Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Integrase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 613 records · Page 34Linked to original sources
PubMed1997

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

Caffeic Acids↗
PubMed2000

Structure-activity relationships and binding mode of styrylquinolines as potent inhibitors of HIV-1 integrase and replication of HIV-1 in cell culture.

Our prior studies showed that polyhydroxylated styrylquinolines are potent HIV-1 integrase (IN) inhibitors that block the replication of HIV-1 in cell culture at nontoxic concentrations. To explore the mechanism of action of these inhibitors, various novel styrylquinoline derivatives were synthesized and tested against HIV-1 IN and in cell-based assays. Regarding the in vitro experiments, the structural requirements for biological activity are a carboxyl group at C-7, a hydroxyl group at C-8 in the quinoline subunit, and an ancillary phenyl ring. However the in vitro inhibitory profile tolerates deep alterations of this ring, e.g. by the introduction of various substituents or its replacement by heteroatomic nuclei. Regarding the ex vivo assays, the structural requirements for activity are more stringent than for in vitro inhibition. Thus, in addition to an o-hydroxy acid group in the quinoline, the presence of one ortho pair of substituents at C-3' and C-4', particularly two hydroxyl groups, in the ancillary phenyl ring is imperatively required for inhibitory potency. Starting from literature data and the SARs developed in this work, a putative binding mode of styrylquinoline inhibitors to HIV-1 IN was derived.

Anti-HIV Agents↗
PubMed2006

Experimental/theoretical electrostatic properties of a styrylquinoline-type HIV-1 integrase inhibitor and its progenitors.

We have established that polyhydroxylated styrylquinolines are potent inhibitors of HIV-1 integrase (IN). Among them, we have identified (E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-methoxyphenyl)-ethenyl]-7-quinolinecarboxylic acid (1) as a promising lead. Previous molecular dynamics simulations and docking procedures have shown that the inhibitory activity involves one or two metal cations (Mg2+), which are present in the vicinity of the active center of the enzyme. However, such methods are generally based on a force-field approach and still remain not as reliable as ab initio calculations with extended basis sets on the whole system. To go further in this area, the aim of the present study was to evaluate the predictive ability of the electron density and electrostatic properties in the structure-activity relationships of this class of HIV-1 antiviral drugs. The electron properties of the two chemical progenitors of 1 were derived from both high-resolution X-ray diffraction experiments and ab initio calculations. The twinning phenomenon and solvent disorder were observed during the crystal structure determination of 1. Molecule 1 exhibits a planar s-trans conformation, and a zwitterionic form in the crystalline state is obtained. This geometry was used for ab initio calculations, which were performed to characterize the electronic properties of 1. The electron densities, electrostatic potentials, and atomic charges of 1 and its progenitors are here compared and analyzed. The experimental and theoretical deformation density bond peaks are very comparable for the two progenitors. However, the experimental electrostatic potential is strongly affected by the crystal field and cannot straightforwardly be used as a predictive index. The weak difference in the theoretical electron densities between 1 and its progenitors reveals that each component of 1 conserves its intrinsic properties, an assumption reinforced by a 13C NMR study. This is also shown through an excellent correlation of the atomic charges for the common fragments. The electrostatic potential minima in zwitterionic and nonzwitterionic forms of 1 are discussed in relation with the localization of possible metal chelation sites.

Carbon Isotopes↗
PubMed2000

Cyclodidemniserinol trisulfate, a sulfated serinolipid from the Palauan ascidian Didemnum guttatum that inhibits HIV-1 integrase.

[structure--see text] Bioassay-guided fractionation of extracts of the Palauan ascidian Didemnum guttatum led to the isolation of cyclodidemniserinol trisulfate (1) as an inhibitor of HIV-1 integrase, which is an attractive target for anti-retroviral chemotherapy. The structure of cyclodidemniserinol trisulfate (1), the stereochemistry of which was only partially determined, was elucidated by interpretation of NMR and mass spectral data.

