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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

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

Elena L Sokolinskaya¹, Lidia V Putlyaeva¹, Vasilisa S Polinovskaya¹

¹Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, Bolshoy Blvd 30, bld. 1, 121205 Moscow, Russia.

International Journal of Molecular Sciences

|July 27, 2022

Summary

Scientists developed two fluorescent sensors to detect the activity of SARS-CoV-2 papain-like protease (PLpro) in real-time within live cells. These sensors enable early detection of viral spread and aid in screening for PLpro inhibitors.

Keywords:

COVID-19 FRET SARS-CoV-2 coronavirus genetically encoded probes live cell imaging protease PLpro translocation

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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.