Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Demonstrating the importance of porcine reproductive and respiratory syndrome virus papain-like protease 2 deubiquitinating activity in viral replication by structure-guided mutagenesis.

Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUB-specific mutations faces significant selective pressure to revert to wild-type virus in MARC-145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication.

Identification, expression profiling, and functional characterization of cystatin C from big-belly seahorse (Hippocampus abdominalis).

Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. Emerging evidence suggests the immune regulatory roles of CSTC in antigen presentation, the release of different inflammatory mediators, and apoptosis in various pathophysiologies. In this study, the 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse (Hippocampus abdominalis) was cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in ovaries. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 14.29-kDa protein of recombinant HaCSTC (rHaCSTC) was expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, HaCSTC overexpression strongly decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Our findings imply the profound role of HaCSTC against pathogen infections by modulating fish immune responses.

Comparative analysis of two cathepsin L genes in Asiatic hard clam (Meretrix meretrix): Similar in sequence features, different in expression profiles.

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5' untranslated region (UTR) of 31 bp, a 3' UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5' UTR of 50 bp, a 3' UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

Origin and Early Diversification of the Papain Family of Cysteine Peptidases.

Peptidases of the papain family play a key role in protein degradation, regulated proteolysis, and the host-pathogen arms race. Although the papain family has been the subject of many studies, knowledge about its diversity, origin, and evolution in Eukaryota, Bacteria, and Archaea is limited; thus, we aimed to address these long-standing knowledge gaps. We traced the origin and expansion of the papain family with a phylogenomic analysis, using sequence data from numerous prokaryotic and eukaryotic proteomes, transcriptomes, and genomes. We identified the full complement of the papain family in all prokaryotic and eukaryotic lineages. Analysis of the papain family provided strong evidence for its early diversification in the ancestor of eukaryotes. We found that the papain family has undergone complex and dynamic evolution through numerous gene duplications, which produced eight eukaryotic ancestral paralogous C1A lineages during eukaryogenesis. Different evolutionary forces operated on C1A peptidases, including gene duplication, horizontal gene transfer, and gene loss. This study challenges the current understanding of the origin and evolution of the papain family and provides valuable insights into their early diversification. The findings of this comprehensive study provide guidelines for future structural and functional studies of the papain family.

Heterologous expression of a papain-like protease inhibitor (SnuCalCpI17) in the E. coli and its mode of inhibition.

The effect of the Escherichia coli (E. coli) Rosetta (DE3) system on the expression of recombinant papain-like cysteine protease inhibitors (SnuCalCpIs) was evaluated, and the inhibition mode of the expressed inhibitor was determined. SnuCalCpI08 and SnuCalCpI17, which previously had not been expressed in the E. coli BL21 (DE3) system due to rare codons of more than 10%, were successfully expressed in E. coli Rosetta (DE3) since the strain provides tRNAs for six rare codons. Initially, both inhibitors were expressed as inclusion bodies; however, the water solubility of SnuCalCpI17 could be improved by lowering the incubation temperature, reducing the IPTG concentration, and increasing the induction time. In contrast, the other inhibitor could not be solubilized in water. To validate whether the inhibitor was expressed with correct protein folding, a papain inhibition assay was performed with SnuCalCpI17. SnuCalCpI17 showed a half-maximal inhibitory concentration (IC50) of 105.671 ± 9.857 µg/mL and a slow-binding inhibition mode against papain at pH 7.0 with a Kiapp of 75.80 µg/mL. The slow-binding inhibitor has a slow dissociation from the inhibitor-target complex, resulting in a long residence time in vivo, and thus can effectively inhibit the target at doses far below the IC50 of the inhibitor. KEY POINTS: • Propeptide inhibitor (SnuCalCpI17) containing rare codons was expressed in E. coli Rosetta (DE3). • The slow-binding inhibition was shown by plotting the apparent first-order rate constant (kobs). • Protein-protein interaction between SnuCalCpIs and papain was verified by docking simulation.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Genome-wide survey of soybean papain-like cysteine proteases and their expression analysis in root nodule symbiosis.

BACKGROUND: Plant papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes and play important roles in root nodule symbiosis (RNS), while the whole-genome studies of PLCP family genes in legume are quite limited, and the roles of Glycine max PLCPs (GmPLCPs) in nodulation, nodule development and senescence are not fully understood. RESULTS: In the present study, we identified 97 GmPLCPs and performed a genome-wide survey to explore the expansion of soybean PLCP family genes and their relationships to RNS. Nineteen paralogous pairs of genomic segments, consisting of 77 GmPLCPs, formed by whole-genome duplication (WGD) events were identified, showing a high degree of complexity in duplication. Phylogenetic analysis among different species showed that the lineage differentiation of GmPLCPs occurred after family expansion, and large tandem repeat segment were specifically in soybean. The expression patterns of GmPLCPs in symbiosis-related tissues and nodules identified RNS-related GmPLCPs and provided insights into their putative symbiotic functions in soybean. The symbiotic function analyses showed that a RNS-related GmPLCP gene (Glyma.04G190700) really participate in nodulation and nodule development. CONCLUSIONS: Our findings improved our understanding of the functional diversity of legume PLCP family genes, and provided insights into the putative roles of the legume PLCPs in nodulation, nodule development and senescence.

