SARS hCoV papain-like protease is a unique Lys48 linkage-specific di-distributive deubiquitinating enzyme

Abstract

Ubiquitin (Ub) and the Ub-like (Ubl) modifier interferon-stimulated gene 15 (ISG15) participate in the host defence of viral infections. Viruses, including the severe acute respiratory syndrome human coronavirus (SARS hCoV), have co-opted Ub-ISG15 conjugation pathways for their own advantage or have evolved effector proteins to counter pro-inflammatory properties of Ub-ISG15-conjugated host proteins. In the present study, we compare substrate specificities of the papain-like protease (PLpro) from the recently emerged Middle East respiratory syndrome (MERS) hCoV to the related protease from SARS, SARS PLpro. Through biochemical assays, we show that, similar to SARS PLpro, MERS PLpro is both a deubiquitinating (DUB) and a deISGylating enzyme. Further analysis of the intrinsic DUB activity of these viral proteases revealed unique differences between the recognition and cleavage specificities of polyUb chains. First, MERS PLpro shows broad linkage specificity for the cleavage of polyUb chains, whereas SARS PLpro prefers to cleave Lys48-linked polyUb chains. Secondly, MERS PLpro cleaves polyUb chains in a 'mono-distributive' manner (one Ub at a time) and SARS PLpro prefers to cleave Lys48-linked polyUb chains by sensing a di-Ub moiety as a minimal recognition element using a 'di-distributive' cleavage mechanism. The di-distributive cleavage mechanism for SARS PLpro appears to be uncommon among USP (Ub-specific protease)-family DUBs, as related USP family members from humans do not display such a mechanism. We propose that these intrinsic enzymatic differences between SARS and MERS PLpro will help to identify pro-inflammatory substrates of these viral DUBs and can guide in the design of therapeutics to combat infection by coronaviruses.

Figure 1. MERS PLpro is a broad-specificity isopeptidase

(A) Relative apparent k_cat/K_m values for SARS and MERS PLpro for Ub- and ISG15-AMC hydrolysis, plotted as compared to SARS PLpro on Ub-AMC. Error bars represent SEM, calculated from Michealis-Menten kinetics as shown in Supplementary Figure 1 B–C). Inlet – SARS and MERS PLpro were labeled by the ubiquitin activity based probe (Ub-PA). (B) P2, P3 and P4 specificity of MERS PLpro. (C) Comparison of the PS-SCL library consensus specificities for P2-P4 with the native cleavage sites used by MERS PLpro in the viral polypeptide. Amino acids are listed as their three letter codes (Φ, hydrophobic; +, positively charged); □ indicates cleavage by MERS PLpro. (D) Comparison of k_cat/K_m values for MERS PLpro on individual tetra-peptide substrates, that were synthetized based on the PS-SCL results in Figure 1. (E) A panel of wild-type and DUB-resistant (UbL73P) di-ubiquitin chains (M1-, K11-, K48- and K63-linked, displayed in the top left, top right, bottom left and bottom right corners, respectively) assayed with SARS PLpro, MERS PLpro and USP2_CD; showing how MERS PLpro can tolerate Pro in P4 in the context of full-length ubiquitin when it is part of a di-ubiquitin chain (F) MERS PLpro (at 100 nM) and SARS PLpro (at 100 nM) were assayed against a panel of di-ubiquitin chains of all 8 Ub-Ub linkages. Dotted lines indicate cropped images from different gels ran side-by-side (see also Supplementary Fig 1F).

Figure 2. Enhanced activity of SARS PLpro on longer K48-linked ubiquitin chains

(A) K48-linked wild-type and DUB-resistant (UbL73P) tetra-ubiquitin chains assayed with a 1/5-fold serial-dilution SARS PLpro, MERS PLpro and USP2_CD (starting at 2μM DUB), showing how both MERS and SARS PLpro can tolerate Pro in P4 in the context of full-length ubiquitin when it is part of a tetra-ubiquitin chain. (B) K48-linked ubiquitin chains (tetra-, tri- and di-Ub) were cleaved in a time-course assay by SARS PLpro (at 2nM, top panels) and by MERS PLpro (at 20nM, bottom panels); highlighting the different cleavage patterns of the two DUBs. Dotted lines indicate cropped images from three gels, ran side by side. (C) Quantification of cleavage rates by SARS and MERS PLpro from (B). (D) K48-linked penta-ubiquitin chains were cleaved in a time-course assay by SARS PLpro (20nM) at 0°C (on ice) and 37°C, and visualized by SDS-PAGE and SYPRO-staining. Dotted lines are included for clarity. (E) Comparison of tetra-Ub cleavage by SARS PLpro of K48- versus K63-linked chains.

Figure 3. SARS PLpro is a di-UbK48-binding, tri-UbK48-cleaving DUB

(A) and (B) Catalytic Cys/Ala mutants of SARS PLpro, MERS PLpro and USP2_CD were analyzed for their ability to bind mono- versus K48-di-Ub (A) or ISG15 versus K48-di-Ub (B). (C) Ubiquitin-activity-based probe (ABP) labeling of SARS and MERS PLpro and their ΔUBL mutants. The position of warhead (the reactive group for covalently labeling the active site Cys of the DUB) within the Ub-ABP is indicated by a star. Note the absence of labeling for SARS PLpro by the di-UbK48-ABP. Dotted lines are included for clarity. (D) Time-course cleavage assay of IFNβ-/MG132-treated cell lysates by SARS and MERS PLpro. Box is shown to highlight the different initial cleavage products released from poly-Ub chains by the two viral DUBs. (E) Schematic representation of the model highlighting the different mechanism of cleavage for ubiquitin chains by SARS (in black) and MERS (in grey) PLpro, showing the presence of a unique di-Ub-recognizing surface within SARS PLpro (dark grey curve), which is lost from MERS PLpro. S2, S1 and S1′ indicate the sub-sites occupied by Ub chains when bound across the active site: S2 is the distal-distal Ub in a tri-Ub; S1 is the distal Ub (with its C-terminus in the active site); and S1′ is the proximal Ub (the one cleaved off).

Figure 4. SARS PLpro is a unique “distal di-distributive” deubiquitinating enzyme

(A) Schematics of the purification strategy of poly-Ub-conjugated Cdc34 in order to asses the directionality of cleavage of Ub chains. (B) Time-course cleavage of _HisCdc34-poly-Ub^FLAG conjugates by SARS and MERS PLpro, revealing stabilization of mono-Ub-conjugated Cdc34 as a cleavage product by SARS PLpro and not by MERS PLpro. (C) Time-course cleavage of _HisCdc34-poly-Ub^FLAG conjugates by USP-family DUBs. (D) Quantification of the relative abundance of the _HisCdc34-mono-Ub^FLAG species from the time-course cleavage assay in (C). (E) Schematic representation of the mono-(for MERS PLpro, USP2 and USP21) and the di-distributive cleavage mechanism (for SARS PLpro), indicating the accumulation of mono-Ub-conjugated substrates only in the case of the di-distributive cleavage mechanism displayed by SARS PLpro.