Versatile SARS-CoV-2 Reverse-Genetics Systems for the Study of Antiviral Resistance and Replication

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Reverse Genetics to Engineer Positive-Sense RNA Virus Variants
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Reverse Genetics to Engineer Positive-Sense RNA Virus Variants

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Ulrik Fahnøe1,2, Long V Pham1,2, Carlota Fernandez-Antunez1,2

  • 1Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Copenhagen University Hospital, 2650 Hvidovre, Denmark.

Viruses
|February 26, 2022
PubMed

Insights

Researchers developed a versatile SARS-CoV-2 reverse-genetics system for studying viral mutations and drug efficacy. This system enables high-throughput testing of antiviral treatments against COVID-19, aiding in the development of new therapeutics.

Area of Science:

  • Virology and Molecular Biology
  • Infectious Diseases
  • Drug Discovery and Development

Background:

  • The COVID-19 pandemic necessitates ongoing research into SARS-CoV-2, driven by limited treatments and emerging variants.
  • Reverse-genetics systems are crucial for understanding viral mechanisms and developing effective therapeutics.
  • Previous methods may lack the versatility required for comprehensive analysis of viral mutations and drug responses.

Purpose of the Study:

  • To develop a functional and versatile reverse-genetics system for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
  • To create tools for high-throughput screening of antiviral drugs and characterization of viral mutants.
  • To investigate the impact of specific mutations on SARS-CoV-2 susceptibility to remdesivir.

Main Methods:

  • Cloning of a SARS-CoV-2 isolate (DK-AHH1) into a bacterial artificial chromosome (BAC) to create an infectious system.
  • Insertion of reporter genes (GFP, nanoluciferase) into the viral genome for monitoring and high-throughput screening.
  • Development of a non-infectious subgenomic replicon system by deleting structural genes (S, E, M).

Main Results:

  • Recovered viruses exhibited growth kinetics and remdesivir susceptibility comparable to the original isolate.
  • Reporter gene insertion facilitated high-throughput treatment experiments and demonstrated no decrease in remdesivir susceptibility with specific nsp12 mutations.
  • A non-infectious subgenomic replicon system showed efficient replication and inhibition by remdesivir.

Conclusions:

  • The developed SARS-CoV-2 reverse-genetics systems provide a powerful platform for studying viral biology and drug efficacy.
  • These systems enable rapid introduction of mutations and assessment of their impact on antiviral treatment effectiveness.
  • The research facilitates fundamental understanding of coronaviruses and supports the development of novel COVID-19 therapies.
Keywords:
GFPRNA virusSARS-CoV-2molecular clonenanoluciferasepolymeraseremdesivirreplicon

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