Respiratory Syndrome Coronavirus Papain-like Protease Research Articles

Encapsulation of MERS antigen into α-GalCer-bearing-liposomes elicits stronger effector and memory immune responses in immunocompetent and leukopenic mice

ObjectivesHere, we prepared a liposome-based vaccine formulation containing Middle East Respiratory Syndrome Coronavirus papain-like protease (MERS-CoV-PLpro). MethodsA persistent leukopenic condition was induced in mice by injecting cyclophosphamide (CYP) three days before each dose of immunization. Mice were immunized on days 0, 14 and 21 with α-GalCer-bearing MERS-CoV PLpro-encapsulated DPPC-liposomes (α-GalCer-MERS-PLpro-liposomes or MERS-CoV PLpo-encapsulated DPPC-liposomes (MERS-PLpro-liposomes), whereas the antigen emulsified in Alum (MERS-PLpro-Alum) was taken as a control. On day 26, the blood was taken from the immunized mice to analyze IgG titer, whereas the splenocytes were used to analyze the lymphocyte proliferation and the level of cytokines. In order to assess the memory immune response, mice were given a booster dose after 150 days of the last immunization. ResultsThe higher levels of MERS-CoV-PLpro-specific antibody titer, IgG2a and lymphocyte proliferation were noticed in mice immunized with α-GalCer-MERS-PLpro-liposomes. Besides, the splenocytes from mice immunized with α-GalCer-MERS-PLpro-liposomes produced larger amounts of IFN-γ as compared to the splenocytes from MERS-PLpro-liposomes or MERS- PLpro-Alum immunized mice. Importantly, an efficient antigen-specific memory immune response was observed in α-GalCer-MERS-PLpro-liposomes immunized mice. ConclusionsThese findings suggest that α-GalCer-MERS-PLpro-liposomes may substantiate to be a successful vaccine formulation against MERS-CoV infection, particularly in immunocompromised individuals.

Delivery of MERS antigen encapsulated in α-GalCer-bearing liposomes elicits stronger antigen-specific immune responses

Alpha-Galactosylceramide (α-GalCer) effectively activates the natural killer T (NKT) cells to secrete remarkable amounts of Th1 and Th2 cytokines and therefore, acts as a potential immunoadjuvant in vaccine formulation. In the present study, we prepared α-GalCer-bearing or α-GalCer-free liposomes and loaded them with Middle East Respiratory Syndrome Coronavirus papain-like protease (α-GalCer-Lip-MERS-CoV PLpro or Lip-MERS-CoV PLpro). These formulations were injected in mice to investigate the antigen-specific humoral and cell-mediated immune responses. The immunisation with α-GalCer-Lip-MERS-CoV PLpro or Lip-MERS-CoV PLpro did not induce any notable toxicity in immunised mice. The results demonstrated that mice immunised with α-GalCer-Lip-MERS-CoV PLpro showed greater antigen-specific antibody titre, switching of IgG isotyping to IgG2a subclass and higher lymphocyte proliferation. Moreover, the splenocytes from α-GalCer-Lip-MERS-CoV PLpro immunised mice secreted greater levels of IFN-γ, IL-4, IL-2 and IL-12. Interestingly, a booster dose induced stronger memory immune responses in mice previously immunised with α-GalCer-Lip-MERS-CoV PLpro. In summary, α-GalCer-Lip-MERS-CoV PLpro may prove to be a promising vaccine formulation to protect the individuals against MERS-CoV infection.

Liposome-Mediated Delivery of MERS Antigen Induces Potent Humoral and Cell-Mediated Immune Response in Mice.

The advancements in the field of nanotechnology have provided a great platform for the development of effective antiviral vaccines. Liposome-mediated delivery of antigens has been shown to induce the antigen-specific stimulation of the humoral and cell-mediated immune responses. Here, we prepared dried, reconstituted vesicles (DRVs) from DPPC liposomes and used them as the vaccine carrier system for the Middle East respiratory syndrome coronavirus papain-like protease (DRVs-MERS-CoV PLpro). MERS-CoV PLpro emulsified in the Incomplete Freund’s Adjuvant (IFA-MERS-CoV PLpro) was used as a control. Immunization of mice with DRVs-MERS-CoV PLpro did not induce any notable toxicity, as revealed by the levels of the serum alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the blood of immunized mice. Immunization with DRVs-MERS-CoV PLpro induced greater antigen-specific antibody titer and switching of IgG1 isotyping to IgG2a as compared to immunization with IFA-MERS-CoV PLpro. Moreover, splenocytes from mice immunized with DRVs-MERS-CoV PLpro exhibited greater proliferation in response to antigen stimulation. Moreover, splenocytes from DRVs-MERS-CoV PLpro-immunized mice secreted significantly higher IFN-γ as compared to splenocytes from IFA-MERS-CoV PLpro mice. In summary, DRVs-MERS-CoV PLpro may prove to be an effective prophylactic formulation to prevent MERS-CoV infection.

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2.

The coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), warranting urgent study of the molecular mechanisms of SARS-CoV-2 infection and host immune response. Type I interferon (IFN-I) is a key component of host innate immune system responsible for eliminating the virus at the early stage of infection. In contrast, SARS-CoV-2 has evolved multiple strategies to evade innate immune response to facilitate viral replication, transmission, and pathogenesis. This review summarizes the recent progresses on SARS-CoV-2 proteins that antagonize host IFN-I production and/or signaling. These progresses have provided knowledge for new vaccine and antiviral development to prevent and control COVID-19.

Phytochemical profile of Paulownia tomentosa (Thunb). Steud.

