Decoupling deISGylating and deubiquitinating activities of the MERS virus papain-like protease

Area of Science:

• Virology
• Structural Biology
• Immunology

Background:

• Middle East Respiratory Syndrome (MERS) virus papain-like proteases (PLpro) possess protease, deubiquitinating (DUB), and deISGylating (deISG) activities.
• These activities contribute to viral infection and suppression of innate immune responses, but their distinct roles remain unclear.
• Existing research faces challenges in separating the DUB and deISG activities of PLpro.

Purpose of the Study:

• To determine the structure of MERS PLpro in complex with human ISG15.
• To design and characterize MERS PLpro mutants with selectively impaired DUB and/or deISG activities.
• To provide tools for investigating the specific roles of DUB and deISG activities in viral pathogenesis and immune evasion.

Main Methods:

• Determined the crystal structure of MERS PLpro-ISG15 complex at 2.3 Å resolution.
• Utilized structural information to design 13 MERS PLpro mutants targeting key amino acid residues.
• Employed fluorescence-based assays to evaluate protease, DUB, and deISG activities of the wild-type and mutant enzymes.

Main Results:

• Identified specific amino acid positions (1649, 1653) for selective modulation of DUB activity.
• Demonstrated that mutations at Val1691 or His1652 completely abolish both DUB and deISG activities.
• Successfully generated MERS PLpro mutants with distinct enzymatic profiles.

Conclusions:

• Selective DUB and deISG activity modulation is achievable through targeted mutagenesis of MERS PLpro.
• The developed mutants serve as valuable tools for dissecting the roles of DUB versus deISG activities in MERS virus infection.
• These findings may inform the development of novel MERS virus attenuation strategies for vaccine design.