Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.     

Product inhibition of the hepatitis C virus NS3 protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.     

Studies on the C-terminal of hexapeptide inhibitors of the hepatitis C virus serine protease

Replacement of the C-terminal carboxylic acid functionality of peptide inhibitors of hepatitis C virus (HCV) NS3 protease (complexed with NS4A peptide cofactor) by activated carbonyl groups does not produce any substantial increase in potency. These latter inhibitors also inhibit a variety of other serine and cysteine proteases whereas the carboxylic acids are specific. Norvaline was identified as a chemically stable replacement for the P1 residue of Ac-DDIVPC-OH which was also compatible with activated carbonyl functionalities.     

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.     

Novel anti-respiratory syncytial(RS) viral compounds: benzodithiin derivatives

Benzodithiin derivatives are highly potent and specific inhibitors of respiratory syncytial virus (RSV) replication in vitro. The most potent and selective congener of a benzodithiin derivative is 1,4-dihydro-2,3-benzodithiin(RD3-0028). According to the modified 3-(4,5-dimethylthiazole-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay developed in our laboratories, this compound has a 50% effective concentration of 4.5 microM and a 50% cytotoxic concentration of 271.0 microM, which is superior to that of ribavirin. This compound also inhibits RSV strain subgroups A and B and clinical isolates. RD3-0028, however, does not inhibit the replication of influenza A virus, measles virus, herpes simplex virus types 1 and 2, or human cytomegalovirus. Two other benzodithiin derivatives [1,4-dihydro-6-methyl-2,3-benzodithiin (RD3-0270) and 1,4-dihydro-5-methyl-1-2,3-benzodithiin (RD3-0284)] also inhibit RSV replication at a selectivity index greater a factor of 20. These results suggest that the benzodithiin skeleton is an important structure for inhibitory activity against RSV replication.     

Characterization of visually similar diffuse diseases from B-scan liver images using nonseparable wavelet transform

BACKGROUND/AIMS: The serine proteinase inhibitors (serpins), alpha-1-antitrypsin (AAT) and alpha-1-antichymotrypsin (ACT), are acute phase proteins synthesized by hepatocytes and excreted to some extent into bile. Their role in gallstone pathogenesis is unclear, and it was the aim of this study to determine their effect on cholesterol crystal growth rate in model bile. METHODS: Purified AAT and ACT were added to model bile at concentrations from 0.5 to 500 microg/ml. Cholesterol crystal growth was analyzed daily by polarizing microscopy and spectrophotometrically at 650 nm. Serpin inhibitory activity was measured spectrophotometrically at 405 nm, and polymerization was studied on 7.5% SDS-PAGE under non-reducing conditions, by immunoelectrophoresis and Western blotting. RESULTS: ACT added to model bile at a concentration of 0.5 microg/ml, inhibited cholesterol crystallization by 30%, had no influence at 5 microg/ml, and increased the crystallization rate 2-3 fold at concentrations of 50 and 500 microg/ml. AAT at a concentration of 0.5 microg/ml had a profound (50%) inhibitory effect on cholesterol crystal growth rate, lacked significant effect at both concentrations of 5 and 50 microg/ml, and showed stimulation of crystal growth up to 30% at a concentration of 500 microg/ml. Both serpins incubated in model bile polymerized and totally lost their inhibitory activity. CONCLUSIONS: Serpins can exhibit both inhibiting and promoting effects on the cholesterol crystallization rate in model bile, dependent on their concentrations. Since AAT and ACT are acute phase proteins, their concentrations may vary under certain pathological conditions, which may result in different actions of these serpins in gallstone formation.     

