KDM5A/B contribute to HIV-1 latent infection and survival of HIV-1 infected cells

Abstract

Combinational antiretroviral therapy (cART) suppresses human immunodeficiency virus type 1 (HIV-1) viral replication and pathogenesis in acquired immunodeficiency syndrome (AIDS) patients. However, HIV-1 remains in the latent stage of infection by suppressing viral transcription, which hinders an HIV-1 cure. One approach for an HIV-1 cure is the "shock and kill" strategy. The strategy focuses on reactivating latent HIV-1, inducing the viral cytopathic effect and facilitating the immune clearance for the elimination of latent HIV-1 reservoirs. Here, we reported that the H3K4 trimethylation (H3K4me3)-specific demethylase KDM5A/B play a role in suppressing HIV-1 Tat/LTR-mediated viral transcription in HIV-1 latent cells. Furthermore, we evaluated the potential of KDM5-specific inhibitor JQKD82 as an HIV-1 "shock and kill" agent. Our results showed that JQKD82 increases the H3K4me3 level at HIV-1 5' LTR promoter regions, HIV-1 reactivation, and the cytopathic effects in an HIV-1-latent T cell model. In addition, we identified that the combination of JQKD82 and AZD5582, a non-canonical NF-κB activator, generates a synergistic impact on inducing HIV-1 lytic reactivation and cell death in the T cell. The latency-reversing potency of the JQKD82 and AZD5582 pair was also confirmed in peripheral blood mononuclear cells (PBMCs) isolated from HIV-1 aviremic patients and in an HIV-1 latent monocyte. In latently infected microglia (HC69) of the brain, either deletion or inhibition of KDM5A/B results in a reversal of the HIV-1 latency. Overall, we concluded that KDM5A/B function as a host repressor of the HIV-1 lytic reactivation and thus promote the latency and the survival of HIV-1 infected reservoirs.

Keywords: AZD5582; CD4(+) T cells; Demethylation; Elimination; HIV-1; JQKD82; KDM5; Latency; Lytic reactivation; Microglia; Monocytes; Reservoir.

Fig. 1.

siRNA knockdown of KDM5A/B increases the HIV-1 Tat/LTR-driven transcription. TZM-bl cells were received from reverse transfection by KDM5A siRNA # 1–2 or KDM5B siRNA #1–2 for 48h, and cells were trypsinized and reverse transfected by pQC empty vector or pQC-HIV-1 Tat for 48h. KDM5A siRNA KD cells were harvested and lysed for RT-qPCR of KDM5A mRNA and IB of anti-KDM5A, GAPDH, and H3K4me3/H3 (A, C). KDM5B siRNA KD cells were harvested and lysed for RT-qPCR of KDM5B and IB of anti-KDM5B, GAPDH, and H3K4me3/H3 (B, D). KDM5A siRNA KD Cells with or without HIV-1 Tat overexpression were harvested for luciferase reporter assay (E). KDM5B siRNA KD cells with or without HIV-1 Tat overexpression were harvested for luciferase reporter assay (F). The readouts of RLU/total protein input (μg) were normalized with the siNT/pQC-empty vector-transfected TZM-bl control group. Results were calculated from at least 3 independent experiments and presented as mean±standard error of the mean (SEM). (**p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA and Tukey’s multiple comparison test compared to the same treated siNT control).

Fig. 2.

KDM5 inhibitor JQKD82 induces the HIV-1 reactivation, cell death, and H3K4me3 at HIV-1 LTR in the latent infected CA5 cells. CA5 cells were treated with 0, 10, or 25 μM JQKD82 for 5 days (A). Cells were harvested for LIVE/DEAD staining and analyzed with flow cytometry to identify the expression of HIV-1 LTR-driven GFP (B) and LIVE/DEAD-APC (C). CA5 cells were treated with DMSO or 25 μM JQKD82 for 5 days and then harvested to perform the ChIP qPCR assay of H3K4me3 focusing on HIV-1 LTR Nuc-0 and Nuc-1 sites (D). Results were calculated from at least 3 independent experiments and presented as mean±standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ****p < 0.0001 by one-way/two-way ANOVA and Tukey’s multiple comparison test compared to untreated (B, D) or parental cells control group (C)).

Fig. 3.

