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A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research
Suzannah J Rihn1, Andres Merits2, Siddharth Bakshi1
1MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.
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.
Insights
Researchers developed a SARS-CoV-2 toolkit, including a reverse genetics system and antibodies, to aid COVID-19 research. This resource facilitates virus manipulation and drug testing for vaccine development.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- The COVID-19 pandemic, caused by SARS-CoV-2, necessitates new research tools for laboratories unfamiliar with coronaviruses.
- There is an urgent need for accessible resources to study SARS-CoV-2 infection and develop countermeasures.
Purpose of the Study:
- To develop and provide a comprehensive toolkit for SARS-CoV-2 research.
- To facilitate genetic manipulation of SARS-CoV-2 and study viral protein expression.
- To enable drug screening and vaccine development for COVID-19.
Main Methods:
- Development of a single plasmid SARS-CoV-2 reverse genetics system for facile genetic manipulation and virus rescue.
- Generation of a panel of antibodies targeting nearly all SARS-CoV-2 proteins.
- Collection of SARS-CoV-2 clinical isolates and establishment of permissive cell lines.
Main Results:
- Demonstrated the expression of the SARS-CoV-2 ORF10 protein in infected cells using the developed tools.
- Confirmed that the antiviral activity of apilimod is dependent on TMPRSS2 expression.
- Successfully rescued infectious SARS-CoV-2 using the reverse genetics system via transient transfection.
Conclusions:
- The provided SARS-CoV-2 toolkit is a valuable resource for the scientific community, accelerating COVID-19 research.
- The toolkit supports advancements in vaccine design, antiviral drug testing, and fundamental discovery science related to SARS-CoV-2.
- The open availability of these tools promotes collaborative research efforts in combating the COVID-19 pandemic.
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