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PubMed2001

Molecular genetics and target site specificity of retroviral integration.

Integration is an essential step in the life cycle of retroviruses, resulting in the stable joining of the viral cDNA to the host cell chromosomes. While this critical process makes retroviruses an attractive vector for gene delivery, it also presents a potential hazard. The sites where integration occurs are nonspecific. Therefore,it is possible that integration of retroviral DNA will affect host gene expression and disrupt normal cellular functions. The mechanism by which integration sites are chosen is not well understood, and is influenced by several factors, including DNA sequence and structure, DNA-binding proteins, DNA methylation, and transcription. Integrase, the viral enzyme responsible for catalyzing integration, also plays a key role in controlling the choice of target sites. The integrase domain responsible for target site selection has been mapped to the central core region. A better understanding of the interaction between the target-specifying motif of integrase and the target DNA may allow a means to manipulate integration into particular chromosomal sites. Another approach to directing integration is to fuse integrase with a sequence-specific DNA-binding protein, which results in a bias of integration in vitro into the recognition site of the fusion partner. Successful incorporation of the fusion protein into infectious virions and the identification of optimal proteins that can be fused to integrase will advance the development of site-specific vectors. Retroviruses are promising for the delivery of genes in experimental and therapeutic protocols. A better understanding of integration will aid in the design of safer and more effective gene transfer vectors.

DNA, Viral↗
PubMedDate not supplied

Archaeal integrative genetic elements and their impact on genome evolution.

Integrases encoded in archaeal genomes can be classified into seven families on the basis of their sequences. They constitute a super-family of tyrosine DNA recombinases together with a number of bacterial integrases and they are likely to be responsible for the formation of integrated elements in archaeal chromosomes. An integrated element is defined as possessing an integrase, a block of foreign genes, and a direct repeat at the two ends with one repeat unit overlapping a tRNA gene. There are two types of archaeal integrated elements, the SSV viral type, including those that carry the partitioned integrase gene, intN and intC, and the pNOB8 type, including those with a tRNA gene overlapping the attL site 5' prior to an integrase gene. Both known and unknown genes are present in these integrated elements and their encoded proteins may have facilitated the adaptation of archaea during evolution.

Chromosomes, Archaeal↗
PubMed1995

Target DNA capture by HIV-1 integration complexes.

BACKGROUND: The early steps of human immunodeficiency virus 1 (HIV-1) replication involve reverse transcription of the viral RNA and integration of the resulting cDNA into a host chromosome. The DNA integration step requires the integration machinery ('preintegration complex') to bind to the host DNA before connecting the viral and host DNAs. Here, we present experiments that distinguish among three possible pathways of target-DNA capture: repeated binding and release of target DNA prior to the chemical strand-transfer step; binding followed by facilitated diffusion along target DNA (sliding); and integration at the initial target-capture site. The mechanism of target-DNA capture has implications for the design of gene therapy methods, and influences the interpretation of results on the selection of integration target sites in vivo. RESULTS: We present new in vitro conditions that allow us to assemble HIV-1 integrase--the virus-encoded recombination enzyme--with a viral DNA and then to trap assembled complexes bound to target DNA. We find that complexes of integrase and viral DNA do not slide along target DNA substantially after binding. We confirm and extend these results by analyzing target capture by a hybrid protein composed of HIV-1 integrase linked to a sequence-specific DNA-binding domain. We find that the integrase domain binds quickly and tightly under the above conditions, thereby obstructing function of the fused sequence-specific DNA-binding domain. We also monitor target-DNA capture by HIV-1 preintegration complexes purified from freshly infected cells. Partially purified complexes commit quickly and stably to the first target DNA added, whereas preintegration complexes in crude cytoplasmic extracts do not. The addition of extracts from uninfected cells to partially purified complexes blocks quick commitment. CONCLUSIONS: Under new conditions favorable for the analysis of target-DNA capture in vitro, HIV-1 integrase complexes bind quickly and stably to target DNA without subsequent sliding. Parallel studies of preintegration complexes support a model in which target-site capture in vivo is reversible as a result of the action of cellular factors.

