A CRISPR screen of HIV dependency factors reveals CCNT1 is non-essential in T cells but required for HIV-1 reactivation from latency [CRISPR]

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV- 1 dependency factors that play a key role in HIV-1 latency reactivation including ELL, UBE2M, TBL1XR1, HDAC3, AMBRA1, and ALYREF. Knockout of Cyclin T1 (CCNT1), a component of the P-TEFb complex important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is redundant in T cells but is absolutely required for HIV latency reversal. Overall design: We performed a CRISPR screen to understand the host genes involved in latency reversal, by targeting a library of putative HIV Dependency Factors previously described using latently infected Jurkat T cells (J-Lats) as a model. In this screen, we transduced a library containing guides targeting 527 genes (8 guides per gene, and 210 non-targeting controls), used puromycin to select for cells which have the vector and treated cells with either a DMSO control or an LRA (latency reversal agent). We are interested in understanding the genes which are depleted in the viral supernatant relative to the genomic DNA pool.