A Dual-Color Fluorescent Probe Allows Simultaneous Imaging of Main and Papain-like Proteases of SARS-CoV-2-Infected...

Area of Science:

• Biochemistry
• Virology
• Molecular Biology

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication relies on main protease (Mpro) and papain-like protease (PLpro).
• These viral proteases are key targets for developing antiviral drugs.
• Simultaneous detection of Mpro and PLpro is crucial for SARS-CoV-2 research but has been technically challenging due to a lack of suitable probes.

Purpose of the Study:

• To develop a novel dual-color fluorescent probe for simultaneous visualization of SARS-CoV-2 Mpro and PLpro.
• To validate the probe's efficacy in detecting and monitoring these proteases in various biological systems.
• To establish a tool for rapid screening of potential antiviral inhibitors targeting SARS-CoV-2 proteases.

Main Methods:

• Development of a dual-color probe (3MBP5) utilizing Förster resonance energy transfer (FRET).
• The probe incorporates Cy3 and Cy5 fluorophores, cleaved specifically by Mpro and PLpro, respectively.
• Validation involved using recombinant proteins, inhibitors, transfected HEK 293T cells, and SARS-CoV-2-infected TMPRSS2-Vero cells.

Main Results:

• The 3MBP5 probe successfully enabled simultaneous fluorescence detection of both Mpro and PLpro.
• Demonstrated probe specificity and activation in various experimental models, including infected cells.
• Verified the simultaneous intracellular distribution and activity of SARS-CoV-2 Mpro and PLpro.

Conclusions:

• The developed dual-color probe (3MBP5) is an effective tool for simultaneous detection of SARS-CoV-2 Mpro and PLpro.
• This probe facilitates the study of protease activity and intracellular localization.
• It offers significant potential for the rapid screening of antiviral compounds targeting SARS-CoV-2.