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PubMed2005

The cellular protein daxx interacts with avian sarcoma virus integrase and viral DNA to repress viral transcription.

The cellular protein Daxx was identified as an interactor with avian sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells.

In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

Binding Sites↗
PubMed1992

Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases.

Our comparison of deduced amino acid sequences for retroviral/retrotransposon integrase (IN) proteins of several organisms, including Drosophila melanogaster and Schizosaccharomyces pombe, reveals strong conservation of a constellation of amino acids characterized by two invariant aspartate (D) residues and a glutamate (E) residue, which we refer to as the D,D(35)E region. The same constellation is found in the transposases of a number of bacterial insertion sequences. The conservation of this region suggests that the component residues are involved in DNA recognition, cutting, and joining, since these properties are shared among these proteins of divergent origin. We introduced amino acid substitutions in invariant residues and selected conserved and nonconserved residues throughout the D,D(35)E region of Rous sarcoma virus IN and in human immunodeficiency virus IN and assessed their effect upon the activities of the purified, mutant proteins in vitro. Changes of the invariant and conserved residues typically produce similar impairment of both viral long terminal repeat (LTR) oligonucleotide cleavage referred to as the processing reaction and the subsequent joining of the processed LTR-based oligonucleotides to DNA targets. The severity of the defects depended upon the site and the nature of the amino acid substitution(s). All substitutions of the invariant acidic D and E residues in both Rous sarcoma virus and human immunodeficiency virus IN dramatically reduced LTR oligonucleotide processing and joining to a few percent or less of wild type, suggesting that they are essential components of the active site for both reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1996

The retrotransposon Tf1 assembles virus-like particles that contain excess Gag relative to integrase because of a regulated degradation process.

The retrotransposon Tf1, isolated from Schizosaccharomyces pombe, contains a single open reading frame with sequences encoding Gag, protease, reverse transcriptase, and integrase (IN). Tf1 has previously been shown to possess significant transposition activity. Although Tf1 proteins do assemble into virus-like particles, the assembly does not require readthrough of a translational reading frame shift or stop codon, common mechanisms used by retroelements to express Gag in molar excess of the polymerase proteins. This study was designed to determine if Tf1 particles contain equal amounts of Gag and polymerase proteins or whether they contain the typical molar excess of Gag. After using two separate methods to calibrate the strength of our antibodies, we found that both S. pombe extracts and partially purified Tf1 particles contained a 26-fold molar excess of Gag relative to IN. Knowing that Gag and IN are derived from the same Tf1 primary translation product, we concluded that the excess Gag most likely resulted from specific degradation of IN. We obtained evidence of regulated IN degradation in comparisons of Tf1 protein extracted from log-phase cells and that extracted from stationary-phase cells. The log-phase cells contained equal molar amounts of Gag and IN, whereas cells approaching stationary phase rapidly degraded IN, leaving an excess of Gag. Analysis of the reverse transcripts indicated that the bulk of reverse transcription occurred within the particles that possess a molar excess of Gag.

Base Sequence↗
PubMed1998

A Ty1 integrase nuclear localization signal required for retrotransposition.

Ty1 retrotransposition in Saccharomyces cerevisiae requires integrase (IN)-mediated insertion of Ty1 cDNA into the host genome. The transposition components are assembled in the cytoplasm and must cross the nuclear envelope to reach the genomic target, since, unlike animal cell nuclear membranes, the yeast cell nuclear membrane remains intact throughout the cell cycle. We have identified a bipartite nuclear localization signal (NLS) in IN required for Ty1 transposition (Ty1 IN) that directs IN to the nucleus. Mutations in the NLS that specifically abolish nuclear localization inactivate transpositional integration but do not affect reverse transcription, protein processing, or catalytic activity in vitro. No additional Ty1-encoded proteins are required for IN nuclear localization. Intragenic complementation experiments suggest that Ty1 IN functions as a multimer and contains two distinct domains, one required for integration and the other for nuclear localization. Nuclear targeting of the preintegration complex by an IN NLS may prove to be a general strategy used by retrotransposons and retroviruses that infect nondividing cells.

Amino Acid Sequence↗
PubMed2001

Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p.

