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Accelerated Communication

Nuclear Import of HIV-1 Integrase Is Inhibited in Vitro by Styrylquinoline Derivatives

Institut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Universités Paris 6 et 7, Paris, France (A.M., C.D.); Bioalliance Pharma, Paris, France (H.L.); and Laboratoire de Biotechnologies et Pharmacologie génétique Appliquée, Unité Mixte Recherche 8113, Centre National de la Recherche Scientifique, Ecole Normale Supérieure de Cachan, Cachan, France (J.-F.M.)

Received April 22, 2004; accepted July 8, 2004

	Abstract

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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.

	Materials and Methods

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Cells and Culture Conditions. Adherent or S3 suspension HeLa cells were maintained in exponential growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Preparation of HeLa Cell Cytosolic Extracts. HeLa cell cytosol was prepared as described by Paschal and Gerace (1995). Ten billion exponentially growing HeLa S3 cells were collected by centrifugation at 300g for 5 min. Cells were washed twice with phosphate-buffered saline and once with lysis buffer [5 mM HEPES, pH 7.4, 5 mM potassium acetate, pH 7.4, 2 mM magnesium acetate, 1 mM EGTA, 2 mM DTT, and the following protease inhibitors: 10 µg/ml each aprotinin, leupeptin, and pepstatin and 200 µg/ml Pefabloc SC (Roche)]. The cell pellet was resuspended in 1 volume of lysis buffer and disrupted in a tight-fitting stainless steel homogenizer (as judged by phase contrast microscopy). The homogenate was diluted with 0.1 volume of 10x transport buffer (transport buffer: 20 mM HEPES, pH 7.4, 110 mM potassium acetate, pH 7.4, 2 mM magnesium acetate, 0.5 mM EGTA, 1 mM DTT, and protease inhibitors) and centrifuged at 40,000g for 30 min at 4°C. The supernatant was further centrifuged at 100,000g for 1 h. The resulting supernatant (10 mg/ml as measured with the protein assay kit from Bio-Rad) was aliquoted, frozen in liquid nitrogen, and stored at -80°C.

Preparation of Transport Substrates. Recombinant IN produced in Escherichia coli was a generous gift from S. Escaich (Avantis Pharma, Ivry, France). 200 µg of recombinant IN was added to 1 mg of N-hydroxysuccinimide ester modified Cy3 (Amersham Biosciences) resuspended in 0.1 M sodium borate, pH 9. The reaction was allowed to proceed for 2 h at room temperature. Labeled IN was separated from unconjugated dye by extensive dialysis against a buffer containing 3 mM sodium phosphate pH 7.4, 1 mM EDTA, 0.3 M NaCl, 0.1% CHAPS, 10% glycerol, and 1 mM DTT. Molar concentration of the labeled protein was estimated by measuring its absorbance at 280 nm and correcting the calculated value for the absorbance of the Cy3 dye at 280 nm according to the manufacturer's instructions.

BSA (1 mg) was labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (85 µg; FLUOS, Roche) in 100 mM sodium borate, pH 8.5. Free fluorochrome was removed by chromatography on a Sephadex G-50 column equilibrated with 50 mM sodium borate, pH 7.6. Resulting fluorescein-BSA was concentrated using Centricon 30 K (Millipore Corporation, Bedford, MA) and coupled to sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate as a cross-linker (500 µg; SulfoSMCC, Pierce) in 50 mM sodium borate, pH 7.6. Excess cross-linker was removed by gel filtration on a Sephadex G-50 column equilibrated in 100 mM sodium phosphate, pH 6.0. Fluorescein-BSA-SulfoSMCC was concentrated and finally cross-linked to a peptide containing the SV40 large T antigen NLS (cggg-DEVKRKVED; 1 mg) in 100 mM sodium phosphate, pH 6.0. Non-coupled peptide was eliminated by gel filtration on a Sephadex G-50 column equilibrated in transport buffer and resulting in BSA-NLS-Fluorescein (BSA-NLS-Fluo) was concentrated.

