Showing papers on "Integrase published in 2002"

Diketo acid inhibitor mechanism and HIV-1 integrase: implications for metal binding in the active site of phosphotransferase enzymes.

Abstract: The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3′ end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg2+. The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Site-specific genomic integration produces therapeutic Factor IX levels in mice

Abstract: We used the integrase from phage φC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was coinjected, hFIX serum levels increased more than tenfold to ∼4 µg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration. Lasting gene therapy may be facilitated by the permanent introduction of exogenous DNA into the chromosomes of patients. Here we describe the use of bacteriophage φC31 integrase to achieve site-specific genomic integration of therapeutic genes into the genomes of adult mice. In nature, the integrase mediates unidirectional recombination of the phage genome into the bacterial chromosome through a site-specific reaction between the phage attP site and the host attB site1,2 .W e have shown that this integrase is functional in mammalian cells3, and that it can efficiently integrate plasmids containing an attB site into pseudo-attP sites in mammalian genomes 4 .T hese pseudo sites have enough identity with wild-type attP to support integrase-mediated recombination, and persistent gene expression occurs from vectors integrated at such sites 4 .I n this study, we have applied these principles in vivo to integrate attB vectors bearing the gene for human blood clotting Factor IX (hFIX) into the chromosomes of mice at native pseudoattP sites, resulting in the generation of persistent normal levels of the protein. Results Integrase-mediated expression of hAAT in mouse liver. We constructed plasmids that contained a φC31 attB site and either a cDNA expression construct of the human α 1-antitrypsin gene (hAAT) 5

Integration sites for genetic elements in prokaryotic tRNA and tmRNA genes: sublocation preference of integrase subfamilies

Abstract: Most classical integrases of prokaryotic genetic elements specify integration into tRNA or tmRNA genes. Sequences shared between element and host integration sites suggest that crossover can occur at any of three sublocations within a tRNA gene, two with flanking symmetry (anticodon-loop and T-loop tDNA) and the third at the asymmetric 3′ end of the gene. Integrase phylogeny matches this classification: integrase subfamilies use exclusively either the symmetric sublocations or the asymmetric sublocation, although tRNA genes of several different aminoacylation identities may be used within any subfamily. These two familial sublocation preferences imply two modes by which new integration site usage evolves. The tmRNA gene has been adopted as an integration site in both modes, and its distinctive structure imposes some constraints on proposed evolutionary mechanisms.

IntI2 Integron Integrase in Tn7

Abstract: Integrons are genetic elements that permit tandem integration and expression of mobile cassettes coding for antibiotic resistance genes (18, 31, 37). The gene cassettes move by a conservative site-specific recombination mechanism, catalyzed by an integrase encoded by the integron 5′-conserved segment (5′-CS) (29, 37). The integrase belongs to the phage integrase family of recombinases (3), now called tyrosine recombinases (13). Integration of cassettes generally occurs into a target site attI, at the junction between the 5′-CS and the first cassette (Fig. ​(Fig.1)1) (11, 32). A second type of cassette recombination sites are the attC sites (59-base elements) which occur either at the 3′ end of each inserted cassette (17, 18, 31, 38) or in a free cassette circle (10). The initially described type of integron (referred to as class 1) is generally borne on elements similar to Tn5090 (30) or on related defective transposons (8), which sometimes “piggyback” on competent transposons to form Tn21 and its relatives. In addition, a second class of integron occurs on the nonreplicative transposon Tn7. Tn7 preferentially inserts into a unique site in bacterial chromosomes (16, 21, 23) from which it is transferred to other bacterial cells by less-specific transposition onto a conjugative plasmid (46). The question of whether or not Tn7 carries a functional integron is interesting because it would add to the multiplicity of systems for gene exchange among microorganisms. FIG. 1. Maps of the integron regions in Tn7 and in the related transposons Tn1825 and Tn1826. Integron sequences are represented by boxes. Black boxes indicate the conserved sequence (5′-CS-2) with the integrase gene, intI2. Cassettes are shown by white ... The integron of Tn7 (Fig. ​(Fig.1)1) has an organization similar to that of the class I integrons and carries three resistance gene cassettes—dfrA1, sat, and aadA1 (38, 39, 40)—close to an open reading frame, IntI2*, that is related to the class 1 integrase, IntI1, of Tn21 (Fig. ​(Fig.1)1) (19, 29). A variation of the cassette content in Tn7 was previously observed among related elements such as Tn1825 and Tn1826 (42) and Tn4132 (49). Since two cassettes in Tn7, dfrA1 and aadA1, have also been observed in class 1 integrons (14, 40), some cassettes appear to have been transferred among integron classes. Part of the integrase gene on Tn7 was included in a sequence by Simonsen et al. (36). The nucleotide sequence included an internal stop codon (TAA) in the intI2 gene. We reverified the presence of the internal stop codon which we also found to be present in the intI2 genes of Tn1825 and Tn1826 (Fig. ​(Fig.1).1). The sequence for intI2 in Tn7 was completed here by sequencing the previously unknown 3′ end of the gene, confirming that intI2 is closely related to intI1 of Tn21 and also resembles an integrase gene from a third integron class (intI3) (1). We show here experimentally that the second class of integrase in Tn7 can promote recombination between cassette recombination sites present in the same plasmid (excision) or in different plasmids (cointegration), but only after changing the termination codon to a sense codon. Recombination activity was assayed in vivo by expressing the Tn7 integrase from a repaired form of intI2, intI2*179E, in which the internal stop codon has been altered into a triplet (GAG) encoding glutamic acid. Analysis of cassette recombination involving the class-specific attI sites revealed that the Tn7 integrase seems to be more restricted to recognition of its own attI site than the Tn21 integrase. The three previously defined cassettes borne on Tn7 mediate resistance to trimethoprim (dfrA1 [15, 40]), streptothricin (sat [39, 42]) and spectinomycin (aadA1 [14]). We checked for further excisable units and found a fourth cassette downstream of those described previously. This new cassette lacked the palindromic attC site that is generally found at the 3′ end of cassettes.

