Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.

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 sub...

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Autores principales:	Uri Mbonye, Konstantin Leskov, Meenakshi Shukla, Saba Valadkhan, Jonathan Karn
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/7dce3404a8e3416ab07b260d03a6cd39
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spelling	oai:doaj.org-article:7dce3404a8e3416ab07b260d03a6cd392021-12-02T20:00:10ZBiogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.1553-73661553-737410.1371/journal.ppat.1009581https://doaj.org/article/7dce3404a8e3416ab07b260d03a6cd392021-09-01T00:00:00Zhttps://doi.org/10.1371/journal.ppat.1009581https://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374The 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.Uri MbonyeKonstantin LeskovMeenakshi ShuklaSaba ValadkhanJonathan KarnPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 17, Iss 9, p e1009581 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Uri Mbonye Konstantin Leskov Meenakshi Shukla Saba Valadkhan Jonathan Karn Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
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.
format	article
author	Uri Mbonye Konstantin Leskov Meenakshi Shukla Saba Valadkhan Jonathan Karn
author_facet	Uri Mbonye Konstantin Leskov Meenakshi Shukla Saba Valadkhan Jonathan Karn
author_sort	Uri Mbonye
title	Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
title_short	Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
title_full	Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
title_fullStr	Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
title_full_unstemmed	Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.
title_sort	biogenesis of p-tefb in cd4+ t cells to reverse hiv latency is mediated by protein kinase c (pkc)-independent signaling pathways.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/7dce3404a8e3416ab07b260d03a6cd39
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Biogenesis of P-TEFb in CD4+ T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways.

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