A CRISPR-screening approach to increase HIV latency reversal to improve an HIV cure strategy

Abstract

Despite the availability of effective antivirals, HIV remains a global burden. Although virus replication can be suppressed, people living with HIV (PLWH) need to continue life-long antiviral therapy as there is currently no way to eradicate the long-lived and stable HIV reservoir that evades the immune system but retains the ability to reactivate and spread virus. Understanding mechanisms that govern HIV latency within these reservoirs, such as host and viral factors that influence transcription initiation, transcription elongation, and chromatin dynamics, has led to the discovery of a number of small molecule drugs called latency reversal agents (LRAs) capable of stimulating HIV transcription. One strategy being explored to target the HIV reservoir is called “shock-and-kill” where the silent provirus in latently infected cells is transcriptionally stimulated such that the immune system can recognize the subsequently synthesized viral proteins and clear infected cells. However, LRAs used to date in this strategy have not been potent or specific enough on their own to sufficiently activate and lead to clearing of infected cells. While pairing together LRAs that function through differing mechanisms of action have proven synergistic in their ability to activate HIV transcripts, these combinations in relevant model systems, such as ex vivo systems from cells from people living with HIV (PLWH), have demonstrated there are more blocks to latency reversal present in primary cells that prevent full virus reactivation. These results highlight a need to find ways to uncover these blocks and improve reactivation. In my thesis, I used a CRISPR-based screening approach with the LRA combination AZD5582 and I-BET151. This combination synergizes on HIV RNA synthesis by targeting the noncanonical NF-kB pathway to increase transcription initiation and targeting BET proteins to increase available cellular PTEF-b to aid in HIV specific transcription elongation in conjunction with HIV Tat. This combination was effective in in vitro models, but not in an ex vivo system. With this screening approach, I was able to identify a gene target, INTS12, that is a part of a larger complex called the Integrator Complex that could be targeted to improve reactivation in an ex vivo system. These studies provide a framework for predicting in vivo blocks to HIV latency reversal and strategically improving LRAs and identifying novel targets for which small molecule drugs should be developed.