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Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.

Le Rouzic E, Bonnard D, Chasset S, Bruneau JM, Chevreuil F, Le Strat F, Nguyen J, Beauvoir R, Amadori C, Brias J, Vomscheid S, Eiler S, Lévy N, Delelis O, Deprez E, Saïb A, Zamborlini A, Emiliani S, Ruff M, Ledoussal B, Moreau F, Benarous R - Retrovirology (2013)

Bottom Line: However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step.Infectivity of viral particles produced in presence of Mut101 was severely decreased.This latter effect also required the binding of the compound to the LEDGF-binding pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biodim Mutabilis, Romainville 93230, France. richard.benarous@mutabilis.fr.

ABSTRACT

Background: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.

Results: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.

Conclusion: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

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Binding to IN-CCD wt, T174I mutant, treatment during production of wt and T174I mutant viruses. (A) Binding kinetics of Mut101 to IN-CCD wt. Serial dilutions of Mut101 (between 9.8 nM and 5 μM) were injected on immobilized GST-Flag-CCD wt. (B) Binding kinetics of Mut101 to IN-CCD T174I. Serial dilutions of Mut101 (between 1.3 μM and 5.0 μM) were injected on immobilized GST-Flag-CCD T174I. (C) Diagram of the experimental setup to study the infectivity of NL4-3 wt and IN T174I virions produced after transfection of 293 T cells. The indicated compounds were added during virus production for 48 h. Supernatants were tested for virus production by p24 assay, and for virus infectivity. (D) Infectivity of wt NL4-3 (blue bars) and IN T174I NL4-3 mutant (red bars) virions harvested from 293 T transfected cells after treatment with the indicated compounds and infection of MT4 cells by cytopathic assay using CellTiter-Glo®.
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Figure 7: Binding to IN-CCD wt, T174I mutant, treatment during production of wt and T174I mutant viruses. (A) Binding kinetics of Mut101 to IN-CCD wt. Serial dilutions of Mut101 (between 9.8 nM and 5 μM) were injected on immobilized GST-Flag-CCD wt. (B) Binding kinetics of Mut101 to IN-CCD T174I. Serial dilutions of Mut101 (between 1.3 μM and 5.0 μM) were injected on immobilized GST-Flag-CCD T174I. (C) Diagram of the experimental setup to study the infectivity of NL4-3 wt and IN T174I virions produced after transfection of 293 T cells. The indicated compounds were added during virus production for 48 h. Supernatants were tested for virus production by p24 assay, and for virus infectivity. (D) Infectivity of wt NL4-3 (blue bars) and IN T174I NL4-3 mutant (red bars) virions harvested from 293 T transfected cells after treatment with the indicated compounds and infection of MT4 cells by cytopathic assay using CellTiter-Glo®.

Mentions: The post-integration block promoted by Mut101 cannot be explained by impaired IN-LEDGF interaction or the inhibition of IN catalytic activity. It could be suggested that such a post-integration defect might be related to an unknown Mut101 target, in addition to IN. We generated an NL4-3 HIV-1 virus bearing the T174I mutation in the LEDGF-binding pocket of IN to rule out this hypothesis. We (E. Le Rouzic unpublished results) and others [36] have selected the T174I mutation for resistance to IN-LEDGF inhibitors: Mut101 had an EC50 > 50 μM on this mutant compared to an EC50 = 0.49 μM on NL4-3 wt. We used Surface Plasmon Resonance (SPR) to confirm that Mut101 was less able to bind to the mutated IN-CCD T174I than to IN-CCD wt. Mut101 bound to IN-CCD wt with high affinity (Kd = 0.12 μM) in a similar range to the IC50 or AC50 found in HTRF assays for inhibition of the IN-LEDGF interaction or enhancement of the IN-IN interaction, respectively (Figure 7A). Mut101 had no significant binding to the mutated IN-CCD T174I (Figure 7B). HIV-1 NL4-3 wt and the NL4-3 IN T174I mutant virus were produced by HEK293T cell transfection in the presence of Mut101, SQV, Raltegravir, Mut063 or DMSO. Virions were harvested and used to infect MT4 cells (as schematized in Figure 7C); their infectivity was tested using a cytopathic CellTiter-Glo® assay. As shown in Figure 7D, NL4-3 wt virus (blue bars) produced in the presence of Mut101 was inactivated and the viability of MT4 cells infected by this virus was preserved. In contrast, the mutant virus T174I (red bars) was insensitive to Mut101 treatment and MT4 cells were fully infected and their viability abrogated. Both wt and T174I viruses were sensitive to and inactivated by SQV treatment. Raltegravir treatment during virus production had no effect on either virus; these retained full infectivity which was comparable to that observed after DMSO or Mut063 treatment. These results demonstrate that integrase is indeed the unique target of Mut101 for its ARV activity, both at the integration and post-integration steps of the HIV-1 replication cycle.


Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage.

