The propeptide of Clostridium septicum alpha toxin functions as an intramolecular chaperone and is a potent inhibitor of alpha toxin-dependent cytolysis

Clostridium septicum alpha toxin is activated by a proteolytic cleavage at Arg-398 in its carboxy terminus, which yields a 41.3-kDa cytolytically active toxin and a 5.1-kDa propeptide. Studies were performed to determine when the propeptide dissociated from the toxin after proteolytic activation of the protoxin (AT^pro) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the toxin after activation with trypsin (AT^act) when analysed by gel filtration or affinity chromatography of a polyhistidine-tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the toxin was decreased significantly when a mutation was introduced in which Val-400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the toxin and the propeptide was found to dissociate from the toxin under the same gel-filtration conditions used for the wild-type toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of alpha toxin activity, which inhibited the oligomerization of alpha toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from AT^act. The importance of the propeptide to the solution stability of alpha toxin was also demonstrated. When AT^pro was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14 000 × g, was made up of both SDS-sensitive and -resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16-fold) were observed when alpha toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric AT^pro and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre-pore complex and forms a pore. The data suggest that oligomerization of the toxin displaces the propeptide from the monomer form of alpha toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.     

Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein

The human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac⁺), all of which produce the 87kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac⁻) also carry the gene, Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33-amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C-termlnal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease-type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.     

Alanine-162 of Bacillus thuringiensis Cyt2Aa2 toxin is essential for membrane binding and oligomerisation

Cyt2Aa2 is a cytolytic toxin produced by Bacillus thuringiensis subsp. darmstadiensis. It is specifically toxic to dipteran larvae in vivo and is also active against several cell types, such as erythrocytes. The active toxin is proposed to bind to the cell membrane, and membrane pore formation by toxin oligomerisation leads to cell lysis. This study aimed to characterise the role of residues (I139, S159, L160, S161, A162, D209 and V215) potentially involved in the membrane binding of Cyt2Aa2. All mutants, except I139A and V215A, showed similar characteristics to the wild-type toxin after proteinase K cleavage. Three mutants, S159A, L160A and S161A, showed high haemolytic activity but low toxicity against Aedes aegypti. Membrane interaction assays showed that these mutants could bind to rat red blood cells (rRBCs) and oligomerise. The mutant D209N had no haemolytic activity but was still mildly toxic to A. aegypti. The mutant A162V could not lyse rRBCs, even at high concentrations, and showed no toxicity against A. aegypti. Our data suggest that alanine 162 of the Cyt2Aa2 toxin is involved in membrane binding and oligomerisation. Substitution of this amino acid altered the conformation of the toxin and affected its biological activity.     

Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin

Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin. When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures ≤15°C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4°C. Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4°C and 37°C. The slight differences in these two processes at 4°C and 37°C could not account for the loss of cytolytic activity at low temperature. At 4°C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes. In contrast, at temperatures ≥25°C both processes proceeded rapidly. Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature. Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane. These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.     

Pore-forming and haemolytic properties of the Gardnerella vaginalis cytoiysin

The pleomorphic bacterium Gardnerella vaginalis releases in the culture broth a haemolytic exotoxin (Gvh) which is probably a virulence determinant of this unique bacterium, implicated in gynaecological and urological disorders. This 59kDa cytolysin was purified to homogeneity in just one chromatographic step directly from the culture supernatant, a final specific activity up to 1.9 × 10⁶ HU mg^?1 being obtained. The toxin-induced lesion on human erythrocytes results from the formation of a pore whose radius is approximately 2.4 nm. The damage is inhibited by osmotic protectants and shows a sigmoidal dose-response profile suggesting an aggregation process of haemolysin molecules on the target membrane to create the functional lesion. The extent and the kinetics of haemolysis are strongly dependent on temperature and an activation energy of 64.0 kJ mol^?1 has been derived. Lipid membranes can be very efficient inhibitors of Gvh-haemolysis, being able to bind the toxin quite avidly. The inhibitory effect requires the presence of cholesterol and it is stronger when cholesterol is mixed with negatively charged phospholipids rather than with zwitterionic phospholipids, suggesting that a negative surface potential increases the affinity of the toxin for the lipid bilayer. The functional properties of Gvh have been compared with those of Clostridium perfringens theta-toxin (PFO) and Escherichia coli haemolysin (HlyA), which are representative of widespread haemolysins produced by Gram-positive and Gram-negative bacteria, respectively. The toxin shares several features with the family of the so-called ‘sulphydryl-activated’ cytolysins produced by Gram-positive bacteria, although Gvh does not truly belong to this family, being deactivated by β-mercaptoethanol and being antigenically distinct from them. We report here for the first time the detection in the vaginal fluid of infected women of a specific IgA response against the toxin.     

