GSE167916
Protein kinase C (PKC)-independent signaling pathways regulate P-TEFb biogenesis in CD4+ T cells to reverse HIV latency
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. Overall design: Approximately 1.0 X 10^7 memory CD4+ T cells were routinely isolated by negative bead selection from previously frozen healthy donor leukapheresis packs containing 1.0 X 10^8 peripheral blood mononuclear cells (PBMCs) using the EasySepTM CD4+ T-cell enrichment kit (StemCell). After isolation cells were allowed to recover overnight in complete RPMI media lacking IL-2. Purity and resting status of the isolated memory T cells were confirmed by immunofluorescence flow cytometry analysis of CD45-RO, cyclin D3 and Ki67 expression. Cells were challenged with Dynabeads CD3/CD28 to activate the T-cell receptor (TCR) at a 1:1 bead-to-cell ratio. 0.6 X 10^6 cells were taken at 24 h and 72 h after TCR stimulation for Drop-seq along with unstimulated control cells.