Abstract After HIV integration of the proviral DNA into the host genome, the virus can remain latent or activate transcription. The viral Tat protein, which enhances transcript elongation from the HIV-1 promoter, is the switch between these two states. Since Tat resides under the control of the same promoter, it enhances its own transcription via a positive feedback loop. We identified didehydro-Cortistatin A (dCA) as a very potent inhibitor of Tat (1, 2). In human CD4 +T cells isolated from aviremic individuals, combining dCA with ART accelerates HIV-1 suppression and prevents viral rebound during treatment interruption, as the HIV-1 promoter remains epigenetically repressed. HIV-1 transcriptional inhibitors have the unique property of reducing particle production from infected cells. dCA is the proof-of-concept that this novel class of molecules is amenable to block-and-lock functional cure approaches, which aim at reducing residual viremia during ART and limit viral rebound. It is thus important to understand the mechanisms that explain not only dCA's inhibition of reactivation, but also mechanisms regulating HIV-1 latency in CD4+T memory T cells in general, to expand on “block-and-lock” approaches, and explore alternative options for retroviral suppression. There are approximately 320 human chromatin regulators, which “write”, “erase”, or “read” chromatin modifications, or remodel nucleosome topology. Specificity in gene expression derives from the combinatorial nature of chromatin modifications, and assembly of related chromatin regulator subunits. The rationale for this proposal is that factors that establish HIV-1 latency are important for viral reactivation, and that by identifying and inhibiting them, a “locked” state of silencing that is exceedingly resistant to reactivation can be achieved. We propose to combine a comprehensive high-resolution mapping of the nucleosome organization and positioning of chromatin remodeling complexes at the HIV promoter during HIV latency, with a robust pooled shRNAs screening approach to interrogate all chromatin regulatory factors in parallel during a single experiment. Primary and secondary screens will be performed in a newly developed primary cell system that captures bona fide HIV-1 latency, and departs from CD4+T cells from successfully treated HIV infected donors. During the R61 phase of the project we will be able to correlate high-resolution nucleosome architecture data with their binding to all chromatin remodeling machine families and develop a comprehensive picture of the signals and factors that drive chromatin activity at the HIV-1 genome during latency. This data will support robust hypotheses and targets to test in detail during the R33 phase. We anticipate that from these candidates, we can infer how HIV-latency is controlled and develop rational therapeutic approaches to modulate HIV latency.