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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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Model accounting for the discrepancy between the dual ARV activities of INLAIs at integration and post-integration. The full replication cycle of HIV is represented with steps occurring in target cells (1) separated from the steps occurring in virus-producing cells (2) by a red line. (1) Inhibitory activity at integration and competition by LEDGF in HIV-1 target cells. LEDGF is present as a chromatin-bound protein in the nucleus but is absent in the cytoplasm. After infection of target cells, IN associated with the entering virus (in blue) is imported into the nucleus as part of the pre-integration complex (PIC). Mut101 and INLAIs (yellow triangles) can bind to IN, inhibiting integration by allosteric inhibition of IN strand transfer activity and preventing IN-LEDGF complex formation. In the nucleus of target cells, LEDGF will compete with Mut101 and INLAIs for binding to IN, thus lowering their apparent affinity for IN and counteracting their antiretroviral activity at the integration stage of the replication cycle. (2) After integration, progeny virions are assembled in the cytoplasm and at the plasma membrane. INLAIs can bind to the Pol polyprotein precursor containing IN or to the matured IN, in the absence of competing LEDGF. Upon binding, these inhibitors promote conformational modification and enhancement of the IN-IN interaction resulting in IN inactivation (in red). Mut101 and INLAIs activity at the post-integration stage is stronger than their activity at integration as there is no competition with LEDGF in the cytoplasmic cellular compartment.
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Figure 8: Model accounting for the discrepancy between the dual ARV activities of INLAIs at integration and post-integration. The full replication cycle of HIV is represented with steps occurring in target cells (1) separated from the steps occurring in virus-producing cells (2) by a red line. (1) Inhibitory activity at integration and competition by LEDGF in HIV-1 target cells. LEDGF is present as a chromatin-bound protein in the nucleus but is absent in the cytoplasm. After infection of target cells, IN associated with the entering virus (in blue) is imported into the nucleus as part of the pre-integration complex (PIC). Mut101 and INLAIs (yellow triangles) can bind to IN, inhibiting integration by allosteric inhibition of IN strand transfer activity and preventing IN-LEDGF complex formation. In the nucleus of target cells, LEDGF will compete with Mut101 and INLAIs for binding to IN, thus lowering their apparent affinity for IN and counteracting their antiretroviral activity at the integration stage of the replication cycle. (2) After integration, progeny virions are assembled in the cytoplasm and at the plasma membrane. INLAIs can bind to the Pol polyprotein precursor containing IN or to the matured IN, in the absence of competing LEDGF. Upon binding, these inhibitors promote conformational modification and enhancement of the IN-IN interaction resulting in IN inactivation (in red). Mut101 and INLAIs activity at the post-integration stage is stronger than their activity at integration as there is no competition with LEDGF in the cytoplasmic cellular compartment.

Mentions: Mut101 displays weak activity at early stage integration and potent activity at late stage production of defective virions. We then explored how a compound acting on a unique target (IN) and on a unique binding site (the LEDGF-binding pocket), displays such a difference between its potency on two ARV activities. The ARV activity of Mut101 series INLAIs and their inhibition of the IN-LEDGF interaction are clearly linked. There is a tight correlation between their action on IN-LEDGF interaction inhibition and their activity on IN-IN interaction enhancement and IN conformational change. Further studies are required to resolve this issue. However, some clues are provided by Wang et al., who studied the ARV activity of a tBPQA compound (racemic BI-D) on wt and LEDGF KO mouse cells infected with a VSV-G-pseudotyped HIV-1 luciferase virus in SR infection experiments [40]. The EC50 of racemic BI-D ARV activity was between 2.4 μM and 2.9 μM when tested on wt cells but between 0.16 μM and 0.20 μM (15 to 18 times lower) on LEDGF KO cells, a result not significantly altered by HRP2 disruption. In contrast, the EC50 of Raltegravir was similar in each cell type. The authors suggest that LEDGF, present in wt cells but not in LEDGF KO cells, can compete with BI-D for binding to the LEDGF-binding pocket of IN. In the presence of a LEDGF competitor in wt cells, the concentrations of BI-D required to achieve similar ARV activity are higher than when LEDGF is absent in KO cells. Strikingly, we found that the EC50 of BI-D ARV activity on MT4 human cells infected with HIV-1 NL4-3 was 2.4 μM ± 0.5 in SR and 0.17 μM ± 0.03 in MR infection assays. This is very similar to the result found by Wang et al. (Table 5), although they worked with mouse cells and we worked with human cells. The data strongly suggest that a mechanism similar to that observed by Wang et al. (LEDGF competition in SR assay and no competition by LEDGF in MR