F-101 Signaling Pathways and Epigenetic Mechanisms Controlling HIV Latency

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Background: HAART must be continuously maintained prevent HIV-1 rebound from latent reservoirs. Factors that are responsible for maintenance of HIV-1 latency were identified by independent genome wide shRNA library screens. The mechanism of action of compounds that reactivate HIV proviruses was studied by comparison to T-cell receptor activation pathways. Methods: shRNA library screens were performed in Jurkat T-cells. Cells carrying reactivated proviruses were purified by sorting after sequential passages and the shRNA sequences identified by next-generation sequencing and classified by systems biology tools. Naive T cells were polarized into Th17, Th1, Th2, and Treg cells, infected with a single round HIV-1 proviral clone and forced into quiescence by cytokine restriction to generate latently infected cell populations. Results: The screen identified many of the shRNAs associated with epigenetic silencing mechanisms, as well as some novel targets including the estrogen receptor, ESR1. In cells where PRC2 (EZH2) was knocked down, all of the inducible proviruses were found in the H3K27me3 population suggesting that PRC2 complex is required not only for maintenance of latency but also for entry of HIV proviruses into latency. High content screens have shown the need to activate both P-TEFb and reverse epigenetic blocks. Evaluation of the mechanisms of action of the T-cell receptor, cytokines, disulfiram, farnesyl transferase and ESR1 inhibitors highlights how activation of unique pathways can lead to proviral reactivation. Conclusions: The reactivation of latent proviruses requires both P-TEFb and transcription initiation. Reversing any number of rate limiting steps can lead to proviral reactivation but in a limited number of cells. This suggests that there will be many opportunities to identify synergies between different classes of proviral activators and design efficient reactivation strategies.