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

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: To investigate the role of CCNT1 in host gene regulation as well as its effect on HIV-1 transcripts, we performed bulk RNA sequencing of J-Lat 10.6 cells that were either wild-type for CCNT1 or knocked out for CCNT1. We performed the RNA sequencing on two clonal knockouts of CCNT1. J-Lat cells were either unstimulated or treated with TNFa. We also wanted to investigate the role of CCNT1 in host gene regulation by knocking out CCNT1 or AAVS1 (control) in primary CD4+ T cells, assessing whether knockout affects T cell activation, and performing bulk RNA sequencing. In this case, we isolated CD4+ T cells from three healthy donors, activated T cells, then performed CRISPR/Cas9 knockout by electroporating Cas9 ribonucleoproteins containing guide RNAs targeting CCNT1 or AAVS1. Cells were then activated with CD3/CD28 antibody co-stimulation and RNA was harvested after 24 hours.