Effects of prior exercise on exercise-induced arterial hypoxemia in young women

Insulin-like growth factor binding proteins (IGFBP) proteases have been proposed to be involved in changes of serum IGFBP pattern during pregnancy. IGFBP-4 and -5 are degraded specifically by proteases in pregnancy serum in vitro, whereas IGFBP-3 proteolytic activity was also detected in nonpregnancy serum. To identify and characterize IGFBP proteases, human pregnancy serum was fractionated by size exclusion chromatography revealing IGFBP-4 protease activities in fractions coeluting with proteins of approximately 600-kDa and 50- to 100-kDa molecular mass. In both fractions, a predominant 50-kDa gelatinase was found, suggesting that parts of the gelatinase activity might aggregate or are complexed with other proteins forming a higher molecular complex. Hydroxyapatite chromatography and chromatofocusing of the 50- to 100-kDa serum fraction showed that the IGFBP-4 protease and the 50-kDa gelatinase activity were copurified. When the 50-kDa gelatinase-containing band was excised from the polyacrylamide gel, it exhibited IGFBP-4 proteolytic activity, resulting in the formation of 17- and 10-kDa fragments. [125I] IGFBP substrate zymography combined with fragment blotting showed that the 1,10-phenanthroline-sensitive 50-kDa protease activity purified by chromatofocusing also cleaved IGFBP-3 and -5. Other proteases detected in pregnancy serum fractions with Mr estimates of 79-, 30-, and 22-kDa degraded IGFBP-3 and -5 but not IGFBP-4. [125I] IGFBP-5 substrate zymography revealed that the 30-kDa IGFBP protease was inhibited by serine protease inhibitors. Whereas 1,10-phenanthroline inhibited the IGFBP proteolytic activity in the solution assay, serine protease inhibitors failed to affect proteolysis, indicating the predominant contribution of the metalloproteinase to IGFBP proteolysis. Tissue inhibitors of matrix metalloproteinases-1 and -2 revealed weak or no inhibition of IGFBP-4 and -5 proteolytic activity, whereas a hydroxamic acid-based inhibitor, potentially inhibiting disintegrin metalloproteases, completely prevented the proteolysis of IGFBPs. Whereas no specific immunoreactivity of the 50-kDa protein with antimatrix metalloproteinase-1, -2, -3, -9, or -13 antibodies was observed, antidisintegrin domain-specific antibodies bound to the 50-kDa gelatinase. These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.     

Oxyanion-mediated inhibition of serine proteases

Novel aryl derivatives of benzamidine were synthesized and tested for their inhibitory potency against bovine trypsin, rat skin tryptase, human recombinant granzyme A, human thrombin, and human plasma kallikrein. All compounds show competitive inhibition against these proteases with Ki values in the micromolar range. X-ray structures were determined to 1.8 A resolution for trypsin complexed with two of the para-substituted benzamidine derivatives, 1-(4-amidinophenyl)-3-(4-chlorophenyl)urea (ACPU) and 1-(4-amidinophenyl)-3-(4-phenoxyphenyl)urea (APPU). Although the inhibitors do not engage in direct and specific interactions outside the S1 pocket, they do form intimate indirect contacts with the active site of trypsin. The inhibitors are linked to the enzyme by a sulfate ion that forms an intricate network of three-centered hydrogen bonds. Comparison of these structures with other serine protease structures with noncovalently bound oxyanions reveals a pair of highly conserved oxyanion-binding sites in the active site. The positions of noncovalently bound oxyanions, such as the oxygen atoms of sulfate, are distinct from the positions of covalent oxyanions of tetrahedral intermediates. Noncovalent oxyanion positions are outside the "oxyanion hole." Kinetics data suggest that protonation stabilizes the ternary inhibitor/oxyanion/protease complex. In sum, both cations and anions can mediate Ki. Cation mediation of potency of competitive inhibitors of serine proteases was previously reported by Stroud and co-workers [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612].     

Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease

The HCV genome encodes, within the NS3 gene, a serine protease whose activity specifically cleaves the viral polyprotein precursor. Proteolytic processing of HCV polyprotein precursor by the viral NS3 proteinase is essential for virion maturation and designing specific inhibitors of this protease as possible anti-viral agents is a desirable and practical objective. With a view to studying both the function of HCV NS3 protease and to designing inhibitors of this enzyme, we directed our interest towards engineering macromolecular inhibitors of the viral protease catalytic activity. We describe here the affinity-selection and biochemical characterization of one inhibitor, cV(H)E2, a 'camelized' variable domain antibody fragment, isolated from a phage displayed synthetic repertoire, which is a potent and selective inhibitor of proteolysis by the NS3 enzyme. In addition to being useful as a biological probe to study the function of HCV protease, this inhibitor can serve as a potential pharmacophore model to design antivirals. Moreover, the results suggest a way of engineering improved human-derived small recognition units tailored for enzyme inhibition.     

Hepatitis G virus encodes protease activities which can effect processing of the virus putative nonstructural proteins

The genome of a recently identified virus, hepatitis G virus (HGV), shows considerable homology to hepatitis C virus (HCV). Two HGV proteases similar to nonstructural proteins NS2 and NS3 of HCV were identified, and their cleavage site specificity was investigated. Amino acids essential for the protease activities were determined by mutation analysis. NS4A of HGV was demonstrated to be a cofactor for NS3-mediated proteolysis, with a region critical for activity residing between Leu1561, and Ala1598.     

Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus

The hepatitis C virus (HCV) nonstructural 3 protein (NS3) contains at least two domains associated with multiple enzymatic activities; a serine protease activity resides in the N-terminal one-third of the protein, whereas RNA helicase activity and RNA-stimulated nucleoside triphosphatase activity are associated with the C-terminal portion. To study the possible mutual influence of these enzymatic activities, a full-length NS3 polypeptide of 67 kDa was expressed as a nonfusion protein in Escherichia coli, purified to homogeneity, and shown to retain all three enzymatic activities. The protease activity of the full-length NS3 was strongly dependent on the activation by a synthetic peptide spanning the central hydrophobic core of the NS4A cofactor. Once complexed with the NS4A-derived peptide, the full-length NS3 protein and the isolated N-terminal protease domain cleaved synthetic peptide substrates with comparable efficiency. We show that, as in the case of the isolated protease domain, the protease activity of full-length NS3 undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B and NS5A-NS5B. We have also characterized and quantified the NS3 ATPase, RNA helicase, and RNA-binding activities under optimized reaction conditions. Compared with the isolated N-terminal and C-terminal domains, recombinant full-length NS3 did not show significant differences in the three enzymatic activities analyzed in independent in vitro assays. We have further explored the possible interdependence of the NS3 N-terminal and C-terminal domains by analyzing the effect of polynucleotides on the modulation of all NS3 enzymatic functions. Our results demonstrated that the observed inhibition of the NS3 proteolytic activity by single-stranded RNA is mediated by direct interaction with the protease domain rather than with the helicase RNA-binding domain.     

Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium

Endogenous proteolytic enzymes have been shown to be potential sources of airway inflammation inducing proinflammatory cytokine release from respiratory epithelial cells; however, whether any of the exogenous proteases from important allergen sources such as the house dust mite present in our environment behave in a similar fashion is unclear. To this end, we have investigated whether the mite cysteine and serine proteolytic allergens, Der p 1 and Der p 9, respectively, induced cytokine production from primary human bronchial epithelial cells and from the epithelial cell line BEAS-2B. Cells were exposed to mite proteases, and cells or supernatants were assayed for cytokine release, cytokine mRNA expression, and modulation of intracellular calcium ion concentration. Both proteases induced concentration- and time-dependent increases in the release of granulocyte-macrophage (GM)-CSF, IL-6, and IL-8 as well as an increase in the expression of IL-6 mRNA. Cytokine release and mRNA expression were first observed at 8 h and 2 h after protease exposure, respectively. The minimum concentration of each protease that was required to stimulate GM-CSF, IL-6, and IL-8 release was approximately 10 ng/ml. Cytokine release was initiated by 1 to 2 h of protease exposure, although maximum concentrations were detected only after a 24-h incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by both proteases at concentrations of > 2 microg/ml. The proteases also stimulated changes in the intracellular calcium ion concentration. All mite protease-induced phenomena were inhibited using appropriate protease inhibitors. These results suggest that the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium.     

Age-related changes in the inner zone of the adrenal cortex of the rat--a morphologic and biochemical study

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.     

In vitro study of the NS2-3 protease of hepatitis C virus

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.     

Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases. The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein. This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents. Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain. This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex. Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor. The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants. These are important steps towards developing effective anti-protease compounds.     

Pneumocystis carinii infections in patients with neoplasms and AIDS--clinical-pathologic changes in the lungs

