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PubMed2006

Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51.

HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection.

Chromosomes, Fungal↗
PubMed2006

Pathogenicity island integrase cross-talk: a potential new tool for virulence modulation.

Instability and excision of pathogenicity islands (PAIs) have already been described in Escherichia coli 536. In this edition of Molecular Microbiology, Bianca Hochhut and colleagues from the University of Würzburg in Germany have shown that the instability of four of the E. coli 536 PAIs is mediated by a P4-type integrase encoded within the specific PAI by a site-specific recombination mechanism. The integrase encoded on PAI II(536) is able to mediate excision and integration of both PAI II(536), and also PAI V(536). The att sites of both these PAIs have a region of sequence similarity, which is also found in several other PAIs and in tRNA genes in several bacterial species. The cross-PAI activity of this integrase (Int(PAI II)) suggests that it plays an important role in both genome evolution and horizontal transfer of pathogenicity elements, possibly even across species barriers. Deletion of PAIs that carry genes for adhesins and other traits might lead to a phase variation-like phenomenon. Differential regulation of integrase activity or production might add a further level of fine-tuning during bacterial infection.

Biological Evolution↗
PubMedDate not supplied

Solution conformation of a peptide corresponding to residues 151-172 of HIV-1 integrase using NMR and CD spectroscopy.

The solution structure of a synthetic peptide corresponding to residues 151-172 of HIV-1 integrase has been determined by NMR and CD spectroscopy. Residues 151-172 of HIV-1 integrase were predicted to be an alpha-helix and to be responsible for the oligomerization of HIV-1 integrase. Two-dimensional 1H NMR and CD studies indicate that this synthetic peptide adopts an amphipathic alpha-helical conformation in TFE-containing solution. However, concentration-dependent CD studies reveal that this peptide motif does not form dimers or oligomers in solution as predicted. These results are in agreement with the crystal structure of the catalytic domain of HIV-1 integrase reported recently.

Amino Acid Sequence↗
PubMed1992

Reversal of integration and DNA splicing mediated by integrase of human immunodeficiency virus.

In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

Base Sequence↗
PubMed1995

Inhibition of human immunodeficiency virus integrase by bis-catechols.

The human immunodeficiency virus type 1 (HIV-1) integrase protein is required for the productive infection of T-lymphoid cells in culture (R. L. LaFemina, C. L. Schneider, H. L. Robbins, P. L. Callahan, K. LeGrow, E. Roth, W. A. Schleif, and E. A. Emini, J. Virol. 66:7414-7419, 1992). This observation suggests that chemical inhibitors of integrase may prevent the spread of HIV in infected individuals. In our search for such potential chemotherapeutic agents, we observed that beta-conidendrol inhibits both the sequence-dependent and sequence-independent endonucleolytic activities of integrase with comparable potencies in vitro (50% inhibitory concentration, 500 nM). Structurally related compounds tested for their abilities to inhibit integrase generated a limited structure-activity analysis which demonstrated that potency is associated with the bis-catechol structure: two pairs of adjacent hydroxyls on separate benzene rings. beta-Conidendrol did not inhibit several other endonucleases and/or phosphoryltransferases. Although beta-conidendrol was not effective in preventing HIV-1 infection in cell culture, the in vitro data demonstrate that it is possible to identify selective agents targeted against this essential HIV-1 function.

Base Sequence↗
PubMed1992

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.

Base Sequence↗
PubMed2002

The IntI-like tyrosine recombinase of Shewanella oneidensis is active as an integron integrase.

We have found an integron-like integrase gene and an attI site in Shewanella oneidensis as part of a small chromosomal integron. We have cloned this gene and tested the ability of the integrase to excise cassettes from various integrons. Most cassettes flanked by two attC sites are readily excised, while cassettes in the "first" position, with an attI1 or attI3 site on one end, are not excised. An exception is a cassette with attI2 on one end. The attI2 site, from Tn7, has greater similarity to the attI site adjacent to the integrase of S. oneidensis than do attI1 or attI3. We cloned the attI site of S. oneidensis and observed the integration of two different cassettes. We have, therefore, demonstrated the function of this integron-like integrase.

Amino Acid Sequence↗
PubMed1993

Characterization of human immunodeficiency virus type 1 integrase expressed in Escherichia coli and analysis of variants with amino-terminal mutations.

Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.

Amino Acid Sequence↗
PubMed1994

Integrase mutants of human immunodeficiency virus type 1 with a specific defect in integration.