Paulownia tomentosa, a member of the plant family Paulowniaceae and a rich source of biologically active secondary metabolites, is traditionally used in Chinese herbal medicine. Flavonoids, lignans, phenolic glycosides, quinones, terpenoids, glycerides, phenolic acids, and miscellaneous other compounds have been isolated from different parts of P. tomentosa plant. Recent interest in this species has focused on isolating and identifying of prenylated flavonoids, that exhibit potent antioxidant, antibacterial, and antiphlogistic activities and inhibit severe acute respiratory syndrome coronavirus papain-like protease. They show cytotoxic activity against various human cancer cell lines and inhibit the effects of human cholinesterase, butyrylcholinesterase, and bacterial neuraminidases. Most of the compounds considered here have never been isolated from any other species of plant. This review summarizes the information about the isolated compounds that are active, their bioactivities, and the structure–activity relationships that have been worked out for them.

Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease

The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-β by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.

Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease

Papain-like protease (PLpro) from severe acute respiratory syndrome (SARS) coronavirus is one of the two proteases involved in the proteolytic processing of the virion polyproteins. In addition, PLpro shows significant in vitro deubiquitinating and de-ISGylating activities. All these findings demonstrated the multifunctional nature of the PLpro. Here we report the sensitivity of PLpro to denaturant urea. An increase in urea concentration induced a reversible biphasic unfolding of the enzyme. Differently, the unfolding of the catalytic triad region located within the palm and thumb domains followed a monophasic unfolding curve. Further observations suggest that the zinc-binding domain may start to unfold during the first transition. An 80% lost of its enzymatic activity at a urea concentration lower than 1M showed a close correlation with unfolding of the zinc-binding domain. The enzyme was also characterized in terms of hydrophobicity and size-and-shape distribution. We have demonstrated that PLpro displayed differential domain structure stability and molten globule state in its folding. These studies will not only assist in our understanding of the folding of this viral enzyme, but also that of other deubiquitinating enzymes with a similar scaffold.

Severe acute respiratory syndrome coronavirus papain-like protease suppressed alpha interferon-induced responses through downregulation of extracellular signal-regulated kinase 1-mediated signalling pathways

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly I : C-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-α-induced responses such as interferon-stimulated response element- and AP-1-driven promoter activation, protein kinase R, 2'-5'-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-α-induced responses. IFN-α treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-α and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-α-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-α-induced expression of 2'-5'-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.

Novel Influenza Virus NS1 Antagonists Block Replication and Restore Innate Immune Function

The innate immune system guards against virus infection through a variety of mechanisms including mobilization of the host interferon system, which attacks viral products mainly at a posttranscriptional level. The influenza virus NS1 protein is a multifunctional facilitator of virus replication, one of whose actions is to antagonize the interferon response. Since NS1 is required for efficient virus replication, it was reasoned that chemical inhibitors of this protein could be used to further understand virus-host interactions and also serve as potential new antiviral agents. A yeast-based assay was developed to identify compounds that phenotypically suppress NS1 function. Several such compounds exhibited significant activity specifically against influenza A virus in cell culture but had no effect on the replication of another RNA virus, respiratory syncytial virus. Interestingly, cells lacking an interferon response were drug resistant, suggesting that the compounds block interactions between NS1 and the interferon system. Accordingly, the compounds reversed the inhibition of beta interferon mRNA induction during infection, which is known to be caused by NS1. In addition, the compounds blocked the ability of NS1 protein to inhibit double-stranded RNA-dependent activation of a transfected beta interferon promoter construct. The effects of the compounds were specific to NS1, because they had no effect on the ability of the severe acute respiratory syndrome coronavirus papainlike protease protein to block beta interferon promoter activation. These data demonstrate that the function of NS1 can be modulated by chemical inhibitors and that such inhibitors will be useful as probes of biological function and as starting points for clinical drug development.

Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS–PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k cat/ K M of 602,000 M −1 s −1, PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-A crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitin-like N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitin-aldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.

The Papain-Like Protease from the Severe Acute Respiratory Syndrome Coronavirus Is a Deubiquitinating Enzyme

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.

Deubiquitination, a New Function of the Severe Acute Respiratory Syndrome Coronavirus Papain-Like Protease?

A new coronavirus has been identified as the infectious agent of severe acute respiratory syndrome (SARS). Although SARS was successfully contained by quarantine measures, the reconstructed itinerary of the virus through 30 countries and its high mortality rate illustrate the global threat that this newly emerging disease represents (17). During the expression of the SARS coronavirus (SCoV) genome, two viral cysteine proteases, a papain-like protease (PLpro) and a chymotrypsin-like protease (3CLpro), process the encoded polyprotein precursor to release most of the proteins required for virus replication. PLpro refers to a domain of nonstructural protein 3, whose boundaries are defined by homology to the papain-like fold (7). The PLpro domain can be regarded as the catalytic core behind PLpro-mediated cleavages, even though processing by PLpros has been reported to be modulated by additional amino acid residues outside of these boundaries (15, 18). The SCoV utilizes a single PLpro, whereas most coronaviruses contain two paralogous enzymes, termed PL1pro and PL2pro (14). PLpros in general are not as well characterized as 3CLpros and have not generated as much interest as pharmaceutical targets. However, further structure-to-function annotations might refocus the attention on PLpros. For this purpose, we mined the current Protein Data Bank (PDB) content using the Structure Prediction Meta Server (http://www.bioinfo.pl/meta ), which assembles state-of-the-art fold recognition methods and provides a consensus sequence-to-structure hyperscore with the 3D-Jury method (4). The only highly reliable prediction (3D-Jury score > 100) obtained for the SCoV PLpro sequence from K1632 to E1847 was the structure of the catalytic core domain of the herpesvirus-associated ubiquitin-specific protease (HAUSP), also known as USP7 (8).