li1, Gauthier Lieber1, Xiang Liu5, Rajendra Lingala8, Colin Loney1, Daniel Mair1, Marion J. McElwee1, Steven McFarlane1, Jenna Nichols1, Kyriaki Nomikou1, Anne Orr1, Richard J. Orton1, Massimo Palmarini1, Yasmin A. Parr1, Rute Maria Pinto1, Samantha Raggett3, Elaine Reid3, David L. Robertson1, Jamie Royle1, Natalia Cameron-Ruiz1, James G. Shepherd1, Katherine Smollett1, Douglas G. Stewart1, Meredith Stewart1, Elena Sugrue1, Agnieszka M. Szemiel1, Aislynn Taggart1, Emma C. Thomson1, Lily Tong1, Leah S. Torrie9, Rachel Toth3, Margus Varjak1, Sainan Wang2, Stuart G. Wilkinson3, Paul G. Wyatt9, Eva Zusinaite2, Dario R. Alessi3‡, Arvind H. Patel1‡,
Ali Zaid5,10‡, Sam J. Wilson1‡*, Suresh Mahalingam5‡*