The Ty5 retrotransposons of Saccharomyces cerevisiae integrate preferentially into regions of silent chromatin at the telomeres and silent mating loci (HMR and HML). We define a Ty5-encoded targeting domain that spans 6 amino acid residues near the C terminus of integrase (LXSSXP). The targeting domain establishes silent chromatin when it is tethered to a weakened HMR-E silencer, and it disrupts telomeric silencing when it is overexpressed. As determined by both yeast two-hybrid and in vitro binding assays, the targeting domain interacts with the C terminus of Sir4p, a structural component of silent chromatin. This interaction is abrogated by mutations in the targeting domain that disrupt integration into silent chromatin, suggesting that recognition of Sir4p by the targeting domain is the primary determinant in Ty5 target specificity.

Amino Acid Motifs↗
PubMed2001

Integrase mediates nuclear localization of Ty3.

Retroviruses in nondividing cells and yeast retrotransposons must transit the nuclear membrane in order for integration to occur. Mutations in a bipartite basic motif in the carboxyl-terminal domain of the Ty3 integrase (IN) protein were previously shown to block transposition at a step subsequent to 3'-end processing of Ty3 extrachromosomal DNA. In this work, the Ty3 IN was shown to be sufficient to target green fluorescent protein to the nucleolus. Mutations in the bipartite basic motif abrogated this localization. The region containing the motif was shown to be sufficient for nuclear but not subnuclear localization of a heterologous protein. Viruslike particles (VLPs) from cells expressing a Ty3 element defective for nuclear localization were inactive in an in vitro integration assay, suggesting that nuclear entry is required to form active VLPs or that this motif is required for post-nuclear entry steps. Ty3 inserts at transcription initiation sites of genomic tRNA genes and plasmid-borne 5S and U6 RNA genes transcribed by RNA polymerase III. In situ hybridization with Ty3- and Ty3 long terminal repeat-specific probes showed that these elements which are associated with tRNA genes do not colocalize with the ribosomal DNA (rDNA). However, a PCR assay of cells undergoing transposition showed that Ty3 insertion does occur into the 5S genes, which, in yeast, are interspersed with the rDNA and therefore, like Ty3 IN, associated with the nucleolus.

Amino Acid Motifs↗
PubMed1999

Integrating DNA: transposases and retroviral integrases.

Transposable elements appear quite disparate in their organization and in the types of genetic rearrangements they promote. In spite of this diversity, retroviruses and many transposons of both prokaryotes and eukaryotes show clear similarities in the chemical reactions involved in their transposition. This is reflected in the enzymes, integrases and transposases, that catalyze these reactions and that are essential for the mobility of the elements. In this chapter, we examine the structure-function relationships between these enzymes and the different ways in which the individual steps are assembled to produce a complete transposition cycle.

Catalytic Domain↗
PubMed2005

Reverse transcriptase and integrase of the Saccharomyces cerevisiae Ty1 element.

Integrase (IN) and reverse transcriptase (RT) play a central role in transposition of retroelements. The mechanism of integration by IN and the steps of the replication process mediated by RT are briefly described here. Recently, active recombinant forms of Ty1 IN and RT have been obtained. This has allowed a more detailed understanding of their biochemical and structural properties and has made possible combined in vitro and in vivo analyses of their functions. A focus of this review is to discuss some of the results obtained thus far with these two recombinant proteins and to propose future directions.

Amino Acid Sequence↗
PubMed2005

A novel small molecular weight compound with a carbazole structure that demonstrates potent human immunodeficiency virus type-1 integrase inhibitory activity.

