Mechanistic insights into the activity of SARS-CoV-2 RNA polymerase inhibitors using single-molecule FRET

Area of Science:

• Molecular biology
• Virology
• Biochemistry

Background:

• The COVID-19 pandemic caused by SARS-CoV-2 necessitates understanding viral replication mechanisms.
• Highly mutated SARS-CoV-2 variants continue to emerge, underscoring the need for effective antiviral strategies.
• The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a key target for antiviral development.

Purpose of the Study:

• To develop and utilize a single-molecule Förster resonance energy transfer (smFRET) assay for real-time analysis of SARS-CoV-2 RNA synthesis.
• To elucidate the molecular mechanisms of SARS-CoV-2 replication and inhibition by antiviral compounds.
• To investigate the specific mechanism of action of remdesivir against the SARS-CoV-2 RdRp.

Main Methods:

• Purification of the minimal SARS-CoV-2 replication complex (nsp12, nsp7, nsp8).
• Development of a smFRET assay using fluorescently labeled RNA substrates to monitor RNA primer elongation in real-time.
• In vitro analysis of RNA synthesis and inhibitor activity at the single-molecule level.

Main Results:

• The smFRET assay successfully visualized and analyzed in vitro RNA synthesis by the SARS-CoV-2 RdRp complex.
• The study provided direct evidence for remdesivir's mechanism of action, involving polymerase stalling and chain termination.
• Remdesivir's inhibitory effect was shown to be dependent on the concentration of competing nucleotide triphosphates.

Conclusions:

• Single-molecule FRET is a powerful technique for dynamic insights into viral RNA replication.
• The developed smFRET platform is valuable for antiviral screening and mechanistic studies of viral RdRp.
• Understanding remdesivir's mechanism offers insights for developing next-generation SARS-CoV-2 inhibitors.