Animals↗
PubMed1997

Solution structure of the N-terminal zinc binding domain of HIV-1 integrase.

The solution structure of the N-terminal zinc binding domain (residues 1-55; IN1-55) of HIV-1 integrase has been solved by NMR spectroscopy. IN1-55 is dimeric, and each monomer comprises four helices with the zinc tetrahedrally coordinated to His 12, His 16, Cys 40 and Cys 43. IN1-55 exists in two interconverting conformational states that differ with regard to the coordination of the two histidine side chains to zinc. The different histidine arrangements are associated with large conformational differences in the polypeptide backbone (residues 9-18) around the coordinating histidines. The dimer interface is predominantly hydrophobic and is formed by the packing of the N-terminal end of helix 1, and helices 3 and 4. The monomer fold is remarkably similar to that of a number of helical DNA binding proteins containing a helix-turn-helix (HTH) motif with helices 2 and 3 of IN1-55 corresponding to the HTH motif. In contrast to the DNA binding proteins where the second helix of the HTH motif is employed for DNA recognition, IN1-55 uses this helix for dimerization.

Bacterial Proteins↗
PubMed1998

Helical and coiled-coil-forming properties of peptides derived from and inhibiting human immunodeficiency virus type 1 integrase assessed by 1H-NMR--use of NH temperature coefficients to probe coiled-coil structures.

Human immunodeficiency virus type 1 integrase (HIV-1 IN) which catalyzes viral DNA integration into the host genome of infected cells represents an attractive target for AIDS therapy. We have previously demonstrated the ability of the IN-(147-175)-peptide derived from the catalytic core domain of HIV-1 IN to inhibit the enzyme activity in vitro. IN-(147-175)-peptide contains four heptad repeats and displays a high propensity for coiled-coil formation while its [P159]IN-(147-175)-peptide analog (Lys159-->Pro in the protein, Lys13-->Pro in the peptide) is unable to form a stable coiled-coil and is devoid of inhibitory activity [Sourgen, F., Maroun, R. G., Frère, V., Bouziane, M., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765-773]. Now, we report results from an NMR study on IN-(147-175)-peptide and [P159]IN-(147- 175)-peptide as well as on an optimized [E156, A163, A167]IN-(147-175)-peptide that is a better inhibitor of IN than IN-(147-175)-peptide. While in aqueous solution, IN-(147-175)-peptide and [P159]IN-(147-175)-peptide display only nascent helical features, [E156, A163, A167]IN-(147-175)-peptide exhibits 20% of helical content. In 20% trifluoroethanol/80% H2O, the helix content is the highest for [E156, A163, A167]IN-(147-175)-peptide (approximately 70%) and the lowest for [P159]IN-(147-175)-peptide (approximately 40%), due to a local helix break caused by the Pro residue. The NHs of residues in the two central helical heptads (a-g) of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide display a regular periodic variation of their temperature coefficients in 20% trifluoroethanol. The b, c and f residues on the hydrophilic face of the amphipathic helix show high coefficients reflecting hydrogen bonded NHs, while the a and d residues on the hydrophobic face exhibit low coefficients, near random-coil values. The particular arrangement of the hydrophobic side-chains of a and d residues at the coiled-coil interface reduces the access of trifluoroethanol molecules to their amide groups. The inability of trifluoroethanol molecules to create interactions with the amide C=O groups, these being required to strengthen the intrahelical C=O...H-N hydrogen bonds, is the main cause for observation of heptadic a and d residues with low NH temperature coefficients. Such effects concern mostly the two central helical heptads of IN-(147-175)-peptide and [E156, A163, A167]IN-(147-175)-peptide implying that these ones are engaged in stable parallel coiled coils. Our results provide a link between the propensity of peptides for helix formation, their coiled-coil properties and their efficiency to inhibit IN.

Amino Acid Sequence↗
PubMed1999

Conformational aspects of HIV-1 integrase inhibition by a peptide derived from the enzyme central domain and by antibodies raised against this peptide.

