The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.     

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

Recent studies from our laboratory have showed that resveratrol, a polyphenol found predominantly in grapes rendered strong cardioprotection in animal models of heart disease. The cardioprotection which was observed was primarily associated with the ability of resveratrol to reduce oxidative stress in these models. The aim of the current study was to corroborate the role of resveratrol as an inhibitor of oxidative stress and explore the underlying mechanisms of its action in heart disease. For this purpose, we used a cell model of oxidative stress, the hydrogen peroxide (H₂O₂) exposed adult rat cardiomyocytes, which was treated with and without resveratrol (30 μM); cardiomyocytes which were not exposed to resveratrol served as controls. Cell injury, cell death and oxidative stress measurements as well as the activities of the major endogenous antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were carried out in control and H₂O₂ exposed cardiomyocytes, treated with and without resveratrol. Pharmacological blockade using specific blockers of the antioxidant enzymes were used to confirm their role in mediating resveratrol action in H₂O₂ exposed cardiomyocytes. The status of H₂O₂ and antioxidant enzymes in serum samples from spontaneously hypertensive rats (SHR) treated with and without resveratrol (2.5 mg/kg body weight) was also examined.Our results showed significant cell injury and death in H₂O₂ exposed cardiomyocytes which was prevented upon resveratrol treatment. SOD and CAT activities were decreased in H₂O₂ exposed adult rat cardiomyocytes; treatment with resveratrol significantly prevented this reduction. However, GPx activity was not altered in the H₂O₂ exposed cardiomyocytes in comparison to controls. Pharmacological blockade of SOD and/or CAT prevented the beneficial effect of resveratrol. In SHR, H₂O₂ levels were increased, but CAT activity was decreased, while SOD remained unchanged, when compared to WKY rats; resveratrol treatment significantly prevented the increase in H₂O₂ levels and the decrease in CAT activities in SHR.Based on our results, we conclude that treatment with resveratrol prevents oxidative stress induced cardiomyocyte injury mainly by preserving the activities of critical antioxidant enzymes. This may be a crucial mechanism by which resveratrol confers cardioprotection.     

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.     

Structural and functional characterization of MERS coronavirus papain-like protease

Backgrounds

A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PL^pro) is expressed, and its structural and functional consequences are elucidated.

Results

Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PL^pro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PL^pro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PL^pro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PL^pro. A natural mutation, Leu105, is the major reason for this difference.

Conclusions

Overall, MERS-CoV PL^pro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PL^pro family, which is applicable to develop strategies inhibiting PL^pro against highly pathogenic coronaviruses.     

Genetic screen for monitoring severe acute respiratory syndrome coronavirus 3C-like protease

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the beta-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.     

Quaternary structure of the severe acute respiratory syndrome (SARS) coronavirus main protease

SARS (severe acute respiratory syndrome) has been one of the most severe viral infectious diseases last year and still remains as a highly risky public health problem around the world. Exploring the types of interactions responsible for structural stabilities of its component protein molecules constitutes one of the approaches to find a destabilization method for the virion particle. In this study, we performed a series of experiments to characterize the quaternary structure of the dimeric coronavirus main protease (M(pro), 3CL(pro)). By using the analytical ultracentrifuge, we demonstrated that the dimeric SARS coronavirus main protease exists as the major form in solution at protein concentration as low as 0.10 mg/mL at neutral pH. The enzyme started to dissociate at acidic and alkali pH values. Ionic strength has profound effect on the dimer stability indicating that the major force involved in the subunit association is ionic interactions. The effect of ionic strength on the protease molecule was reflected by the drastic change of electrostatic potential contour of the enzyme in the presence of NaCl. Analysis of the crystal structures indicated that the interfacial ionic interaction was attributed to the Arg-4...Glu-290 ion pair between the subunits. Detailed examination of the dimer-monomer equilibrium at different pH values reveals apparent pK(a) values of 8.0 +/- 0.2 and 5.0 +/- 0.1 for the Arg-4 and Glu-290, respectively. Mutation at these two positions reduces the association affinity between subunits, and the Glu-290 mutants had diminished enzyme activity. This information is useful in searching for substances that can intervene in the subunit association, which is attractive as a target to neutralize the virulence of SARS coronavirus.     

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride

Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.     

Molecular biology of severe acute respiratory syndrome coronavirus

The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. Coronaviruses and their closest relatives possess extremely large plus-strand RNA genomes and employ unique mechanisms and enzymes in RNA synthesis that separate them from all other RNA viruses. The SARS epidemic prompted a variety of studies on multiple aspects of the coronavirus replication cycle, yielding both rapid identification of the entry mechanisms of SARS-CoV into host cells and valuable structural and functional information on SARS-CoV proteins. These recent advances in coronavirus research have important implications for the development of anti-SARS drugs and vaccines.     

