Characterization of Recombinant Integrase of Human Immunodeficiency Virus Type 1 (Isolate Bru)

Integration of the human immunodeficiency virus type 1 (HIV-1) DNA into the human genome requires the virusencoded integrase protein. The recombinant integrase protein of HIV-1 (isolate Bru) was prepared by constructing a plasmid based on pET-15b encoding the integrase gene. Integrase of HIV-1 was purified using a bacterial expression system (Escherichia coli). The main kinetic parameters of HIV-1 integrase (K _m = (3.7 ± 0.2)·10^–10 M, k _cat = (1.2 ± 0.3)·10^–7 sec^–1) were determined using an oligonucleotide duplex constructed on the basis of the U5-terminal sequence of proviral HIV-1 DNA as the substrate. Inhibition of integrase by aurintricarbonic acid ([I]₅₀ = 6.3 ± 0.4 M) and dependence of integrase activity on Mg²⁺ and Mn²⁺ concentration were studied.     

Irreversible inhibition of human immunodeficiency virus type 1 integrase by dicaffeoylquinic acids

Human immunodeficiency virus type 1 (HIV-1) and other retroviruses require integration of a double-stranded DNA copy of the RNA genome into the host cell chromosome for productive infection. The viral enzyme, integrase, catalyzes the integration of retroviral DNA and represents an attractive target for developing antiretroviral agents. We identified several derivatives of dicaffeoylquinic acids (DCQAs) that inhibit HIV-1 replication in tissue culture and catalytic activities of HIV-1 integrase in vitro. The specific step at which DCQAs inhibit the integration in vitro and the mechanism of inhibition were examined in the present study. Titration experiments with different concentrations of HIV-1 integrase or DNA substrate found that the effect of DCQAs was exerted on the enzyme and not the DNA. In addition to HIV-1, DCQAs also inhibited the in vitro activities of MLV integrase and truncated variants of feline immunodeficiency virus integrase, suggesting that these compounds interacted with the central core domain of integrase. The inhibition on retroviral integrases was relatively specific, and DCQAs had no effect on several other DNA-modifying enzymes and phosphoryltransferases. Kinetic analysis and dialysis experiments showed that the inhibition of integrase by DCQAs was irreversible. The inhibition did not require the presence of a divalent cation and was unaffected by preassembling integrase onto viral DNA. The results suggest that the irreversible inhibition by DCQAs on integrase is directed toward conserved amino acid residues in the central core domain during catalysis.     

Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration

Integrase is the key enzyme that mediates integration of retroviral DNA into cellular DNA which is essential for viral replication. Inhibitors of HIV‐1 that target integrase recognize the nucleoprotein complexes formed by integrase and viral DNA substrate (intasomes) rather than the free enzyme. Atomic resolution structures of HIV‐1 intasomes are therefore required to understand the mechanisms of inhibition and drug resistance. To date, prototype foamy virus (PFV) is the only retrovirus for which such structures have been determined. We show that PFV strand transfer complexes (STC) can be assembled on product DNA without going through the normal forward reaction pathway. The finding that a retroviral STC can be assembled in this way may provide a powerful tool to alleviate the obstacles that impede structural studies of nucleoprotein intermediates in HIV‐1 DNA integration.     

Analysis of the Site-Specific Integration System of the <Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> Phage μ1/6

The bacteriophage μ1/6 integrates its DNA into the chromosome of tetracycline producing strains of Streptomyces aureofaciens by a site-specific recombination process. A bioinformatic analysis of the μ1/6 genome revealed that orf5 encodes a putative integrase, a basic protein of 416 amino acids. The μ1/6 integrase was found to belong to the integrase family of site-specific tyrosine recombinases. The phage attachment site (attP) was localized downstream of the int gene. The attachment junctions (attL and attR) were determined, allowing identification of the bacterial attachment site (attB). All attachment sites shared a 46-bp common core sequence within which a site-specific recombination occurs. This core sequence comprises the 3′ end of a putative tRNA^Thr gene (anticodon TGT) which is completely restored in attL after integration of the phage into the host genome. An integration vector containing μ1/6 int-attP region was inserted stably into the S. aureofaciens B96, S. lividans TK24, and S. coelicolor A3. The μ1/6 integrase was shown to be functional in vivo in heterologous Escherichia coli without any other factors encoded by Streptomyces. In vitro recombination assay using purified μ1/6 integrase demonstrated its ability to catalyze integrative recombination in the presence of a crude extract of E. coli cells.     