Monospecific antibodies were raised against a synthetic peptide K159 (SQGVVESMNKELKKIIGQVRDQAEHLKTA) reproducing the segment 147-175 of HIV-1 integrase (IN). Synthesis of substituted and truncated analogs of K159 led us to identify the functional epitope reacting with antibodies within the C-terminal portion 163-175 of K159. Conformational studies combining secondary structure predictions, CD and NMR spectroscopy together with ELISA assays, showed that the greater is the propensity of the epitope for helix formation the higher is the recognition by anti-K159. Both the antibodies and the antigenic peptide K159 exhibited inhibitory activities against IN. In contrast, neither P159, a Pro-containing analog of K159 that presents a kink around proline but with intact epitope conformation, nor the truncated analogs encompassing the epitope, were inhibitors of IN. While the activity of antibodies is restricted to recognition of the sole epitope portion, that of the antigenic K159 likely requires interactions of the peptide with the whole 147-175 segment in the protein [Sourgen F., Maroun, R.G., Frère, V., Bouziane, A., Auclair, C., Troalen, F. & Fermandjian, S. (1996) Eur. J. Biochem. 240, 765-773]. Actually, of all tested peptides only K159 was found to fulfill condition of minimal number of helical heptads to achieve the formation of a stable coiled-coil structure with the IN 147-175 segment. The binding of antibodies and of the antigenic peptide to this segment of IN hampers the binding of IN to its DNA substrates in filter-binding assays. This appears to be the main effect leading to inhibition of integration. Quantitative analysis of filter-binding assay curves indicates that two antibody molecules react with IN implying that the enzyme is dimeric within these experimental conditions. Together, present data provide an insight into the structure-function relationship for the 147-175 peptide domain of the enzyme. They also strongly suggest that the functional enzyme is dimeric. Results could help to assess models for binding of peptide fragments to IN and to develop stronger inhibitors. Moreover, K159 antibodies when expressed in vivo might exhibit useful inhibitory properties.

Amino Acid Sequence↗
PubMed2006

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site.

Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide (128)AACWWAGIK(136) was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.

4-Hydroxycoumarins↗
PubMed2002

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

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 Mg(2+). 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.

Anti-HIV Agents↗
PubMed1996

Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase.

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.

Amino Acid Sequence↗
PubMed2000

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

Base Composition↗
PubMed2005

Peptides derived from the reverse transcriptase of human immunodeficiency virus type 1 as novel inhibitors of the viral integrase.

Recent studies have shown that the integrase (IN) of HIV-1 is inhibited in vitro by HIV-1 reverse transcriptase (RT). We further investigated the specific protein sequences of RT that were involved in this inhibition by screening a complete library of RT-derived peptides for their inhibition of IN activities. Two 20-residue peptides, peptide 4286, derived from the RT DNA polymerase domain, and the one designated 4321, from the RT ribonuclease H domain, inhibit the enzymatic activities of IN in vitro. The former peptide inhibits all three IN-associated activities (3'-end processing, strand transfer, and disintegration), whereas the latter one inhibits primarily the first two functions. We showed the importance of the sequences and peptide length for the effective inhibition of IN activities. Binding assays of the peptides to IN (with no DNA substrate present) indicated that the two inhibitory peptides (as well as several non-inhibitory peptides) interact directly with IN. Moreover, the isolated catalytic core domain of IN also interacted directly with the two inhibitory peptides. Nevertheless, only peptide 4286 can inhibit the disintegration activity associated with the IN core domain, because this activity is the only one exhibited by this domain. This result was expected from the lack of inhibition of disintegration of full-length IN by peptide 4321. The data and the three-dimensional models presented suggested that the inhibition resulted from steric hindrance of the catalytic domain of IN. This information can substantially facilitate the development of novel drugs against HIV INs and thus contribute to the fight against AIDS.

Catalytic Domain↗
PubMed2005

Synthetic approaches to nuclease-resistant, nonnatural dinucleotides of anti-HIV integrase interest.