Binding Sites↗
PubMed2002

Total synthesis and evaluation of lamellarin alpha 20-Sulfate analogues.

In order to explore the influence of sulfate groups on the bioactivity profiles of marine alkaloids of the lamellarin class, three such alkaloids, lamellarin alpha, lamellarin alpha 13,20-disulfate and lamellarin H, were synthesized and their activities against HIV-1 integrase and cancer cell lines were compared with those of lamellarin alpha 20-sulfate, which is a selective inhibitor of HIV-1 integrase. Lamellarin alpha does not inhibit HIV-1 integrase but shows moderate cytotoxicity with good cell line selectivity. Lamellarin alpha 13,20-disulfate is a moderate inhibitor of both HIV-1 integrase and cancer cell lines. Lamellarin H is a more potent inhibitor of HIV-1 integrase but lacked the specificity required to be medicinally useful.

Antineoplastic Agents↗
PubMed2006

Retroviral DNA integration: reaction pathway and critical intermediates.

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.

Blotting, Western↗
PubMed2005

Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific genome integration of murine phenylalanine hydroxylase cDNA.

We explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine phenylalanine hydroxylase cDNA in the livers of phenylketonuric (PKU) mice. The phiBT1 phage integrase is an enzyme that catalyses the efficient recombination between unique sequences in the phage and bacterial genomes, leading to the site-specific integration of the former into the latter in a unidirectional manner. Here we showed that this phage integrase functions efficiently in mouse cells, and several naturally occurring pseudo-attP sites located in the intergenic regions of the mouse genome have been identified and molecularly characterized. We further demonstrated that the addition of nuclear localization signal sequences to the C terminus of the phage integrase enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system, we delivered mouse phenylalanine hydroxylase cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that the severity of the hyperphenylalaninemic phenotype in the treated mice decreased significantly. After three applications, serum phenylalanine levels in all treated PKU mice were reduced to the normal range and remained stable thereafter. Their fur color also changed from gray to black, indicating the reconstitution of melanin biosynthesis as a result of available tyrosine derived from reconstituted phenylalanine hydroxylation in the liver. Thus, the phiBT1 bacteriophage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in DNA-mediated gene therapy for a multitude of genetic disorders.

Animals↗
PubMed1998

In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.

The genome of the broad host range Streptomyces temperate phage, phiC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites. The results show that directionality in phiC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.

Amino Acid Sequence↗
PubMed2000

Stability-activity relationships of a family of G-tetrad forming oligonucleotides as potent HIV inhibitors. A basis for anti-HIV drug design.

Recently, we have demonstrated that T30695, a G-tetrad-forming oligonucleotide, is a potent inhibitor of human immunodeficiency virus, type I (HIV-1) integrase and the K(+)-induced loop folding of T30695 plays a key role in the inhibition of HIV-1 integrase (Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). Here we have modified T30695 by introducing a hydrophobic bulky group, propynyl dU, or a positively charged group, 5-amino dU, into the bases of T residues of the loops, and by substitution of the T-G loops by T-T loops. Physical measurements have demonstrated that the substitution of propynyl dU or 5-amino dU for T in the T residues of the loops did not alter the structure of T30695, and these derivatives also formed an intramolecular G-quartet structure, which is an essential requirement for anti-HIV activity. Measured IC(50) and EC(50) values show that these substitutions did not induce an apparent decrease in the ability to inhibit HIV-1 integrase activity and in the inhibition of HIV-1 replication in cell culture. However, the substitution of T-T loops for T-G loops induced a substantial decrease in both thermal stability and anti-HIV activity. The data analysis of T30695 and the 21 derivatives shows a significant, functional correlation between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 integrase activity and between thermal stability of the G-tetrad structure and the capacity to inhibit HIV-1 replication, as assessed with the virus strains HIV-1 RF, IIIB, and MN in cell culture. This relationship between thermostability and activity provides a basis for improving the efficacy of these compounds to inhibit HIV-1 integrase activity and HIV-1 replication in cell culture.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Disruption of the terminal base pairs of retroviral DNA during integration.

Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA. These two different polynucleotidyl transfer reactions are apparently carried out by a single active site. The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome. A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions. We propose that integrase distorts DNA substrates to accommodate both reactions within the active site. Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing. Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end. This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites. The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses.

Base Composition↗
PubMed2003

Traffic at the tmRNA gene.

A partial screen for genetic elements integrated into completely sequenced bacterial genomes shows more significant bias in specificity for the tmRNA gene (ssrA) than for any type of tRNA gene. Horizontal gene transfer, a major avenue of bacterial evolution, was assessed by focusing on elements using this single attachment locus. Diverse elements use ssrA; among enterobacteria alone, at least four different integrase subfamilies have independently evolved specificity for ssrA, and almost every strain analyzed presents a unique set of integrated elements. Even elements using essentially the same integrase can be very diverse, as is a group with an ssrA-specific integrase of the P4 subfamily. This same integrase appears to promote damage routinely at attachment sites, which may be adaptive. Elements in arrays can recombine; one such event mediated by invertible DNA segments within neighboring elements likely explains the monophasic nature of Salmonella enterica serovar Typhi. One of a limited set of conserved sequences occurs at the attachment site of each enterobacterial element, apparently serving as a transcriptional terminator for ssrA. Elements were usually found integrated into tRNA-like sequence at the 3' end of ssrA, at subsites corresponding to those used in tRNA genes; an exception was found at the non-tRNA-like 3' end produced by ssrA gene permutation in cyanobacteria, suggesting that, during the evolution of new site specificity by integrases, tropism toward a conserved 3' end of an RNA gene may be as strong as toward a tRNA-like sequence. The proximity of ssrA and smpB, which act in concert, was also surveyed.

Attachment Sites, Microbiological↗
PubMed1997

Tethering human immunodeficiency virus type 1 preintegration complexes to target DNA promotes integration at nearby sites.

Integration of retroviral cDNA in vivo is normally not sequence specific with respect to the integration target DNA. We have been investigating methods for directing the integration of retroviral DNA to predetermined sites, with the dual goal of understanding potential mechanisms governing normal site selection and developing possible methods for gene therapy. To this end, we have fused retroviral integrase enzymes to sequence-specific DNA-binding domains and investigated target site selection by the resulting proteins. In a previous study, we purified and analyzed a fusion protein composed of human immunodeficiency virus integrase linked to the DNA-binding domain of lambda repressor. This fusion could direct selective integration in vitro into target DNA containing lambda repressor binding sites. Here we investigate the properties of a fusion integrase in the context of a human immunodeficiency virus provirus. We used a fusion of integrase to the DNA binding domain of the zinc finger protein zif268 (IN-zif). Initially we found that the fusion was highly detrimental to replication as measured by the multinuclear activation of a galactosidase indicator (MAGI) assay for infected centers. However, we found that viruses containing mixtures of wild-type integrase and IN-zif were infectious. We prepared preintegration complexes from cells infected with these viruses and found that such complexes directed increased integration near zif268 recognition sites.

Cell Line, Transformed↗
PubMed2002

Subcellular localization and integration activities of rous sarcoma virus reverse transcriptase.

Reverse transcriptases (RTs) alphabeta and beta from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT alphabeta and the beta subunit, when expressed independently, can be detected in the nucleus, whereas the separate alpha subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of beta, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT alphabeta with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT alphabeta is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT alphabeta could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).

3T3 Cells↗
PubMed1994

Genetic structures associated with spread of the type Ia trimethoprim-resistant dihydrofolate reductase gene amongst Escherichia coli strains isolated in the Nottingham area of the United Kingdom.

