DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION

Selection of hRAD51 chemical modulators affecting its HIV-1 integrase inhibition ability

hRAD51 inhibits HIV-1 integration in vitro (SI1) and enhancement of the hRAD51/DNA active nucleofilaments formation by the RAD51 stimulatory compound 1, RS-1, (chemical structure shown in figure 1A) improves the integration inhibition by the recombinase both in vitro and in vivo [7]. These data highlighted the role of the nucleocomplex in the inhibition process. To better characterize the mechanism of inhibition, we searched for compounds capable of affecting the hRAD51/DNA binding properties and, thus, the formation of the active nucleofilament. Stilbenes, like DIDS, having previously been shown to affect hRAD51 activity [12], we tested natural stilbenes derivatives recently identified in our laboratory [13] in a hRAD51/DNA interaction assay. As reported in SI2, most of the 22 molecules tested were found to inhibit the hRAD51/DNA association but only one, the E-pterostilbene (P-ter) (chemical structure shown in figure 1A), stimulated binding. Given that hRAD51 stimulatory compounds are attractive candidates as antiviral agents [7], we first focused our interest on the stimulatory molecule. To verify its possible stimulation effect on hRAD51 strand exchange activity, P-ter was assayed in an in vitro recombination activity. As shown in SI3A, P-ter stimulates the recombinase activity at least as efficiently as RS-1, (EC50 for P-ter = 19 VSports注册入口. 2 ± 2. 4 µM and EC50 for RS-1 = 25 ± 3 µM).

The hRAD51 stimulatory compounds were then tested on in vitro hRAD51 mediated inhibition of HIV-1 IN activity using the concerted integration assay described in SI1. As reported in SI4A and [13], P-ter was previously shown to inhibit HIV-1 IN activity with an IC50 = 47. 5 ± 2 VSports在线直播. 5 µM. Since P-ter showed hRAD51 stimulatory effect between 5–20 µM (see SI3) without significantly affecting HIV-1 IN activity, it was tested at concentrations up to 15 µM on hRAD51-mediated IN inhibition. As reported in figure 1C, a strong improvement in integration restriction was observed in the presence of P-ter, as observed in the case of RS-1 (figure 1D). This indicates that the stimulation of the hRAD51 activity not only by RS-1 but also by other stimulatory compounds as P-ter can enhance the HIV-1 IN inhibition properties of the recombinase. To determine whether the active hRAD51 nucleofilament could serve as a target for modulating hRAD51 mediated inhibition of HIV-1 IN, we next examined whether inhibition of the recombinase could also affect its integration restriction properties. Two hRAD51 chemical inhibitors: DIDS [12] and RI-1 [14, 15] and several DNA aptamers previously selected against hRAD51, [16] and figure 1B, were tested. These molecules were assayed on the in vitro hRAD51 recombination activity shown in SI3A. Both RI-1 and DIDS displayed an inhibitory activity on the strand exchange catalyzed by hRAD51 (IC50 determined for RI-1 and DIDS were respectively 50 ± 5 µM and 90 ± 2. 5 µM, SI3B). A47 and A30 aptamers [16] were also found to strongly inhibit hRAD51 under these conditions (IC50 for A47 = 75 ± 4 nM and IC50 for A30 = 30 ± 2 nM), while their shortened control versions A47c and A30c did not (SI3C). Among the hRAD51 inhibitory molecules assayed, DIDS showed a significant IN inhibitory effect (IC50 = 5 ± 2 µM, SI4B) and, thus, was excluded from further analyses. We next compared the effect of RS-1 and P-ter with that of RI-1 and A30, which showed the best inhibitory effect, on hRAD51-mediated integration restriction. As reported in figure 1E and F good correlation was observed between hRAD51 activity and IN inhibition. Indeed, all the hRAD51 inhibitors induced a significant decrease in the hRAD51-mediated integration inhibition, which is in sharp contrast to the potentiation observed with the hRAD51 stimulatory compounds RS-1 and P-ter. No effect of molecules on the IN/hRAD51 interaction was detected (SI5) confirming that the modulation of hRAD51-mediated inhibition by the drugs was mainly due to their effect on the active hRAD51 nucleofilaments. This suggested that modulators of the nucleocomplex could be used as tools to explore its biological regulatory function in infected cells.

