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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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Effect of INLAIs on the oligomeric state of IN. (A-B) IN-IN HTRF interaction: (A) IN-IN interaction activation dose–response curves. Data represent the means of three independent experiments done in duplicate with standard deviations shown as error bars. (B) Correlation between AC50 of IN-IN interaction and EC50 of ARV activity on MT4 cells infected with HxB2 HIV-1 (R2 = 0.78). This study used 21 of the set of 51 compounds. AC50 = concentration required to activate IN-IN interaction by 50% of the maximum effect. (C-F) Size exclusion chromatography of IN: Binding of INLAIs BI-D or Mut101, to IN NL4-3 wt (C-D) or IN NL4-3 A128T (E-F), promotes a shift toward higher IN oligomeric state, independently of LEDGF. Blue peaks: elution of IN wt and IN A128T in the absence of compound. Red peaks: elution of IN wt and IN A128T in the presence of BI-D (C, E) or Mut101 (D, F).The elution volume and identification of each peak (numbered 1 to 7) are indicated in supplementary table S2.
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Figure 4: Effect of INLAIs on the oligomeric state of IN. (A-B) IN-IN HTRF interaction: (A) IN-IN interaction activation dose–response curves. Data represent the means of three independent experiments done in duplicate with standard deviations shown as error bars. (B) Correlation between AC50 of IN-IN interaction and EC50 of ARV activity on MT4 cells infected with HxB2 HIV-1 (R2 = 0.78). This study used 21 of the set of 51 compounds. AC50 = concentration required to activate IN-IN interaction by 50% of the maximum effect. (C-F) Size exclusion chromatography of IN: Binding of INLAIs BI-D or Mut101, to IN NL4-3 wt (C-D) or IN NL4-3 A128T (E-F), promotes a shift toward higher IN oligomeric state, independently of LEDGF. Blue peaks: elution of IN wt and IN A128T in the absence of compound. Red peaks: elution of IN wt and IN A128T in the presence of BI-D (C, E) or Mut101 (D, F).The elution volume and identification of each peak (numbered 1 to 7) are indicated in supplementary table S2.

Mentions: We evaluated the ability of IN-LEDGF inhibitors to promote modifications in the interaction between IN subunits as these inhibitors act at the IN dimer interface. We designed an HTRF-based assay to monitor the interaction between His6-IN/Flag-IN subunits. In the presence of compound concentrations the HTRF signal corresponding to the His6-IN/Flag-IN interaction was more than twice as strong as the signal obtained in the absence of compound (Figure 4A). The concentration required to activate the IN-IN interaction by 50% (AC50) closely correlated with the inhibition of the IN-LEDGF interaction and the antiretroviral activity EC50 (Figure 4B). Raltegravir had no effect on either the IN-LEDGF interaction or IN-IN interaction (data not shown). These results are in agreement with previously reported observations on the effect of some LEDGINs and tBPQAs on IN-IN interactions [35-37]. In order to determine if this enhancement of IN-IN interaction corresponds to a change toward higher IN oligomerization state, we performed size exclusion chromatography of IN that has been or not preincubated with Mut101 or with the related compound BI-D. As shown in Figure 4C-D and on Additional file 1: Table S2 for the elution volumes of the different peaks, while IN wt in the absence of INLAIs behaves as an IN dimer (blue peaks), pre-incubation with Mut101 or BI-D results in higher IN oligomerization state (red peaks), that likely corresponds to a partial formation of IN tetramer. Raltegravir had no effect (data not shown). In contrast with some LEDGINs previously described [18], Mut101 and BI-D conserved full ARV activity on the HIV-1 mutant IN A128T and full in vitro activity on the IN NL4-3 A128T protein mutant. So, we performed similar experiments with this IN A128T protein. As shown in Figure 4E-F and on Additional file 1: Table S2, the higher IN oligomerization state promoted by binding of Mut101 or BI-D to the LEDGF binding pocket, corresponds clearly to a shift from IN dimer (blue peaks) toward IN tetramer (red peaks). This slight difference between the results obtained with IN wt and the IN A128T mutant is likely due to a more soluble behavior of the IN A128T mutant protein compared to IN wt. In both experiments we did not observe the formation of IN aggregates of very high molecular weight, except for a very minor peak (peak 7) after incubation of IN A128T with Mut101, which elution volume (see Additional file 1: Table S2) could correspond to the formation of such aggregates. However, we cannot exclude that insoluble aggregates are formed but do not enter the gel filtration matrix.


