Valadkhan, S. | bioRxiv Science

bioRxiv2021

The Nerve Growth Factor IB-like Receptor Nurr1 (NR4A2) recruits CoREST transcription repressor complexes to silence HIV following proviral reactivation in microglial cells

Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells (h{micro}glia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND. AUTHOR SUMMARYHIV enters the brain almost immediately after infection where it infects perivascular macrophages, microglia and, to a less extent, astrocytes. In previous work using an immortalized human microglial cell model, we observed that integrated HIV constantly underwent cycles of reactivation and subsequent silencing. In the present study, we found that the Nurr1 nuclear receptor is a key mediator of HIV silencing. The functional activation of Nurr1 by specific agonists, or the over expression of Nurr1, resulted in rapid silencing of activated HIV in microglial cells. Global gene expression analysis confirmed that Nurr1 not only repressed HIV expression but also regulated numerous genes involved in microglial homeostasis and inflammation. Thus, Nurr1 is pivotal for HIV silencing and repression of inflammation in the brain and is a promising therapeutic target for treatment of HAND.

microbiology↗

bioRxiv2021

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 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.

molecular biology↗