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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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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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. |
format | Online Article Text |
id | pubmed-8478230 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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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