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

Abstract

Background: HIV-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.

Results: Here 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.

Conclusions: Although 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.

Keywords: Allosteric integrase inhibitor; HIV-1; INLAI; Immunoreactivity; Integrase; LEDGF; LEDGIN.

Fig. 1

Structure, biochemical and antiretroviral activities of MUT-A. a Chemical structure of MUT-A. b Titration of MUT-A ARV activity on HIV-1 NL4-3 and HxB2 strains in MT4 cell infection assay. c Biochemical activities of MUT-A on IN-LEDGF/p75 interaction (IC₅₀) and of IN multimerization (activation concentration AC₅₀ and maximum signal increase plateau). d EC₅₀ of ARV activities of MUT-A and references HIV-1 NL4-3 and HxB2 strains in MT4 cell infection assay. DTG: INSTI Dolutegravir. e–g Correlations between IN multimerization (IN–IN) AC₅₀ and IN-LEDGF/p75 IC₅₀ (e), ARV EC₅₀ and IN-LEDGF IC₅₀ (f), ARV EC₅₀s and IN–IN AC₅₀s (g) of MUT-A compound series

Fig. 2

Resistance profile of MUT-A. a Comparative resistance profile of MUT-A and references (EVG, BI-D and BI-224436). The color code subdivides the fold change in resistance into five levels of magnitude. b Long-term culture of infected MT4 cells with escalating concentrations of drugs. Cells were initially infected at 0.005 MOI with HIV-1 and were passaged twice weekly in the presence of RAL (filled circle), NVP (filled diamond), or MUT-A (filled upward triangle). The serial passage was carried out over 67 days. The highest concentration of compounds at which the virus could replicate in culture are shown. Error bars represent the variation obtained in one experiment done in triplicate. c Frequency of mutations in IN sequences. Cloned sequences were analyzed at day 23, 43 and 67 from 30 to 40 individual clones (% of total clones analyzed)

Fig. 3

Antiviral activity in infection assays with HIV-1 and SIV. Dose–response curves for RAL (a), BI-D (b) and MUT-A (c) activity in MT4 cells infected with either HIV-1 NL4-3 (filled upward triangle) or SIVmac239 (filled circle). Error bars represent the variation obtained from three independent experiments done in duplicate

Fig. 4

Immunoreactivity of HIV-1 produced in the presence or absence of MUT-A. a Schematic of the antibody capture assay of native HIV-1 particles produced. The capacity of the different antibodies to capture native HIV-1 particles was assessed by ELISA. HIV-1 particles retained by the antibodies were lysed and quantified by CA-p24 detection by ELISA. b The different monoclonal and polyclonal antibodies used in virus-capture assays are listed together with their specific target and their ability to neutralize HIV-1. Nonspecific antibodies (polyclonal IgG HIV- and mAb Syn = Synagis) were used as negative controls. c Quantitation by CA-p24 ELISA of native HIV-1 particles produced in the presence (red bars) or absence (blue bars) of MUT-A that have been captured with the different antibodies used at three different concentrations

Fig. 5

T-cell proliferation induced by autologous MDDCs exposed to MUT-A-inactivated HIV-1 assessed by CFSE proliferation assay. Proliferation in response to autologous MDDCs exposed to wt HIV-1 (NL4-3 DMSO) or MUT-A or AT-2 inactivated HIV-1 in a 6-day co-culture was assessed in triplicates using the CFSE proliferation assay. MDDCs were pulsed with virus particles corresponding to 1 μg/mL of HIV-1 Gag CA-p24. A representative (out of 3 independent experiments) flow cytometry dot plots showing lymphocyte gating strategy by FSC/SSC and CD3⁺ positive staining, as well as dot plots showing CFSE dilution in gated CD3⁺ CD4⁺ T lymphocytes after in vitro stimulation with the above mentioned virus or negative (medium) and positive SEA controls

Fig. 6

CD4⁺ and CD8⁺ T-cell proliferation induced by autologous MDDCs exposed to MUT-A-inactivated HIV-1. a Proliferation in response to autologous MDDC exposed to wt HIV-1 (NL4-3 DMSO) or MUT-A inactivated HIV-1 or AT-2-inactivated HIV-1 in a 6-day co-culture was assessed in triplicates using the CFSE proliferation assay. MDDCs were pulsed with either 5 μg/mL HIV-1 (NL4-3 DMSO or MUT-A) or 1 μg/mL HIV-1 (NL4-3 AT-2) of HIV-1 gag CA-p24. A representative (out of three independent experiments) flow cytometry dot plots showing CFSE dilution in gated CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T lymphocytes after in vitro stimulation with the above mentioned viruses or negative (medium) and positive SEA controls. b, c Quantitation of T CD4⁺ (B) and CD8⁺ (C) proliferation in response to autologous MDDC exposed to wt HIV virus (NL4-3 DMSO) or inactivated HIV virus (MUT-A or NL4-3 AT-2) in a 6-day co-culture was assessed in triplicates using the CFSE proliferation assay. In all viruses used, MDDCs were pulsed with 1 µg/mL of HIV gag CA-p24. Graph showing mean ± SD of percentages of CFSE low CD3⁺ CD4⁺ (b) and CD3⁺ CD8⁺ (c) T cells from three different co-cultures are represented

Fig. 7

Secretion of IL-12, IFN-γ, IL2-R and MIP-1β induced by autologous MDDCs exposed to MUT-A-inactivated HIV-1. Cytokine and chemokine secretion in response to autologous MDDCs exposed to wt HIV-1 (NL4-3 DMSO) or MUT-A-inactivated HIV-1 (MUT-A 1 µM) in supernatants from a 6-day co-culture was assessed in duplicates by Luminex assay. Graph representing mean ± SD concentrations (pg/mL) of IL-12 (a), IFN-γ (b), IL2-R (c), and MIP-1β (d) analyzed after three different co-cultures

Fig. 8

IL-5, IL-6, IL-10, IL-13, MIP-1α, MCP-1 and IP-10 secretion induced by MUT-A-HIV-1-exposed autologous MDDCs. Cytokine and chemokine secretion in response to autologous MDDCs exposed to wt HIV-1 (NL4-3 DMSO) or MUT-A-inactivated HIV-1 (MUT-A 1 µM) in supernatants from a 6-day co-culture was assessed in duplicates by Luminex assay. Graph representing mean ± SD concentrations (pg/mL) of IL-10 (a), IL-6 (b), IL-13 (c), MIP-1α (d), MCP-1 (e), IL-5 (f) and IP-10 (g) analyzed after three different co-cultures