Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

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Science Advances  12 Aug 2020:
Vol. 6, no. 33, eaba6617
DOI: 10.1126/sciadv.aba6617
  • Fig. 1 Medium-throughput screen of fungal secondary metabolites combined with orthogonal fractionation and MS strategy coupled to latency reversal bioassays identifies GTX from growth supernatant of A. fumigatus to reverse HIV-1 latency.

    (A) Phylogenetic tree representing the main orders of the fungal kingdom with strains used in the current study, collapsed per order. Orders selected from the tree published (57), with some of the lower orders included for structural reasons. Approximate ecological trends in the orders are summarized by symbols, as follows: Embedded Image vertebrate pathogenicity prevalent, Embedded Image climatic extremotolerance prevalent, Embedded Image frequent production of extracellular metabolites or mycotoxins, Embedded Image frequent osmotolerance or growth in sugary fluids, Embedded Image numerous members with soilborne lifestyle, Embedded Image numerous members inhabiting decaying wood rich in hydrocarbons, Embedded Image frequent insect association, Embedded Image frequent mushroom decomposition or hyperparasitism on fungi or lichens, and Embedded Image frequent inhabitants of foodstuffs or vertebrate intestinal tracts. (B) Latency reversal bioassay performed by treatment of J-Lat A2 cells with increasing volumes of growth [normalized by O.D. (optical density)] supernatants obtained from selected fungal strains. (C) Latency reversal bioassay in J-Lat A2 cells with growth supernatants obtained from members of the Aspergillus genus. Cells were treated as in (B). (D) Schematic representation of the orthogonal MS strategy coupled to latency reversal bioassays used to identify putative LRA. See main text for full description. (E) Three preconcentration cartridges (HLC, SCX, and MAX) were combined with variable content of extracting solvent (A: 5% MeOH, B: 45% MeOH, and C: 95% MeOH; FT, flowthrough). Latency reversal potential of fractionated secondary fungal metabolites was tested via treatment of J-Lat A2 cells. Latency reversal (fold increase percentage of GFP, left axis, black bars) and cell viability (percentage of viability, right axis, empty bars) were assessed by flow cytometry analysis. (F) Commercially obtained versions of five common molecules identified in active fractions were tested for LRA activity in J-Lat A2 cells. Data are presented as fold increase percentage of GFP expression and percentage of viability as indicated, ±SD from at least three independent experiments.

  • Fig. 2 GTX (20 nM) reverses HIV-1 latency in ex vivo infected primary CD4+ T cells without associated cytotoxicity, T cell activation, or inhibition of proliferative capacity.

    (A) Latency reversal after treatment of latently infected primary CD4+ T cells with increasing concentrations of GTX for 24 hours as indicated, shown as fold induction over untreated control luciferase activity. Statistical analysis was calculated using Kruskal-Wallis one-way analysis of variance (ANOVA) (****P < 0.0001). (B) Data presented in (A) normalized and shown as percent latency reversal activity relative to treatment with PMA/Ionomycin. (C) Latency reversal after treatment of HIV-1–infected latent primary CD4+ T cells with 20 nM GTX isolated from growth supernatant of A. fumigatus CBS 100074, measured as increased luciferase activity normalized to and shown as percentage of activation of PMA/ionomycin (n = 6). Wide horizontal lines represent average, and shorter horizontal lines represent SD. (D) Viability of the primary CD4+ T cells treated for 24 (red line) and 48 (green line) hours with indicated increasing concentrations of GTX, assessed by flow. (E) Unstimulated or αCD3/αCD28-stimulated PBMCs were treated with 20 and 100 nM GTX for 72 hours, stained with AnnexinV, and cell death (AnnexinV+ CD3+CD8+) was determined by flow cytometry. Treatment-specific cell cytotoxicity was calculated as indicated in the Materials and Methods section. Each symbol represents one healthy donor (n = 6), and horizontal lines depict means. (F) Representative fluorescence-activated cell sorting (FACS) plot overlay showing the proliferation of unstimulated or αCD3/CD28-stimulated CD8+ T cells in the presence or absence of GTX. Cells were stained with a proliferation dye and analyzed 72 hours later by flow cytometry to define nonproliferating cells (proliferation dye undiluted, bright stain intensity) and dividing cells (proliferation dye diluted, reduced stain intensity). (G) Activation status of primary CD4+ T cells upon GTX treatment as indicated (n = 6).

  • Fig. 3 GTX strongly synergizes with HDAC and BAF inhibitors and reverses latency in primary CD4+ T cells obtained from all tested aviremic participants.

    (A and B) HIV-1 latency reversal in latent ex vivo HIV-1–infected primary CD4+ T cells in response to 24-hour cotreatment with 20 nM GTX and distinct LRA class compounds [2 nM RMD, 350 nM SAHA, 1 μM CAPE, 2 μM OTX-01, 0.5 μM JQ-1, and 0.2 μM prostratin (A) and 1 μM CAPE and 5 μM BRD-K98645985 (B)] and shown as fold increase in luciferase activity. S indicates compound synergism in latency reversal according to the Bliss independence score. (C) Cytotoxicity of GTX alone and combined with indicated LRAs in CD4+ T cells. Unstimulated or αCD3/αCD28-stimulated PBMCs were cotreated as indicated for 72 hours followed by annexin V staining, and cell death (AnnexinV+ CD3+CD4+) was determined by flow cytometry. (D) GTX (20 nM) does not alter activation of CD4+ T cells. PBMCs from healthy donors were incubated with the indicated LRAs for 72 hours, either unstimulated or stimulated with αCD3/αCD28 antibodies. Panels depict pooled data showing the frequency of CD25+ cells within CD4+ T cells. (E) Absolute, cell-associated (CA) pol copy number in CD4+ T cells isolated from five aviremic participants that were treated in vitro with vehicle control (Untr), GTX (20 and 40 nM), or positive control for 24 hours as indicated. Statistical significance was calculated using t test (*P < 0.05; **P < 0.005; ***P < 0.0005). ns, not significant. (F) Data presented in (D) had been averaged and plotted together. Each symbol represents aviremic participant: green, participant 1; blue, participant 2; white, participant 3; yellow, participant 4; and pink, participant 5. Statistical significance was calculated using unpaired Mann-Whitney test (*P < 0.05).