The integration of reverse transcribed proviral DNA into a host genome is an essential event in the human immunodeficiency virus type 1 (HIV-1) replication life cycle. Therefore, the viral enzyme integrase (IN), which plays a crucial role in the integration event, has been an attractive target of anti-retroviral drugs. Several IN inhibitory compounds have been reported previously, yet none has been successful in clinical use. To find a new, more successful IN inhibitor, we screened a diverse library of 12 000 small molecular weight compounds randomly by in vitro strand-transfer assay. We identified a series of substituted carbazoles that exhibit strand-transfer inhibitory activity at low micromolar concentrations. Of these, the most potent compound exhibited an IC50 of 5.00+/-3.31 microM (CA-0). To analyse the structural determinants of strand-transfer inhibitory activity of the carbazole derivatives, we selected 23 such derivatives from our compound library and performed further analyses. Of these 23 compounds, six showed strong strand-transfer inhibition. The inhibition kinetics analyses and ethidium bromide displacement assays indicated that the carbazole derivatives are competitive inhibitors and not intercalators. An HeLa4.5/LTR-nEGFP cell line was employed to evaluate in vitro virus replication inhibition of the carbazole derivatives, and IC50 levels ranged from 0.48-1.52 microM. Thus, it is possible that carbazole derivatives, which possess structures different from previously-reported IN inhibitors, may become novel lead compounds in the development of IN inhibitors.

Anti-HIV Agents↗
PubMed2005

Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import.

BACKGROUND: In addition to mediating the integration process, HIV-1 integrase (IN) has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s) and/or motif(s) within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. RESULTS: Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV) and sequence Q (211KELQKQITK) in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA) exhibited more severe defect of induction of beta-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-beta-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C-terminal mutant infection, even though they displayed various low levels of nucleus-associated viral DNA, suggesting that these C-terminal mutants also impaired viral DNA integration ability. CONCLUSION: All of these results indicate that, in addition to being involved in HIV-1 reverse transcription and integration, the C-terminal tri-lysine regions of IN also contribute to efficient viral DNA nuclear import during the early stage of HIV-1 replication.

Active Transport, Cell Nucleus↗
PubMed2006

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody.

BACKGROUND: To further our understanding of the structure and function of HIV-1 integrase (IN) we developed and characterized a library of monoclonal antibodies (mAbs) directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. RESULTS: We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A) and IN (I267A/I268A), exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. CONCLUSION: It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather our findings are most consistent with a model whereby mAb33 binding distorts or constrains the structure of the C-terminal domain and/or blocks substrate binding indirectly. The DNA binding properties and non-specific nuclease activity of the I267A derivatives suggest that the C-terminal domain of IN normally plays an important role in aligning the viral DNA end for proper processing.

Alanine↗
PubMed2005

Identification of the LEDGF/p75 HIV-1 integrase-interaction domain and NLS reveals NLS-independent chromatin tethering.

To investigate the basis for the LEDGF/p75 dependence of HIV-1 integrase (IN) nuclear localization and chromatin association, we used cell lines made stably deficient in endogenous LEDGF/p75 by RNAi to analyze determinants of its location in cells and its ability to interact with IN. Deletion of C-terminal LEDGF/p75 residues 340-417 preserved nuclear and chromatin localization but abolished the interaction with IN and the tethering of IN to chromatin. Transfer of this IN-binding domain (IBD) was sufficient to confer HIV-1 IN interaction to GFP. HRP-2, the only other human protein with an identifiable IBD domain, was found to translocate IN to the nucleus of LEDGF/p75(-) cells. However, in contrast to LEDGF/p75, HRP-2 is not chromatin bound and does not tether IN to chromatin. A single classical nuclear localization signal (NLS) in the LEDGF/p75 N-terminal region ((146)RRGRKRKAEKQ(156)) was found by deletion mapping and was shown to be transferable to pyruvate kinase. Four central basic residues in the NLS are critical for its activity. Strikingly, however, stable expression studies with NLS(+/-) and IBD(+/-) mutants revealed that the NLS, although responsible for LEDGF/p75 nuclear import, is dispensable for stable, constitutive nuclear association of LEDGF/p75 and IN. Both wild-type LEDGF/p75 and NLS-mutant LEDGF/p75 remain entirely chromatin associated throughout the cell cycle, and each tethers IN to chromatin. Thus, these experiments reveal stable nuclear sequestration of a transcriptional regulator by chromatin during the nuclear-cytosolic mixing of cell division, which additionally enables stable tethering of IN to chromatin. LEDGF/p75 is a multidomain adaptor protein that interacts with the nuclear import apparatus, lentiviral IN proteins and chromatin by means of an NLS, an IBD and additional chromatin-interacting domains.

Active Transport, Cell Nucleus↗
PubMed1995

Intracellular transport of the murine leukemia virus during acute infection of NIH 3T3 cells: nuclear import of nucleocapsid protein and integrase.