Opposite effects of hRAD51 chemical modulation on HIV-1 integration step in 293T cells

RS-1 has previously been shown to stimulate hRAD51 activity both in vitro and in vivo [8] and to inhibit HIV-1 replication in single- and multiple-round infection assays performed in different cell types including primary PBMC resting cells [7]. To better characterize the mechanism of action of RS-1, especially at the integration step, we compared it to P-ter using a typical 293T single-round replication assay, in order to focus on the early steps of infection V体育2025版. Cytotoxicity measurement in cells treated with increasing concentrations of drug showed no significant effect on cells viability (CC50 > 250 µM, SI6A and B). We next tested the drugs for their effect on cellular hRAD51-mediated DNA repair activity using a typical cisplatin resistance assay in a non-toxic concentration range. As reported in SI7A and B, a 24-hour treatment of 293T cells with either RS-1 or P-ter induced an increase in the cisplatin resistance, as expected from the stimulation of the active hRAD51 nucleofilament. To determine the effect of the molecule on intracellular hRAD51 protein, we performed an immunolocalization analysis of the recombinase. As reported in SI7C, cells treated with either RS-1or P-ter showed an increased number of hRAD51 nuclear foci as compared to untreated cells. Quantification of the cytoplasmic and nuclear foci (SI7D) confirmed that treatment with the hRAD51 stimulatory compounds induced a nuclear relocalization of hRAD51, consistent with a stimulation of the formation of active nucleofilaments in the nuclear compartment. Since i) hRAD51 activity could be altered during cell cycle and ii) the effect of the compounds on the viral replication might depend on the alternation of cell cycle we analyzed the impact of drugs treatments on the cellular cycle. As reported in SI7E, propidium iodide labeling of the cells showed no significant change in the cell cycle alternation of the treated versus untreated cells. This allowed further analyzes of drugs effects on early steps of retroviral replication.

As shown in figure 2A a 24-hour treatment of 293T cells with RS-1 prior to transduction with pNL4. 3 based pRRLsin-PGK-eGFP-WPRE VSV-G pseudo-typed viruses induced an inhibition of transduction efficiency. Quantification of the different viral DNA populations indicated that this phenotype was due to an inhibition of integration, as shown by an increase of the amount of unintegrated two-LTR DNA circles, and a decrease of the integrated DNA form, while the total DNA amount remained unchanged (figure 2B). In contrast, treatments performed 5 hours after transduction induced an opposite phenotype, showing a stimulation of the viral replication correlated with an increased integration. To determine whether this dual effect was specific to the RS-1 molecule, we tested the newly selected P-ter. Assays performed on 293T cells transduced with the lentiviral vector produced results similar to those obtained with RS-1 (figure 2C and 2D) VSports.

The RI-1 compound, exerting an opposite effect to RS-1 on hRAD51, was then tested. As reported in SI6C, no significant RI-1 toxicity was observed in a 1–50 µM range, though a slight decrease in cell viability could be observed at concentrations higher than 50 µM (CC₅₀ = 200 ± 15 µM). A 24-hour pre-treatment of 293T cells with RI-1 was found to induce a decrease in both the cisplatin resistance (SI8A) and the nuclear foci formation (SI8B). These results confirmed that RI-1 could negatively modulate the hRAD51 DNA repair activity. The drug was next tested on early steps of retroviral replication. As reported in figure 3A, a 24-hour pre-treatment led to an increase of the percentage of eGFP-positive 293T cells transduced with the lentiviral vector (EC₅₀ = 50 ± 12 µM). Quantification of the viral DNA species indicated that the phenotype was due to a stimulation of integration, as shown by the significant increase in integrated DNA forms and the decrease of two-LTR circles, the global viral DNA amount remaining unchanged (figure 3B). Strikingly, a 5-hour post-transduction treatment with RI-1 induced an opposite effect. Indeed, the viral replication was inhibited (EC50 = 50 ± 6 µM), with a decrease in the integrated DNA forms accompanied by slight increase of the two-LTR circles. In contrast, a 16-hour post-transduction treatment had no significant effect on replication.