Structure activity of 3-aryl-1,3-diketo-containing compounds as HIV-1 integrase inhibitors.

Abstract: The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl β-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3‘-processing (3‘-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the “left” and “right” sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3‘-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent ...

Inhibition of HIV-1 integrase by galloyl glucoses from Terminalia chebula and flavonol glycoside gallates from Euphorbia pekinensis.

Abstract: The bioassay-directed isolation of Terminalia chebula fruits afforded four human immunodeficiency virus type 1 (HIV-1) integrase inhibitors, gallic acid ( 1) and three galloyl glucoses ( 2 - 4). In addition, four flavonol glycoside gallates ( 5 - 8) from Euphorbia pekinensis containing the galloyl moiety also showed the inhibitory activity at a level comparable to those of 2 - 4. By comparison with the activities of the compounds not bearing this moiety, it is proposed that the galloyl moiety plays a major role for inhibition against the 3'-processing of HIV-1 integrase of these compounds.

Reassessment of the Roles of Integrase and the Central DNA Flap in Human Immunodeficiency Virus Type 1 Nuclear Import

Abstract: Human immunodeficiency virus type 1 (HIV-1) can infect nondividing cells productively because the nuclear import of viral nucleic acids occurs in the absence of cell division. A number of viral factors that are present in HIV-1 preintegration complexes (PICs) have been assigned functions in nuclear import, including an essential valine at position 165 in integrase (IN-V165) and the central polypurine tract (cPPT). In this article, we report a comparison of the replication and infection characteristics of viruses with disruptions in the cPPT and IN-V165. We found that viruses with cPPT mutations still replicated productively in both dividing and nondividing cells, while viruses with a mutation at IN-V165 did not. Direct observation of the subcellular localization of HIV-1 cDNAs by fluorescence in situ hybridization revealed that cDNAs synthesized by both mutant viruses were readily detected in the nucleus. Thus, neither the cPPT nor the valine residue at position 165 of integrase is essential for the nuclear import of HIV-1 PICs.

Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells.

Abstract: The integrase of the phage ΦC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of ΦC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved ΦC31-integrase as a new tool for genome engineering.

Identification and characterization of phage-resistance genes in temperate lactococcal bacteriophages.

Abstract: The sie2009 gene, which is situated between the genes encoding the repressor and integrase, on the lysogeny module of the temperate lactococcal bacteriophage Tuc2009, was shown to mediate a phage-resistance phenotype in Lactococcus lactis against a number of bacteriophages. The Sie2009 protein is associated with the cell membrane and its expression leaves phage adsorption, transfection and plasmid transformation unaffected, but interferes with plasmid transduction, as well as phage replication. These observations indicate that this resistance is as a result of DNA injection blocking, thus representing a novel superinfection exclusion system. A polymerase chain reaction (PCR)-based strategy was used to screen a number of lactococcal strains for the presence of other prophage-encoded phage-resistance systems. This screening resulted in the identification of two such systems, without homology to sie2009, which were shown to mediate a phage-resistance phenotype similar to that conferred by sie2009. To our knowledge, this is the first description of a phage-encoded super-infection exclusion/injection blocking mechanism in the genus Lactococcus.

Phage TP901-1 site-specific integrase functions in human cells.

