The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme

TLDR

The SARS‑CoV papain‑like protease (PLpro) processes the viral polyprotein and is related to the herpesvirus ubiquitin‑specific protease HAUSP, suggesting it may also deubiquitinate host proteins. The study aimed to confirm the predicted deubiquitinating activity of SARS‑CoV PLpro. The authors expressed and purified the PLpro catalytic domain (aa 1507–1858) from *Escherichia coli* for biochemical assays. Purified PLpro hydrolyzed ubiquitin‑AMC with a catalytic efficiency of 13,100 M⁻¹ s⁻¹, was inhibited by ubiquitin aldehyde (Ki 210 nM), cleaved branched polyubiquitin chains (Ub₂–₇, Ub₄), processed ISG15‑fused proteins in vitro and in HeLa cells, and thus was confirmed as a deubiquitinating enzyme, revealing a novel viral strategy to manipulate host ubiquitination. Published in J.

Abstract

ABSTRACT The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79: 4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli . The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M −1 s −1 , 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.