These results demonstrated that the hRAD51 activity modulation can have opposite effects on integration depending on the chronology of the treatment. Especially, stimulation of the recombinase prior to transduction rendered the cells resistant to integration, whereas stimulation of hRAD51 post-transduction had a positive effect on integration. Thus, depending on its catalytic status, hRAD51 can either positively or negatively influence the early steps of HIV-1 replication by acting at the integration step. In view of these data, one important conclusion is that cells with higher hRAD51 intracellular concentrations would be expected to be more resistant to HIV-1 infection. To verify this hypothesis, we studied the effect of the hRAD51 intracellular concentration on integration and viral replication.

Modulation of hRAD51 expression affects both cellular DNA repair activity and HIV-1 integration

In order to increase the hRAD51 intracellular content, a hRAD51-FLAG tagged protein was overexpressed in 293T cells by transfection with the pcDNA-hRAD51 expression vector. Expression of the heterologous recombinase was checked by western blotting using an anti-FLAG antibody (figure 4A). The global hRAD51 expression level was evaluated by western blotting using an anti-hRAD51 antibody allowing detection of both the expressed and the endogenous hRAD51. Under our experimental conditions, we reached a 3 to 5-fold increase in hRAD51 expression as compared to nontransfected cells or cells transfected with a BAP-FLAG control vector (figure 4B). Immunolocalization experiments of the hRAD51-FLAG protein using an anti-FLAG antibody showed the formation of typical nuclear foci specific of the active DNA repair recombinase while the BAP-FLAG control protein showed a more diffuse localization (figure 4C). This was confirmed by measuring the hRAD51-mediated DNA repair activity through a cisplatin resistance assay, indicating a significant enhancement of the resistance to cisplatin of the cells overexpressing hRAD51-FLAG (figure 4D). Altogether these data demonstrate that hRAD51-FLAG overexpression stimulates the intrinsic cellular DNA repair activity.

Early steps of HIV-1 replication were then analyzed in cells overexpressing hRAD51-FLAG by transduction with pRRLsin-PGK-eGFP-WPRE VSV-G pseudotyped viruses and measurement of the eGFP expression from the integrated gene. As reported in figure 4E, hRAD51-FLAG overexpression induced a significant 40–50 % inhibition of HIV-1 replication, in contrast to BAP-FLAG. Under these conditions, no significant change in the total viral DNA amount was detected, while a strong decrease in the integrated DNA forms was observed in addition to an increase of the unintegrated two-LTR circles (figure 4F). This confirmed the effect of hRAD51 on the integration step following nuclear entry of the viral cDNA. To better characterize the relationship between the intracellular concentration of hRAD51 and HIV-1 integration efficiency, we tested whether decreasing the hRAD51 expression level could induce an opposite phenotype. For this purpose, a pharmacological approach was used to reduce hRAD51 expression. Imatinib (figure 5A) was previously reported to decrease the hRAD51 protein levels and increase tumor cell radiosensitivity [17]. In vitro control experiments showed that this compound did not affect HIV-1 IN and hRAD51 catalytic activities (SI9A), validating our approach. As reported in SI9B, treatment with imatinib concentrations above 10 µM led to a significant cellular toxicity (EC₅₀ = 30 ± 8 µM). The effect of the drug on hRAD51 expression was thus evaluated using concentrations below 10 µM. Western blot quantifications of hRAD51 expression following imatinib treatment confirmed that the drug could induce an efficient 40–50% decrease 10 hour post-treatment, while the initial level of hRAD51 was recovered after 72 h (figure 5B). The decrease was also associated with a reduced hRAD51 DNA repair activity as measured by the quantification of cisplatin resistance (figure 5C). Based on these data, HIV-1 replication was tested 24-hour post-treatment. As reported in figure 5D, imatinib treatment 24 hours before transduction of the cells led to an increase in eGFP expression associated with a typical enhancement of the integration efficiency (figure 5E). Taken together, these results demonstrate that an up-regulation of hRAD51 expression both stimulates endogenous DNA repair and induces HIV-1 integration restriction.

hRAD51 expression and intracellular localization are modulated during HIV-1 early steps of replication