Abstract: We demonstrate that the site-specific integrase encoded by phage TP901-1 of Lactococcus lactis subsp. cremoris has potential as a tool for engineering mammalian genomes. We constructed vectors that express this integrase in Escherichia coli and in mammalian cells and developed a simple plasmid assay to measure the frequency of intramolecular integration mediated by the integrase. We used the assay to document that the integrase functions efficiently in E. coli and determined that for complete reaction in E. coli, the minimal sizes of attB and attP are 31 and 50 bp, respectively. We carried out partial purification of TP901-1 integrase protein and demonstrated its functional activity in vitro in the absence of added cofactors, characterizing the time course and temperature optimum of the reaction. Finally, we showed that when expressed in human cells, the TP901-1 integrase carries out efficient intramolecular integration on a transfected plasmid substrate in the human cell environment. The TP901-1 phage integrase thus represents a new reagent for manipulating DNA in living mammalian cells.

λ Integrase and the λ Int Family

Abstract: This chapter starts with Allan Campbell’s insightful proposal for the pathway by which the chromosome of bacteriophage λ is integrated into and excised from the chromosome of its Escherichia coli host. The chapter discusses different levels of λ Int family complexity. There are presently four classes of integron integrases, IntI1 to -4, sharing approximately 50% identity. In contrast to the IntI1 integrase, which has not suggested any striking deviations from its cousins, the integron recombination targets, attI1 and attC, present several new variations on λ Int family themes. Recombination between att sites with identical 7-bp spacer regions is not any more efficient than that for two sites differing at five positions. Recent evidence suggests that λ Int may have gone even further in evolving a dependence on arm binding. Whereas full Int binds very poorly to core-type DNA sites, C65 (lacking the N-terminal domain) binds very well. The amino acid and nucleotide residues responsible for distinction have been variously identified by genetic selections, construction of chimeric integrases (via recombination or site-directed mutagenesis), and alteration of core sites. Superimposing the crystal structure of the catalytic domain of vaccinia virus topoisomerase on the coordinates of the previously reported HP1 and Cre recombinase structures, it is apparent that the order and topology of the secondary and tertiary structural elements are strikingly similar. A unified topological mechanism of site-specific recombination by λ integrase family members has remained elusive until fairly recently, in contrast to the well-characterized mechanisms for several enzymes of the resolvase/invertase family.

N-substituted hydroxypyrimidinone carboxamide inhibitors of HIV integrase

Abstract: N-substituted 5-hydroxypyrimidin-6-one-4-carboxamides of formula (I): are described as inhibitors of HIV integrase and inhibitors of HIV replication, wherein R1, R2, R3 and R4 are defined herein. These compounds are useful in the prevention and treatment of infection by HIV and in the prevention, delay in the onset, and treatment of AIDS. The compounds are employed against HIV infection and AIDS as compounds per se or in the form of pharmaceutically acceptable salts. The compounds and their salts can be employed as ingredients in pharmaceutical compositions, optionally in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of preventing, treating or delaying the onset of AIDS and methods of preventing or treating infection by HIV are also described.

Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration, not PIC nuclear import.

Abstract: Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types In this study we confirmed that HIV-1V165A and HIV-1R166A were replication defective and that less mutant viral cDNA localized to infected cell nuclei However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1V165A and HIV-1R166A, suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli Based on these results, we conclude that HIV-1V165A and HIV-1R166A are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells

Wild-type levels of nuclear localization and human immunodeficiency virus type 1 replication in the absence of the central DNA flap.

Abstract: Numerous factors have been implicated in the nuclear localization of retroviral preintegration complexes. Whereas sequences in human immunodeficiency virus type 1 (HIV-1) matrix, Vpr, and integrase proteins were initially reported to function specifically in nondividing cells, other recently identified sequences apparently function in dividing cells as well. One of these, the central DNA flap formed during reverse transcription, is specific to lentiviruses. It was previously reported that flap-negative (F(-)) HIV-1(LAI) was completely defective for viral spread in the MT-4 T-cell line, yet F(-) HIV-1 vectors were only 2- to 10-fold defective in various single-round transduction assays. To address these different findings, we analyzed the infectivity and nuclear localization phenotypes of two highly related T-cell-tropic strains, HIV-1(NL4-3) and a derivative of HIV-1(HXBc2) deficient for both Vpr and Nef. In stark contrast to the previous report, F(-) derivatives of both strains replicated efficiently in MT-4 cells. F(-) HIV-1(NL4-3) also spread like wild-type HIV-1(NL4-3) in infected Jurkat and primary T-cell cultures. In contrast, F(-) HIV-1(HXBc2) was replication defective in primary T cells. Results of real-time quantitative PCR assays, however, indicated that F(-) HIV-1(HXBc2) entered primary T-cell nuclei as efficiently as its wild-type counterpart. Thus, the F(-) HIV-1(HXBc2) growth defect did not appear to correlate with defective nuclear import. Consistent with this observation, wild-type nef restored replication to F(-) HIV-1(HXBc2) in primary T cells. Our results indicate that the central DNA flap does not play a major role in either preintegration complex nuclear import or HIV-1 replication in a variety of cell types.