assay), could explain the difference in ARV activity we found for INLAIs assayed in SR and MR infection assays. These data, and our in vitro data showing that LEDGF can compete with Mut101 for binding to IN, support the model illustrated in Figure 8 concerning the considerable difference in the potency of INLAIs between their low ARV activity at integration and their much higher activity inhibiting the production of infectious particles at post-integration stages, although both activities are due to the occupation of the same binding site on IN. The inhibition of HIV-1 integration by INLAIs, measured in SR infection assays, is based on the impairment of the IN-LEDGF interaction and allosteric inhibition of IN. This takes place in the nucleus of HIV-1 target cells. In this cellular compartment LEDGF is abundant and can compete effectively with INLAIs for binding to IN, limiting ARV activity of these inhibitors at this stage. In contrast, the activity of INLAIs at the virus production stage, as measured in MR assays, takes place in the cytoplasm of virus-producer cells after integration. LEDGF, a chromatin-bound nuclear protein, is absent from this cellular compartment and cannot compete with INLAIs for binding to IN or to the Pol polyprotein containing IN [42]. INLAIs are able to target both the IN associated with incoming virions at the step of integration in target cells (in the nucleus, in the presence of competing LEDGF) and the newly synthesized IN in producer cells (associated with progeny virions in the cytoplasm or at the plasma membrane, in the absence of LEDGF). This model suggests that the activity of a protein-protein interaction inhibitor (in this case, concerning the interaction between a viral and a cellular protein) is governed not only by its intrinsic affinity for its target, but also by the cellular compartment in which it is acting. It is the presence or absence of the partner protein of the inhibitor target that could, by competitive binding, negatively affect the level of inhibitor activity.


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)

Model accounting for the discrepancy between the dual ARV activities of INLAIs at integration and post-integration. The full replication cycle of HIV is represented with steps occurring in target cells (1) separated from the steps occurring in virus-producing cells (2) by a red line. (1) Inhibitory activity at integration and competition by LEDGF in HIV-1 target cells. LEDGF is present as a chromatin-bound protein in the nucleus but is absent in the cytoplasm. After infection of target cells, IN associated with the entering virus (in blue) is imported into the nucleus as part of the pre-integration complex (PIC). Mut101 and INLAIs (yellow triangles) can bind to IN, inhibiting integration by allosteric inhibition of IN strand transfer activity and preventing IN-LEDGF complex formation. In the nucleus of target cells, LEDGF will compete with Mut101 and INLAIs for binding to IN, thus lowering their apparent affinity for IN and counteracting their antiretroviral activity at the integration stage of the replication cycle. (2) After integration, progeny virions are assembled in the cytoplasm and at the plasma membrane. INLAIs can bind to the Pol polyprotein precursor containing IN or to the matured IN, in the absence of competing LEDGF. Upon binding, these inhibitors promote conformational modification and enhancement of the IN-IN interaction resulting in IN inactivation (in red). Mut101 and INLAIs activity at the post-integration stage is stronger than their activity at integration as there is no competition with LEDGF in the cytoplasmic cellular compartment.
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Figure 8: Model accounting for the discrepancy between the dual ARV activities of INLAIs at integration and post-integration. The full replication cycle of HIV is represented with steps occurring in target cells (1) separated from the steps occurring in virus-producing cells (2) by a red line. (1) Inhibitory activity at integration and competition by LEDGF in HIV-1 target cells. LEDGF is present as a chromatin-bound protein in the nucleus but is absent in the cytoplasm. After infection of target cells, IN associated with the entering virus (in blue) is imported into the nucleus as part of the pre-integration complex (PIC). Mut101 and INLAIs (yellow triangles) can bind to IN, inhibiting integration by allosteric inhibition of IN strand transfer activity and preventing IN-LEDGF complex formation. In the nucleus of target cells, LEDGF will compete with Mut101 and INLAIs for binding to IN, thus lowering their apparent affinity for IN and counteracting their antiretroviral activity at the integration stage of the replication cycle. (2) After integration, progeny virions are assembled in the cytoplasm and at the plasma membrane. INLAIs can bind to the Pol polyprotein precursor containing IN or to the matured IN, in the absence of competing LEDGF. Upon binding, these inhibitors promote conformational modification and enhancement of the IN-IN interaction resulting in IN inactivation (in red). Mut101 and INLAIs activity at the post-integration stage is stronger than their activity at integration as there is no competition with LEDGF in the cytoplasmic cellular compartment.
