Structural and functional characterization of MERS coronavirus papain-like protease

Abstract

BackgroundsA new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.ResultsCircular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.ConclusionsOverall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.

Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

“…In vitro studies have shown that the papain like Cys protease of MERS also has a relatively low k cat /K m when compared with papain (72). This suggested the absence or substitution of an active site residue(s), or the absence of an intracellular activator (e.g.…”

“…Interestingly, the VEEV nsP2 cysteine protease is not the only viral cysteine protease lacking a residue homologous to Gln-19 in papain. Trp-107 in the SARS PLpro is thought to stabilize the oxyanion (72) in a manner similar to Gln-19 in papain. Trp-107 was mutated to Ala and no activity was observed (67) (Figure 5B).…”

“…Trp-107 was mutated to Ala and no activity was observed (67) (Figure 5B). Lin et al noted that in the MERS PLpro the Trp is replaced by a Leu, a residue which cannot function as a hydrogen bond donor (Figure 5C), and then showed that the mutation of the Leu to Trp actually increased the k cat /K m 23-fold (72). Consequently, the WT MERS PLpro has a lower k cat /K m than the SARS PLpro (72).…”

“…Asn-475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. Notably, the corona virus motif (DNN C YL) of the Middle East respiratory syndrome (MERS-CoV PL pro ) and severe acute respiratory syndrome (SARS) papain like proteases (66;72) also contains an Asn at the n-2 position relative to the nucleophilic Cys, and this Asn aligns with Asn-475 in the Clan CN motif ( 475 NV C WAK 480 ) when the Cys-His ion pairs of these proteases are superposed (Figure 5). …”

Synonymous and Biased Codon Usage by MERS CoV Papain-Like and 3CL-Proteases

“…MERS CoV PLpro has de-ISGylation (removal of Interferon Stimulated gene 15) and deubiquitination activities. [12][13][14] These activities are essential to block host interferon regulation and to interfere with host innate immunity.…”