Identification and characterization of dominant helper T-cell epitopes in the nucleocapsid protein of severe acute respiratory syndrome coronavirus

By using a series of overlapping synthetic peptides covering 98% of the amino acid sequence of the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus (SARS-CoV), four helper T-cell (Th) epitopes (NP11, residues 11 to 25; NP51, residues 51 to 65; NP61, residues 61 to 75; and NP111, residues 111 to 125) in C57BL mice (H-2(b)), four (NP21, residues 21 to 35; NP91, residues 91 to 105; NP331, residues 331 to 345; and NP351, residues 351 to 365) in C3H mice (H-2(k)), and two (NP81, residues 81 to 95; and NP351, residues 351 to 365) in BALB/c mice (H-2(d)) have been identified. All of these peptides were able to stimulate the proliferation of NP-specific T-cell lines or freshly isolated lymph node cells from mice immunized with recombinant NP. Immunization of mice with synthetic peptides containing appropriate Th epitopes elicited strong cellular immunity in vivo, as evidenced by delayed-type hypersensitivity. Priming with the helper peptides (e.g., NP111 and NP351) significantly accelerated the immune response induced by recombinant NP, as determined by the production of NP-specific antibodies. When fused with a conserved neutralizing epitope (SP1143-1157) from the spike protein of SARS-CoV, NP111 and NP351 assisted in the production of high-titer neutralizing antibodies in vivo. These data provide useful insights regarding immunity against SARS-CoV and have the potential to help guide the design of peptide-based vaccines.     

On the mechanisms of bananin activity against severe acute respiratory syndrome coronavirus

In a previous study, severe acute respiratory syndrome coronavirus (SARS-CoV) was cultured in the presence of bananin, an effective adamantane-related molecule with antiviral activity. In the present study, we show that all bananin-resistant variants exhibit mutations in helicase and membrane protein, although no evidence of bananin interference on their mutual interaction has been found. A structural analysis on protein sequence mutations found in SARS-CoV bananin-resistant variants was performed. The S259/L mutation of SARS-CoV helicase is always found in all the identified bananin-resistant variants, suggesting a primary role of this mutation site for bananin activity. From a structural analysis of SARS-CoV predicted helicase structure, S259 is found in a hydrophilic surface pocket, far from the enzyme active sites and outside the helicase dimer interface. The S/L substitution causes a pocket volume reduction that weakens the interaction between bananin and SARS-CoV mutated helicase, suggesting a possible mechanism for bananin antiviral activity.     

Study on substrate specificity at subsites for severe acute respiratory syndrome coronavirus 3CL protease

Autocleavage assay and peptide-based cleavage assay were used to study the substrate specificity of 3CL protease from the severe acute respiratory syndrome coronavirus. It was found that the recognition between the enzyme and its substrates involved many positions in the substrate, at least including residues from P4 to P2＇. The deletion of either P4 or P2＇ residue in the substrate would decrease its cleavage efficiency dramatically. In contrast to the previous suggestion that only small residues in substrate could be accommodated to the S 1＇ subsite, we have found that bulky residues such as Tyr and Trp were also acceptable. In addition, based on both peptide-based assay and autocleavage assay, Ile at the PI＇ position could not be hydrolyzed, but the mutant L27A could hydrolyze the Ile peptide fragment. It suggested that there was a stereo hindrance between the S 1＇ subsite and the side chain of Ile in the substrate. All 20 amino acids except Pro could be the residue at the P2＇ position in the substrate, but the cleavage efficiencies were clearly different. The specificity information of the enzyme is helpful for potent anti-virus inhibitor design and useful for other coronavirus studies.     

Characterization of severe acute respiratory syndrome coronavirus membrane protein

The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.     

Mosaic evolution of the severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a deadly form of pneumonia caused by a novel coronavirus, a viral family responsible for mild respiratory tract infections in a wide variety of animals including humans, pigs, cows, mice, cats, and birds. Analyses to date have been unable to identify the precise origin of the SARS coronavirus. We used Bayesian, neighbor-joining, and split decomposition phylogenetic techniques on the SARS virus replicase, surface spike, matrix, and nucleocapsid proteins to reveal the evolutionary origin of this recently emerging infectious agent. The analyses support a mammalian-like origin for the replicase protein, an avian-like origin for the matrix and nucleocapsid proteins, and a mammalian-avian mosaic origin for the host-determining spike protein. A bootscan recombination analysis of the spike gene revealed high nucleotide identity between the SARS virus and a feline infectious peritonitis virus throughout the gene, except for a 200- base-pair region of high identity to an avian sequence. These data support the phylogenetic analyses and suggest a possible past recombination event between mammalian-like and avian-like parent viruses. This event occurred near a region that has been implicated to be the human receptor binding site and may have been directly responsible for the switch of host of the SARS coronavirus from animals to humans.     

The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication

The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function.     

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.     

Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.