A previous genetic analysis of the human immunodeficiency virus type 1 integrase protein failed to identify single amino acid substitutions that only block the integration of viral DNA (C.-G. Shin, B. Taddeo, W.A. Haseltine, and C.M. Farnet, J. Virol. 68:1633-1642, 1994). Additional substitutions of amino acids that are highly conserved among retroviral integrases were constructed in human immunodeficiency virus type 1 and analyzed for their effects on viral protein synthesis and processing, virion morphology, and viral DNA synthesis and integration in an attempt to identify mutants with a specific defect in integration. Four single amino acid substitutions resulted in replication defective viruses. Conservative, single amino acid substitutions of the two invariant aspartic acid residues found in all retroviral integrases prevented the integration of viral DNA and had no detectable effect on the other stages in the viral replication cycle, indicating that these mutants exhibited a specific defect in integration. Mutations at two positions, S-81 and P-109, blocked the integration of viral DNA but also resulted in the production of viral particles that exhibited reduced reverse transcriptase activity, suggesting additional defects in viral replication. Substitution of the highly conserved amino acid T66 had no effect on viral replication in a CD4+ human T-cell line. This analysis extends the range of possible phenotypes that may be produced by single amino acid substitutions in conserved residues of the integrase protein.

Animals↗
PubMed1994

The core and carboxyl-terminal domains of the integrase protein of human immunodeficiency virus type 1 each contribute to nonspecific DNA binding.

The integrase protein of human immunodeficiency virus type 1 removes two nucleotides from the 3' ends of reverse-transcribed human immunodeficiency virus type 1 DNA (3' processing) and covalently inserts the processed ends into a target DNA (DNA strand transfer). Mutant integrase proteins that lack the amino-and/or carboxyl-terminal domains are incapable of catalyzing 3' processing and DNA strand transfer but are competent for an apparent reversal of the DNA strand transfer reaction (disintegration) in vitro. Here, we investigate the binding of integrase to DNA by UV cross-linking. Cross-linked complexes form with a variety of DNA substrates independent of the presence of divalent metal ion. Analysis with amino- and carboxyl-terminal deletion mutant proteins shows that residues 213 to 266 of the 288-residue protein are required for efficient cross-linking in the absence of divalent metal ion. Carboxyl-terminal deletion mutants that lack this region efficiently cross-link only to the branched disintegration DNA substrate, and this reaction is dependent on the presence of metal ion. Both the core and C-terminal domains of integrase therefore contribute to nonspecific DNA binding.

Base Sequence↗
PubMed1995

Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes.

Base Sequence↗
PubMed1996

The role of manganese in promoting multimerization and assembly of human immunodeficiency virus type 1 integrase as a catalytically active complex on immobilized long terminal repeat substrates.

The integration of a DNA copy of the viral genome into the genome of the host cell is an essential step in the replication of all retroviruses. Integration requires two discrete biochemical reactions; specific processing of each viral long terminal repeat terminus or donor substrate, and a DNA strand transfer step wherein the processed donor substrate is joined to a nonspecific target DNA. Both reactions are catalyzed by a virally encoded enzyme, integrase. A microtiter assay for the strand transfer activity of human immunodeficiency virus type 1 integrase which uses an immobilized oligonucleotide as the donor substrate was previously published (D. J. Hazuda, J. C. Hastings, A. L. Wolfe, and E. A. Emini, Nucleic Acids Res. 22;1121-1122, 1994). We now describe a series of modifications to the method which facilitate study of both the nature and the dynamics of the interaction between integrase and the donor DNA. The enzyme which binds to the immobilized donor is shown to be sufficient to catalyze strand transfer with target DNA substrates added subsequent to assembly; in the absence of the target substrate, the complex was retained on the donor in an enzymatically competent state. Assembly required high concentrations of divalent cation, with optimal activity achieved at 25 mM MnCl2. In contrast, preassembled complexes catalyzed strand transfer equally efficiently in either 1 or 25 mM MnCl2, indicating mechanistically distinct functions for the divalent cation in assembly and catalysis, respectively. Prior incubation of the enzyme in 25 mM MnCl2 was shown to promote the multimerization of integrase in the absence of a DNA substrate and alleviate the requirement for high concentrations of divalent cation during assembly. The superphysiological requirement for MnCl2 may, therefore, reflect an insufficiency for functional self-assembly in vitro. Subunits were observed to exchange during the assembly reaction, suggesting that multimerization can occur either before or coincident with but not after donor binding. These studies both validate and illustrate the utility of this novel methodology and suggest that the approach may be generally useful in characterizing other details of this biochemical reaction.

Base Sequence↗
PubMed1996

Human endogenous retrovirus K10 encodes a functional integrase.

We cloned a human endogenous retrovirus K1O DNA fragment encoding integrase and expressed it as a fusion protein with Escherichia coli maltose-binding protein. Integrase activities were measured in vitro by using a double-stranded oligonucleotide as a substrate mimicking viral long terminal repeats (LTR). The fusion protein was highly active for both terminal cleavage and strand transfer in the presence of Mn2+ on the K1O LTR substrate. It was also active on both Rous sarcoma virus and human immunodeficiency virus type 1 LTR substrates, whereas Rous sarcoma virus and human immunodeficiency virus type 1 integrases were active only on their corresponding LTR substrates. The results strongly suggest that K1O encodes a functional integrase with relaxed substrate specificity.

