Retinoic acid enhances HIV-1 reverse transcription and transcription in macrophages via mTOR-modulated mechanisms

Abstract

The intestinal environment facilitates HIV-1 infection via mechanisms involving the gut-homing vitamin A-derived retinoic acid (RA), which transcriptionally reprograms CD4⁺ T cells for increased HIV-1 replication/outgrowth. Consistently, colon-infiltrating CD4⁺ T cells carry replication-competent viral reservoirs in people with HIV-1 (PWH) receiving antiretroviral therapy (ART). Intriguingly, integrative infection in colon macrophages, a pool replenished by monocytes, represents a rare event in ART-treated PWH, thus questioning the effect of RA on macrophages. Here, we demonstrate that RA enhances R5 but not X4 HIV-1 replication in monocyte-derived macrophages (MDMs). RNA sequencing, gene set variation analysis, and HIV interactor NCBI database interrogation reveal RA-mediated transcriptional reprogramming associated with metabolic/inflammatory processes and HIV-1 resistance/dependency factors. Functional validations uncover post-entry mechanisms of RA action including SAMHD1-modulated reverse transcription and CDK9/RNA polymerase II (RNAPII)-dependent transcription under the control of mammalian target of rapamycin (mTOR). These results support a model in which macrophages residing in the intestine of ART-untreated PWH contribute to viral replication/dissemination in an mTOR-sensitive manner.

Keywords: ART; CCR5; CDK9; CP: Immunology; CP: Microbiology; HIV-1; RARα; RNAPII; SAMHD1; mTOR; monocyte-derived macrophages; retinoic acid.

Figure 1.. ATRA increases CCR5 expression and R5 HIV-1 replication in macrophages

(A) The experimental flowchart. Briefly, monocyte-derived macrophages (MDMs) were obtained by culturing monocytes in medium containing M-CSF (20 ng/mL) for 6 days. MDMs were exposed (ATRA-MDMs) or not (DMSO-MDMs) to ATRA (10 nM) before and after HIV-1 exposure. (B and C) Prior to HIV-1 exposure, MDMs were analyzed by flow cytometry upon staining with CD4, CCR5, and CXCR4 antibodies. Shown are histograms for CD4, CCR5, and CXCR4 expression on MDMs from one representative donor (B), and statistical analysis of CD4, CCR5, and CXCR4 MFI expression on MDMs from n = 14 participants. In parallel, MDMs were exposed to replication-competent CCR5-tropic HIV-1 strains (HIV_NL4.3BaL; T/F HIV_THRO) and cultured in medium containing M-CSF in the presence/absence of ATRA for 15 additional days. Cell-culture supernatants were collected, and fresh medium containing M-CSF and/or ATRA was added every 3 days. (D–G) MDMs exposed to HIV_NL4.3BaL (D and E) and T/F HIV_THRO (F and G) were analyzed for HIV-DNA integration by PCR at day 3 post infection (D and F) and viral replication by HIV-p24 ELISA every 3 days up to 15 days post infection (E and G). Shown are the kinetics of HIV-1 replication in one representative donor (E and G, left panels) and statistical analysis performed at day 9 post infection for n = 8 participants (E and G, middle and right panels). Wilcoxon p values are on the graphs.

Figure 2.. HIV-1 entry assay in ATRA-treated macrophages

The Blam-Vpr HIV-1 entry assay was performed in DMSO-MDMs and ATRA-MDMs, using the single-round CCR5-tropic ADA-Env HIV-1 (NL4.3Env⁻Vpr⁻/ADA-Env/Blam-Vpr; HIV_Blam-ADA) or VSV-G-pseudotyped HIV-1 (NL4.3Env⁻Vpr⁻/VSV-G-Env/Blam-Vpr; HIV_Blam-VSV-G) viruses containing the β-lactamase (Blam)-Vpr protein chimera. MDMs were loaded with CCF2-AM and analyzed by flow cytometry for the change in CCF2 fluorescence from green (520 nm; AmCyan channel; uncleaved) to blue (447 nm; Alexa Fluor 405 channel; cleaved) upon Blam-mediated cleavage. Shown are representative dot plots for cleaved and uncleaved CCF2-AM in DMSO-MDMs versus ATRA-MDMs pretreated or not with MVC (A). Statistical analyses of HIV entry in MDMs exposed to HIV_Blam-ADA (B) or HIV_Blam-VSV-G (C). Friedman and uncorrected Dunn’s p values are on the graphs.

Figure 3.. ATRA increases HIV-1 replication at post-entry levels before and after integration

(A) Shown is the experimental flowchart, when MDMs were exposed to single-round VSV-G pseudotyped HIV-1 (HIV_VSV-G) and treated with ATRA (10 nM) before and after infection (A–C). HIV-DNA integration and HIV-p24 levels were quantified at day 3 post infection. Shown are the levels of early reverse transcripts (RU5) (B, left panel), late reverse transcripts (Gag) (B, middle panel), and integrated HIV-DNA (B, right panel), as well as (C) HIV-p24 levels in cell-culture supernatants. (D) Shown is the experimental flowchart when MDMs were exposed to ATRA (10 nM) at day 2 post infection. At day 3 post -infection, cells were collected and analyzed for HIV-DNA integration by real-time nested PCR and for GFP and intracellular HIV-p24 expression by flow cytometry. Shown are statistical analysis of HIV-DNA integration (E), GFP, and HIV-p24 co-expression in MDMs from one representative donor (F), as well as statistical analysis of the frequency of viable (G) infected (GFP⁺HIV-p24⁺) MDMs (H), the GFP (I, left panel), and HIV-p24 (I, right panel) MFI expression from n = 9 participants. Finally, shown are statistical analysis of HIV-p24 levels in cell-culture supernatants measured by ELISA (J). Experiments were performed on MDMs from n = 14 (A–C) or n = 9 (E–J) HIV-uninfected participants. Wilcoxon p values are indicated on the graphs.

