Gliotoxin, identified from a screen of Aspergillus fumigatus metabolites, reverses HIV-1 latency via release of P-TEFb
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA557682 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9870580.fastq.gz | Fastqsanger.gz | |||
| SRR9870590.fastq.gz | Fastqsanger.gz | |||
| SRR9870581.fastq.gz | Fastqsanger.gz | |||
| SRR9870591.fastq.gz | Fastqsanger.gz | |||
| SRR9870582.fastq.gz | Fastqsanger.gz |
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Project description:A leading pharmacological strategy towards HIV cure requires “shock” or activation of expression of the HIV genome in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. As a source of bio-active molecules we used fungal secondary metabolites (extrolites) in a screen for novel LRAs. We used orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, and identified gliotoxin (GTX) to potently reverse latency. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA to be most significantly downregulated in independent donors upon GTX treatment of CD4+ T cells. GTX disrupted the 7SK snRNP complex causing release of P-TEFb, which induced phosphorylation of RNA Pol II CTD, and increased HIV transcription. Our data highlight the power of combining low throughput bioassays, mycology and orthogonal mass spectrometry as an approach to screen for and characterize novel LRAs.
2020-08-31 | GSE135184 | GEO
Project description:The switch between HIV latency and productive transcription is regulated by an auto-feedback mechanism initiated by the viral trans-activator Tat, which functions to recruit the host transcription elongation factor P-TEFb to proviral HIV. A heterodimeric complex of CDK9 and one of three cyclin T subunits, P-TEFb is expressed at vanishingly low levels in latently infected memory CD4+ T cells and cellular mechanisms controlling its availability for proviral transcription are not well-defined. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we sought to identify specific T-cell receptor (TCR) signaling pathways that regulate the generation of transcriptionally active P-TEFb, defined as the coordinate expression of cyclin T1 and phospho-Ser175 CDK9. Unexpectedly, cellular treatment with diacylglycerol-mimicking protein kinase C (PKC) agonists in the absence of intracellular calcium mobilization with an ionophore was sufficient to stimulate active P-TEFb expression and reactivate latent HIV with minimal cytotoxicity. Furthermore, inhibition-based experiments demonstrated that PKC agonists and TCR-mobilized diacylglycerol signal through MAP kinases ERK1/2 rather than through PKC to effect the reactivation of both P-TEFb and latent HIV. Single-cell RNA-seq analysis revealed that of the four known isoforms of RasGRP, the diacylglycerol-dependent Ras guanine nucleotide exchange factor RasGRP1 is by far the predominantly expressed isoform in memory CD4+ T cells and should therefore mediate the activation of ERK1/2 upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-AKT-mTORC1/2 pathway alongside the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and strongly suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells in response to either TCR co-stimulation or PKC agonist challenge is largely independent of PKC activity but rather primarily involves two complementary signaling arms of the TCR cascade namely RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-AKT-mTORC1/2.
2021-09-07 | GSE167916 | GEO
Project description:A leading pharmacological strategy toward HIV cure requires "shock" or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.
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Project description:The transcription factor T-bet induces differentiation of CD4+ T cells into the Th1 lineage and also allows for a degree of functional plasticity. Here, we show that T-bet acts through super-enhancers to recruit the elongation factor P-TEFb. Th1-specific genes are poised for activation in Th2 cells and P-TEFb recruitment activates transcriptional elongation. T-bet also induces extensive P-TEFb binding at super-enhancers, where it acts to stimulate enhancer RNA transcription. P-TEFb inhibition selectively blocks activation of lineage-specific genes and reverses Th1-associated retinitis pathology. T-bet-mediated recruitment of P-TEFb to super-enhancers at otherwise poised genes provides a model for how lineage-specifying factors promote differentiation towards specific cell fates whilst maintaining a degree of functional plasticity. Transcriptional profiling of mouse Th1 and Th2 cells treated with JQ1, which inhibits P-TEFb recruitment, or the P-TEFb inhibitor Flavopiridol vs DMSO conrol treated cells. Goal was to identify genes which are sensitive to P-TEFb inhibition.
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Project description:The transcription factor T-bet induces differentiation of CD4+ T cells into the Th1 lineage and also allows for a degree of functional plasticity. Here, we show that T-bet acts through super-enhancers to recruit the elongation factor P-TEFb. Th1-specific genes are poised for activation in Th2 cells and P-TEFb recruitment activates transcriptional elongation. T-bet also induces extensive P-TEFb binding at super-enhancers, where it acts to stimulate enhancer RNA transcription. P-TEFb inhibition selectively blocks activation of lineage-specific genes and reverses Th1-associated retinitis pathology. T-bet-mediated recruitment of P-TEFb to super-enhancers at otherwise poised genes provides a model for how lineage-specifying factors promote differentiation towards specific cell fates whilst maintaining a degree of functional plasticity.
2016-06-08 | GSE62483 | GEO
Project description:The transcription factor T-bet induces differentiation of CD4+ T cells into the Th1 lineage and also allows for a degree of functional plasticity. Here, we show that T-bet acts through super-enhancers to recruit the elongation factor P-TEFb. Th1-specific genes are poised for activation in Th2 cells and P-TEFb recruitment activates transcriptional elongation. T-bet also induces extensive P-TEFb binding at super-enhancers, where it acts to stimulate enhancer RNA transcription. P-TEFb inhibition selectively blocks activation of lineage-specific genes and reverses Th1-associated retinitis pathology. T-bet-mediated recruitment of P-TEFb to super-enhancers at otherwise poised genes provides a model for how lineage-specifying factors promote differentiation towards specific cell fates whilst maintaining a degree of functional plasticity. Transcriptional profiling of human Th1 and Th2 cells
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Project description:In patients with chronic pulmonary disease colonization with the mold Aspergillus fumigatus is associated with declining pulmonary function and obstructive airway disease. One potential effector of this inflammatory response is the pulmonary mast cell. In vitro studies have demonstrated that A. fumigatus contact induces IgE-independent mast cell degranulation. Conversely, the Aspergillus secondary metabolite gliotoxin has been shown to suppress mast cell activation. These contradictory results emphasize the need for a better understanding of the interactions between A. fumigatus and mast cells. Thus, the objective of this work was to identify A. fumigatus genes that are differentially regulated upon exposure to mast cells. Transcriptional profiling experiments indicated that, in addition to genes encoding for iron acquisition systems, allergens and putative virulence factors, genes from the gliotoxin biosynthesis cluster were significantly down-regulated upon exposure to mast cells. Globally, the results from this study provide insight into the A. fumigatus response to mast cells and suggest that one mechanism by which the host may circumvent the effects of gliotoxin is via the suppression of fungal gliotoxin synthesis by mast cells.
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Project description:Pharmacological Inhibition of PPARγ Reverses HIV Latency but Prevents Viral Production/Infectivity while Boosting Th17 Functions
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