Analysis of Coronavirus Temperature-Sensitive Mutants Reveals an Interplay between the Macrodomain and Papain-Like Protease Impacting Replication and Pathogenesis.

Analysis of temperature-sensitive (ts) mutant viruses is a classic method allowing researchers to identify genetic loci involved in viral replication and pathogenesis. Here, we report genetic analysis of a ts strain of mouse hepatitis virus (MHV), tsNC11, focusing on the role of mutations in the macrodomain (MAC) and the papain-like protease 2 (PLP2) domain of nonstructural protein 3 (nsp3), a component of the viral replication complex. Using MHV reverse genetics, we generated a series of mutant viruses to define the contributions of macrodomain- and PLP2-specific mutations to the ts phenotype. Viral replication kinetics and efficiency-of-plating analysis performed at permissive and nonpermissive temperatures revealed that changes in the macrodomain alone were both necessary and sufficient for the ts phenotype. Interestingly, mutations in the PLP2 domain were not responsible for the temperature sensitivity but did reduce the frequency of reversion of macrodomain mutants. Coimmunoprecipitation studies are consistent with an interaction between the macrodomain and PLP2. Expression studies of the macrodomain-PLP2 portion of nsp3 indicate that the ts mutations enhance proteasome-mediated degradation of the protein. Furthermore, we found that during virus infection, the replicase proteins containing the MAC and PLP2 mutations were more rapidly degraded at the nonpermissive temperature than were the wild-type proteins. Importantly, we show that the macrodomain and PLP2 mutant viruses trigger production of type I interferon in vitro and are attenuated in mice, further highlighting the importance of the macrodomain-PLP2 interplay in viral pathogenesis.IMPORTANCE Coronaviruses (CoVs) are emerging human and veterinary pathogens with pandemic potential. Despite the established and predicted threat these viruses pose to human health, there are currently no approved countermeasures to control infections with these viruses in humans. Viral macrodomains, enzymes that remove posttranslational ADP-ribosylation of proteins, and viral multifunctional papain-like proteases, enzymes that cleave polyproteins and remove polyubiquitin chains via deubiquitinating activity, are two important virulence factors. Here, we reveal an unanticipated interplay between the macrodomain and the PLP2 domain that is important for replication and antagonizing the host innate immune response. Targeting the interaction of these enzymes may provide new therapeutic opportunities to treat CoV disease.

An exclusive 42 amino acid signature in pp1ab protein provides insights into the evolutive history of the 2019 novel human-pathogenic coronavirus (SARS-CoV-2).

The city of Wuhan, Hubei province, China, was the origin of a severe pneumonia outbreak in December 2019, attributed to a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]), causing a total of 2761 deaths and 81109 cases (25 February 2020). SARS-CoV-2 belongs to genus Betacoronavirus, subgenus Sarbecovirus. The polyprotein 1ab (pp1ab) remains unstudied thoroughly since it is similar to other sarbecoviruses. In this short communication, we performed phylogenetic-structural sequence analysis of pp1ab protein of SARS-CoV-2. The analysis showed that the viral pp1ab has not changed in most isolates throughout the outbreak time, but interestingly a deletion of 8 aa in the virulence factor nonstructural protein 1 was found in a virus isolated from a Japanese patient that did not display critical symptoms. While comparing pp1ab protein with other betacoronaviruses, we found a 42 amino acid signature that is only present in SARS-CoV-2 (AS-SCoV2). Members from clade 2 of sarbecoviruses have traces of this signature. The AS-SCoV2 located in the acidic-domain of papain-like protein of SARS-CoV-2 and bat-SL-CoV-RatG13 guided us to suggest that the novel 2019 coronavirus probably emerged by genetic drift from bat-SL-CoV-RaTG13. The implication of this amino acid signature in papain-like protein structure arrangement and function is something worth to be explored.