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N<Superscript>pro</Superscript>) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N^pro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N^pro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N^pro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N^pro rabbit serum. When rabbits were immunized with the N^pro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N^pro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N^pro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N^pro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.     

A broad-spectrum cytolytic toxin from Bacillus thuringiensis var. kyushuensis.

Bacillus thuringiensis (Bt) var. kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa. We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer. This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var. darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var. israelensis. The cytolytic activity of these Bt var. kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.     

Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly

A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA. The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene. The toss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin. Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type. The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.     

Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2^pro) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2^pro activity by the NS3 protease domain. NS2^pro activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2^pro activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2^pro activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2^pro could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2^pro activity mimic structural changes occurring during NS3-mediated NS2^pro activation. Introducing these activating NS2^pro mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2^pro activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2^pro activation by NS3.     

Amino acid replacements in the Serratia marcescens haemolysin ShlA define sites involved in activation and secretion

The haemolysin of Serratia marcescens (ShlA) is translocated through the cytoplasmic membrane by the signal peptide-dependent export apparatus. Translocation across the outer membrane (secretion) is mediated by the ShIB protein. Only the secreted form of ShlA is haemolytic. ShIB also converts in vitro inactive ShlA (ShlA*), synthesized in the absence of ShIB, into the haemolytic form (a process termed activation). To define regions in ShlA involved in both processes, ShlA derivatives were isolated and tested for secretion and activation. Analysis of C-terminally truncated proteins (ShlA) assigned the secretion signal to the amino-terminal 238 residues of ShlA. Trypsin cleavage of a secreted ShlA' derivative yielded a 15kDa N-terminal fragment, by which a haemolytically inactive ShlA* protein could be activated in vitro. It is suggested that the haemolysin activation site is located in this N-terminal fragment. Replacement of asparagine-69 and asparagine-109 by isoleucine yielded inactive haemolysin derivatives. Both asparagine residues are part of two short sequence motifs, reading Ala-Asn-Pro-Asn, which are critical to both activation and secretion. These point mutants as well as N-terminal deletion derivatives which were not activated by ShIB were activated by adding a non-haemolytic N-terminal fragment synthesized in an ShIB⁺ strain (complementation). Apparently the activated N-terminal fragment substituted for the missing activation of the ShlA derivatives and directed them into the erythrocyte membrane, where they formed pores. It is concluded that activation is only required for initiation of pore formation, and that in vivo activation and secretion are tightly coupled processes. Complementation may also indicate that haemolysin oligomers form the pores.     

Chalcones isolated from Angelica keiskei inhibit cysteine proteases of SARS-CoV

Two viral proteases of severe acute respiratory syndrome coronavirus (SARS-CoV), a chymotrypsin-like protease (3CL^pro) and a papain-like protease (PL^pro) are attractive targets for the development of anti-SARS drugs. In this study, nine alkylated chalcones (1–9) and four coumarins (10–13) were isolated from Angelica keiskei, and the inhibitory activities of these constituents against SARS-CoV proteases (3CL^pro and PL^pro) were determined (cell-free/based). Of the isolated alkylated chalcones, chalcone 6, containing the perhydroxyl group, exhibited the most potent 3CL^pro and PL^pro inhibitory activity with IC₅₀ values of 11.4 and 1.2?µM. Our detailed protein-inhibitor mechanistic analysis of these species indicated that the chalcones exhibited competitive inhibition characteristics to the SARS-CoV 3CL^pro, whereas noncompetitive inhibition was observed with the SARS-CoV PL^pro.     

