衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

Chemical modification of arginine residues of rat liver S-adenosylhomocysteinase

Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by phenylglyoxal following pseudo-first order kinetics. The dependence of the apparent first order rate constant for inactivation on the phenylglyoxal concentration shows that the inactivation is second order in reagent. This fact together with the reversibility of inactivation upon removal of excess reagent and the lack of reaction at residues other than arginine as revealed by amino acid analysis and incorporation of phenylglyoxal into the protein indicate that the inactivation is due to the modification of arginine residue. The substrate adenosine largely but not completely protects the enzyme against inactivation. Although the modification of two arginine residues/subunit is required for complete inactivation, the relationship between loss of enzyme activity and the number of arginine residues modified, and the comparison of the numbers of phenylglyoxal incorporated into the enzyme in the presence and absence of adenosine indicate that one residue which reacts very rapidly with the reagent compared with the other is critical for activity. Although the phenylglyoxal treatment does not result in alteration of the molecular size of the enzyme or dissociation of the bound NAD+, the intrinsic protein fluorescence is largely lost upon modification. The equilibrium binding study shows that the modified enzyme apparently fails to bind adenosine.     

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host-pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein--its binding partner within replication complexes--leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity.     

Initial selectivity of the antiplatelet covalent action of biogenic chloramines on platelet-rich plasma

To describe in full the peculiarities of the antiplatelet action of covalent inhibitors on platelet-rich plasma, we have proposed to take into account the initial selectivity that determines the elevated efficacy of inactivation of platelet molecular target (receptor). The quantitative index of initial selectivity is the ratio of rate constant of inactivation of the platelet molecular target to the rate constant of the chemical reaction of an inhibitor with reactive atomic groups in plasma proteins. For the important case of the domination of the inhibitor expenditure in the reaction with plasma proteins, a formula was derived which depicts the dependence of the share of inactivated targets on the concentration of the inhibitor introduced and reactive atomic groups contained in plasma. In the case of chloramine derivatives of amino acids, evidence was obtained indicating that the degree of inhibition of platelet aggregation measured by the turbidimetric method is equal to the square of the share of inactivated receptors. The index of initial selectivity can be evaluated by measuring the degree of inhibition of platelet aggregation and the operating concentration of the inhibitor. According to experimental evidence, the effects of a number of chloramine derivatives of amino acids (biochloramines) on aggregation of platelets stimulated by ADP show selectivity at the molecular target level, so that the index of initial selectivity is greater than 1. The mechanism of the selective action of the biochloramines having significant molecular masses (150-200 Da) probably consists in the inactivation of the molecular target via chemical modification of several reactive atomic groups in its different sites. One may suppose that the biochloramines with lower molecular masses (150-100 Da) exhibit a high anti-aggregatory capacity owing to another mechanism of initial selectivity, which involves the modification of highly sensitive sulfur-containing atomic groups.     

Affinity labeling of catalytic subunit of bovine heart muscle cyclic AMP-dependent protein kinase by 5'-p-fluorosulfonylbenzoyladenosine.

Incubation of 5'-p-fluorosulfonylbenzoyladenosine with the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase led to the formation of an inactive enzyme irreversibly modified with approximately one mol of reagent per mol of subunit. The inactivation reaction followed pseudofirst order kinetics. The rate of inactivation at various reagent concentrations exhibited saturation kinetics implying that the reagent reversibly binds to the enzyme prior to inactivation. The addition of MgATP, MgADP, or MgAMP-PNP to the reaction mixture fully protected the enzyme from inactivation by 5'-p-fluorosulfonylbenzoyladenosine. The reagent was demonstrated to be a competitive inhibitor of MgATP with a Ki of 0.235 mM. Metal-free nucleotides were without effect upon the reaction rate while metal ions alone accelerated the inactivation rate up to 7-fold. The inclusion of casein or synthetic peptide substrate in the incubation mixture did not affect the reaction kinetics. Reaction of 5'-p-fluorosulfonylbenzoyladenosine with the kinase subunit exhibits all of the characteristics of affinity labeling of the MgATP-binding site.     

Affinity labelling of rat-muscle hexokinase type II by a glucose-derived alkylating agent.

The glucose-derived alkylating agent N-bromoacetylglucosamine (GlcNBrAc) is shown to cause a time-dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme-inhibitor complex prior to modification, indicating that the reagent is active-site-directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N-propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme-glucose complex from these studies is in agreement with the kinetically-determined Km of 0.2 mM. The substrate-unrelated but chemically similar alkylating agents bromoacetic acid and N-bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7--9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo-first-order rate constant) of 1.5 x 10(-3) S-1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active-site-directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I.     

