Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

Abstract

Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), came the realization of the multifunctional nature of these enzymes. Structural and enzymatic studies revealed that SARS-CoV PLpro can act as both a protease to cleave peptide bonds and also as a deubiquitinating (DUB) enzyme to cleave the isopeptide bonds found in polyubiquitin chains. Furthermore, viral DUBs can also remove the protective effect of conjugated ubiquitin-like molecules such as interferon stimulated gene 15 (ISG15). Extension of these studies to other coronaviruses and arteriviruses led to the realization that viral protease/DUB activity is conserved in many family members. Overexpression studies revealed that viral protease/DUB activity can modulate or block activation of the innate immune response pathway. Importantly, mutations that alter DUB activity but not viral protease activity have been identified and arteriviruses expressing DUB mutants stimulated higher levels of acute inflammatory cytokines after infection. Further understanding of the multifunctional nature of the Nidovirus PLP/DUBs may facilitate vaccine development. Here, we review studies describing the PLPs' enzymatic activity and their role in virus pathogenesis.

Keywords: Arterivirus; Coronavirus; DeISGylating activity; Deubiquitinating enzyme; MERS-CoV; Papain-like protease.

Fig. 1

Coronavirus and arterivirus papain-like proteases. Schematic depiction of the Nterminal region of the replicase polyprotein of selected coronaviruses (A) and arteiviruses (B). The papain-like protease domains, termed PLpro, PLP1 or PLP2 for CoV, or Pα, Pβ or PLP2 for arteriviruses are depicted with correspondingly colored arrowheads indicating predicted or confirmed cleavage sites.

Fig. 2

Structures of SARS-CoV PLpro and EAV PLP2 compared to USP14 and yeast OTU1. (A) Structural superposition of SARS-CoV PLpro (magenta, PDB code 2FE8) to USP14 (cyan, PDB code 2AYO). The zinc atom from the zinc finger of SARS-CoV PLpro is shown as a gray sphere. (B) Close-up view of the active site in SARS-CoV PLpro with the catalytic triad residues shown as sticks. Numbering of the residues is based on (26) (C) Structural overlay of EAV PLP2 (blue, PDB code 4IUM) with yeast OTU1 (yellow, PDB code 3BY4) bound to ubiquitin. The ubiquitin molecules have been omitted for clarity. The zinc atom from the zinc finger of EAV PLP2 is shown in gray sphere. (D) Close-up view of the active site in EAV PLP2. Numbering of the residues is based on (49). Asn263 in EAV PLP2 catalytic triad has two alternative conformations. Images were generated using PyMOL.