Antioxidant activity of oxygen-containing aromatic compounds

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H₂O₂-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) or the inhibition degree (%). K _i values varied in the range of 4 to 500 μM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds of guaiacyl and syringal series exhibited high antioxidant activity (K _i in the range of 10–300 μM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycoumaric acid.     

Site-specific genome integration in alphaproteobacteria mediated by TG1 integrase

The serine-type phage integrase is an enzyme that catalyzes site-specific recombination between two attachment sites of phage and host bacterial genomes (attP and attB, respectively) having relatively short but distinct sequences without host auxiliary factor(s). Previously, we have established in vivo and in vitro site-specific recombination systems based on the serine-type integrase produced by actinophage TG1 and determined the minimal sizes of attP _TG1 and attB _TG1 sites required for the in vitro TG1 integrase reaction as 43- and 39-bp, respectively. Here, DNA databases were surveyed by FASTA program with the authentic attB _TG1 sequence of Streptomyces avermitilis as a query. As a result, possible attB _TG1 sequences were extracted from genomes of bacterial strains belonging to Class Alphaproteobacteria in addition to those of Class Actinobacteria. Those sequences extracted with a high similarity score and high sequence identity (we took arbitrarily more than 80% identity) turned out to be located within a conserved region of dapC or related genes encoding aminotransferases and proved to be actually recognized as the cognate substrate of attP _TG1 site by the in vitro TG1 integrase assay. Furthermore, the possible attB _TG1 site of Rhodospirillum rubrum revealed to be used actually as a native (endogenous) attachment site for the in vivo TG1-based integration system. These features are distinct from other serine-type phage integrases and advantageous for a tool of genome technology in varied industrially important bacteria belonging to Class Alphaproteobacteria.     

Kinetics of deactivation for thermostable DNA polymerase enzymes

Summary The kinetics of thermal deactivation for thermostable DNA polymerase enzymes were investigated by using the experimental data published elsewhere (Nielson et al. 1996. Strategies. 9, 7–8). The order of deactivation (a) and the deactivation rate constants (k _d) were determined for different Taq DNA polymerase enzymes and were found to be of first order.     

6-OXOCYTIDINE CONTAINING OLIGONUCLEOTIDES INHIBIT THE HIV-1 INTEGRASE IN VITRO

Integration of the proviral DNA into the genome of infected cells is a key step of HIV-1 replication. Integration is catalyzed by the viral enzyme integrase (IN). 6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors of integrase in vitro. The inhibitory effect is sequence-specific and strictly requires the presence of the 6-oxocytidine base. It is due to the impairment of the integrase binding to its substrate and does not involve an auto-structure of the oligonucleotide.     

Designed zinc finger protein interacting with the HIV‐1 integrase recognition sequence at 2‐LTR‐circle junctions

Integration of HIV‐1 cDNA into the host genome is a crucial step for viral propagation. Two nucleotides, cytosine and adenine (CA), conserved at the 3′ end of the viral cDNA genome, are cleaved by the viral integrase (IN) enzyme. As IN plays a crucial role in the early stages of the HIV‐1 life cycle, substrate blockage of IN is an attractive strategy for therapeutic interference. In this study, we used the 2‐LTR‐circle junctions of HIV‐1 DNA as a model to design zinc finger protein (ZFP) targeting at the end terminal portion of HIV‐1 LTR. A six‐contiguous ZFP, namely 2LTRZFP was designed using zinc finger tools. The designed motif was expressed and purified from E. coli to determine its binding properties. Surface plasmon resonance (SPR) was used to determine the binding affinity of 2LTRZFP to its target DNA. The level of dissociation constant (K_d) was 12.0 nM. The competitive SPR confirmed that 2LTRZFP specifically interacted with its target DNA. The qualitative binding activity was subsequently determined by EMSA and demonstrated the aforementioned correlation. In addition, molecular modeling and binding energy analyses were carried out to provide structural insight into the binding of 2LTRZFP to the specific and nonspecific DNA target. It is suggested that hydrogen‐bonding interactions play a key role in the DNA recognition mechanisms of the designed ZFP. Our study suggested an alternative HIV therapeutic strategy using ZFP interference of the HIV integration process.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Some kinetic properties of the β- -glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β- -glucosidase (cellobiase, β- -glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β- -glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. -Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. -Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while -fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β- -glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

DNA-induced polymerization of HIV-1 integrase analyzed with fluorescence fluctuation spectroscopy

Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 microm(-1) under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 +/- 0.6 x 10(8) m(-1). Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.     