Affinity Purification of Angiotensin Type 2 Receptors from N1E-115 cells: Evidence for Agonist-Induced Formation of Multimeric Complexes

Abstract: The murine neuroblastoma N1E-115 cell line possesses type 1 and type 2 angiotensin II (Angll) receptor subtypes. In vitro differentiation of these cells substantially increases the density of the AT₂-receptor subtype, whereas the density of the AT₁ receptors remains unchanged. In the present study, we report that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonate (CHAPS) selectively solubilized AT₂receptors from N1E-115 cell membranes and that these receptors could be purified further to near homogeneity by affinity chromatography. More specifically, the presence of an agonist (Angll) during affinity purification of AT₂ receptors resulted in the elution of high (110-kDa) and low (66-kDa) molecular mass proteins as determined by gel electrophoresis under nonreducing conditions. In contrast, when the nonselective antagonist Sar¹, lle⁸-Angll was used during purification, only the lower 66-kDa protein was observed. Affinity purification in the presence of the peptide and nonpeptide AT₂-receptor antagonists CGP42112A and PD123319 also resulted in elution of the same 66-kDa protein, but unlike that in the presence of Sar¹, lle⁸-Angll, some of the high molecular weight site was observed as well. On the other hand, Losartan, an AT₁-receptor antagonist, was completely ineffective in eluting any Angll receptors from the affinity column, further confirming their AT₂ identity. After agonist elution, the 110-kDa band dissociated into two low molecular mass bands of 66 kDa and 54 kDa when sodium dodecyl sulfate-gel electrophoresis was run under reducing conditions. The 110-kDa and 66-kDa proteins, but not smaller, affinity-purified proteins, specifically bound ¹²⁵l-Angll as determined by covalent cross-linking of ¹²⁵l-Angll to the receptors with the homobifunctional cross-linker disuccinimidyl suberate, or by size exclusion chromatography on a TSK 3000 SW column. Lastly, immunoblot analysis of affinity- purified material with antibodies selective for AT₂ receptors revealed major immunoreactive proteins of 110 kDa and 66 kDa in the presence of an agonist, whereas the same 66-kDa protein, as well as a smaller (54-kDa) immunoreactive protein, was detected under reducing conditions. Collectively, these data suggest that CHAPS-solubilized AT₂ receptors from N1E-115 cells may consist of a binding protein of approximately 66 kDa, which in the presence of an agonist readily associates with other smaller proteins to form larger multimeric complexes.     

Crystal structure of BinB: A receptor binding component of the binary toxin from Lysinibacillus sphaericus

The binary toxin (Bin), produced by Lysinibacillus sphaericus, is composed of BinA (42 kDa) and BinB (51 kDa) proteins, which are both required for full toxicity against Culex and Anopheles mosquito larvae. Specificity of Bin toxin is determined by the binding of BinB component to a receptor present on the midgut epithelial membranes, while BinA is proposed to be a toxic component. Here, we determined the first crystal structure of the active form of BinB at a resolution of 1.75 Å. BinB possesses two distinct structural domains in its N‐ and C‐termini. The globular N‐terminal domain has a β‐trefoil scaffold which is a highly conserved architecture of some sugar binding proteins or lectins, suggesting a role of this domain in receptor‐binding. The BinB β‐rich C‐terminal domain shares similar three‐dimensional folding with aerolysin type β‐pore forming toxins, despite a low sequence identity. The BinB structure, therefore, is a new member of the aerolysin‐like toxin family, with probably similarities in the cytolytic mechanism that takes place via pore formation. Proteins 2014; 82:2703–2712. © 2014 Wiley Periodicals, Inc.     

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

The 3C-like proteinase (3CL^pro) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CL^pro is active. However, as the internally encoded 3CL^pro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CL^pro (XX), SARS 3CL^pro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CL^pro using fluorescence resonance energy transfer. In contrast to the matured 3CL^pro, the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CL^pro in the polyprotein, and a modified model for the 3CL^pro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.     

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A^pro) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A^pro is a major contributor to Nup62 processing. The ability of purified 2A^pro to cleave bacterially expressed and purified Nup62 demonstrated that 2A^pro directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A^pro in vitro. This analysis revealed that 2A^pro cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.     

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture

An insecticidal protein gene from Bacillus thuringiensis var. aizawal was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteloytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resist-ant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CFI ceils but not dipteran (Aedes albopictus) calls. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawal toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran ceil membranes, which may be the receptor for this toxin.     

