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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

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: Mbonye, Uri, Leskov, Konstantin, Shukla, Meenakshi, Valadkhan, Saba, Karn, Jonathan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478230/
https://www.ncbi.nlm.nih.gov/pubmed/34529720
http://dx.doi.org/10.1371/journal.ppat.1009581
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author Mbonye, Uri
Leskov, Konstantin
Shukla, Meenakshi
Valadkhan, Saba
Karn, Jonathan
author_facet Mbonye, Uri
Leskov, Konstantin
Shukla, Meenakshi
Valadkhan, Saba
Karn, Jonathan
author_sort Mbonye, Uri
collection PubMed
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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spelling pubmed-84782302021-09-29 Biogenesis of P-TEFb in CD4(+) T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways Mbonye, Uri Leskov, Konstantin Shukla, Meenakshi Valadkhan, Saba Karn, Jonathan PLoS Pathog Research Article 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. Public Library of Science 2021-09-16 /pmc/articles/PMC8478230/ /pubmed/34529720 http://dx.doi.org/10.1371/journal.ppat.1009581 Text en © 2021 Mbonye et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Mbonye, Uri
Leskov, Konstantin
Shukla, Meenakshi
Valadkhan, Saba
Karn, Jonathan
Biogenesis of P-TEFb in CD4(+) T cells to reverse HIV latency is mediated by protein kinase C (PKC)-independent signaling pathways
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8478230/
https://www.ncbi.nlm.nih.gov/pubmed/34529720
http://dx.doi.org/10.1371/journal.ppat.1009581
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