The entry and intracellular transport of Moloney-murine leukemia virions inside mouse NIH 3T3 cells have been followed by electron microscopy techniques. Five viral proteins--matrix (MA, p15), capsid (CA, p30), nucleocapsid (NC, p10), integrase (IN), and the envelope glycoprotein (SU, gp70)--were located by immunolabeling using gold probes. After entering the cells, viral particles were frequently detected inside cytoplasmic vesicles of variable size. Their viral envelope was apparently lost during intracytoplasmic transport. When the unenveloped viral cores reached the nuclear membrane or its vicinity, they were disrupted. Two of the immunolabeled proteins, NC and IN, were detected entering the nucleus of non-dividing cells, where both were targeted to the nucleolus. However, MA and CA were found only in the cytoplasm. NC is a nucleic acid-binding protein which contains potential nuclear localization signals. We suggest that NC could enter the nucleus as part of a nucleoprotein complex, associated with IN, and possibly, also with viral DNA.

3T3 Cells↗
PubMed2002

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

Two new flavanone glucosides, (2R)- and (2S)-5-O-beta-D-glucopyranosyl-7,4'-dihydroxy-3',5'-dimethoxyflavanone[pervianoside I (3), peruvianoside II(4)] and a new flavonol glycoside, quercetin 3-O-[beta-D-glucopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->6)]-beta-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.

Flavonoids↗
PubMed2006

HIV-1 integrase inhibition of biscoumarin analogues.

Nineteen biscoumarins bearing free and modified hydroxyl substituents at benzoyloxyphenyl linker have been synthesized by multiple step synthesis. Among these biscoumarins, thirteen were found to be active molecules against HIV-1 integrase (HIV-1 IN). The structure-activity relationship of the nineteen compounds on HIV IN may be useful for the design of potent therapeutic agents.

Acetylation↗
PubMed2001

Structure-based HIV-1 integrase inhibitor design: a future perspective.

The pol gene of HIV-1 encodes for three essential enzymes, protease (PR), reverse transcriptase (RT) and integrase (IN). More than 16 drugs, targeting two of these enzymes, PR and RT have been approved by the FDA. At present, there are no clinically useful agents that inhibit the third enzyme, IN. Combination chemotherapy consisting of PR and RT inhibitors has shown remarkable success in the clinic and has benefited many patients. It is thought that a combination of drugs targeting all three enzymes should further incapacitate the virus. Discovery of highly selective PR inhibitors owe their success to the recent development in structure-guided drug design. During the past several years a plethora of structures of HIV-1 PR in complex with an inhibitor have been solved by x-ray crystallography. This incredible wealth of information provided opportunities for the discovery of second and third generation inhibitors. Due to the inherent nature of IN and insufficient structural information, structure-based inhibitor design selective for IN has not kept pace. However, because of recent developments in the field such information could soon become available. In this review, emphasis is placed on inhibitors with identified or proposed drug binding sites on IN.

Amino Acid Sequence↗
PubMed2003

Peptides as new inhibitors of HIV-1 reverse transcriptase and integrase.

Current treatments of human immunodeficiency virus type 1 (HIV-1) infection consist in the combination of drugs targeting reverse transcriptase (RT) and protease (PR). Despite the multiple clinical benefits of this combination therapy, the emergence of resistance highlights the need for new anti-HIV agents. Agents able to interfere with additional steps of viral replication, such as integration of viral DNA in the host genome, would improve the antiviral potency of the treatment. In this regard, we have focused our interest on peptide-based compounds that have been shown to exhibit potential inhibition of RT and integrase (IN) activities in vitro and in vivo. Recently, the expansion of powerful technologies which allow the selection of peptides exhibiting high affinity for a target protein have provided a new approach to selecting potential anti-HIV drugs. Furthermore, efforts to characterize the protein-protein interactions involved in efficient reverse transcription and integration, as well as the determination of the enzyme structure, have generated a very useful source of data for the development of peptide inhibitors. Finally, while this class of compounds has long been considered as poor drug candidates, current knowledge on improving the stability and bioavailability of these agents would lead to the effective use of peptides in therapy.

Anti-HIV Agents↗