New, nonnatural dinucleotide 5'-monophosphates with a surrogate isonucleoside component of L-related stereochemistry, have been synthesized. Structures of the target compounds were confirmed by multinuclear NMR spectra (1H, 13C, 31P, COSY), UV hypochromicity, FAB HRMS data and X-ray crystallography. These compounds are totally resistant to cleavage by 3'- and 5'-exonucleases. Dinucleotides of this study with a terminal L-isonucleoside component showed remarkable selectivity for inhibition of the strand transfer step of HIV-1 integrase. To the best of our knowledge, these compounds represent only the second example of this type of selectivity of inhibition of the strand transfer step.

Crystallography, X-Ray↗
PubMed2001

Structure-activity of inhibition of HIV-1 integrase and virus replication by G-quartet oligonucleotides.

As novel anti-HIV agents, the G-tetrad-forming oligonucleotides have been explored for their structure-activity relations with regard to inhibition of integrase (IN) (N. Jing, Expert Opin. Investig. Drugs (2000) 9, 1777-1785). We have now developed two families of G-quartet oligonucleotides: T40217-T40222, with potential formation of a tail-to-tail G-quartet dimer, and T40224-T40227, with phosphorothioate (PT) linkages in the guanine loops. The results obtained from biophysical measurements and the assays of the inhibition of HIV-1 IN and virus replication demonstrated that an increase in the length of the G-quartet structure from a monomer (15A) to a tail-to-tail dimer (47A) does not distinctly disrupt the inhibition of HIV-1 IN activity or the inhibition of HIV-1 replication in cell cultures. G-quartet oligonucleotides were observed to induce molecular aggregation of HIV-1 IN and interrupt the binding of viral DNA to HIV-1 IN. Also, PT substitutions did not confer any advantages compared with the regular phosphodiesters for the inhibition of HIV-1 replication by intramolecular G-quartets. The G-quartet motif is the primary requirement for the remarkable nuclease resistance and pronounced biological efficacy of these oligonucleotides.

Cell Line↗
PubMed1999

Inhibition of replication of HIV-1 at both early and late stages of the viral life cycle by single-chain antibody against viral integrase.

Retroviruses including HIV-1 integrates a DNA copy of their RNA genome into cellular DNA of the infected cell. This reaction, essential and unique to replication of retroviruses, is mediated by the viral enzyme, integrase (IN). We constructed a recombinant gene encoding a single-chain, antigen-binding peptide (scAb2-19), which interacted with a carboxyl terminal part of HIV-1 IN. HeLa CD4 cells expressing scAb2-19 localized in either cytoplasmic or nuclear compartment were resistant to HIV-1 infection at an multiplicity of infection (MOI) of 0.25 or 0.063, but the resistance was overcome when MOI was increased to 1. To determine whether this resistance was due to inhibition of the early events, transduction experiments were performed with a replication-incompetent HIV-1 vector carrying bacterial lacZ driven by an internal Tat-independent cytomegalovirus immediate early promoter. Both cytoplasmic and nuclear expressions of scAb2-19 resulted in decrease in the transduction efficiency on HeLa CD4 cells. This implies that an early step of replication--before or during integration--was affected by the scAb2-19. Furthermore, cytoplasmic expression of scAb2-19 did not affect the viral amount released from the cells transfected with HIV-1 infectious clone DNA (pLAI). However, infectivity relative to reverse transcriptase activity was lower for virions released from the 293T cells cotransfected with pLAI and the cytoplasmic scAb2-19 expression plasmid than for those released from the 293T cells transfected with pLAI alone. This implies that scAb2-19 reduced infectivity of released virions by interfering a late step of the viral replication. The single-chain, antigen-binding peptide molecule may prove useful not only for studies of the functions of IN and its role in the viral life cycle but also for developing a gene therapy strategy against AIDS.

