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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.

SUBMITTER: Rihn SJ

PROVIDER: S-EPMC7906417 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

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

PLoS biology 20210225 2

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 ...[more]

PMID: 33630831

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Project description:BackgroundThe COVID-19 agent, SARS-CoV-2, is conspecific with SARS-CoV, the causal agent of the severe acute respiratory syndrome epidemic in 2002-03. Although the viruses share a completely homologous repertoire of proteins and use the same cellular entry receptor, their transmission efficiencies and pathogenetic traits differ. We aimed to compare interferon antagonism by SARS-CoV and SARS-CoV-2.MethodsFor this functional study, we infected Vero E6 and Calu-3 cells with strains of SARS-CoV and SARS-CoV-2. We studied differences in cell line-specific replication (Vero E6 vs Calu-3 cells) and analysed these differences in relation to TMPRSS2-dependent cell entry based on inhibition with the drug camostat mesilate. We evaluated viral sensitivity towards type I interferon treatment and assessed cytokine induction and type I interferon signalling in the host cells by RT-PCR and analysis of transcription factor activation and nuclear translocation. Based on reverse genetic engineering of SARS-CoV, we investigated the contribution of open reading frame 6 (ORF6) to the observed phenotypic differences in interferon signalling, because ORF6 encodes an interferon signalling antagonist. We did a luciferase-based interferon-stimulated response element promotor activation assay to evaluate the antagonistic capacity of SARS-CoV-2 wild-type ORF6 constructs and three mutants (Gln51Glu, Gln56Glu, or both) that represent amino acid substitutions between SARS-CoV and SARS-CoV-2 protein 6 in the carboxy-terminal domain.FindingsOverall, replication was higher for SARS-CoV in Vero E6 cells and for SARS-CoV-2 in Calu-3 cells. SARS-CoV-2 was reliant on TMPRSS2, found only in Calu-3 cells, for more efficient entry. SARS-CoV-2 was more sensitive to interferon treatment, less efficient in suppressing cytokine induction via IRF3 nuclear translocation, and permissive of a higher level of induction of interferon-stimulated genes MX1 and ISG56. SARS-CoV-2 ORF6 expressed in the context of a fully replicating SARS-CoV backbone suppressed MX1 gene induction, but this suppression was less efficient than that by SARS-CoV ORF6. Mutagenesis showed that charged amino acids in residues 51 and 56 shift the phenotype towards more efficient interferon antagonism, as seen in SARS-CoV.InterpretationSARS-CoV-2 ORF6 interferes less efficiently with human interferon induction and interferon signalling than SARS-CoV ORF6. Because of the homology of the genes, onward selection for fitness could involve functional optimisation of interferon antagonism. Charged amino acids at positions 51 and 56 in ORF6 should be monitored for potential adaptive changes.FundingBundesministerium für Bildung und Forschung, EU RECOVER project.

Dataset Information

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

Publications

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

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