A CRISPR Screen of HIV Dependency Factors to Identify Host Proteins Necessary for Activation of Latent HIV Proviruses

Abstract

The human immunodeficiency virus (HIV) integrates into host DNA and therefore persists as a long-lived pathogen in people living with HIV (PLHIV). One of the greatest challenges to achieving a cure for human immunodeficiency virus (HIV) is the presence of latently infected long-lived immune cells that endure throughout the course of antiretroviral treatment. If antiretroviral therapy is stopped, these immune cells will sporadically produce virions which contribute to viral rebound and progressive infection. There are several approaches to targeting the latently infected cell population. One approach, called “shock and kill”, relies on small molecule drugs called latency reversal agents (LRAs) that stimulate transcription of HIV proviral genes which could lead to the infected cells being targeted by the immune system for elimination. The second approach, which is predominantly the focus of this thesis, is called “block and lock” which seeks to permanently silence the viral reservoir to prevent viral reactivation from the latent cell population. In this thesis, I performed a CRISPR screen in latently infected Jurkat T cell lymphocytes to identify host factors required for latency reactivation using a gene set of putative HIV dependency factors previously described. The goal was to broadly screen for these factors in Jurkat T cells but ultimately validate these hits in primary CD4+ T cells. I identified several factors that are novel for reactivation from latency including ALYREF, UBE2M, TBL1XR1 and AMBRA1. The top hit, Cyclin T1 (CCNT1) is a member of the P-TEFb complex with cyclin-dependent kinase 9 (CDK9) known to required for HIV transcription elongation but also for host gene transcriptional elongation. I found that CCNT1 knockout prevents reactivation using LRAs that target a broad spectrum of pathways, and that CCNT1 knockout in primary CD4+ T cells dramatically prevents latency reactivation without affecting T cell receptor activation. RNA sequencing of CCNT1 knockout cells revealed minimal effects on host gene transcription but dramatic effects on HIV transcription. I hypothesize that CCNT1 knockout is compensated by its paralogs CCNT2 or CCNK for host cell function, and thus CCNT1 may be a promising target in a block and lock model of HIV cure. Moreover, I hypothesize that HIV dependency factors also play a key role in latency reactivation.