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)

Effect of INLAIs on the oligomeric state of IN. (A-B) IN-IN HTRF interaction: (A) IN-IN interaction activation dose–response curves. Data represent the means of three independent experiments done in duplicate with standard deviations shown as error bars. (B) Correlation between AC50 of IN-IN interaction and EC50 of ARV activity on MT4 cells infected with HxB2 HIV-1 (R2 = 0.78). This study used 21 of the set of 51 compounds. AC50 = concentration required to activate IN-IN interaction by 50% of the maximum effect. (C-F) Size exclusion chromatography of IN: Binding of INLAIs BI-D or Mut101, to IN NL4-3 wt (C-D) or IN NL4-3 A128T (E-F), promotes a shift toward higher IN oligomeric state, independently of LEDGF. Blue peaks: elution of IN wt and IN A128T in the absence of compound. Red peaks: elution of IN wt and IN A128T in the presence of BI-D (C, E) or Mut101 (D, F).The elution volume and identification of each peak (numbered 1 to 7) are indicated in supplementary table S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Effect of INLAIs on the oligomeric state of IN. (A-B) IN-IN HTRF interaction: (A) IN-IN interaction activation dose–response curves. Data represent the means of three independent experiments done in duplicate with standard deviations shown as error bars. (B) Correlation between AC50 of IN-IN interaction and EC50 of ARV activity on MT4 cells infected with HxB2 HIV-1 (R2 = 0.78). This study used 21 of the set of 51 compounds. AC50 = concentration required to activate IN-IN interaction by 50% of the maximum effect. (C-F) Size exclusion chromatography of IN: Binding of INLAIs BI-D or Mut101, to IN NL4-3 wt (C-D) or IN NL4-3 A128T (E-F), promotes a shift toward higher IN oligomeric state, independently of LEDGF. Blue peaks: elution of IN wt and IN A128T in the absence of compound. Red peaks: elution of IN wt and IN A128T in the presence of BI-D (C, E) or Mut101 (D, F).The elution volume and identification of each peak (numbered 1 to 7) are indicated in supplementary table S2.
Mentions: We evaluated the ability of IN-LEDGF inhibitors to promote modifications in the interaction between IN subunits as these inhibitors act at the IN dimer interface. We designed an HTRF-based assay to monitor the interaction between His6-IN/Flag-IN subunits. In the presence of compound concentrations the HTRF signal corresponding to the His6-IN/Flag-IN interaction was more than twice as strong as the signal obtained in the absence of compound (Figure 4A). The concentration required to activate the IN-IN interaction by 50% (AC50) closely correlated with the inhibition of the IN-LEDGF interaction and the antiretroviral activity EC50 (Figure 4B). Raltegravir had no effect on either the IN-LEDGF interaction or IN-IN interaction (data not shown). These results are in agreement with previously reported observations on the effect of some LEDGINs and tBPQAs on IN-IN interactions [35-37]. In order to determine if this enhancement of IN-IN interaction corresponds to a change toward higher IN oligomerization state, we performed size exclusion chromatography of IN that has been or not preincubated with Mut101 or with the related compound BI-D. As shown in Figure 4C-D and on Additional file 1: Table S2 for the elution volumes of the different peaks, while IN wt in the absence of INLAIs behaves as an IN dimer (blue peaks), pre-incubation with Mut101 or BI-D results in higher IN oligomerization state (red peaks), that likely corresponds to a partial formation of IN tetramer. Raltegravir had no effect (data not shown). In contrast with some LEDGINs previously described [18], Mut101 and BI-D conserved full ARV activity on the HIV-1 mutant IN A128T and full in vitro activity on the IN NL4-3 A128T protein mutant. So, we performed similar experiments with this IN A128T protein. As shown in Figure 4E-F and on Additional file 1: Table S2, the higher IN oligomerization state promoted by binding of Mut101 or BI-D to the LEDGF binding pocket, corresponds clearly to a shift from IN dimer (blue peaks) toward IN tetramer (red peaks). This slight difference between the results obtained with IN wt and the IN A128T mutant is likely due to a more soluble behavior of the IN A128T mutant protein compared to IN wt. In both experiments we did not observe the formation of IN aggregates of very high molecular weight, except for a very minor peak (peak 7) after incubation of IN A128T with Mut101, which elution volume (see Additional file 1: Table S2) could correspond to the formation of such aggregates. However, we cannot exclude that insoluble aggregates are formed but do not enter the gel filtration matrix.

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