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

Erwann Le Rouzic,Céline Amadori,Alessia Zamborlini,Olivier Delelis,Marc Ruff,Sophie Chasset,Frédéric Le Strat,Eric Deprez,Nicolas Lévy,Francis Chevreuil,Julie Brias,Juliette Nguyen,Jean-Michel Bruneau,Damien Bonnard,Sophie Vomscheid,Sylvia Eiler,Roxane Beauvoir,Stéphane Emiliani,Benoît Ledoussal ,F Moreau ,Ali Saı̈b ,Richard Bénarous

doi:10.1186/1742-4690-10-144

Abstract

BackgroundLEDGF/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.ResultsWe 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.ConclusionMut101 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.

Highlights

LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN)
MT4 cells were infected with HxB2 HIV-1 and a subset of 51 compounds showed a good correlation between their ARV activity and their ability to inhibit IN-catalytic domain (CCD)/LEDGF-integrase binding domain (IBD) or IN-LEDGF interactions (Figures 1B and D)
Effect of IN-LEDGF inhibitors on IN strand transfer and 3’ processing activities is independent of LEDGF We found that these compounds inhibited the IN strand transfer activity as quantitated by ELISA assay (Figure 3A), in agreement with previously reported data, with IC50 values in a similar range to those found for inhibition of the IN-LEDGF interaction (Table 1)

Summary

Introduction

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 second generation of INSTIs, less sensitive to drugresistance mutations, has been approved (Dolutegravir (DTG) from GSK-Shionogi-ViiV). DTG belongs to the same class of compounds and remains sensitive to the strongest INSTI resistance mutations [5,6]. This highlights the need for integration inhibitors with completely different mechanism of action

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