Generation and characterization of an inter-generic bivalent alpha domain fusion protein αCS from Clostridium perfringens and Staphylococcus aureus for concurrent diagnosis and therapeutic applications

Aim: To evaluate an inter‐generic recombinant alpha domain fusion protein for simultaneous detection and neutralization of Clostridium perfringens and Staphylococcus aureus alpha toxins. Methods and Results: Truncated portions of clostridial and staphylococcal alpha haemolysin genes were PCR amplified and linked to each other through a hydrophilic flexible Glycine linker sequence using overlap‐extension PCR to form a chimeric gene αCS. The recombinant αCS fusion protein was expressed and characterized for its toxicity, cell binding capacity and haemolysis inhibition properties. The fusion protein was nontoxic and effectively retarded staphylococcal alpha haemolysis, probably by competitively interacting with putative staphylococcal alpha haemolysin receptors on erythrocytes. Murine hyperimmune polysera raised against r‐αCS specifically detected 42‐kDa and 33‐kDa proteins when culture supernatants of Cl. perfringens (clostridial alpha toxin) and Staph. aureus (staphylococcal alpha toxin), respectively, were analysed in Western blot. The polyclonal antisera effectively diminished the haemolytic action of both the wild‐type toxins in vitro. Conclusions: The r‐αCS fusion protein was nontoxic competitive inhibitor of staphylococcal alpha haemolysin. The protein elicited specific immune response against Cl. perfringens and Staph. aureus alpha toxins. The antisera also neutralized the toxicities of both the native wild‐type toxins in vitro. Significance of the Study: The bivalent recombinant αCS protein could be a novel intervention in the field of diagnostics and therapeutics against Cl. perfringens and Staph. aureus infections, particularly, in case of co‐infections like gangrenous ischaemia, gangrenous mastitis, etc.     

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 20-50-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3C^pro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3C^pro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3C^pro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^pro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3C^pro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3C^pro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3C^pro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.     

Processing of the Human Coronavirus 229E Replicase Polyproteins by the Virus-Encoded 3C-Like Proteinase: Identification of Proteolytic Products and Cleavage Sites Common to pp1a and pp1ab

Replicase gene expression by the human coronavirus 229E involves the synthesis of two large polyproteins, pp1a and pp1ab. Experimental evidence suggests that these precursor molecules are subject to extensive proteolytic processing. In this study, we show that a chymotrypsin-like enzyme, the virus-encoded 3C-like proteinase (3CL^pro), cleaves within a common region of pp1a and pp1ab (amino acids 3490 to 4068) at four sites. trans-cleavage assays revealed that polypeptides of 5, 23, 12, and 16 kDa are processed from pp1a/pp1ab by proteolysis of the peptide bonds Q3546/S3547, Q3629/S3630, Q3824/N3825, and Q3933/A3934. Relative rate constants for the 3CL^pro-mediated cleavages Q2965/A2966, Q3267/S3268, Q3824/N3825, and Q3933/A3934 were derived by competition experiments using synthetic peptides and recombinant 3CL^pro. The results indicate that coronavirus cleavage sites differ significantly with regard to their susceptibilities to proteolysis by 3CL^pro. Finally, immunoprecipitation with specific rabbit antisera was used to detect the pp1a/pp1ab processing end products in virus-infected cells, and immunofluorescence data that suggest an association of these polypeptides with intracellular membranes were obtained.     

Effects of magnesium ion and chelating agents on enzymatic production of ATP from adenine

Summary As reported previously, enzymatic production of ATP from adenine by resting cells of Brevibacterium ammoniagenes (Fujio and Furuya 1983) accumulated 13.0 mg of ATP · Na₂ · 3H₂O/ml, but ATP formation ceased within 6–8 h. Simultaneous addition of magnesium ion and phytic acid, a chelator of divalent cations, allowed ATP formation to continue longer, and 24.2 mg of ATP · Na₂ · 3H₂O/ml was accumulated in 10 h. However, ATP formation ceased thereafter.This second cessation was found to be caused by the lack of magnesium ion active as a co-factor (Mg^act). The Mg^act was tentatively taken as the difference between soluble magnesium ion (Mg^sol) and the ion chelated by an equimolar amount of ATP (Mg^ATP), namely Mg^act=Mg^sol-Mg^ATP. In order to provide Mg^act, sufficient phytic acid had to be added at the beginning of the reaction and magnesium ion was also added intermittently. Under these conditions ATP formation continued further, and the rate of ATP formation was increased; 37.0 mg of ATP · Na₂ · 3H₂O/ml was accumulated in 13 h.Since whole culture broth is preferable to frozen cells as a practical enzyme source, the conditions neccessary for use of whole culture broth of B. ammoniagenes were also investigated.