Amino Acid Sequence↗
PubMed2004

Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites.

In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.

Base Sequence↗
PubMed2000

DNA integration by Ty integrase in yku70 mutant Saccharomyces cerevisiae cells.

In the present work we examined nonhomologous integration of plasmid DNA in a yku70 mutant. Ten of 14 plasmids integrated as composite elements, including Ty sequences probably originating from erroneous strand-switching and/or priming events. Three additional plasmids integrated via Ty integrase without cointegrating Ty sequences, as inferred from 5-bp target site duplication and integration site preferences. Ty integrase-mediated integration of non-Ty DNA has never been observed in wild-type cells, although purified integrase is capable of using non-Ty DNA as a substrate in vitro. Hence our data implicate yKu70 as the cellular function preventing integrase from accepting non-Ty DNA as a substrate.

Antigens, Nuclear↗
PubMed1994

Biophysical and enzymatic properties of the catalytic domain of HIV-1 integrase.

A deletion derivative of the integrase protein from human immunodeficiency virus type-1 (HIV-1) consisting of the central core domain (amino acids 50-212) has been characterized biophysically and biochemically. This deletion mutant is of particular interest for structural studies as it can carry out the disintegration reaction suggesting the presence of an active site and, under certain conditions, is more soluble than full-length integrase. The circular dichroism and fluorescence of the deletion mutant and the 288-residue full-length integrase were similar, indicating that the core residues maintain similar overall conformations in both proteins. The deletion mutant is approximately 10% more alpha-helical than the full-length protein. Analytical centrifugation demonstrated that both proteins undergo monomer-dimer association although the truncated protein showed slightly less tendency to dimerize; the dissociation constants were 2.5 x 10(-5) M for the full-length protein and 8.0 x 10(-5) M for the truncated protein. The disintegration activity of both proteins was also compared. Although a higher concentration of the truncation mutant was required for optimal activity, the mutant did not have altered pH or Mn2+ requirements relative to the full-length protein. The combined biophysical and enzymatic studies suggest that this truncated form of HIV-1 integrase is likely to be useful for structural studies.

Amino Acid Sequence↗
PubMed1994

Binding sites for bacteriophage HP1 integrase on its DNA substrates.

The temperate phage HP1 integrates its genome into the chromosome of Haemophilus influenzae by site-specific recombination between host and phage DNA segments, the attachment sites. This reaction is promoted by the HP1-encoded integrase. The interactions of HP1 integrase with its DNA substrates have been characterized by DNase I footprinting. Two classes of binding sites were identified. At sites of type I, integrase binding almost completely eliminated cleavage by DNase I; type I sites shared the consensus sequence 5'-AGGGATTTWW. At type II sites, integrase binding produced alternating regions of protection from and enhancement of cleavage, suggesting that binding at these sites distorted the DNA. The consensus sequence for type II sites was 5'-ACTGGCGRTW. Each binding site contained two copies of the relevant consensus. The host attachment site (attB) contains an inverted pair of type I consensus sequences surrounding the strand exchange points. The phage attachment site (attP) includes six binding sites, three of type I and three of type II, distributed along its 500 nucleotide pairs. All type I sites contain two consensus motifs arranged as inverted repeats. One of these surrounds the strand exchange points in this substrate, one is located internally, and the third coincides with the right boundary of the attP sequence. One type II site, consisting of an inverted repeat of two type II consensus motifs, coincides with the left boundary of the attP sequence. The other two type II sites contain directly repeated pairs of the consensus and are internally located.

Attachment Sites, Microbiological↗
PubMed1997

Discovery and analysis of inhibitors of the human immunodeficiency integrase.

An essential step in the replication of retroviruses is the integration of a DNA copy of the viral genome into the genome of the host cell. Integration encompasses a series of ordered endonucleolytic and DNA strand transfer reactions catalyzed by the viral enzyme, integrase. The requirement for integrase activity in the propagation of HIV-1 in cell culture defines the enzyme as a potential target for chemotherapeutic intervention. We have therefore developed a non-radioisotopic microtiter plate assay which can be used to identify novel inhibitors of integrase from random chemical screens and for the bioassay driven isolation of inhibitors from natural products. This assay uncouples various steps in the reaction pathway and therefore can be exploited to characterize inhibitors. In this monograph we describe a series of modifications to the method which facilitate such mechanistic studies using as an example a series of previously described integrase inhibitors.

Antiviral Agents↗