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Construction and characterization of two SARS-CoV-2 minigenome replicon systems
Hu Zhang1,2, Douglas K Fischer1,2, Masahiro Shuda1,2
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Journal of Medical Virology
|February 9, 2022
Summary
Researchers developed two SARS-CoV-2 minigenome replicon systems for studying the virus and discovering antivirals. These systems operate safely and show promise for future research and antiviral development.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- The COVID-19 pandemic necessitates advanced tools for studying SARS-CoV-2.
- Current research requires safe and efficient systems for viral replication studies and antiviral screening.
- A biosafety level 2 (BSL-2) compliant system is crucial for widespread accessibility.
Purpose of the Study:
- To construct and characterize novel SARS-CoV-2 minigenome replicon systems.
- To establish systems suitable for basic virology research and antiviral drug discovery.
- To evaluate the performance and limitations of different replicon system designs.
Main Methods:
- Construction of two SARS-CoV-2 minigenome replicon systems: IVT-CoV2-Rep (in vitro transcribed mRNA) and BAC-CoV2-Rep (DNA-launched).
- Utilized nanoluciferase (NLuc) reporter for quantifying viral RNA replication.
- Performed transient transfection assays in various cell lines and assessed replication inhibition by known SARS-CoV-2 inhibitors.
Main Results:
- Both IVT-CoV2-Rep and BAC-CoV2-Rep systems demonstrated transient replication of SARS-CoV-2 RNA.
- Replication in the IVT-CoV2-Rep system was sensitive to known antiviral inhibitors and not abrogated by host innate antiviral responses.
- The DNA-launched BAC-CoV2-Rep system showed enhanced and prolonged replicon signals compared to IVT-CoV2-Rep, with a portion of the signal resistant to antivirals due to spliced mRNA.
Conclusions:
- The developed SARS-CoV-2 transient replicon systems are valuable tools for fundamental research on viral replication.
- These systems facilitate the discovery and testing of novel antiviral compounds against SARS-CoV-2.
- Further optimization of these systems holds potential for developing stable replicon cell lines for advanced research applications.
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