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 and are 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 NFkB 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, and the combination of ING3 knockout with the activation of non-canonical NFkB via AZD5582 act together to dramatically increase initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters. We used Cleavage under targets and Tagmentation (Cut-and-Tag), a chromatin profiling strategy in which antibody-targeted controlled integration of DNA sequencing adapters produces libraries for paired-end DNA sequencing.