Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal [CRISPR]

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo. We also detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Overall design: Latency HIV-CRISPR is performed by generating a pool of ZAP-knockout J-Lat 10.6 and 5A8 cells that are each knocked out for human epigenome genes using the HuEpi guide RNA library or a NFkB-related gene library (NFkB) with all Integrator complex members added in. The dosage and timing of drugs varied by screen: all HuEpi screens were performed with 10 nM AZD5582 & 100 nM I-BET151 for 48 hrs whereas all NFkB screens were performed with 1 nM AZD5582 & 2.5uM I-BET151 for 24hrs. Each screen is performed in two biological replicates. Afterwards, for each condition, the cell pellets were harvested, genomic DNA (gDNA) was extracted, and sequencing was performed to evaluate the single guide RNA (sgRNA) representation of the library. Simultaneously, the viral supernatant was harvested, viral RNA (vRNA) was extracted, and sequencing was performed. By comparing the sgRNA representation of the viral RNA to the library representation of the genomic DNA, the sgRNAs that are enriched and depleted in the viral population are associated with potential HIV-1 latency factors. The plasmid library of the HIV-CRISPR HuEpi or NFkB guide RNA library was also sequenced.