By phosphorylating elongation factors and the C-terminal domain of RNA polymerase II, the positive transcription elongation factor b (P-TEFb) is the critical kinase for transcription elongation and co-transcriptional processing of eukaryotic genes. It exists in inactive small nuclear ribonucleoprotein (7SK snRNP) and active (free P-TEFb) complexes in cells. The P-TEFb equilibrium determines the state of cellular activation, proliferation, and differentiation. Free P-TEFb, which is required for growth, can be recruited to RNA polymerase II via transcription factors, BRD4, or the super elongation complex (SEC). UV light, various signaling cascades, transcriptional blockade, or compounds such as hexamethylene bisacetamide (HMBA), suberoylanilide hydroxamic acid (SAHA), and other histone deacetylase inhibitors lead to a rapid release of free P-TEFb, followed by its reassembly into the 7SK snRNP. As a consequence, transcription of HEXIM1, a critical 7SK snRNP subunit, and HIV is induced. In this study, we found that a bromodomain and extra-terminal (BET) bromodomain inhibitor, JQ1, which inhibits BRD4 by blocking its association with chromatin, also leads to the rapid release of free P-TEFb from the 7SK snRNP. Indeed, JQ1 transiently increased levels of free P-TEFb and BRD4·P-TEFb and SEC·P-TEFb complexes in cells. As a consequence, the levels of HEXIM1 and HIV proteins rose. Importantly, the knockdown of ELL2, a subunit of the SEC, blocked the ability of JQ1 to increase HIV transcription. Finally, the effects of JQ1 and HMBA or SAHA on the P-TEFb equilibrium were cooperative. We conclude that HMBA, SAHA, and JQ1 affect transcription elongation by a similar and convergent mechanism.

Human immunodeficiency virus (HIV) persists in a latent form in infected individuals treated effectively with highly active antiretroviral therapy (HAART). In part, these latent proviruses account for the rebound in viral replication observed after treatment interruption. A major therapeutic challenge is to purge this reservoir. In this study, we demonstrate that suberoylanilide hydroxamic acid (SAHA) reactivates HIV from latency in chronically infected cell lines and primary cells. Indeed, P-TEFb, a critical transcription cofactor for HIV, is released and then recruited to the viral promoter upon stimulation with SAHA. The phosphatidylinositol 3-kinase/Akt pathway is involved in the initiation of these events. Using flow cytometry-based single cell analysis of protein phosphorylation, we demonstrate that SAHA activates this pathway in several subpopulations of T cells, including memory T cells that are the major viral reservoir in peripheral blood. Importantly, SAHA activates HIV replication in peripheral blood mononuclear cells from individuals treated effectively with HAART. Thus SAHA, which is a Food and Drug Administration-approved drug, might be considered to accelerate the decay of the latent reservoir in HAART-treated infected humans.

Hexamethylene bisacetamide (HMBA) is a potent inducer of cell differentiation and HIV production in chronically infected cells. However, its mechanism of action remains poorly defined. In this study, we demonstrate that HMBA activates transiently the PI3K/Akt pathway, which leads to the phosphorylation of HEXIM1 and the subsequent release of active positive transcription elongation factor b (P-TEFb) from its transcriptionally inactive complex with HEXIM1 and 7SK small nuclear RNA (snRNA). As a result, P-TEFb is recruited to the HIV promoter to stimulate transcription elongation and viral production. Despite the continuous presence of HMBA, the released P-TEFb reassembles rapidly with 7SK snRNA and HEXIM1. In contrast, a mutant HEXIM1 protein that cannot be phosphorylated and released from P-TEFb and 7SK snRNA via the PI3K/Akt pathway antagonizes this HMBA-mediated induction of viral production. Thus, our studies reveal how HIV transcription is induced by HMBA and suggest how modifications in the equilibrium between active and inactive P-TEFb could contribute to cell differentiation. The reservoir of HIV in infected people remains an insurmountable problem in the era of highly active antiretroviral therapy. Thus, the virus persists despite the best possible treatment. HIV hides in many cells and tissues, where its genome is not expressed. Thus, neither drugs nor the immune system can eradicate it from the body. One hope is to activate the production of HIV in these reservoirs in the presence of optimal treatment. Strategies aimed at activating hematopoetic cells and thus viral replication have been tried and failed. In this report, we targeted a specific host transcriptional complex that is essential for the transcription of HIV genome. Its activation should not lead to generalized stimulation of the immune system. Indeed, paradoxically, hexamethylene bisacetamide (HMBA) and related compounds lead to cellular differentiation and apoptosis. By studying properties of these differentiation agents, we discovered that they activate transiently transcription of HIV, be it in stable cell lines or in primary infected cells. Thus, compounds related to HMBA, some of which have now been approved for clinical use, could be tried to diminish or eliminate the reservoir of HIV in optimally treated infected individuals.