KINETICS OF THROMBIN INACTIVATION AS INFLUENCED BY PHYSICAL CONDITIONS,TRYPSIN, AND SERUM

1. A new technique for studying the progressive inactivation of thrombin is described. 2. Thrombin inactivation follows the kinetics of a first order reaction. 3. The rate constant of the inactivation reaction increases with temperature and pH (5.0 → 10.0), and also with the presence of crystalline trypsin, or serum. The rate varies for different thrombin preparations, even under the same experimental conditions. 4. The temperature characteristics of the reaction indicate that thrombin is associated with protein. 5. Thrombin preparations are most stable at pH 4 to 5, even when trypsin or serum is added. 6. The progressive inactivation is believed to be due to two mechanisms: (1) a major effect, thought to be the action of a "serum-tryptase," which is usually present in the thrombin preparations, and (2) a minor effect, probably attributable to denaturation of thrombin-protein. 7. Sources of the thrombinolytic factor (serum-tryptase) and its implications in the general theory and practical problems of blood coagulation and antithrombic action are briefly discussed.     

Chemical modification of chalcone isomerase by diethyl pyrocarbonate: histidine residues are not essential for catalysis

Chalcone isomerase form soybean is inactivated by treatment with diethyl pyrocarbonate (DEP). The competitive inhibitor 4',4-dihydroxychalcone provides kinetic protection against inactivation by DEP with a binding constant at the site of protection in agreement with its binding constant at the active site. Very high concentrations of the competitive inhibitors 4',4-dihydroxychalcone or morin hydrate offer a 10- to 40-fold maximal protection, suggesting a second slower mechanism for inactivation which cannot be prevented by blockage of the active site. Blockage of the only cysteine residue in chalcone isomerase with p-mercuribenzoate does not affect the rate constant for DEP-dependent inactivation and indicates that the modification of the cysteine residue is not responsible for the activity loss observed in the presence of DEP. Treatment of inactivated enzyme with hydroxylamine does not restore catalytic activity, indicating that the modification of histidine or tyrosine residues is not responsible for the activity loss. All five histidines of chalcone isomerase are modified by DEP at pH 5.7 and ionic strength 1.0 M. The rate constant for the modification of the histidine residues of chalcone isomerase is close to that for the reaction of N-acetyl histidine with DEP, indicating that the histidine residues are quite accessible to the modifying reagent. The rate of histidine modification is the same in native enzyme, in urea-denatured enzyme, and in the presence of a competitive inhibitor. In the presence of the competitive inhibitor morin hydrate, all of the histidine residues of chalcone isomerase can be modified without significant loss in catalytic activity. These results demonstrate that the histidine residues of chalcone isomerase are not essential for catalysis and therefore cannot function as nucleophilic catalysts as previously proposed.     

Chemical modification of rabbit skeletal muscle phosphorylase kinase with phenylglyoxal

Nonactivated phosphorylase kinase from rabbit skeletal muscle is inactivated by treatment with phenylglyoxal. Under mild reaction conditions, a derivative that retains 10-15% of the pH 8.2 catalytic activity is obtained. The kinetics of inactivation profile, differential effects of modification on pH 6.8 and 8.2 catalytic activities, and the insensitiveness of the modified enzyme to activation by ADP reveal that the 10-15% of catalytic activity remaining is very likely due to intrinsic catalytic activity of the derivative rather than to the presence of unmodified enzyme molecules. The kinetic results also suggest that the inactivation is correlatable with the reaction of one molecule of the reagent with the enzyme without any prior binding of phenylglyoxal. The phenylglyoxal modification reduces the autophosphorylation rate of the kinase. Autophosphorylated phosphorylase kinase is inactivated by phenylglyoxal at a much slower rate than the inactivation of nonactivated kinase. Thus, phenylglyoxal modification influences the phosphorylation and vice versa. The modified enzyme can be reactivated by treatment with trypsin or by dissociation using chatropic salts. The activity of the phenylglyoxal-modified enzyme after trypsin digestion or dissociation with LiBr reaches the same level as that of the native enzyme digested with trypsin or treated with LiBr under identical conditions. The results suggest that the effect of modification is overcome by dissociation of the subunits of phosphorylase kinase and that the catalytic site is not modified under conditions when 85% of the pH 8.2 catalytic activity is lost. Among various nucleotides and metal ions tested, only ADP, with or without Mg2+, afforded effective protection against inactivation with phenylglyoxal. At pH 6.8, 1 mM ADP afforded complete protection against inactivation. Experiments with 14C-labeled phenylglyoxal revealed that ADP seemingly protects one residue from modification. This result is in agreement with the kinetic result that the inactivation seemingly is due to reaction of one molecule of the reagent with the enzyme. The results confirm the existence of a high-affinity ADP binding site on nonactivated phosphorylase kinase and suggest the involvement of a functional arginyl residue at or near the ADP binding site in the regulation of of pH 8.2 catalytic activity of the enzyme.     

Infectivity of RNA from inactivated poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Purification and cDNA cloning of a novel protease inhibitor secreted into culture supernatant by MDCK cells.