Figure 4.. ATRA transcriptionally reprograms MDMs for increased expression of HIV permissiveness transcripts and pathways

(A) Shown is the experimental flowchart. Briefly, RNA sequencing was performed on total RNA extracted from MDMs of n = 6 HIV-uninfected participants generated in the presence/absence of ATRA (10 nM) before infection. (B) Differentially expressed genes (DEGs) were analyzed for the presence of RA-responsive elements (RAREs) in their promoters, using the ENCODE bioinformatic tool (https://www.encodeproject.org). This allowed the identification of n = 2,271 DEGs that may represent putative direct RA transcriptional targets in ATRA-treated MDMs. (C and D) Further, gene set variation analysis (GSVA) was performed to identify signaling pathways modulated by ATRA in MDMs. Heatmaps depict top modulated signaling pathways in ATRA-treated MDMs (C), as well as gene sets (shown are selected transcripts) associated with three top modulated pathways: mTORC1, PI3K AKT mTOR, and Wnt/β-Catenin (D), as well as gene sets and selected transcripts modulated by ATRA in MDMs (p < 0.05; FC cutoff 1.3) matching the lists of genes included on the NCBI HIV interaction database (E). Heatmap cells are scaled by the expression level Z scores for each probe individually. Results from each donor are indicated with a different color code.

Figure 5.. mTOR inhibition counteracts the effect of ATRA on CCR5 expression and HIV replication and integration

MDMs generated as in Figure 1A were treated with the mTOR inhibitor INK128 (50 nM) 2 days before infection and the day of infection. (A) The representative flow cytometry histograms of extracellular CD4, CCR5, and CXCR4 expressions. (B) Statistical analyses of the relative MFI of CD4, CCR5, and CXCR4 expressions. (C and D) A fraction of MDMs, pretreated or not with INK128, were exposed to T/F HIV_THRO. Cells and cell-culture supernatants were harvested on day 3 post infection for the quantification of integrated HIV-DNA by nested real-time PCR (C) and soluble HIV-p24 by ELISA (D). (E and F) In parallel, another fraction of ATRA-MDMs and DMSO-MDMs pretreated with INK128 were exposed to HIV_VSV-G for 3 days. Shown are the levels of early reverse transcripts (RU5; E, left panel), late reverse transcripts (Gag; E, center panel), integrated HIV-DNA (Alu; E, right panel), as well as the levels of HIV-p24 (F). Experiments were performed on MDMs from n = 8 HIV-uninfected individuals. Repeated measures (RM) one-way ANOVA, Tukey’s test, and Wilcoxon p values are indicated on the graphs.

Figure 6.. ATRA modulates SAMHD1 phosphorylation in a mTOR-dependent manner

(A–D) MDMs were generated in the presence/absence of ATRA (10 nM) and pretreated or not with INK128 (50 nM), as in Figure 5. Prior to HIV-1 infection, cells were harvested for RT-PCR (A–C) and western blotting investigations (D–F). Shown are mRNA levels of Cyclin D2 (CCND2) (A), CDK1 (B), and SAMHD1 expressions (C), as well as levels of total (molecular weight [MW], 72 kDa) and phosphorylated (MW, 72 kDa) SAMHD1 protein, relative to β-actin (MW, 42 kDa) expression (D) in MDMs from eight different HIV-uninfected donors. (E and F) Graphs depict total (E) and phosphorylated SAMHD1 protein levels (F) normalized to β-actin expression. Experiments were performed on MDMs from n = 8 HIV-uninfected individuals. Friedman and uncorrected Dunn’s test p values are indicated on the graphs.

Figure 7.. ATRA promotes HIV-1 transcription via RARα/CDK9/RNAPII-dependent and/or mTOR-dependent mechanisms

(A) The THP1 monocytic cell line productively infected with a full-length NL4.3 HIV-1 was cultured in the presence/absence of INK128 (50 nM) 1 day before treatment with ATRA (10 nM) for 24 h. Chromatin samples were immunoprecipitated with RARα, CDK9, the phosphorylated RNA polymerase II (RNAPII) on its serine 2 or 5 antibodies, or with rabbit immunoglobulin (Ig) G as negative control. qPCRs were performed with primers amplifying specifically the 5′ LTR HIV-1, in the Nuc-1 region. Fold increases relative to IgG are presented, where fold enrichments for each immunoprecipitated DNA were calculated by the relative standard curve on input DNA. Values represent means of duplicate samples ± SD. One representative experiment out of three is presented. (B) In parallel, total RNA preparations from THP-1 cells were reverse transcribed. Unspliced HIV-1 RNAs (unspliced transcripts) were quantified by RT-qPCR using GAPDH and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as first normalizer and the DMSO-treated condition as second normalizer. Shown are results representative of two different experiments on THP-1 cells. Means from duplicate ± SD are indicated.