Coronavirus Papain-like Peptidases

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.

Author Biographies

Dr. Kiira Ratia received her BSc from Haverford College, Haverford, PA, where she studied the biophysical properties of peptide oligomerization under Dr. Robert Fairman. She then moved to Chicago, IL and began work on several enzyme-related projects under Dr. Andrew Mesecar in the Departments of Medicinal Chemistry and Pharmacognosy at the University of Illinois. She received her PhD in 2009 for work on the characterization of two coronaviral papain-like proteases: SARS-CoV PLpro and HCoV-NL63 PLP2. Her studies on SARS-CoV PLpro led to the first structure of a coronaviral papain-like protease, the discovery of non-covalent PLpro inhibitors with antiviral activity, and detailed characterization of the de-ubiquitinating activity of PLpro. Dr. Ratia is currently the director of a UIC Research Resources Center core, the High-Throughput Screening Facility.

Professor Andrew Mesecar completed his BSc in Chemistry degree at Purdue University. He received his Ph.D. in Biochemistry in 1995 from the University of Notre Dame, South Bend, IN, which is also his hometown. He then went onto the University of California, Berkeley, and completed a post-doctoral fellowship in structural enzymology under the direction of Prof. Daniel E. Koshland Jr. In 1999, he started his assistant professorship at the University of Illinois Chicago where he started collaborating with Professor Susan Baker on the structure and function of coronavirus papain-like proteases. In collaboration with Professor Baker, their labs were the first to determine the X-ray structure of coronavirus papain-like protease, demonstrate that these enzymes can deUbiquitinate and deISGylate host-cell proteins, and that it is possible to develop non-covalent inhibitors of papain-like proteases with antiviral activity. In August of 2010, Professor Mesecar moved to Purdue University as the Walther Professor of Cancer Structural Biology. He research focus is on the roles of deUbiqutination and Ubiquitination processes in cancer and viral pathogenesis. He continues to collaborate with Professor Baker on the roles of papain-like proteases in viral replication and the innate immune response and on development of anti-coronaviral therapeutics.

Dr. Amornrat O’Brien (formerly Kanjanahaluethai) completed her BSc degree in Medical Technology and MSc degree in Microbiology at Chiang Mai University in Chiang Mai, Thailand. She received her PhD in Microbiology in 2002 from the Department of Microbiology and Immunology at Loyola University Chicago Stritch School of Medicine under the direction of Professor Susan Baker. When Amornrat returned to Thailand after her PhD, the National Research Council of Thailand honored her by ranking her dissertation number one among all science dissertations completed that year by Thai students. Amornrat accepted a position as a faculty member in the Department of Microbiology, Faculty of Medicine at Chiang Mai University. Her faculty responsibilities include teaching medical students, undergraduate students in medical science and mentoring the graduate students’ research projects. Much of her research has focused on the development of dengue vaccines, but she is now shifting focus to explore the mechanisms of interferon resistance of hepatitis C virus. In addition, Amornrat continues to collaborate with Professor Baker studying the role of viral proteases in the replication of coronaviruses, including the coronavirus responsible for severe acute respiratory syndrome (SARS-CoV).

Professor Susan C. Baker completed her BSc in Biology at St. Olaf College in Northfield, MN and her Ph.D. in Microbiology from Vanderbilt University in Nashville, TN. She then went on to the University of Southern California in Los Angeles and completed a post-doctoral fellowship in molecular virology under the direction of Dr. Michael Lai, who was an Investigator in the Howard Hughes Medical Institute. Dr. Baker joined the faculty at Loyola University Stritch School of Medicine in 1990 as an Assistant Professor and rose to the rank of full Professor. Professor Susan Baker worked with her student Amonrnat Kanjanahaluthai O’Brien on the characterization on the papain-like protease activity of murine coronavirus, and with enzymologists and structural biologists Dr. Andrew Mesecar and Kiira Ratia on the structure and function of SARS coronavirus papain-like protease. Dr. Baker’s ongoing research focus is on the roles of protease and isopeptidase activity in coronavirus replication and pathogenesis. She continues to collaborate with Drs. O’Brien, Ratia, and Mesecar on the function of papain-like proteases in viral replication and the innate immune response and on development of anti-coronaviral therapeutics.

