Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration

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

International audienceBACKGROUND: 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

The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity

International audienceAbstractBackgroundHIV-1 Integrase (IN) interacts with the cellular co-factor LEDGF/p75 and tethers the HIV preintegration complex to the host genome enabling integration. Recently a new class of IN inhibitors was described, the IN-LEDGF allosteric inhibitors (INLAIs). Designed to interfere with the IN-LEDGF interaction during integration, the major impact of these inhibitors was surprisingly found on virus maturation, causing a reverse transcription defect in target cells.ResultsHere we describe the MUT-A compound as a genuine INLAI with an original chemical structure based on a new type of scaffold, a thiophene ring. MUT-A has all characteristics of INLAI compounds such as inhibition of IN-LEDGF/p75 interaction, IN multimerization, dual antiretroviral (ARV) activities, normal packaging of genomic viral RNA and complete Gag protein maturation. MUT-A has more potent ARV activity compared to other INLAIs previously reported, but similar profile of resistance mutations and absence of ARV activity on SIV. HIV-1 virions produced in the presence of MUT-A were non-infectious with the formation of eccentric condensates outside of the core. In studying the immunoreactivity of these non-infectious virions, we found that inactivated HIV-1 particles were captured by anti-HIV-specific neutralizing and non-neutralizing antibodies (b12, 2G12, PGT121, 4D4, 10-1074, 10E8, VRC01) with efficiencies comparable to non-treated virus. Autologous CD4+ T lymphocyte proliferation and cytokine induction by monocyte-derived dendritic cells (MDDC) pulsed either with MUT-A-inactivated HIV or non-treated HIV were also comparable.ConclusionsAlthough strongly defective in infectivity, HIV-1 virions produced in the presence of the MUT-A INLAI have a normal protein and genomic RNA content as well as B and T cell immunoreactivities comparable to non-treated HIV-1. These inactivated viruses might form an attractive new approach in vaccine research in an attempt to study if this new type of immunogen could elicit an immune response against HIV-1 in animal models

MOESM1 of The HIV-1 integrase-LEDGF allosteric inhibitor MUT-A: resistance profile, impairment of virus maturation and infectivity but without influence on RNA packaging or virus immunoreactivity

Additional file 1. Supplementary methods. Chemical synthesis process of MUT-A,HTRF®-based IN-LEDGF interaction assay, HTRF®-based IN multimerization assay, Viral RNA isolation, Northern blot analyses, primer extension assays, RT activity assays, Cryo-electron microscopy of HIV particles, Determination of HIV-1 replication capacity. Figure S1: 1H NMR spectrum of MUT-A. Figure S2: Impact of MUT-A on HIV-1 replication and production. Figure S3: Analysis of genomic RNA, Reverse transcriptase and tRNALys3 primer in MUT-A-treated HIV-1. A-D. HIV-1 RNA packaging and thermal stability of HIV-1 RNA dimers. Figure S4: HIV-1 NL4-3 particles observed by cryo-EM. A. Figure S5: Replicative capacity of HIV-1 NL4-3 viruses bearing resistance mutations to INLAIs or INSTIs. Table S1: Immunoreactivity of HIV-1 NL4-3 produced in the presence of MUT-A, or Saquinavir, or after AT2 treatment, or in the presence of DMSO. Figure S6: Results of virus immunocapture from Table S1 represented in bar graphs