Native Mass Spectrometry Reveals Binding Interactions of SARS-CoV-2 PLpro with Inhibitors and Cellular Targets

Area of Science:

• Biochemistry
• Structural Biology
• Virology

Background:

• The SARS-CoV papain-like protease (PLpro) is crucial for viral replication and immune evasion.
• Understanding PLpro's structure and inhibitor interactions is key for developing antiviral therapies.

Purpose of the Study:

• To investigate the structural effects of PLpro inhibitors using native mass spectrometry.
• To compare SARS-CoV-1 and SARS-CoV-2 PLpro interactions with inhibitors and ISG15.
• To identify potential therapeutic strategies targeting coronavirus proteases.

Main Methods:

• Native mass spectrometry (native MS) was employed to study PLpro.
• Ultraviolet photodissociation (UVPD) and variable temperature electrospray ionization (vT ESI) were used to map inhibitor binding sites.
• Thermodynamic measurements and analysis of a PLpro mutant were conducted.

Main Results:

• Inhibitors were found to stabilize the tertiary structure of PLpro.
• Specific binding sites for PLpro inhibitors were localized.
• Differences in inhibitor and ISG15 interactions between SARS-CoV-1 and SARS-CoV-2 PLpro were observed.
• The inhibitor PR-619 demonstrated a stabilizing effect on the folded PLpro structure.

Conclusions:

• Native MS, UVPD, and vT ESI are effective tools for characterizing protease-inhibitor interactions.
• PLpro inhibitors stabilize the protease's tertiary structure, offering a therapeutic avenue.
• Comparative analysis of SARS-CoV PLpro variants provides insights into protease function and inhibitor specificity.
• These findings support the development of PLpro as a therapeutic target for SARS-CoV-2 and related viruses.