In vitro characterization of the site-specific recombination system based on actinophage TG1 integrase

We have previously shown that, in vivo, the integration system based on the gene encoding the TG1 integrase and the corresponding attB _TG1 and attP _TG1 sites works well not only in Streptomyces strains, but also in Escherichia coli. Furthermore, the attachment sites for TG1 integrase are distinct from those of ϕC31 integrase. In this report, we expressed TG1 integrase as a GST-TG1 integrase fusion protein and then used affinity separation and specific cleavage to release purified integrase. Conditions for in vitro recombination were established using the purified TG1 integrase and its cognate attP _TG1 and attB _TG1 sites. TG1 integrase efficiently catalyzed a site-specific recombination between attB _TG1 and attP _TG1 sites irrespective of their substrate topology. The minimal sequences of attP _TG1 and attB _TG1 sites required for the substrates of TG1 integrase were demonstrated to be 43 and 39-bp, respectively. These results provide the basic features of the TG1 integrase system to be used as biotechnological tools, as well as to unravel the mechanism of the serine integrase.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

Carbonyl J Derivatives: A New Class of HIV-1 Integrase Inhibitors

Integration of a DNA copy of the HIV-1 genome is required for viral replication and pathogenicity, and this highly specific molecular process is mediated by the virus-encoded integrase protein. The requirement for integration, combined with the lack of a known analogous process in mammalian cells, makes integrase an attractive target for therapeutic inhibitors of HIV-1 replication. While many reports of HIV-1 IN inhibitors exist, no such compounds have yet emerged to treat HIV-1 infection. As such, new classes of integrase inhibitors are needed. We have combined molecular modeling and combinatorial chemistry to identify and develop a new class of HIV-1 integrase inhibitors, the Carbonyl J [N,N'-bis(2-(5-hydroxy-7-naphthalenesulfonic acid)urea] derivatives. This new class includes a number of compounds with sub-micromolar IC(50) values for inhibiting purified HIV-1 integrase in vitro. Herein we describe the chemical characteristics that are important for integrase inhibition and cell toxicity within the Carbonyl J derivatives. Copyright 2000 Academic Press.     

Substrate features important for recognition and catalysis by human immunodeficiency virus type 1 integrase identified by using novel DNA substrates.

The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.     

Peroxidase-catalyzed Oxidation of 3,3",5,5"-Tetramethylbenzidine in the Presence of 2,4-Dinitrosoresorcinol and Polydisulfide Derivatives of Resorcinol and 2,4-Dinitrosoresorcinol

A comparative study of the kinetics of peroxidase-catalyzed oxidation of 3,3",5,5"-tetramethylbenzidine (TMB) in the presence of 2,4-dinitrosoresorcinol (DNR), its polydisulfide derivative [poly(DNRDS)], and resorcinol polydisulfide [poly(RDS)], substances that competitively inhibit the formation of TMB conversion product, was carried out. The inhibition constants K _i for DNR, poly(DNRDS), and poly(RSD) were determined at 20°C and pH 6.4 to be 110, 13.5, and 0.78 M, respectively. The stoichiometric coefficients of inhibition were calculated to be 0.38 and 76 for poly(DNRDS) and poly(RDS), respectively. In the pH range 6.4–7.0, the initial rates of the peroxidative oxidation of TMB, and its mixtures with DNR and poly(DNRDS) and the K _i value for poly(RDS) substantially decreased with increasing pH. The kinetic parameters of poly(RDS) (K _i 0.22–0.78 M and f 76) suggest that it is the most efficient inhibitor of peroxidase oxidation of TMB: in micromolar concentrations, it completely stops this process and can be used in EIA.