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

Abstract

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. Author SummaryDissecting the cellular pathways through which HIV emerges from latency is a key step in the development of therapeutically viable approaches for latency reversal and eventual clearance of persistent HIV in infected individuals. The essential host transcription elongation factor P-TEFb, a heterodimer of CDK9 kinase and a regulatory cyclin T subunit, is a critical mediator of the trans-activation of latent HIV. Availability of P-TEFb for proviral transcription is highly limited due to a posttranscriptional restriction in cyclin T1 expression and dephosphorylation of CDK9 on its activation loop. Using a well-characterized primary T-cell model of HIV latency alongside healthy donor memory CD4+ T cells, we have now defined the signaling pathways that are essential for the generation of transcriptionally active P-TEFb and, consequently, proviral reactivation. Crucial among these findings is the demonstration that protein kinase C (PKC) agonists signal through off-target activation of RasGRP1-Ras-Raf-MEK-ERK1/2 to effect the reactivation of both P-TEFb and proviral HIV. Understanding these pathways should lead to the discovery of novel highly selective activators of P-TEFb to improve the efficiency of HIV reactivation in the memory T-cell population of virally suppressed individuals.