  • Fig. 4 GTX treatment of resting CD4+ T cells causes decrease in 7SK RNA and activation of P-TEFb activity.

    (A) Heatmap of differentially expressed genes obtained from RNA sequencing analysis of primary CD4+ T cells treated as indicated for 4 hours (top). RNA sequencing indicates 7SK RNA to be the most differentially decreased gene in response to GTX treatment of CD4+ T cells in two independent donors (bottom). (B) GTX treatment of primary CD4+ T cells for 4 hours leads to specific depletion of 7SK RNA levels and not mRNA levels of other components of the 7SK snRNP complex. (C) Representative Western blot (WB) analysis of CTD Ser2 phosphorylation of RNA Pol II mediated by CDK9 in primary CD4+ T cells treated for 6 hours with GTX as indicated. PMA was used as a positive control, and FPD was used as a negative control. (D) Quantification of bands representing phosphorylated RNA Pol II normalized to tubulin and relative to untreated control from three independent experiments (C; fig. S9C). Statistical significance was calculated using unpaired t test (*P < 0.05; **P < 0.01). (E and F) GTX treatment causes release of P-TEFb from sequestration within the 7SK snRNP complex. (E) Schematic of glycerol gradient experiments, in which cell lysates (from GTX-treated or untreated cells) are loaded on top of generated glycerol gradients (10 to 30%) and ultracentrifuged, followed by collection of fractions, trichloroacetic acid protein precipitation, and subsequent analysis by SDS–polyacrylamide gel electrophoresis Western blotting. (F) Representative Western blot analysis of glycerol gradient sedimentation of lysates from primary CD4+ T cell, which were treated (6 hours) with GTX (20 nM) or vehicle control as indicated using antibodies specific for the P-TEFb component CDK9. As control, untreated lysates were treated with RNase for 1 hour to digest 7SK RNA and release P-TEFb. (G) Quantification of free versus total CDK9 in primary CD4+ T cells treated as indicated, as shown in (F) and fig. S9E, from three independent donors. Statistical significance was calculated using unpaired t test (*P < 0.05).

  • Fig. 5 GTX disrupts 7SK snRNP, causing release of P-TEFb and enhanced HIV-1 transcription.

    (A) Crystal structure of human LARP7 CTD in complex with 7SK RNA SL4 (Protein Data Bank ID code 6D12). LARP7 is shown in space-filling representation (gray) with bound RNA as yellow cartoon representation with bases indicated as flat rings (oxygen in red). (B) Proposed binding mode of GTX into the deep pocket on the surface of LARP7 as predicted by Chimera’s AutoDock Vina function and the Achilles Blind Docking server. Color code for GTX: carbon (turquoise), oxygen (red), sulfur (yellow), hydrogen (white), and nitrogen (blue). (C) Schematic representation of the 7SK snRNP coimmunoprecipitation CDK9 release experiment. (D) 7SK snRNP complex is immunoprecipitated using beads coated with anti-LARP7. Beads are divided and left either untreated or treated with GTX 20 nM or 1 μM or RNase A as indicated. Bead-bound fractions are separated and subjected to Western blot analysis to detect presence of LARP7 and CDK9. IgG, immunoglobulin G. (E) Quantification of CDK9 abundance normalized to immunoprecipitated LARP7 and relative to untreated control (n = 3). Statistical significance was calculated using unpaired t test (*P < 0.05; **P < 0.01). (F) 7SK RNA release assay from immunoprecipitated 7SK snRNP complex, as represented in (C). Bound and released fractions are subjected to reverse transcription quantitative polymerase chain reaction to quantitate the levels of bead-bound and released 7SK RNA in reaction supernatant. Input 7SK RNA (prereaction beads) was used for normalization. Statistical significance was calculated with unpaired t test (n = 3, **P < 0.01; ***P < 0.001; n.d., not detected). (G) Proposed model for GTX-mediated transcription activation of the latent HIV-1 LTR via degradation of 7SK RNA and release of CDK9 from the 7SK snRNP complex. Free P-TEFb is then recruited to the HIV-1 Tat-TAR axis, leading to phosphorylation of RNA Pol II at Ser2 and subsequent stimulation of transcription elongation.

Supplementary Materials

  • Supplementary Materials

    Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency

    Mateusz Stoszko, Abdullah M. S. Al-Hatmi, Anton Skriba, Michael Roling, Enrico Ne, Raquel Crespo, Yvonne M. Mueller, Mohammad Javad Najafzadeh, Joyce Kang, Renata Ptackova, Elizabeth LeMasters, Pritha Biswas, Alessia Bertoldi, Tsung Wai Kan, Elisa de Crignis, Miroslav Sulc, Joyce H.G. Lebbink, Casper Rokx, Annelies Verbon, Wilfred van Ijcken, Peter D. Katsikis, Robert-Jan Palstra, Vladimir Havlicek, Sybren de Hoog, Tokameh Mahmoudi

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