Nuclear Import Assay. Digitonin-permeabilized HeLa cells were prepared according to Adam et al. (1990). Cells were plated onto glass coverslips 24 h before the assay and grown to 80% confluence. Cells were washed once in phosphate-buffered saline containing 0.5 mM CaCl₂ and 1 mM MgCl₂ and once in transport buffer before permeabilization with 55 µg/ml digitonin (Sigma) in transport buffer for 5 min at 4°C. Nuclear import assays in the absence of cytosolic extracts were performed in transport buffer containing an energy-regenerating system (1 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, and 8 U/ml creatine phosphokinase), 25 µl of BSA (10 mg/ml in transport buffer), and 1 µg/ml of Cy3-coupled recombinant IN in a total volume of 50 µl. For nuclear import assays in the presence of cytosolic extracts, BSA was replaced by 25 µl of HeLa cell cytosolic extracts (10 mg/ml), and 15 µg/ml of BSA-NLS-Fluo was added to the 50 µl reaction mix. The assays in the presence of IN inhibitors were performed using the tested molecules at concentrations ranging from 10 to 100 µM. Transport reactions were allowed to proceed at 30°C for 30 min. Cells were then washed with transport buffer and fixed with 2% paraformaldehyde and 0.1% glutaraldehyde. Coverslips were subsequently mounted in phosphate-buffered saline containing 50% glycerol.

Images were acquired with a Leica DMRB epifluorescence microscope equipped with a CCD camera (Princeton) controlled by Metaview software (Universal Imaging Corporation). For each condition, fluorescence intensity per surface unit was quantified in 150 to 300 nuclei from three independent experiments using Image J software.

	Results and Discussion

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IN Import Is Specifically Inhibited by Active SQ in Vitro. Active and nonactive members of the SQ family of inhibitors represented on Fig. 1 were defined according to their ability to inhibit IN integration activity in vitro (Zouhiri et al., 2000; Polanski et al., 2002; Bénard et al., 2004; Deprez et al., 2004). The archetypal strand transfer inhibitor L-731,988 (Hazuda et al., 2000) is also shown. The anti-integrase effect of khd161, fz41, and BioA53 was linked in vitro to the presence of the salicylic motif on the quinoline ring (Bénard et al., 2004). In contrast, fz117 represents a structural analog lacking the 8-hydroxyl group and devoid of anti-integrase activity (Deprez et al., 2004). Likewise, addition of a carboxyl group at position 10 prevents khd227 from affecting the enzyme activity (Polanski et al., 2002).

View larger version (19K): [in this window] [in a new window]   Fig. 1. Chemical structure of the compounds tested for their effect on IN import. Khd161, fz41 and BioA53 are active SQs, whereas fz117 and khd227 are nonactive members of the SQ family. Active and nonactive SQs are defined according to their ability to inhibit IN integration activity in vitro. L-731,988 is a specific strand transfer inhibitor.

We previously characterized properties of IN nuclear import using an in vitro transport assay (Depienne et al., 2001). In this experimental system (Fig. 2A), the plasma membrane of HeLa cells is selectively permeabilized with digitonin, which at low concentrations selectively perforates the plasma membrane, thus releasing soluble cytosolic components, but does not affect the integrity of the nuclear envelope (Adam et al., 1990). Indeed, digitonin preferentially perforates the plasma membrane compared with internal cellular membranes because of its proportionally higher cholesterol content (Colbeau et al., 1971). A fluorescent import substrate can readily enter the resulting leaky plasma membrane, and its uptake into the nucleus can be followed by fluorescence microscopy. Using this assay, we have previously reported that Cy3-labeled IN (IN-Cy3) rapidly accumulates into nuclei of digitonin-permeabilized cells. This accumulation results not from passive diffusion but from an active transport that occurs through nuclear pore complexes. This import results from a saturable mechanism indicating that a limiting cellular factor is involved in this process. However, we found that IN import is not mediated by a classic import pathway involving karyopherin family members or the Ran GTPase and, more generally, does not require addition of cytosolic extracts. Cellular factors involved in this atypical pathway are thus likely to remain associated with the nuclear compartment of permeabilized cells. In contrast to classic import pathways, IN import requires energy in the form of ATP hydrolysis.

View larger version (85K): [in this window] [in a new window]   Fig. 2. IN import is specifically inhibited by active SQ in vitro in the absence of cytosolic extracts. A, schematic representation of the in vitro import assay. Plasma membrane of HeLa cells is selectively permeabilized with digitonin, thus leading to the release of cytosolic components without affecting the nuclear envelope integrity. Nuclear import of Cy3-labeled IN (IN-Cy3) can be reconstituted in this system in the presence of an energy-regenerating system. B, HeLa cells were digitonin-permeabilized and then incubated for 30 min at 30°C with IN-Cy3 in the presence of an energy-regenerating system and increasing concentrations of different compounds as indicated. Cells were subsequently fixed and analyzed by direct fluorescence. Images were acquired with an epifluorescence microscope equipped with a CCD camera. C, for each experimental condition shown in B, fluorescence intensity per surface unit was quantified in 150 to 300 nuclei from three independent experiments using ImageJ software. The resulting values were expressed as a percentage of the untreated control value.