Amino Acid Sequence↗
PubMed2006

Antiretroviral activity, pharmacokinetics, and tolerability of MK-0518, a novel inhibitor of HIV-1 integrase, dosed as monotherapy for 10 days in treatment-naive HIV-1-infected individuals.

BACKGROUND: MK-0518 is a novel HIV-1 integrase strand transfer inhibitor with potent in vitro activity against HIV-1 (95% inhibitory concentration [IC95] = 33 nM in 50% human serum) and good bioavailability in uninfected subjects. This study explored the antiretroviral activity and safety of MK-0518 versus placebo for 10 days as monotherapy in antiretroviral therapy-naive HIV-1-infected patients with plasma HIV-1 RNA levels of at least 5000 copies/mL and CD4 T-cell counts of at least 100 cells/mm. METHODS: This was a multicenter, double-blind, randomized, placebo-controlled 2-part study, with the first part using MK-0518 in 1 of 4 doses (100, 200, 400, and 600 mg) versus placebo (randomized 1:1:1:1:1) given twice daily for 10 days of monotherapy. Patients were monitored for safety, pharmacokinetic parameters, and antiretroviral effect. RESULTS: Thirty-five patients were enrolled (6-8 patients per treatment group) and completed 10 days of therapy; the mean baseline log10 HIV RNA level ranged from 4.5 to 5.0 copies/mL in each group. On day 10, the mean decrease from baseline in the log10 HIV RNA level was -0.2 copies/mL for the placebo group and -1.9, -2.0, -1.7 and -2.2 log10 copies/mL for the MK-0518 100-, 200-, 400-, and 600-mg treatment groups, respectively. All dose groups had superior antiretroviral activity compared with placebo (P < 0.001 for comparison of each dose with placebo). At least 50% of patients in each MK-0518 dose group achieved an HIV RNA level <400 copies/mL by day 10. Mean trough MK-0518 concentrations at each dose exceeded the IC95 of 33 nM. Study therapy was generally well tolerated. The most common adverse experiences were headache and dizziness; these were similar between active and control groups. There were no discontinuations because of adverse experiences and no serious adverse experiences. CONCLUSIONS: MK-0518 showed potent antiretroviral activity as short-term monotherapy and was generally well tolerated at all doses. Based on these results, part 2 of the study, a dose-ranging 48-week trial of MK-0518 versus efavirenz in a combination regimen, has been initiated.

Adult↗
PubMed2001

Characterization of chimeric enzymes between caprine arthritis--encephalitis virus, maedi--visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli.

In order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis-encephalitis virus (CAEV), maedi-visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.

Arthritis-Encephalitis Virus, Caprine↗
PubMed2004

Nuclear import of HIV-1 integrase is inhibited in vitro by styrylquinoline derivatives.

Nuclear import of HIV-1 preintegration complexes (PICs) allows the virus to infect nondividing cells. Integrase (IN), the PIC-associated viral enzyme responsible for the integration of the viral genome into the host cell DNA, displays karyophilic properties and has been proposed to participate to the nuclear import of the PIC. Styrylquinolines (SQs) have been shown to block viral replication at nontoxic concentrations and to inhibit IN 3'-processing activity in vitro by competing with the DNA substrate binding. However, several lines of evidence suggested that SQs could have a postentry, preintegrative antiviral effect in infected cells. To gain new insights on the mechanism of their antiviral activity, SQs were assayed for their ability to affect nuclear import of HIV-1 IN and compared with the effect of a specific strand transfer inhibitor. Using an in vitro transport assay, we have previously shown that IN import is a saturable mechanism, thus showing that a limiting cellular factor is involved in this process. We now demonstrate that SQs specifically and efficiently inhibit in vitro nuclear import of IN without affecting other import pathways, whereas a specific strand transfer inhibitor does not affect IN import. These data suggest that SQs not only inhibit IN-DNA interaction but would also inhibit the interaction between IN and the cellular factor required for its nuclear import.

Active Transport, Cell Nucleus↗