To determine whether hRAD51 expression could be modulated during the viral infection we transduced 293T cells with pNL4.3 based pRRLsin-PGK-eGFP-WPRE VSV-G pseudo-typed vectors and analyzed the hRAD51 protein content of cellular extracts obtained at different time points post-transduction. As reported in figure 6A, a decrease in the hRAD51 protein content was detected 4 to 12 hours post-transduction and an increase of the protein level was observed 16–24 hours post-transduction. In contrast, the hRAD51 protein amount was found unchanged in the non-transduced cells. Transcription activity of the RAD51 genes was then analyzed. We used the http://www.peachi.labtelenti.org web resource allowing the querying of cellular responses to infection in SupT1 T cells transduced by HIV-based vectors [18]. Genes encoding for RAD51 and paralogs were found highly downregulated during the early steps of infection (0–6 hours) and upregulated during the integration step (8–16 hours) in contrast to other RAD genes as RAD18 or RAD54 (SI10). Taken together these data indicate that hRAD51 expression is modulated during the early steps of replication at the transcription level. We next analyzed the cellular behavior of the endogenous hRAD51 by immunofluorescence staining of transduced cells at different time points. As shown in figure 6B, a modulation of the cellular localization could also be detected during the early stages of the replication. Indeed while hRAD51 foci were found equally distributed in both cytoplasm and nuclear compartment during the 0 to 8 hours post-transduction a strong relocalization of the recombinase was detected during the 12–24 hours post-transduction. No significant change was observed in the non-transduced cells (S11). Consequently, all these data strongly suggest that viral infection modulates both hRAD51 expression and intracellular localization during the early stages of replication.

Chemicals, proteins and antibodies

RS-1 and RI-1 were previously selected from a 10,000 compounds library (ChembridgeDIVERSet) screened for their capacity to affect the DNA binding properties of hRAD51 [8],[15]. DIDS previously reported as an hRAD51 inhibitor [12] was purchased from Sigma. E-pterostilbene (P-ter) was extracted from Vitis vinifera leaves (isolation and purification protocols described in [13]). Imatinib was purchased from Selleck chemicals (Euromedex, France). Aptamers A30 and A47, previously selected as ligands of hRAD51 [16], and their shortened versions A30c and A47c were purchased from MWG. Cisplatin was purchased from Sigma. Recombinant HIV-1 IN was expressed in yeast and purified using the IN_Hybrid method previously described [26]. Wild-type recombinant hRAD51 was produced as described before [27]. Monoclonal anti-FLAG and polyclonal anti-hRAD51 antibodies were purchased from Sigma.

In vitro enzymatic assays

HIV-1 concerted integration reactions were performed as described previously [26] and in SI1. All IN activities were quantified by scanning the bands (half-site plus full-site integration products) following gel electrophoresis and autoradiography, using the Image J software. hRAD51 activity was evaluated according to the strand exchange reaction previously reported [28] and adapted to our concerted integration conditions (see SI3). Strand exchange products were quantified following autoradiography of the gel, using the Image J software. Measurements of the hRAD51 DNA binding activity was performed as previously described with slight modifications [15] (see SI2).

Transfection assay

293T cells were grown in DMEM Glutamax® medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50 µg/ml gentamicin. The cells were seeded into 48-well plates 24 hours before transfection. Fifteen thousand cells were transfected with 1 µg of either p3X-FLAG-CMV-hRAD51 (hRAD51) expression vector or p3X-FLAG-CMV-7-BAP (BAP) expression control plasmid using lipofectamine 2000 (Invitrogen). DMEM containing 20 % of FCS was added 4 hours post-transfection. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 48 hours and then lysed with SDS loading buffer. Extracted proteins were separated on 12 % SDS-PAGE and analyzed by western blotting.