Mentions: Mut101 displays weak activity at early stage integration and potent activity at late stage production of defective virions. We then explored how a compound acting on a unique target (IN) and on a unique binding site (the LEDGF-binding pocket), displays such a difference between its potency on two ARV activities. The ARV activity of Mut101 series INLAIs and their inhibition of the IN-LEDGF interaction are clearly linked. There is a tight correlation between their action on IN-LEDGF interaction inhibition and their activity on IN-IN interaction enhancement and IN conformational change. Further studies are required to resolve this issue. However, some clues are provided by Wang et al., who studied the ARV activity of a tBPQA compound (racemic BI-D) on wt and LEDGF KO mouse cells infected with a VSV-G-pseudotyped HIV-1 luciferase virus in SR infection experiments [40]. The EC50 of racemic BI-D ARV activity was between 2.4 μM and 2.9 μM when tested on wt cells but between 0.16 μM and 0.20 μM (15 to 18 times lower) on LEDGF KO cells, a result not significantly altered by HRP2 disruption. In contrast, the EC50 of Raltegravir was similar in each cell type. The authors suggest that LEDGF, present in wt cells but not in LEDGF KO cells, can compete with BI-D for binding to the LEDGF-binding pocket of IN. In the presence of a LEDGF competitor in wt cells, the concentrations of BI-D required to achieve similar ARV activity are higher than when LEDGF is absent in KO cells. Strikingly, we found that the EC50 of BI-D ARV activity on MT4 human cells infected with HIV-1 NL4-3 was 2.4 μM ± 0.5 in SR and 0.17 μM ± 0.03 in MR infection assays. This is very similar to the result found by Wang et al. (Table 5), although they worked with mouse cells and we worked with human cells. The data strongly suggest that a mechanism similar to that observed by Wang et al. (LEDGF competition in SR assay and no competition by LEDGF in MR assay), could explain the difference in ARV activity we found for INLAIs assayed in SR and MR infection assays. These data, and our in vitro data showing that LEDGF can compete with Mut101 for binding to IN, support the model illustrated in Figure 8 concerning the considerable difference in the potency of INLAIs between their low ARV activity at integration and their much higher activity inhibiting the production of infectious particles at post-integration stages, although both activities are due to the occupation of the same binding site on IN. The inhibition of HIV-1 integration by INLAIs, measured in SR infection assays, is based on the impairment of the IN-LEDGF interaction and allosteric inhibition of IN. This takes place in the nucleus of HIV-1 target cells. In this cellular compartment LEDGF is abundant and can compete effectively with INLAIs for binding to IN, limiting ARV activity of these inhibitors at this stage. In contrast, the activity of INLAIs at the virus production stage, as measured in MR assays, takes place in the cytoplasm of virus-producer cells after integration. LEDGF, a chromatin-bound nuclear protein, is absent from this cellular compartment and cannot compete with INLAIs for binding to IN or to the Pol polyprotein containing IN [42]. INLAIs are able to target both the IN associated with incoming virions at the step of integration in target cells (in the nucleus, in the presence of competing LEDGF) and the newly synthesized IN in producer cells (associated with progeny virions in the cytoplasm or at the plasma membrane, in the absence of LEDGF). This model suggests that the activity of a protein-protein interaction inhibitor (in this case, concerning the interaction between a viral and a cellular protein) is governed not only by its intrinsic affinity for its target, but also by the cellular compartment in which it is acting. It is the presence or absence of the partner protein of the inhibitor target that could, by competitive binding, negatively affect the level of inhibitor activity.

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