Administration of 0.5 or 1% lyophilized green tea (5 or 10 mg tea solids per ml, respectively) as the sole source of drinking fluid to female Long-Evans rats for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 30-37%, 15-27% and 26-60%, respectively. Oral administration of 0.5% lyophilized green tea to female CD-1 mice for 18 days stimulated liver microsomal glucuronidation of estrone, estradiol and 4-nitrophenol by 33-37%, 12-22% and 172-191%, respectively. The in vitro addition of a green tea polyphenol mixture, a black tea polyphenol mixture or (-)-epigallocatechin gallate inhibited rat liver microsomal glucuronidation of estrone and estradiol in a concentration-dependent manner and their IC50 values for inhibition of estrogen metabolism were approximately 12.5, 50 and 10 microg/ml, respectively. Enzyme kinetic analysis indicates that the inhibition of estrone glucuronidation by 10 microM (-)-epigallocatechin gallate was competitive while inhibition by 50 microM (-)-epigallocatechin gallate was noncompetitive. Similarly, several flavonoids (naringenin, hesperetin, kaempferol, quercetin, rutin, flavone, alpha-naphthoflavone and beta-naphthoflavone) also inhibited rat liver microsomal glucuronidation of estrone and estradiol to varying degrees. Naringenin and hesperetin displayed the strongest inhibitory effects (IC50 value of approximately 25 microM). These two hydroxylated flavonoids had a competitive mechanism of enzyme inhibition for estrone glucuronidation at a 10 microM inhibitor concentration and a predominantly noncompetitive mechanism of inhibition at a 50 microM inhibitor concentration.     

Novel thieno[2,3-d][1,3]oxazin-4-ones as inhibitors of human leukocyte elastase

A series of thieno[2,3-d][1,3]oxazin-4-ones was synthesized and evaluated in vitro for inhibitory activity toward human leukocyte elastase. New synthetic routes to 2-alkoxy-, 2-alkylthio-, and 2-sec-amino-substituted derivatives are reported. This study demonstrates the versatility of 2-aminothiophenes prepared by Gewald reaction as a synthetic entry to serine protease-inhibiting, fused 1,3-oxazin-4-ones. Introduction of ethoxy, n-propoxy, and ethylthio groups at C-2 delivered the most potent inhibitors of this series with Ki values lower than 11 nM. Kinetic studies and product analyses revealed the formation of acyl-enzymes as a result of the attack of the active site serine at the carbon C-4 and subsequent deacylation. This mode of action is similar to the inhibition of serine proteases by 4H-3,1-benzoxazin-4-ones. Replacement of the benzene ring in benzoxazinones by a (substituted) thiophene led to improved hydrolytic stability and retained inhibitory potency.     

Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.     

Insulin-like growth factor-binding protein-5 is cleaved by physiological concentrations of thrombin

Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is cleaved by a serine protease that is secreted by fibroblasts and porcine smooth muscle cells (pSMC) in culture. To investigate whether other serine proteases could cleave this substrate at physiologically relevant concentrations, we determined the proteolytic effects of thrombin on IGFBP-5. Human alpha-thrombin (0.0008 NIH U/ml) cleaved IGFBP-5 into 24-, 23-, and 20-kDa non-IGF-I-binding fragments. Cleavage occurred at a physiologically relevant thrombin concentration. The effect was specific for IGFBP-5, as other forms of IGFBPs, e.g. IGFBP-1, IGFBP-2, and IGFBP-4 were not cleaved by thrombin. Although IGFBP-3 was cleaved by thrombin, this effect required a 50-fold greater thrombin concentration. [35S]Methionine labeling followed by immunoprecipitation confirmed that IGFBP-5 that was constitutively synthesized by pSMC cultures was also degraded by thrombin into 24-, 23-, and 20-kDa fragments. The binding of IGF-I to IGFBP-5 partially inhibited IGFBP-5 degradation by thrombin, and an IGF analog that does not bind to IGFBP-5 had no effect. Thrombin did not account for the serine protease activity that had been shown previously to be present in pSMC-conditioned medium. This was proven by showing that 1) no immunoreactive thrombin could be detected in the pSMC-conditioned medium; 2) the IGFBP-5 fragments that were generated by thrombin showed three cleavage sites (Arg192-Ala193, Arg156-Ile157, and Lys120-His121), whereas the serine protease in conditioned medium cleaves IGFBP-5 at a different site; and 3) hirudin had no effect on IGFBP-5 cleavage by the protease in pSMC medium; however, it inhibited IGFBP-5 degradation by thrombin. To determine the physiological significance of IGFBP-5 cleavage, the effect of an IGFBP-5 mutant that is resistant to cleavage by the pSMC protease and has been shown to inhibit IGF-I actions in pSMC was determined. This mutant inhibited IGF-I-stimulated DNA synthesis, but if thrombin was added simultaneously, IGF-I was fully active. In summary, physiological concentrations of thrombin degrade IGFBP-5. Degradation can be blocked by hirudin and is partially inhibited by IGF-I binding. Generation of active thrombin in vessel walls may be a physiologically relevant mechanism for controlling IGF-I bioactivity.     

Mutagenesis of the NS3 protease of dengue virus type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.