The Influence of Early Integration Events on HIV Latency and Reactivation Potential

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PROJECT SUMMARY Human Immunodeficiency Virus (HIV) persists in long-lived, latent reservoirs in infected patients despite continuous, long-term treatment with highly active antiretroviral drugs. Curative therapies designed to reactivate and clear these latently infected cells through the use of latency reversing agents (LRAs) are known as “shock and kill” approaches, but these approaches have largely failed due to incomplete reactivation of the latent pool. While differences in cellular environment and proviral integration site are thought to be responsible for this variability, very little is actually known about how specific host factors can influence latency and LRA potential in primary models. Do events during early integration and latency establishment ultimately influence latency maintenance and reactivation? A majority of the latent reservoir is thought to be composed of quiescent CD4+ T cells harboring replication competent, but transcriptionally silent proviruses. These cells have traditionally been very difficult to generate ex vivo and have been even harder to manipulate genetically for the study of specific host factors. Recent advancements in primary cell latency models and genome engineering, however, have made these studies feasible for the first time. In this proposal, I intend to test the hypothesis that early events in HIV integration, proviral silencing, and latency establishment help dictate latency maintenance and reactivation potential. To test this hypothesis, I will use a novel platform for CRISPR/Cas9 editing in primary T cells to ablate two host factors involved in integration site preference, LEDGF and CPSF6. These cells will be infected with a dual fluorescent reporter virus to monitor proviral silencing and integration site profiles determined by deep sequencing. These cells will be returned to a quiescent state and treated with representative LRAs targeting distinct functional pathways to interrogate latency maintenance and determine if reactivation potential correlates with differences observed during early infection in each of genetic background (Aim 1). Conversely, LRAs that were originally designed to alter latency maintenance may alter events during early integration and latency establishment as well and therefore serve as molecular probes for identifying novel host factors involved in these processes. Towards this end, primary T cells will be pre-treated with a panel of LRAs, infected with a dual fluorescent reporter virus, and monitored for differences in integration, proviral silencing, and latency establishment (Aim 2). Orthologous small molecules in these pathways and targeted genetic knock-outs will be used to validate these findings and confirm the identity of novel latency host factors. Taken together, these data will be the first to analyze the relationship between host factors, HIV integration, latency establishment, and reactivation potential directly in primary T cells. Understanding how host-dependent events that occur early in infection are linked to therapeutic efficacy during chronic disease will be critical to the development of new personalized therapeutic strategies for the treatment and cure of not only HIV, but other disease states as well.