Protein kinase C (PKC)-independent signaling pathways regulate P-TEFb biogenesis in CD4+ T cells to reverse HIV latency
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ABSTRACT:
PROVIDER: PRJNA705493 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13808297_1.fastq.gz | Fastqsanger.gz | |||
| SRR13808297_2.fastq.gz | Fastqsanger.gz | |||
| SRR13808298_1.fastq.gz | Fastqsanger.gz | |||
| SRR13808298_2.fastq.gz | Fastqsanger.gz | |||
| SRR13808299_1.fastq.gz | Fastqsanger.gz |
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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: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 resting memory CD4+ T cells and cellular mechanisms controlling its availability are central to regulation of the emergence of HIV from latency. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we characterized 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. Protein kinase C (PKC) agonists, such as ingenol and prostratin, stimulated active P-TEFb expression and reactivated latent HIV with minimal cytotoxicity, even in the absence of intracellular calcium mobilization with an ionophore. Unexpectedly, 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 and bulk RNA-seq analyses revealed that of the four known isoforms of the Ras guanine nucleotide exchange factor RasGRP, RasGRP1 is by far the predominantly expressed diacylglycerol-dependent isoform in CD4+ T cells. RasGRP1 should therefore mediate the activation of ERK1/2 via Ras-Raf signaling upon TCR co-stimulation or PKC agonist challenge. Combined inhibition of the PI3K-mTORC2-AKT-mTORC1 pathway and the ERK1/2 activator MEK prior to TCR co-stimulation abrogated active P-TEFb expression and substantially suppressed latent HIV reactivation. Therefore, contrary to prevailing models, the coordinate reactivation of P-TEFb and latent HIV in primary T cells following either TCR co-stimulation or PKC agonist challenge is independent of PKC but rather involves two complementary signaling arms of the TCR cascade, namely, RasGRP1-Ras-Raf-MEK-ERK1/2 and PI3K-mTORC2-AKT-mTORC1.
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Project description:Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore, administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way. Here, we compare the transcriptional profiles of PKC-treated NK and CD4+ T cells to assess their generalized cellular effects. Transcriptomic profiles from both cell types displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. However, the transcriptomic effects of PKC stimulation on NK cells are much milder than that in CD4+ T cells. This finding, combined with our experimental data testing killing capacity of PKC-stimulated NK cells against K562 cells and infected CD4+ T cells suggests that their contribution in “kick and kill” strategies is primarily due to upregulating HIV expression in CD4+ T cells, rather than directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the “kick” rather than the “kill” arm of this HIV cure approach.
2025-01-23 | GSE287754 | GEO
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.
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Project description:Defining the Effects of PKC Modulator HIV Latency-Reversing Agents on Natural Killer Cells
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Project description:Gliotoxin, identified from a screen of Aspergillus fumigatus metabolites, reverses HIV-1 latency via release of P-TEFb
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Project description:Many ncRNAs serve as regulatory molecules in various physiological pathways. Distinct from protein-coding RNA expression, ncRNA expression is regulated solely by transcription and RNA processing/stability. Transcriptional regulation in ncRNA genes is thus important to be understood and is yet to be known completely. Previously, we identified that a subset of mammalian ncRNA genes is transcriptionally regulated by RNA Pol II promoter-proximal pausing and in a tissue-specific manner. In this study, human stimulus-inducible ncRNA genes were monitored to assess the function of P-TEFb, a master Pol II pausing regulator for protein-coding genes, in ncRNA transcription. Our findings indicate that the expression of many ncRNA genes is regulated by P-TEFb. Interestingly, a biphasic characteristic of P-TEFb inhibition in serum responsive ncRNA genes was observed: S2 phospho-Pol II was largely increased in the TSS (–300 to +300) whereas overall, it was decreased in the gene body (> +350) upon chemical inhibition of P-TEFb. In addition, the three representative ncRNAs, MALAT1, NEAT1, and XIST, were further analyzed for determining P-TEFb association. Taken together, we propose the transcriptional activation of many human ncRNAs utilizing pausing and releasing Pol II and the regulatory mechanism of transcriptional elongation of these genes requiring the function of P-TEFb.
2018-07-31 | GSE117848 | GEO
Project description:The positive transcription elongation factor b (P-TEFb) regulates RNA polymerase II elongation. In cells, P-TEFb partitions between small active and larger inactive states. In the latter, HEXIM1 binds to 7SK snRNA and recruits as well as inactivates P-TEFb in the 7SK snRNP. Several stimuli can affect this P-TEFb equilibrium. In this study, we demonstrate that protein kinase C (PKC) phosphorylates the serine at position158 (S158) in HEXIM1. This phosphorylated HEXIM1 protein neither binds to 7SK snRNA nor inhibits P-TEFb. Phorbol esters or the engagement of the T cell antigen receptor, which activate PKC and the expression of the constitutively active (CA) PKCθ protein, which is found in T cells, inhibit the formation of the 7SK snRNP. All these stimuli increase P-TEFb-dependent transcription. In contrast, the kinase-negative PKCθ and the mutant HEXIM1 (S158A) proteins block effects of these PKC-activating stimuli. These results indicate that the phosphorylation of HEXIM1 by PKC represents a major regulatory step of P-TEFb activity in cells.
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Project description:To identify novel host factors as putative targets to reverse HIV-1 latency, we performed an insertional mutagenesis genetic screen in a latently HIV-1-infected pseudohaploid KBM7 cell line (Hap-Lat). Following mutagenesis, insertions were mapped to the genome, and bioinformatic analysis resulted in the identification of 69 candidate host genes involved in maintaining HIV-1 latency.
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