Genetically Encoded Fluorescent Sensors for SARS-CoV-2 Papain-like Protease PLpro.

Area of Science:

• Virology
• Molecular Biology
• Biochemistry

Background:

• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) is essential for viral replication.
• PLpro cleaves viral polyproteins at the earliest stage of the SARS-CoV-2 lifecycle.
• Targeting PLpro is a key strategy for developing antiviral therapies.

Purpose of the Study:

• To design and validate novel genetically encoded fluorescent sensors for real-time detection of PLpro activity.
• To enable visualization of PLpro enzymatic function in live cells.
• To provide tools for screening potential PLpro inhibitors.

Main Methods:

• Development of two Förster resonance energy transfer (FRET)-based fluorescent sensors utilizing mScarlet and miRFP670.
• Engineering of a second sensor tethered to the endoplasmic reticulum (ER) membrane with mNeonGreen and mScarletI.
• Co-expression of sensors with SARS-CoV-2 PLpro in HeLa cells for activity monitoring.

Main Results:

• The FRET-based sensor showed a 1.5-fold increase in donor fluorescence upon PLpro cleavage.
• The ER-tethered sensor demonstrated a two-fold increase in the nucleus/cytoplasm ratio of mScarletI after PLpro action.
• Both sensors successfully detected and quantified PLpro activity in live cells.

Conclusions:

• The developed fluorescent sensors provide sensitive and real-time detection of SARS-CoV-2 PLpro activity.
• These tools can be utilized for early diagnosis of SARS-CoV-2 infection.
• The sensors are valuable for high-throughput screening of antiviral compounds targeting PLpro.