Identifying Genetic Determinants of Latency Establishment and Maintenance

The persistence of HIV in long-lived latent reservoirs of resting CD4+ T cells is the critical barrier to the development of an effective cure. “Shock and kill” strategies designed to eliminate the latent reservoir by forced reactivation of the virus have proved to be untenable due to incomplete penetrance of and heterogeneous responses to currently available latency reversing agents. While this is likely due to complex relationships between cis- and trans- acting host factors at the proviral integration site, the intrinsic host factors underlying latency establishment, maintenance, and reactivation are poorly understood. Here, we propose to leverage a new technology that allows us to edit the genomes of primary resting CD4+ T cells for the first time. First, we will optimize this gene editing platform for downstream use in latency establishment and reversal assays (Aim 1). Next, we will leverage this pipeline to systematically test the impact of knocking out 30 HIV host factors on latency establishment and reactivation potential (Aim 2). Not only will these results develop a powerful new pipeline for understanding the role of host factors in HIV latency in primary cells, but they will also begin to reveal the complex molecular architecture controlling latency maintenance.