Using a CRISPR-based screen to study individual and combination molecular mechanisms of HIV-1 latency

Abstract

Long-lived immune cells harboring transcriptionally silenced, latent Human Immunodeficiency Virus Type 1 (HIV-1) proviruses continues to pose a barrier to HIV-1 cure. HIV-1 latency involves a breadth of host and viral factors that influence transcription initiation, transcription elongation, and chromatin modifications of the long terminal repeat (LTR) of the HIV-1 provirus. These encompass both positive and negative regulators of transcription including factors that influence the activity of transcription factors such as NF-kB, factors that influence the ability of the viral transactivator, Tat, to recruit the elongation complex P-TEFb to the viral promoter, and factors that influence both activating and repressive modifications to histones surrounding the HIV-1 LTR. Thus, to examine the interplay of overlapping mechanisms involved in HIV-1 latency, I have developed a new HIV-1 latency CRISPR-based screening strategy that combines screening for multiple pathways by targeting one pathway while simultaneously activating another transcriptional mechanism. In this thesis, I established a high-throughput CRISPR-based screen named Latency HIV-CRISPR that uses the packaging of guideRNAs into the viral supernatant as a direct readout of factors involved in the maintenance of HIV-1 latency. I devised a strategy for latency reversal that uses a custom generated guideRNA library targeting epigenetic regulatory genes combined with a treatment with or without AZD5582, an activator of the non-canonical NF-kB pathway and a latency reversal agent. This screen identified novel individual and combination pathways that contribute to HIV-1 latency. These studies provide progress to the goal of clearance and ultimately elimination of the latent reservoir of HIV-1 infected cells using pathways that increase both the potency and specificity of latency reversal agents.