JQKD82/AZD5582 combination treatment synergistically increases the HIV-1 reactivation and cell death in CA5 cells. CA5 cells were treated with 0, 10, or 25 μM JQKD82 for 3 days and refreshed the JQKD82-treated medium with or without 0.2 μM AZD5582 for 48h. Cells were performed the LIVE/DEAD staining and analyzed by flow cytometry (A). Treated CA5 cells were analyzed by the GFP expression from the HIV-1 reactivation. Results were calculated from 3 independent experiments and were presented as mean±standard error of the mean (SEM) (B). (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by 2-way ANOVA and Tukey’s multiple comparison test compared to 0 μM JQKD82-treated control). Treated CA5 and parental Jurkat cells were analyzed for the LIVE/DEAD APC expression (C). Results were calculated from 3 independent experiments and were presented as mean±standard error of the mean (SEM). (*p < 0.05; **p < 0.01 by 3-way ANOVA and Tukey’s multiple comparison test compared to Jurkat cells under the same treatment.). Jurkat and CA5 cells were treated with JQKD82/AZD and harvested for immunoblotting (D).

Fig. 4.

JQKD82/AZD5582 combinatory treatment induced HIV-1/SIV reactivation in the primary T cells, HIV-1 patients’ PBMCs, and SIV-infected macaque cells. The primary Tcm latency model was established by DHIV-1 infection. The DHIV-1 infected Tcm cells were back to latency and treated with DMSO or 10 μM JQKD82 for 3 days and then refreshed the treated medium for JQKD82-treated medium for an additional 3 days with or without 0.1 μM AZD5582. Cells were harvested for RNA extraction and RT-qPCR analysis for HIV-1 Gag mRNA level (A). The HIV-1 patients’ PBMC with CD8⁺ T cell-depletion for Donor #1–7 and treated with DMSO or 10 μM JQKD82 for 3 days. Then these cells were refreshed with the JQKD82-treated medium for an additional 3 days with or without 0.1 μM AZD5582. Cultured supernatant was harvested for viral RNA extraction and ultrasensitive nested RT-qPCR analysis and normalized the viral RNA copies with HIV-1 III titration standard curve. Results were presented in combination (B) or for individual donor (C). SIV-infected human T cells were subjected to the same treatment of DHIV-infected Tcm cells, followed by RNA extraction and RT-qPCR analysis for SIV Gag mRNA level (D). SIV-infected macaque PBMCs were subjected to the same treatment of HIV-1 patients’ PBMCs, followed by RNA extraction and RT-qPCR analysis for SIV Gag mRNA level (E). Results were calculated from 3 technical repeats and presented as mean±standard error of the mean (SEM). (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA and Tukey’s multiple comparison test compared higher to mock control group).

Fig. 5.

KDM5 inhibitor JQKD82 or siRNA knockdown of KDM5A/B induced the HIV-1 latency reactivation in HC69 microglia cells. HC69 microglia cells were received from reverse transfection by KDM5A siRNA # 1–2, or KDM5B siRNA #1–2 for 72hr (A). The siRNA-transfected cells were harvested for RNA extraction and RT-qPCR to identify the siRNA KD efficiency. Cells were harvested for LIVE/DEAD staining and analyzed with flow cytometry to identify the expression of HIV-1 LTR-driven GFP (B, C) and cell death (D). HC69 microglial cells were treated with 0, 10, 25, or 50 μM JQKD82 for 5 days (E). Cells were harvested for LIVE/DEAD staining and analyzed with flow cytometry to identify the expression of HIV-1 LTR-driven GFP (F) and LIVE/DEAD-APC (G). HC69 cells were treated with JQKD82 and lysed for IB of total H3K4me3 level (H). Results were calculated from 3 independent experiments and presented as mean±standard error of the mean (SEM). (***p < 0.001; ****p < 0.0001 by one-way ANOVA and Tukey’s multiple comparison test compared to the untreated or siNT control group).

Fig. 6.

JQKD82 in combination with AZD5582 enhanced HIV-1 lytic reactivation in U1/HIV-1 monocytic cell line of HIV-1 latency. U1/HIV-1 cells were treated with JQKD82 alone at the indicated doses for 3 days followed by the JQKD82 ± AZD5582 (0.2 μM) co-treatment for 2 days, were harvested and subjected to flow cytometry analysis of HIV-1 lytic reactivation (A). The percentage of HIV-1 reactivated cells was determined by intracellular immunostaining of HIV-1 Gag protein (B). Cells in (A) were harvested and subjected to RNA extractions and RT-qPCR assays to measure HIV-1 Gag mRNA level (C). Cells were harvested and subjected to LIVE/DEAD assays to measure cell death (D). Results were calculated from at least 3 independent experiments and presented as mean±standard error of the mean (SEM). (*p < 0.05; **p < 0.01; ***p < 0.001; by two-way ANOVA and Tukey’s multiple comparison test).