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. 2015 Jun 18;22(6):712-23.
doi: 10.1016/j.chembiol.2015.04.020. Epub 2015 Jun 4.

V体育官网 - Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration

Sylvain Thierry  1 , Mohamed Salah Benleulmi  2 , Ludivine Sinzelle  2 , Eloise Thierry  1 , Christina Calmels  2 , Stephane Chaignepain  3 , Pierre Waffo-Teguo  4 , Jean-Michel Merillon  4 , Brian Budke  5 , Jean-Max Pasquet  6 , Simon Litvak  2 , Angela Ciuffi  7 , Patrick Sung  8 , Philip Connell  5 , Ilona Hauber  9 , Joachim Hauber  9 , Marie-Line Andreola  2 , Olivier Delelis  1 , Vincent Parissi  10
Affiliations

Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration (V体育平台登录)

Sylvain Thierry et al. Chem Biol. .

Abstract

The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51 VSports手机版. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication. .

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Figures

Figure 1
Figure 1. Effect of hRAD51 modulators on HIV-1 integration
The chemical structure of the stimulatory compounds RS-1 and P-ter (A) and the inhibitory compounds RI-1 and DIDS, as well as the sequence of the aptamers (B) are indicated. Increasing concentrations of wt-hRAD51 were added in a standard concerted integration assay in the presence of 100 µM ATP (w/o molecule or aptamer), and with 7.5 or 15 µM RS-1 (C), P-ter (D), RI-1 (E) or 0.1, 0.25 or 0.5 µM of A30 (F). The data reported represent the mean values of at least three independent experiments ± standard deviation (error bars). The activity in the absence of compounds, corresponding to the total amount of donor DNA integrated into the acceptor plasmid, as detected on agarose gel electrophoresis and shown in SI1, was normalized to 100 %.
Figure 2
Figure 2. Effect of RS-1 and P-ter treatment on early steps of HIV-1 replication and viral DNA populations
Cells were treated for 24 hours prior to, concomitantly, or 5–16 hours post-transduction with either RS-1 (A and B) or P-ter (C and D). The eGFP fluorescence was measured 10 days post-transduction by flow cytometry. The percentage of eGFP-positive cells in the absence of compound was normalized to 100% (A and C). The amount of the total, integrated and 2-LTR circles viral DNA forms was quantified as described in Materials and Methods at a fixed 30 µM concentration (effective and non-cytotoxic) of RI-1 or p-ter, under two distinct treatment conditions (early and late). The amount of each viral DNA species produced in the absence of compound was normalized to 100 % (B and D). Results are the mean of three independent experiments. The p-values calculated using a Student’s t-test are indicated as *p<0.05, **p<0.005.
Figure 3
Figure 3. Effect of hRAD51 inhibitory compounds RI-1 on early steps of HIV-1 replication
Cells were treated for 24 hours prior to, concomitantly, or 5–16 hours post-transduction. eGFP fluorescence was measured 10 days following transduction by flow cytometry. The percentage of GFP-positive cells obtained in the absence of compound was normalized to 100 % (A). The effect of RI-1 on viral DNA production was measured by quantifying the total, integrated and 2-LTR circles viral DNA forms at a fixed 50 µM concentration (non-cytotoxic and effective) of RI-1, under two treatment conditions. The proportion of the different DNA species obtained in the absence of compound was normalized to 100 % (B). Results are represented as the mean values calculated from three independent experiments. The p-values are reported as *p<0.05, **p<0.005.
Figure 4
Figure 4. Effect of hRAD51 overexpression on endogenous DNA repair and early steps of HIV-1 replication and viral DNA productions
The expression of hRAD51-FLAG or BAP-FLAG in 293T cells was checked 48 hours post-transfection by western blotting using anti-FLAG antibodies (A, lane 1: protein extract from cells expressing hRAD51-FLAG, lane 2: protein extract from cells expressing BAP-FLAG). The global hRAD51 level was determined in cells transfected with the hRAD51-FLAG (hRAD51) and BAP-FLAG (BAP) expression vectors in parallel to untransfected control cells (w/o transfection), by western blotting using anti-hRAD51 antibodies. The amounts of protein loaded were normalized to the endogenous actin protein revealed by western blotting using an anti-actin antibody (B). The cellular