Transduction of 293T cells

293T cells were plated in 48-multiwells plates at 50,000 cells/well using 400 µL of DMEM (Invitrogen, Carlsbad, CA) containing 10% (v/v) fetal calf serum (FCS, Invitrogen) and 50 µg/mL of gentamycin (Invitrogen). Infection was assayed using pNL4.3 based pRRLsin-PGK-eGFP-WPRE VSV-G pseudotyped lentiviruses produced as described in [29]. After three washes with PBS (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 pH 7.4), the cells were transduced with the lentiviral vector at an optimized M.O.I of 1, in a final volume of 400 µl (under these conditions, 25–35% of the cells contained one integrated viral cDNA copy). After 10 days, the cells were washed twice with PBS, treated with trypsin for 5 minutes at room temperature and centrifuged 2 minutes at 2000 rpm. The pellet was washed twice with PBS and resuspended in 200 µl PBS/200 mM EDTA. Fluorescence was quantified using 10,000 cells on a FACS Calibur (Beckton-Dickinson, San Jose, CA). Plots were analyzed using the FCS express v3.00.0103. Data are presented as the percentage of cells showing a significant level of fluorescence or as the fluorescence intensity calculated by the X-median fluorescence intensity of all cells.

"V体育ios版" Intracellular hRAD51 immunolocalization

293T cells transfected with the p3X-FLAG-CMV-hRAD51 (hRAD51) or p3X-FLAG-CMV-7-BAP (BAP) expression vectors, or treated with one of our compounds, were washed three times with PBS and incubated for 10 minutes in the fixation buffer containing 2% formaldehyde. After two washes, the cells were incubated 5 minutes in the permeabilization buffer (PBS supplemented with 0.4% saponin and 0.1% Triton X-100). The cells were washed with PBS supplemented with 0.1% saponin and incubated for 1 hour at room temperature in the blocking buffer (PBS 0.1%, 1% BSA, 2% SVF). The cells were incubated with the primary antibody over night at 4 °C. After three washes with PBS, the secondary antibody coupled to Alexa 440 was added and the cells were further incubated 1 hour at 37 °C. DAPI was added at a final concentration of 1 µg/ml for 10 minutes at room temperature. The cells were then washed four times with PBS before fluorescence microscopy analysis. Similar procedure was used for hRAD51 intracellular localization analysis in 293T cells transduced with pRRLsin-PGK-eGFP-WPRE VSV-G pseudotyped lentiviruses.

Cytotoxicity and cell cycle measurements

The cellular cytotoxicity of the compounds was determined by measuring the survival of cells treated with increasing concentrations of molecules for two days with a standard MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (PromegaCellTiter 96 AQueous One Solution Cell Proliferation Assay). Analyses of the cell cycle were performed using the Propidium iodide flow cytometry kit from abcam following the manufacturer instruction.

Quantification of cisplatin resistance

The effect of protein overexpression and chemical treatment on hRAD51-mediated homologous recombination was assessed using the standard cisplatin resistance assay described previously [8].

Quantification of HIV-1 DNA species

The cells were harvested 8, 24 and 48h post-infection by centrifugation, and 2 to 6.10⁶ cells aliquots were kept frozen at −80°C until analysis. Total DNA (including integrated and episomal HIV-1 DNA) was extracted using the QiAmp blood DNA mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions and eluted in 50 µl elution buffer. Quantification of the viral DNA species was performed using the conditions and primers described in [30]. The total HIV-1 DNA was amplified by quantitative real-time PCR using alight Cycler Instrument (Roche Diagnostics, Meylan, France). Quantifications of total HIV-1 DNA, including 2-LTR circles and integrated HIV-1 cDNA, were performed by quantitative PCR on a Light Cycler instrument (Roche Diagnostics) using the fit point method provided in the Light Cycler quantification software, version 4.1 as previously described [31]. The copy number of 2-LTR circles and total viral DNA were determined with reference to a standard curve prepared by amplification of quantities ranging from 10 to 1.10⁶ copies of plasmid comprising the HIV_LAI 2-LTR junction [19]. The integrated HIV-1 cDNA copy number was determined in reference to a standard curve generated by the concomitant two-stage PCR amplification of serial dilutions of an integrated HIV-1 DNA standard from Hela-R7 Neocells [32]. Cell equivalents were calculated according to amplification of the β-globin gene (two copies per diploid cell) with commercially available materials (Control Kit DNA; Roche Diagnostics). 2-LTR circles, total and integrated HIV-1 DNA levels were determined as copy numbers per 10⁶ cells. Two-LTR circles and integrated cDNA were also expressed as a percentage of the total viral DNA.