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

Abstract

A B S T R A C TCoronavirus papain-like proteases (PLPs or PLpro), such as the one encoded in the genome of the infectious Middle East Respiratory Syndrome (MERS) virus, have multiple enzymatic activities that promote viral infection. PLpro acts as a protease and processes the large coronavirus polyprotein for virus replication. PLpro also functions as both a deubiquitinating (DUB) and deISGylating (deISG) enzyme and removes ubiquitin (Ub) and interferon-stimulated gene 15 (ISG15) from cellular proteins. Both DUB and deISG activities are implicated in suppressing innate immune responses; however, the precise role of each activity in this process is still unclear due in part to the difficulties in separating each activity. In this study, we determine the first structure of MERS PLpro in complex with the full-length human ISG15 to a resolution of 2.3 Å. This structure and available structures of MERS PLpro-Ub complexes were used as molecular guides to design PLpro mutants that lack either or both DUB/deISG activities. We tested 13 different PLpro mutants for protease, DUB, and deISG activitites using fluorescence-based assays. Results show that we can selectively modulate DUB activity at amino acid positions 1649 and 1653 while mutation of Val1691 or His1652 of PLpro to a positive charged residue completely impairs both DUB/deISG activities. These mutant enzymes will provide new functional tools for delineating the importance of DUB versus deISG activity in virus-infected cells and may serve as potential candidates for attenuating the MERS virus in vivo for modified vaccine design efforts.

Mechanism and inhibition of the papain‐like protease, PLpro, of SARS‐CoV‐2

“…K232E, Fig EV3F). Similar experiments have recently been described for MERS PLpro (Clasman et al , ).…”

Functional and druggability analysis of the SARS-CoV-2 proteome

“…In agreement with another work [58], it was shown that while SARS-CoV-2 PL pro retained de-ISGylating activity similar to its SARS-CoV counterpart, its hydrolyzing ability of diUb K48 was decreased [59]; this might not be surprising, since specificity for Ub and ISG15 substrates might depend on only one mutation [60][61][62], and while the Ub S1 site is conserved between the PL pro s of SARS-CoV and SARS-CoV-2 (83% similarity, 17% identity), the Ub S2 site has only 67% similarity and 13% identity. Interestingly, SARS-CoV-2 PL pro displays preference for ISG15s from certain species including humans [58].…”

Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity

“…The overall assembly of SCoV2-PLpro and both ISG15 domains was similar to the MERS-PLpro-humanISG15 complex (PDB ID: 6BI8, Extended data Fig. 2a) 15 . The catalytic cysteine residue is also conserved in SARS (Extended data Fig.…”