We thus used this minimal system to evaluate the effect of SQ derivatives on IN import compared with the specific strand transfer inhibitor L-731,988. The import reaction was performed in the presence of increasing concentrations of active SQs, nonactive SQs, or L-731,988. For each condition, IN import was quantified by measuring fluorescence intensity per surface unit in 150 to 300 nuclei from three independent experiments. Nuclear import of IN was clearly and specifically inhibited by the active SQ derivatives fz41 and khd161 in a dose-dependent manner with an IC₅₀ of about 30 µM for fz41 and 55 µM for khd161 (Fig. 2, B and C). BioA-53 prevented only 20% of the nuclear import when used at 100 µM, thus showing a poor inhibitory effect. However, it should be noted that this compound displays a 10-fold lower inhibitory effect in catalytic activity assays compared with fz41 and khd161 (data not shown). In contrast, neither the nonactive SQs fz117 and khd227 nor the archetypal L-731,988 strand transfer inhibitor displayed any significant inhibitory effect on IN import at any tested concentration (Fig. 2, B and C). Together, these results indicate that active SQs specifically inhibit IN import in digitonin-permeabilized cells in the absence of cytosolic extracts, whereas nonactive SQs and the strand transfer inhibitor L-731,988 do not display any significant effect on this transport.

Active SQ Inhibit Specifically IN Import in Vitro in the Presence of Cytosolic Extracts. To test whether SQs selectively affect nuclear import of IN or prevent more generally nuclear import pathways, we tested the effect of SQ derivatives on the import pathway mediated by the classic basic NLS of the SV40 large T antigen. Transport of proteins containing the SV40 large T antigen NLS is insured by a specific heterodimeric receptor, importin /1, as well as the small GTPase Ran. Therefore, in vitro nuclear import of such proteins requires addition of exogenous cytosolic extracts to digitonin-permeabilized cells (Adam et al., 1990). The import assay of both fluorescein-labeled bovine serum albumin fused to the SV40 large T antigen NLS (BSA-NLS-Fluo) and IN-Cy3 was thus performed in the presence of HeLa cell cytosolic extracts in the same cells. As before, import reactions were performed in the presence of increasing concentrations of active SQs, nonactive SQs, or L-731,988. BSA-NLS-Fluo and IN-Cy3 import was quantified by measuring fluorescence intensity per surface unit in 150 to 300 nuclei from three independent experiments. None of the tested molecules presented any significant inhibitory effect on import mediated by the classic SV40 large T antigen NLS (Fig. 3, B and C), whereas in the same cells, nuclear import of IN was specifically inhibited by fz41 (IC₅₀ 17 µM), khd161 (IC₅₀ 33 µM), and BioA-53 (IC₅₀ 60 µM) in a dose-dependent manner (Fig. 3, B and C). The discrepancy of inhibitory activity of BioA-53 measured in the absence or presence of cellular extracts in the import assay is likely to correspond to a stabilization of this compound by the extracts in vitro. Similar to results observed in the absence of cytosolic extracts, neither the nonactive SQs fz117 and khd227 nor the strand transfer inhibitor L-731,988 was able to affect IN import in the presence of cytosolic extracts at any concentration tested (Fig. 3, B and C). Together, these results indicate that active SQs specifically inhibit IN nuclear import in digitonin-permeabilized cells in the presence of cytosolic extracts and do not affect other import pathways, such as the classic basic NLS-mediated transport.

View larger version (79K): [in this window] [in a new window]   Fig. 3. Active SQ inhibit specifically IN import in vitro in the presence of cytosolic extracts. A, schematic representation of the in vitro import assay in the presence of cytosolic extracts. Nuclear import of Cy3-labeled IN (IN-Cy3) and fluorescein-labeled bovine serum albumin fused to the classic basic NLS (BSA-NLS-Fluo) can be reconstituted and followed in the same digitonin-permeabilized HeLa cells in the presence of an energy-regenerating system and cytosolic extracts. B, HeLa cells were digitonin-permeabilized and then incubated for 30 min at 30°C with IN-Cy3 and BSA-NLS-Fluo in the presence of an energy-regenerating system, cytosolic extracts, and increasing concentrations of different compounds as indicated. Cells were subsequently fixed and analyzed by direct fluorescence. Images were acquired with an epifluorescence microscope equipped with a CCD camera. C, for each experimental condition shown in B, fluorescence intensity per surface unit was quantified in 150 to 300 nuclei from three independent experiments. The resulting values were expressed as a percentage of the untreated control value.