Dihydroxypyrimidine carboxamide inhibitors of HIV integrase

Abstract: 4,5-Dihydroxypyrimidine-6-carboxamides of formula (I); are described as inhibitors of HIV integrase and inhibitors of HIV replication, wherein R?1, R2, R3 and R4? are defined herein. These compounds are useful in the prevention and treatment of infection by HIV and in the prevention, delay in the onset, and treatment of AIDS. The compounds are employed against HIV infection and AIDS as compounds per se or in the form of pharmaceutically acceptable salts. The compounds and their salts can be employed as ingredients in pharmaceutical compositions, optionally in combination with other antivirals, immunomodulators, antibiotics or vaccines. Methods of preventing, treating or delaying the onset of AIDS and methods of preventing or treating infection by HIV are also described.

Flavanone and flavonol glycosides from the leaves of Thevetia peruviana and their HIV-1 reverse transcriptase and HIV-1 integrase inhibitory activities.

Abstract: Two new flavanone glucosides, (2R)- and (2S)-5-O-β-D-glucopyranosyl-7,4′-dihydroxy-3′,5′-dimethoxyflavanone[pervianoside I (3), peruvianoside II (4)] and a new flavonol glycoside, quercetin 3-O-{β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside} (peruvianoside III, 13) were isolated from the leaves of Thevetia peruviana SCHUM., together with nine known flavonol glycosides and two known iridoid glucosides. The structures of all compounds were determined on the basis of chemical and spectroscopic methods. Their inhibitory effects against HIV-1 reverse transcriptase and HIV-1 integrase were also investigated.

Metal-dependent inhibition of HIV-1 integrase.

Abstract: Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202−3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN−DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg2+ at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg2+-based assays, while IN inhibition by salicylhydrazides is strictly Mn2+-dependent. Additionally, in target...

Patented small molecule inhibitors of HIV-1 integrase: a 10-year saga

Abstract: Validation of integrase as a suitable therapeutic target for the development of drugs against HIV infection; discovery of diketo acid derivatives as bona fide inhibitors of integrase and the recent news of Phase II clinical trials with S-1360 are major findings fuelling the recent surge of interest in the development of integrase inhibitors. Herein, the first review of the entire patent literature for small molecule intergrase inhibitors is presented. The emphasis is based on compounds with potential to serve as leads, therefore, no attempts are made to include: antibodies, oligonucleotides or polypeptides.

Efficient concerted integration by recombinant human immunodeficiency virus type 1 integrase without cellular or viral cofactors.

Abstract: Replication of retroviruses requires integration of the linear viral DNA genome into the host chromosomes. Integration requires the viral integrase (IN), located in high-molecular-weight nucleoprotein complexes termed preintegration complexes (PIC). The PIC inserts the two viral DNA termini in a concerted manner into chromosomes in vivo as well as exogenous target DNA in vitro. We reconstituted nucleoprotein complexes capable of efficient concerted (full-site) integration using recombinant wild-type human immunodeficiency virus type I (HIV-1) IN with linear retrovirus-like donor DNA (480 bp). In addition, no cellular or viral protein cofactors are necessary for purified bacterial recombinant HIV-1 IN to mediate efficient full-site integration of two donor termini into supercoiled target DNA. At ∼30 nM IN (20 min at 37°C), approximately 15 and 8% of the input donor is incorporated into target DNA, producing half-site (insertion of one viral DNA end per target) and full-site integration products, respectively. Sequencing the donor-target junctions of full-site recombinants confirms that 5-bp host site duplications have occurred with a fidelity of ∼70%, similar to the fidelity when using IN derived from nonionic detergent lysates of HIV-1 virions. A key factor allowing recombinant wild-type HIV-1 IN to mediate full-site integration appears to be the avoidance of high IN concentrations in its purification (∼125 μg/ml) and in the integration assay (<50 nM). The results show that recombinant HIV-1 IN may not be significantly defective for full-site integration. The findings further suggest that a high concentration or possibly aggregation of IN is detrimental to the assembly of correct nucleoprotein complexes for full-site integration.

Chemical modification of coumarin dimer and HIV-1 integrase inhibitory activity.

Abstract: A systematic series of chemically modified coumarin dimmers has been synthesized and tested for their inhibitory activity against HIV-1 integrase. We observed that modified coumarin dimmers containing hydrophobic moiety on the linker display potent inhibitory activities.