DNA probes for specific integrase genes were used to study 122 R plasmids encoding the predominant trimethoprim-insusceptible type Ia dihydrofolate reductase (DHFR) found in clinical isolates of Escherichia coli. The predominance of the type Ia DHFR was thought to result from the location of its gene on transposon Tn7, but of trimethoprim R plasmids carrying this gene that were collected between 1978 and 1983, between 1987 and 1988, and during 1992, only 49/60 (81.6%), 30/43 (69.8%) and 9/19 (47.4%) respectively hybridized with a probe for the Tn7 integrase gene. It has been suggested that novel genetic elements termed 'integrons' may play an important role in the dissemination of antibiotic resistance genes. Known integrons encode an integrase similar to that encoded by transposon Tn21, and 28 Tn7-negative plasmids (10/60 from 1978-83, 10/43 from 1987-8 and 8/19 from 1992) showed homology with a probe specific for the Tn21 integrase gene. Six plasmids were negative with both probes. It is concluded that Tn7 has played an important role in the dissemination of the gene encoding the type Ia DHFR amongst clinical isolates of E. coli in the Nottingham region of the UK, but that other genetic structures, some of which seem to have an integrase function similar to that of known integrons, may be playing an increasingly significant role.

Bacteriuria↗
PubMed2000

Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.

A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway.

Alanine↗
PubMed2002

In search of authentic inhibitors of HIV-1 integration.

Current strategies for the treatment of HIV infection are based on cocktails of drugs that target the viral reverse transcriptase or protease enzymes. At present, the clinical benefit of this combination therapy for HIV-infected patients is considerable, although it is not clear how long this effect will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting additional steps of the viral replication cycle may increase the potency of inhibition and prevent resistance development. During HIV replication, integration of the viral genome into the cellular chromosome is an essential step catalysed by the viral integrase. Although HIV integrase is an attractive target for antiviral therapy, so far all research efforts have led to the identification of only one series of compounds that selectively inhibit the integration step during HIV replication, namely the diketo acids. In this review we try to address the question why it has proven so difficult to find potent and selective integrase inhibitors. We point to potential pitfalls in defining an inhibitor as an authentic integrase inhibitor, and propose new strategies and technologies for the discovery of authentic HIV integration inhibitors.

Anti-HIV Agents↗
PubMed1994

Architectural elements in nucleoprotein complexes: interchangeability of specific and non-specific DNA binding proteins.

Integration host factor (IHF) is required in lambda site-specific recombination to deform the DNA substrates into conformations active for recombination. HU, a homolog of IHF, can also deform DNA but binds without any apparent sequence specificity. We demonstrate that HU can replace IHF by cooperating with the recombinase protein, integrase, to generate a stable and specific complex with electrophoretic mobility and biochemical activity very close to the complex formed by IHF and integrase. The eukaryotic HMG1 and HMG2 proteins differ entirely in structure from HU but they also bind DNA non-specifically and induce or stabilize deformed DNA. We show that the eukaryotic HMG1 and HMG2 proteins cooperate with integrase at least as well as does HU to make a defined structure. We also find that the eukaryotic core histone dimer H2A-H2B can replace IHF, suggesting that the histone dimer is functional outside the context of a nucleosome. HU and the HMG proteins not only contribute to the formation of stable complexes, but they can at least partially replace IHF for the integrative and excisive recombination reactions. These results, together with our analysis of nucleoprotein complexes made with damaged recombination sites, lead us to conclude that the cooperation between HU and integrase does not depend on protein-protein contacts. Rather, cooperation is manifested through building of higher order structures and depends on the capacity of the non-specific DNA binding proteins to bend DNA. While all these non-specific binding proteins appear to fulfil the same bending function, they do so with different efficiencies. This probably reflects subtle structural differences between the assembled complexes.

Allosteric Regulation↗
PubMed2003

The roles of cellular factors in retroviral integration.

A key early step in the retroviral life cycle is the integration of reverse-transcribed viral cDNA into a chromosome of an infected cell. The key protein player in retroviral integration is the viral integrase, which enters the cell as part of the virus. Although purified integrase protein is necessary and sufficient to perform the basic catalytic DNA breakage and joining steps of retroviral integration, a variety of normal cellular proteins have been implicated as playing important roles in establishing the integrated provirus in cells. This chapter reviews the roles of host cell factors that function during integrase catalysis, during the repair of the resulting DNA recombination intermediate, and by potentially guiding viral preintegration complexes to their chromosomal locations for cDNA integration. The potential to interfere with proper integration by blocking either integrase catalysis or the function of cellular integration cofactors is also discussed.

Animals↗
PubMed2001

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

Bacteriophages↗