Together, these data show that IN import is specifically inhibited by active SQs in digitonin-permeabilized cells and suggest that SQs would inhibit the interaction between IN and cellular factor required for its nuclear import. SQs have been reported previously to be competitive inhibitors for IN binding to DNA in vitro that block the 3'-processing activity of the enzyme (Deprez et al., 2004). The in vitro effect of SQs on both IN integration activity and nuclear import could be explained either by a binding of SQs on distinct functional sites of IN or alternatively by a binding on a unique site controlling both functions. We recently found that IN mutants bearing mutations on the V165 residue are resistant toward fz41 (Bonnenfant et al., 2004). This region could be involved in the interaction of IN with LEDGF/p75, a factor recently proposed to be involved in the subcellular localization of IN (Maertens et al., 2003). This region is also located in the vicinity of the actual active site of the enzyme, and mutations of the V165 residue are known to alter the catalytic activity of recombinant IN, thereby pointing to a possible dual effect of the binding to a unique site (Limon et al., 2002). Nevertheless, we cannot rule out a simultaneous binding to different sites on the enzyme as we recently demonstrated by molecular modeling (Deprez et al., 2004). The fact that SQs are less active against nuclear import than against IN catalytic activity would favor this second hypothesis.

In contrast to SQs, the strand transfer inhibitor L-731,988 did not affect IN import in digitonin-permeabilized cells, supporting a distinct binding mode for SQs and L-731,988 to IN. In agreement with this result, viruses resistant to SQs have been selected and found to present either a single mutation (C280Y) or a double mutation (V165I, V249I) in the IN sequence (Bonnenfant et al., 2004). These mutated amino acids are different from those conferring resistance to L-731,988 (T66I and/or S153Y, M154I and/or T66I) (Hazuda et al., 2000), clearly indicating that these two types of inhibitors use distinct binding sites on IN. IN catalyzes the insertion of a donor DNA into an acceptor DNA. Pommier and colleagues proposed that both DNA substrates could bind distinct adjacent sites on IN and that monofunctional -diketo acids such as L-731,988, that specifically inhibit the strand transfer reaction, would bind selectively the acceptor DNA site on IN (Marchand et al., 2002). In contrast, SQs, which preferentially inhibit 3'-processing in a competitive manner (Deprez et al., 2004), would have a higher affinity for the donor DNA site of IN. If SQs preferentially bind a unique site on IN affecting both integration activity and nuclear import, the binding of SQs to the donor DNA site on IN would inhibit the binding of the cellular factor required for its nuclear import, and we could expect that binding of donor DNA would also prevent nuclear import in vitro.

SQs now represent a major tool for the identification of the IN import factor, just as the antifungal antibiotic leptomycin B constituted a major clue to the identification of the Nuclear Export Sequence receptor Crm1. In turn, characterization of the IN import pathway should lead to the identification of new antiviral target.

	Acknowledgements

 
We thank Fatima Zouhiri for the synthesis of anti-integrase compounds. We thank Nathalie Simoes for technical help and members of the laboratory for helpful discussions.

	Footnotes

 
This work was supported by the French National Agency for AIDS Research (ANRS) and SIDAction.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.104.001735.

ABBREVIATIONS: HIV-1, human immunodeficiency virus type 1; PIC, preintegration complex; IN, integrase; NLS, nuclear localization signal; SQ, styrylquinoline; DTT, dithiothreitol; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]propanesulfonate; BSA, bovine serum albumin; L-731,988, 4-[1-(4-fluorobenzyl)-1H-pyrvol1-2-yl]-2,4-dioxo-butyore acid; SV40, simian virus 40.

Address correspondence to: Catherine Dargemont, Institut Jacques Monod, UMR 7592 CNRS, Universités Paris 6 et 7, 2 Place Jussieu, Tour 43, 75251 Paris Cedex 05, France. E-mail: dargemont{at}ijm.jussieu.fr

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This Article Abstract Full Text (PDF) All Versions of this Article: mol.104.001735v1 66/4/783    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mousnier, A. Articles by Dargemont, C. Search for Related Content PubMed PubMed Citation Articles by Mousnier, A. Articles by Dargemont, C.