Middle East Respiratory Syndrome Coronavirus Spike Protein Is Not Activated Directly by Cellular Furin during Viral Entry into Target Cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host cellular proteases to enter cells. A previous report shows that furin, which is distributed mainly in the Golgi apparatus and cycled to the cell surface and endosomes, proteolytically activates the MERS-CoV spike (S) protein following receptor binding to mediate fusion between the viral and cellular membranes. In this study, we reexamined furin usage by MERS-CoV using a real-time PCR-based virus cell entry assay after inhibition of cellular proteases. We found that the furin inhibitor dec-RVKR-CMK blocked entry of MERS-CoV harboring an S protein lacking furin cleavage sites; it even blocked entry into furin-deficient LoVo cells. In addition, dec-RVKR-CMK inhibited not only the enzymatic activity of furin but also those of cathepsin L, cathepsin B, trypsin, papain, and TMPRSS2. Furthermore, a virus cell entry assay and a cell-cell fusion assay provided no evidence that the S protein was activated by exogenous furin. Therefore, we conclude that furin does not play a role in entry of MERS-CoV into cells and that the inhibitory effect of dec-RVKR-CMK is specific for TMPRSS2 and cathepsin L rather than furin.IMPORTANCE Previous studies using the furin inhibitor dec-RVKR-CMK suggest that MERS-CoV utilizes a cellular protease, furin, to activate viral glycoproteins during cell entry. However, we found that dec-RVKR-CMK inhibits not only furin but also other proteases. Furthermore, we found no evidence that MERS-CoV uses furin. These findings suggest that previous studies in the virology field based on dec-RVKR-CMK should be reexamined carefully. Here we describe appropriate experiments that can be used to assess the effect of protease inhibitors on virus cell entry.

Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion.

BACKGROUND: Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. RESULTS: We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caricain, were grouped into the lineage-specific expansion branch in the subfamily III. Mapping PLCP genes on chromosomes of five plant species revealed that lineage-specific expansions of PLCP genes were mostly derived from tandem duplications. We estimated divergence time of papaya PLCP genes of subfamily III. The major duplication events leading to lineage-specific expansion of papaya PLCP genes in subfamily III were estimated at 48 MYA, 34 MYA, and 16 MYA. The gene expression patterns of the papaya PLCP genes in different tissues were assessed by transcriptome sequencing and qRT-PCR. Most of the papaya PLCP genes of subfamily III expressed at high levels in leaf and green fruit tissues. CONCLUSIONS: Tandem duplications played the dominant role in affecting copy number of PLCPs in plants. Significant variations in size of the PLCP subfamilies among species may reflect genetic adaptation of plant species to different environments. The lineage-specific expansion of papaya PLCPs of subfamily III might have been promoted by the continuous reciprocal selective effects of herbivore attack and plant defense.

Papain-like and legumain-like proteases in rice: genome-wide identification, comprehensive gene feature characterization and expression analysis.

BACKGROUND: Papain-like and legumain-like proteases are proteolytic enzymes which play key roles in plant development, senescence and defense. The activities of proteases in both families could be inhibited by a group of small proteins called cystatin. Cystatin family genes have been well characterized both in tobacco and rice, suggesting their potential roles in seed development. However, their potential targets, papain-like and legumain-like proteases, have not been well characterized in plants, especially in rice, a model plant for cereal biology. RESULTS: Here, 33 papain-like and 5 legumain-like proteases have been identified in rice genome, respectively. Gene structure, distribution in rice chromosome, and evolutionary relationship to their counterparts in other plants have been well characterized. Comprehensive expression profile analysis revealed that two family genes display divergent expression pattern, which are regulated temporally and spatially during the process of seed development and germination. Our experiments also revealed that the expression of most genes in these two families is sensitively responsive to plant hormones and different abiotic stresses. CONCLUSIONS: Genome-wide identification and comprehensive gene expression pattern analysis of papain-like and legumain-like proteases in rice suggests their multiple and cooperative roles in seed development and response to environmental variations, which provides several useful cues for further in-depth study.

Papain-like cysteine protease encoding genes in rubber (Hevea brasiliensis): comparative genomics, phylogenetic, and transcriptional profiling analysis.

MAIN CONCLUSION: 43 HbPLCPs representing nine subfamilies or 20 orthologous groups were found in rubber, where paralogs were resulted from the recent WGD and local duplication. Several senescence-associated genes were also identified. Papain-like cysteine proteases (PLCPs) comprise a large family of proteolytic enzymes involved in plant growth and development, seed germination, organ senescence, immunity, and stress response. Despite their importance and the extensive research in the model plant Arabidopsis thaliana, little information is available on rubber tree (Hevea brasiliensis), a rubber-producing plant of the Euphorbiaceae family. This study performed a genome-wide identification of PLCP family genes in rubber, resulting in a relatively high number of 43 members. The phylogenetic analysis assigned these genes into nine subfamilies, i.e., RD21 (6), CEP (4), XCP (4), XBCP3 (2), THI (1), SAG12 (18), RD19 (4), ALP (2), and CTB (2). Most of them were shown to have orthologs in Arabidopsis; however, several members in SAG12, CEP and XBCP3 subfamilies form new groups as observed in other core eudicots such as Manihot esculenta, Ricinus communis, Populus trichocarpa, and Vitis vinifera. Based on an expert sequence comparison, 20 orthologous groups (OGs) were proposed for core eudicots, and rubber paralogs were shown to be resulted from the recent whole-genome duplication (WGD) as well as local duplication. Transcriptional profiling showed distinct expression pattern of different members across various tissues, e.g., root, leaf, bark, laticifer, flower, and seed. By using the senescence-specific HbSAG12H1 as the indicator, the transcriptome of senescent rubber leaves was deeply sequenced and several senescence-associated PLCP genes were identified. Results obtained from this study provide valuable information for future functional analysis and utilization of PLCP genes in Hevea and other species.