distribution of the overexpressed proteins was determined by immunolocalization using an anti-FLAG antibody (C). The hRAD51 activity was determined under each condition by a cisplatin resistance assay as described in Materials and Method (D). The cells were transduced 48 hours post-transfection with the hRAD51 or BAP expression plasmids. HIV-1 replication was evaluated from fluorescence measurement 10 days after transduction by flow cytometry. The percentage of untransfected eGFP-positive cells was normalized to 100 % (E). The amount of total, integrated and 2-LTR circles viral DNAs was measured by quantitative PCR as described in Materials and Methods. The proportion of the different viral DNA species produced in untransfected control cells was normalized to 100 % (F). Results are represented as the mean values calculated from three independent experiments. The p-values are shown as *p<0.05, **p<0.005.
Figure 5
Figure 5. Effect of imatinib treatment on hRAD51 expression levels, endogenous DNA repair and early steps of HIV-1 replication
The chemical structure of imatinib is reported in (A). Total protein fraction was extracted 6 to 72 hours following treatment with imatinib. The hRAD51 protein levels were determined from western blot analyses using an anti-hRAD51 antibody (B). The amount of hRAD51 protein present in untreated cells was normalized to 100%. The ability of imatinib to affect cisplatin resistance was checked in a standard survival analysis performed after 24 hours of treatment with increasing concentrations of the compound (C). Survival was expressed as the ratio of absorbance at 492 nM (Synergy (BioTek) plate reader) of cisplatin-treated cells (pre-incubated with the compound or not) relative to untreated cells. Results represent the means of at least three independent experiments ± standard deviation (error bars).The effect of imatinib on early steps of HIV-1 replication was analyzed following a 24 hours treatment of the cells before transduction with the lentiviral vector. The eGFP fluorescence was measured 10 days post-transduction by flow cytometry. The percentage of eGFP-positive cells in the absence of compound was normalized to 100% (D). The amount of the total, integrated and 2-LTR circles viral DNA forms was quantified at a fixed 10 µM concentration (effective and non-cytotoxic) of imatinib. The amount of each viral DNA species produced in the absence of compound was normalized to 100 % (E). The results are represented as the mean of three independent experiments. The p-values are indicated as *p<0.05, **p<0.005.
Figure 6
Figure 6. Expression (A) and intracellular localization (B) of hRAD51 during the early steps of HIV-1 replication in 293T cells
Proteins were extracted at different time points (0 to 32 hours) after 293T cells transduction with pNL4.3 based pRRLsin-PGK-eGFP-WPRE VSV-G pseudotyped viruses and the hRAD51 expression level was analyzed by western-blot. The amount of hRAD51 was normalized to the amount of actin detected by western-blot. The quantity of hRAD51 detected at time point = 0 was then normalized to 100 %. The results are represented as the mean of three independent experiments (A). Data obtained without transduction (mock) are also reported. The cellular distribution of hRAD51 was determined by immunolocalization at different time points after 297T cells transduction using an anti-hRAD51 antibody. Results are reported as the mean number ± standard deviation of cytoplasmic and nuclear foci observed under each condition for at least 50 cells (B). A typical picture of hRAD51 intracellular localization is also provided for several time periods corresponding to the early and late steps of integration.
Figure 7
Figure 7. Influence of hRAD51 and its modulation on HIV-1 integration
The hRAD51 recombinase activity, namely the formation of the active nucleofilament, plays a negative regulatory role on the steps following PIC nuclear import and preceding strand transfer catalysis, and a positive regulatory role during the steps subsequent to integration, including DNA repair. Chemical modulators targeting these dual effects can exert opposite effects on HIV-1 integration, and hence on viral replication.
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V体育2025版 - References

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