key: cord-349501-p1fttfpr authors: Ratia, Kiira; Mesecar, Andrew; O’Brien, Amornrat; Baker, Susan C. title: Chapter 494 Coronavirus Papain-like Peptidases date: 2013-12-31 journal: Handbook of Proteolytic Enzymes DOI: 10.1016/b978-0-12-382219-2.00493-2 sha: doc_id: 349501 cord_uid: p1fttfpr The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 494 is Coronavirus papain-like endopeptidases. Keywords: Coronavirus, Severe acute respiratory syndrome, SARS-CoV, polyprotein processing, ubiquitin-like domain, noncovalent protease inhibitors, de-ubiquitination, DUB, ISG-15, de-ISGylation. Koonin, E.V., Choi, G.H., Nuss, D.L., Shapira, R., Carrington, J.C. (1991) . Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive- Proteolytic processing of a polyprotein precursor is an event common to the replication cycle of many RNA viruses. For coronaviruses, a family of positive-stranded RNA viruses with large genomes (28À32 kb), the gene encoding the viral non-structural proteins (nsp's), including the RNA-dependent RNA-polymerase, is translated into a large precursor polyprotein, which must be proteolytically processed to mediate viral transcription and replication [1] . Sequence analysis of coronavirus genomic RNA reveals the presence of either 1 or 2 papain-family protease domains that were shown to process the amino-terminal region of the replicase polyprotein ( Figure Cloning and expression of the N-terminal region of the murine coronavirus replicase polyprotein revealed that a predicted papain-family protease (papain-like protease, PLP) domain was responsible for processing the aminoterminal non-structural protein (nsp) from the replicase polyprotein [2À3] . Further studies revealed the murine coronavirus contained two PLP domains, with PLP1 processing at the nsp1/2 site and the nsp2/3 site [5] . The downstream PLP2 domain processes the nsp3/4 cleavage site using a highly conserved cleavage recognition site of LXGG [17] . Analysis of the N-terminal region of the replicase polyprotein of SARS-CoV revealed only one PLP domain, termed PLpro, which was shown to process the nsp1/2, nsp2/3 and nsp3/4 cleavage sites using the LXGG recognition motif [18] . Interestingly, the PLpro cleavage recognition site, LXGG, is homologous to the LRGG site used by cellular de-ubiquitinating enzymes. Based on this observation, Sulea and colleagues proposed that the SARS-CoV PLpro could have both endopeptidase and isopeptidase activity [19] . This dual substrate recognition and catalytic function of SARS-CoV PLpro was validated independently by two groups [12, 13] . These studies showed that a core domain of PLpro could be expressed and purified from E. coli and can catalytically process both polyprotein and polyubiquitin substrates. Ultimately, determination of the high resolution X-ray crystal structure of the core domain of SARS-CoV PLpro revealed a canonical Cys-His-Asp catalytic triad within the active site and an adjacent flexible loop, a zinc-finger domain and a ubiquitin-like domain which was not previously predicted as part of the structure (Figure 494 .2) [9] . Analysis of the structure revealed homology of SARS-CoV PLpro to cellular de-ubiquitinating enzymes such as USP7 (see Chapter 464) and USP 14 (see Chapter 470). Initially, the similarity between viral and cellular protease structures raised questions about the ability to make an inhibitor that was selective exclusively for the viral protease. Highthroughput screening of a modest 50 K compound library and subsequent structureÀactivity relationship analysis of lead compounds led to the identification of non-covalent, specific inhibitors of PLpro that also inhibited the replication of SARS-CoV [10, 11] . These studies validated SARS-CoV PLpro as a therapeutic target and provided the proof of principle for the development of viral and perhaps even cellular DUB-specific inhibitors. A soluble and active form of SARS-CoV PLpro was expressed in E. coli and purified using column chromatography [12] . This 35 kilodalton protein was evaluated for the ability to process a variety of substrates, including a FRET-based peptide representing polyprotein recognition sequences: E Edans -RELNGGkAPIK Dabcyl -S. To test the de-ubiquitinating activity of the enzyme, PLpro was characterized with several fluorescent, ubiquitin (Ub)-related substrates, including full-length Ub-AMC, ISG15-AMC, and the short peptide RLRGG-AMC, representing the 5 Cterminal residues of ubiquitin and ISG15 [10, 12] . The coronavirus PLP domains have two major functions: (1) processing of the replicase polyprotein; and (2) antagonism of the innate immune response via de-ubiquitinating and de-ISGlyating target proteins. Coronavirus PLPs play a critical role in the processing of the precursor polyprotein to generate the non-structural proteins associated with viral replication (Figure 494.1) . The processed replicase products embed into the endoplasmic reticulum and generate convoluted membranes and double membrane vesicles (DMVs) which are the sites of viral RNA synthesis [1, 20, 21] . Furthermore, coronavirus PLP isopeptidase activity mediates de-ubiquitination and de-ISGylation of cellular targets, likely blocking the activation of the innate immune response to viral infection [14À16, 22, 23] . Further studies are needed to determine the role of viral DUB activity in the pathogenesis of coronavirus infections. Either one or two PLP domains have been identified in all coronaviruses sequenced to date (sequences available at www.viperbrc.org). The SARS-CoV PLpro domain is structurally similar to USP7 (see Chapter 464) and USP14 (see chapter 470). In addition, the arteriviruses, which group together with coronaviruses in the order Nidovirales, encode functional papain-like cysteine protease domains as described in detail in Chapters 495, 497 and 498. Recommended papers include those of Ratia et al. [9, 10] , and Perlman & Netland [1] . Coronaviruses post-SARS: update on replication and pathogenesis Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein Identification of the catalytic sites of a papain-like cysteine proteinase of murine coronavirus Proteolytic processing at the amino terminus of human coronavirus 229E gene 1-encoded polyproteins: identification of a papain-like proteinase and its substrate Expression of murine coronavirus recombinant papain-like proteinase: efficient cleavage is dependent on the lengths of both the substrate and the proteinase polypeptides The autocatalytic release of a putative RNA virus transcription factor from its polyprotein precursor involves two paralogous papain-like proteases that cleave the same peptide bond Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage Mechanisms and enzymes involved in SARS coronavirus genome expression Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication Severe acute respiratory syndrome coronavirus papain-like protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-κB signaling Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases Identification of mouse hepatitis virus papain-like proteinase 2 activity Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity Deubiquitination, a new function of the severe acute respiratory syndrome coronavirus papain-like protease? RNA replication of mouse hepatitis virus takes place at double membrane vesicles SARS-Coronavirus replication is supported by a reticulovesiculuar network of modified endoplasmic reticulum Inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63 Email: kratia@uic.edu Andrew Mesecar Walther Professor of Cancer Structural Biology, Deputy Director Email: sbaker1@lumc.edu Handbook of Proteolytic Enzymes, 3rd Edn ©