A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent-AZD5582, an activator of the non-canonical NF kappa B pathway-to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NF kappa B pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters. Author summaryHIV-1 establishes long-lived latent reservoirs that present a barrier for virus eradication and HIV Cure. One approach to reduce the latent reservoir is the use of small molecules called latency reversal agents (LRA) to trigger immune-mediated clearance of virus-producing cells. However, this approach is limited by the inability of current LRAs to reactivate the majority of latent proviruses and a lack of specificity, highlighting the need for a greater understanding of the interplay of mechanisms involved in the maintenance of HIV-1 latency. We developed a modular CRISPR-based screening, called HIV-CRISPR, in a T-lymphocyte (J-Lat) model of latent infection to gain a comprehensive representation of the pathways involved in HIV-1 latency. As the latent state of HIV-1 is controlled by multiple, parallel mechanisms, we used the screen to study the epigenetic factors in the presence of an LRA targeting a transcriptional activation mechanism. The screens identified novel factors controlling HIV-1 latency both in the J-Lat and primary T cell models of HIV-1 latency.

Automated summary

By developing a new CRISPR screening strategy called "Latency HIV-CRISPR", we gained insights into the mechanisms controlling the latency of HIV-1. Our study found that the ING3 component of the NuA4 HAT complex acts in synergy with AZD5582 to activate latent HIV-1 proviruses. This research is important for understanding the maintenance mechanisms of HIV-1 latency.