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

Abstract

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.

A Virus-Packageable CRISPR System Identifies Host Dependency Factors Across Multiple HIV-1 Strains

“…We also added back the known dependency factors that fell below this threshold in the screen including CCR5 and PAPSS1 which are essential for CCR5-dependent strains. Because transcription and chromatin modification/ regulation were major categories of negatively enriched genes in the whole genome screen (Figure 1C), we also performed an additional targeted screen using a custom-designed Human Epigenome (HuEpi) library consisting of 838 human epigenome and epigenetic regulator genes (17) and included any gene with <10% false discovery rate (FDR) from the HuEpi screen that were not already included in the top TKOv3 gene list (Supplemental Figure S1A) as well as smaller number of negatively enriched genes in an ISG-related library (11) (Supplemental Figure S1B), resulting in an additional 131 genes. Finally, 11 genes previously called “non-essential” (18) that were neither depleted nor enriched in our genome-wide screen were included as negative control genes.…”