The infectivity of influenza viruses to host cells depends on the activation of the viral glycoprotein hemagglutinin (HA) by proteases. Starting from the observation that influenza virus replication in MDCK (Madin Darby canine kidney) cells was impaired by inactivation of trypsin in the culture fluids, we demonstrated that the inhibitory activity was resolved into two Trypsin-inactivating factors (TF), TF A (15 kDa) and TF B (11 kDa). N-terminal protein sequences of the factors revealed that TF A was a known Submandibular Protease Inhibitor (SPI) secreted in dog saliva, while TF B was a novel protein (renamed CKPI; canine kidney protease inhibitor). Following peptide mapping and protein sequencing of CKPI we obtained a 390 bp cDNA encoding a 130-amino-acid protein from MDCK cell total RNA. Protein sequence comparison showed a 63.8% identity with human secretory leukocyte protease inhibitor (SLPI), the molecule containing two conserved whey acidic protein (WAP) motifs, and we suggest that CKPI is thought to be the canine analogue of human SLPI. These results suggest that the inhibitory factors are secreted from MDCK cells, which are involved in prevention of virus replication, and applicable to the protection of host cells from virus infection.     

INACTIVATION OF CRYSTALLINE TRYPSIN

1. The rate of inactivation of crystalline trypsin solutions and the nature of the products formed during the inactivation at various pH at temperatures below 37°C. have been studied. 2. The inactivation may be reversible or irreversible. Reversible inactivation is accompanied by the formation of reversibly denatured protein. This denatured protein exists in equilibrium with the native active protein and the equilibrium is shifted towards the denatured form by raising the temperature or by increasing the alkalinity. The decrease in the fraction of active enzyme present (due to the formation of this reversibly denatured protein) as the pH is increased from 8.0 to 12.0 accounts for the decrease in the rate of digestion of proteins by trypsin in this range of pH. 3. The loss of activity at high temperatures or in alkaline solutions, just described, is very rapid and is completely reversible for a short time only. If the solutions are allowed to stand the loss in activity becomes gradually irreversible and is accompanied by the appearance of various reaction products the nature of which depends upon the temperature and pH of the solution. 4. On the acid side of pH 2.0 the trypsin protein is changed to an inactive form which is irreversibly denatured by heat. The course of the reaction in this range is monomolecular and its velocity increases as the acidity increases. 5. From pH 2.0 to 9.0 trypsin protein is slowly hydrolyzed. The course of the inactivation in this range of pH is bimolecular and its velocity increases as the alkalinity increases to pH 10.0 and then decreases. As a result of these two reactions there is a point of maximum stability at about pH 2.3. 6. On the alkaline side of pH 13.0 the reaction is similar to that in strong acid solution and consists in the formation of inactive protein. The course of the reaction is monomolecular and the velocity increases with increasing alkalinity. From pH 9.0 to 12.0 some hydrolysis takes place and some inactive protein is formed and the course of the reaction is represented by the sum of a bi- and monomolecular reaction. The rate of hydrolysis decreases as the solution becomes more alkaline than pH 10.0 while the rate of formation of inactive protein increases so that there is a second point at about pH 13.0 at which the rate of inactivation is a minimum. In general the decrease in activity under all these conditions is proportional to the decrease in the concentration of the trypsin protein. Equations have been derived which agree quantitatively with the various inactivation experiments.     

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

Fetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by HTS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures.     

Infectivity of RNA from Inactivated Poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Rubella virus capsid protein modulates viral genome replication and virus infectivity

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.     

Thermal inactivation of oral polio vaccine: contribution of RNA and protein inactivation.

Heating the Sabin strains of poliovirus at 42 to 45 degrees C caused inactivation, loss of native antigen, and release of the viral RNA (vRNA). The loss of virion infectivity exceeded the loss of vRNA infectivity (as measured by transfection) by roughly 2 log10. Pirodavir inhibited the loss of native antigen and RNA release and reduced the loss of virion infectivity to the same level as the loss of vRNA infectivity. Thermoinactivation thus involves an RNA and a protein component, and pirodavir protected only against the latter.     

2-Bromoacetylaminopentitol 1,5-bisphosphate as an affinity label for ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

2-Bromoacetylaminopentitol 1,5-bisphosphate (BrAcNH-pentitol-P2) (an epimeric mixture of 2-bromoacetylamino-2-deoxy-D-ribitol bisphosphate and 2-bromoacetylamino-2-deoxy-D-arabinitol 1,5-bisphosphate) has been synthesized from D-ribulose 1,5-bisphosphate by reductive amination with sodium cyanoborohydride followed by bromoacetylation of the resultant amine with bromoacetyl bromide. Under conditions that favor full activation of the enzyme, ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum is completely inactivated by BrAcNH-pentitol-P2 in a pseudo-first order process. A rate saturation is observed with a minimal inactivation half-life of 38 min and Kinact for reagent of 0.38 mM. The competitive inhibitor 2-carboxyribitol 1,5-bisphosphate reduces the rate of inactivation, and kinetic analyses are consistent with the protection reflecting true competition of inhibitor and reagent for the same site. As shown with isotopically labeled reagent, complete inactivation is associated with covalent incorporation of 1.1 mol of reagent/mol of subunit. Based on reversibility of inactivation by thiolysis and based on analysis of labeled products in acid hydrolysates of the modified enzyme, a methionyl sulfonium salt is the reaction product. In the absence of CO2 and Mg2+ (ligands required for activation), the enzyme is resistant to BrAcNH-pentitol-P2, which suggests that the site-specific modification of a methionyl residue requires a fully functional catalytic center.