Le Rouzic E, Bonnard D, Chasset S, Bruneau JM, Chevreuil F, Le Strat F, Nguyen J, Beauvoir R, Amadori C, Brias J, Vomscheid S, Eiler S, Lévy N, Delelis O, Deprez E, Saïb A, Zamborlini A, Emiliani S, Ruff M, Ledoussal B, Moreau F, Benarous R - Retrovirology (2013)

Binding to IN-CCD wt, T174I mutant, treatment during production of wt and T174I mutant viruses. (A) Binding kinetics of Mut101 to IN-CCD wt. Serial dilutions of Mut101 (between 9.8 nM and 5 μM) were injected on immobilized GST-Flag-CCD wt. (B) Binding kinetics of Mut101 to IN-CCD T174I. Serial dilutions of Mut101 (between 1.3 μM and 5.0 μM) were injected on immobilized GST-Flag-CCD T174I. (C) Diagram of the experimental setup to study the infectivity of NL4-3 wt and IN T174I virions produced after transfection of 293 T cells. The indicated compounds were added during virus production for 48 h. Supernatants were tested for virus production by p24 assay, and for virus infectivity. (D) Infectivity of wt NL4-3 (blue bars) and IN T174I NL4-3 mutant (red bars) virions harvested from 293 T transfected cells after treatment with the indicated compounds and infection of MT4 cells by cytopathic assay using CellTiter-Glo®.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222603&req=5

Figure 7: Binding to IN-CCD wt, T174I mutant, treatment during production of wt and T174I mutant viruses. (A) Binding kinetics of Mut101 to IN-CCD wt. Serial dilutions of Mut101 (between 9.8 nM and 5 μM) were injected on immobilized GST-Flag-CCD wt. (B) Binding kinetics of Mut101 to IN-CCD T174I. Serial dilutions of Mut101 (between 1.3 μM and 5.0 μM) were injected on immobilized GST-Flag-CCD T174I. (C) Diagram of the experimental setup to study the infectivity of NL4-3 wt and IN T174I virions produced after transfection of 293 T cells. The indicated compounds were added during virus production for 48 h. Supernatants were tested for virus production by p24 assay, and for virus infectivity. (D) Infectivity of wt NL4-3 (blue bars) and IN T174I NL4-3 mutant (red bars) virions harvested from 293 T transfected cells after treatment with the indicated compounds and infection of MT4 cells by cytopathic assay using CellTiter-Glo®.
Mentions: The post-integration block promoted by Mut101 cannot be explained by impaired IN-LEDGF interaction or the inhibition of IN catalytic activity. It could be suggested that such a post-integration defect might be related to an unknown Mut101 target, in addition to IN. We generated an NL4-3 HIV-1 virus bearing the T174I mutation in the LEDGF-binding pocket of IN to rule out this hypothesis. We (E. Le Rouzic unpublished results) and others [36] have selected the T174I mutation for resistance to IN-LEDGF inhibitors: Mut101 had an EC50 > 50 μM on this mutant compared to an EC50 = 0.49 μM on NL4-3 wt. We used Surface Plasmon Resonance (SPR) to confirm that Mut101 was less able to bind to the mutated IN-CCD T174I than to IN-CCD wt. Mut101 bound to IN-CCD wt with high affinity (Kd = 0.12 μM) in a similar range to the IC50 or AC50 found in HTRF assays for inhibition of the IN-LEDGF interaction or enhancement of the IN-IN interaction, respectively (Figure 7A). Mut101 had no significant binding to the mutated IN-CCD T174I (Figure 7B). HIV-1 NL4-3 wt and the NL4-3 IN T174I mutant virus were produced by HEK293T cell transfection in the presence of Mut101, SQV, Raltegravir, Mut063 or DMSO. Virions were harvested and used to infect MT4 cells (as schematized in Figure 7C); their infectivity was tested using a cytopathic CellTiter-Glo® assay. As shown in Figure 7D, NL4-3 wt virus (blue bars) produced in the presence of Mut101 was inactivated and the viability of MT4 cells infected by this virus was preserved. In contrast, the mutant virus T174I (red bars) was insensitive to Mut101 treatment and MT4 cells were fully infected and their viability abrogated. Both wt and T174I viruses were sensitive to and inactivated by SQV treatment. Raltegravir treatment during virus production had no effect on either virus; these retained full infectivity which was comparable to that observed after DMSO or Mut063 treatment. These results demonstrate that integrase is indeed the unique target of Mut101 for its ARV activity, both at the integration and post-integration steps of the HIV-1 replication cycle.

Bottom Line: However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step.Infectivity of viral particles produced in presence of Mut101 was severely decreased.This latter effect also required the binding of the compound to the LEDGF-binding pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biodim Mutabilis, Romainville 93230, France. richard.benarous@mutabilis.fr.

ABSTRACT

Background: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction.

Results: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket.

Conclusion: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place.

Show MeSH
Related in: MedlinePlus