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

Middle East respiratory syndrome coronavirus (MERS CoV) is a recently evolved fatal respiratory disease that poses a concern for a global epidemic. MERS CoV encodes 2 proteases, 3C-like protease (3CLpro) and papain-like protease (PLpro). These proteases share in processing MERS CoV polyproteins at different sites to yield 16 nonstructural proteins. In this work, we provide evidence that MERS CoV 3CLpro and PLpro are subject to different genetic and evolutionary influences that shape the protein sequence, codon usage pattern, and codon usage bias. Compositional bias is present in both proteins due to a preference for AT nucleotides. Thymidine (T) was highly preferred at the third position of codons, preferred and overrepresented codons in PLpro, but was replaced by guanosine (G) in 3CLpro. Compositional constraints were important in PLpro but not in 3CLpro. Directed mutation pressure seems to have a strong influence on 3CLpro codon usage, which is more than 30-fold higher than that in PLpro. Translational selection was evident with PLpro but not with 3CLpro. Both proteins are less immunogenic by showing low CpG frequencies. Correspondence analysis reveals the presence of 3 genetic clusters based on codon usage in PLpro and 3CLpro. Every protein had one common cluster and 2 different clusters. As revealed by correspondence analysis, the number of influences on codon usage are restricted in MERS CoV 3CLpro. In contrast, PLpro is controlled by a broader range of compositional, mutational, and other influences. This may be due to the multifunctional protease, deubiquitination, and innate immunity suppressing profiles of PLpro.

Rational Design of Recombinant Papain-Like Cysteine Protease: Optimal Domain Structure and Expression Conditions for Wheat-Derived Enzyme Triticain-α.

Triticain-α is a papain-like cysteine protease from wheat (Triticumaestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichiapastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-ß mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL(pro)) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL(pro) was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL(pro) domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL(pro), we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL(pro) to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL(pro) DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL(pro) domain was found to suppress IFN-ß promoter activation, PL(pro) variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL(pro), and not its proteolytic activity per se, in the inhibition of IFN-ß promoter activity. The ability to decouple the DUB activity of PL(pro) from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL(pro) as a viral DUB during MERS-CoV infection.

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

UNLABELLED: The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1ß, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1ß were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. IMPORTANCE: SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites for each PLP1 were confirmed by testing mutant constructs. Several unique features of the SHFV PLP1s were discovered. The SHFV PLP1α catalytic Cys63 is unique among arterivirus PLP1s in being adjacent to an Ala instead of a Trp. Other arterivirus PLP1s cleave only in cis at a single downstream site, but SHFV PLP1γ can cleave at both the downstream nsp1γ-nsp2 and upstream nsp1ß-nsp1γ junctions. The three mature nsp1 proteins were produced both in the in vitro reactions and in infected cells.

Structural basis for catalysis and ubiquitin recognition by the severe acute respiratory syndrome coronavirus papain-like protease.

Papain-like protease (PLpro) is one of two cysteine proteases involved in the proteolytic processing of the polyproteins of Severe acute respiratory syndrome coronavirus (SARS-CoV). PLpro also shows significant in vitro deubiquitinating and de-ISGylating activities, although the detailed mechanism is still unclear. Here, the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub) is reported at 1.4 Šresolution. The Ub core makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4-P1 residues and providing the first atomic insights into its catalysis. One of the O atoms of the C-terminal Gly residue of Ub is located in the oxyanion hole consisting of the main-chain amides of residues 112 and 113. Mutations of residues in the PLpro-Ub interface lead to reduced catalytic activity, confirming their importance for Ub binding and/or catalysis. The structure also revealed an N-cyclohexyl-2-aminethanesulfonic acid molecule near the catalytic triad, and kinetic studies suggest that this binding site is also used by other PLpro inhibitors. Overall, the structure provides a foundation for understanding the molecular basis of coronaviral PLpro catalysis.