Databanks

MEROPS name: murine hepatitis coronavirus papain-like peptidase 1

MEROPS classification: clan CA, family C16, subfamily C16A, peptidase C16.001

Species distribution: known only from murine hepatitis virus

Reference sequence from: murine hepatitis virus (UniProt: P19751)

MEROPS name: murine hepatitis coronavirus papain-like peptidase 2

MEROPS classification: clan CA, family C16, subfamily C16B, peptidase C16.006

Species distribution: family Coronaviridae

Reference sequence from: murine hepatitis virus (UniProt: P19751)

MEROPS name: porcine transmissible gastroenteritis coronavirus papain-like peptidase 2

MEROPS classification: clan CA, family C16, subfamily C16B, peptidase C16.008

Species distribution: known only from transmissible gastroenteritis virus

Reference sequence from: transmissible gastroenteritis virus

MEROPS name: SARS coronavirus papain-like peptidase

MEROPS classification: clan CA, family C16, subfamily C16B, peptidase C16.009

Tertiary structure: Available

Species distribution: family Coronaviridae

Reference sequence from: SARS coronavirus (UniProt: P59641)

MEROPS name: human coronavirus 229E papain-like peptidase 2

MEROPS classification: clan CA, family C16, subfamily C16B, peptidase C16.010

Species distribution: known only from human coronavirus

Reference sequence from: human coronavirus (UniProt: Q05002)

Name and History

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 (28–32 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 494.1 ) [2], [3], [4], [5], [6], [7], [8]. The emergence in 2002–2003 of a novel coronavirus with 10% mortality, severe acute respiratory syndrome coronavirus (SARS-CoV), motivated researchers to further characterize the papain-like protease domain as a potential target for antiviral drug development [9], [10], [11]. These studies validated coronavirus PLP domains as targets for antiviral drugs and revealed the dual nature of coronavirus PLPs as both endopeptidases and isopeptidases. The isopeptidase activity of coronavirus PLPs acts as a deubiquitinating (DUB) and de-ISGylating enzyme that is likely important in viral pathogenesis [12], [13], [14], [15], [16].

Figure 494.1.

The amino-terminal end of the coronavirus replicase polyprotein is processed by 1 or 2 papain-like protease domains. Schematic diagram of a coronavirus genome (top) and replicase products (below) generated by translation of the 5′-most open reading frame. The non-structural proteins (nsp’s) are generated by the activity of papain-like proteases 1 and 2. Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes only one papain-like protease domain (PLpro) which acts at 3 sites by recognizing the canonical sequence –LXGG. Human coronavirus NL-63 (HCoV-NL63) replicase polyprotein contains 2 papain like protease domains. PLP1 releases nsp1 from the polyprotein and PLP2 processes the nsp2/3 and nsp3/4 sites. The murine coronavirus mouse hepatitis virus (MHV) encodes 2 PLPs with PLP1 processing the nsp1/2 and nsp2/3 sites and PLP2 processing the nsp3/4 junction.

Structure and Chemistry

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 amino-terminal non-structural protein (nsp) from the replicase polyprotein [2], [3]. 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. High-throughput screening of a modest 50 K compound library and subsequent structure–activity 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.

Figure 494.2.

Structure of severe acute respiratory syndrome papain-like protease domain. (A) The overall structure of the SARS-CoV PLpro domain can be modelled as a left hand, with a fingers domain that coordinates a zinc residue, a palm domain containing the Cys-His-Asp catalytic triad required for endopeptidase and isopeptidase activity, the adjacent thumb domain and the ubiquitin-like domain (ubl) of unknown function; (B) A flexible loop region (purple circle) adjacent to the catalytic triad can be targeted by non-covalent protease inhibitors [10].

Activity and Specificity

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-RELNGG↓APIK^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 C-terminal residues of ubiquitin and ISG15 [10], [12]. All assays were performed at 25°C, in 20 mM HEPES, pH 7.5, 0.1 mg/mL BSA, and 5 mM DTT. With the exception of ISG15-AMC, none of the substrates saturated the enzyme up to the concentrations tested, and therefore pseudo first-order rate constants, k _app were reported (k _app~k _cat/K _m for non-saturable enzymes): E^Edans-RELNGG↓APIK^Dabcyl-S, 0.0244±0 0003 min⁻¹  µM⁻¹; Ub-AMC, 4.48±0.1⁻¹  min⁻¹  µM⁻¹; RLRGG-AMC, 0.61±0^.01 min⁻¹  µM⁻¹. PLpro is considerably more active with the ISG15-AMC substrate, producing k _cat and K _m values of 370±16 min⁻¹ and 2.3±0.3 µM, respectively [10].

Biological Aspects

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 [14], [15], [16], [22], [23]. Further studies are needed to determine the role of viral DUB activity in the pathogenesis of coronavirus infections.

Related Peptidases

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 498Chapter 495Chapter 497Chapter 498.