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

本研究旨在验证如下假说：HIV-1复制所必需的宿主因子，同样在HIV潜伏期逆转过程中发挥功能。本研究使用靶向潜在HIV宿主依赖因子的成簇规律间隔短回文重复序列（CRISPR）基因文库，开展筛选以鉴定参与潜伏期再激活的宿主基因。最终鉴定出多个在HIV-1潜伏期再激活中发挥关键作用的HIV宿主依赖因子，包括ELL、UBE2M、TBL1XR1、HDAC3、AMBRA1及ALYREF。作为转录延伸重要复合物P-TEFb的组成成分，细胞周期蛋白T1（Cyclin T1, CCNT1）的敲除是本次筛选中的头号命中靶点，且在多种潜伏期逆转试剂（latency reversal agent, LRA）处理下，对HIV潜伏期逆转的影响最为显著。此外，在HIV潜伏期的原代CD4+ T细胞模型中，CCNT1敲除可阻断潜伏期再激活，且不会影响这些细胞的活化状态。RNA测序（RNA sequencing）数据显示，CCNT1对HIV-1前病毒基因的调控程度远超其他任何宿主基因，且在原代T细胞活化后，不会对其RNA转录本产生显著影响。综上，本研究认为CCNT1的功能在T细胞中存在冗余，但对于HIV潜伏期逆转则是绝对必需的。整体实验设计：本研究以潜伏感染的Jurkat T细胞（J-Lats）为模型，利用此前已报道的HIV宿主依赖因子文库，开展CRISPR筛选以解析参与潜伏期逆转的宿主基因。本次筛选中，我们将靶向527个基因的向导RNA（guide RNA）文库（每个基因对应8条向导RNA，另设210条非靶向对照）转导至细胞中，使用嘌呤霉素（puromycin）筛选成功携带载体的细胞，随后分别以二甲基亚砜（dimethyl sulfoxide, DMSO）对照或潜伏期逆转试剂（LRA）处理细胞。本研究旨在探究相较于基因组DNA池，病毒上清液中丰度出现下调的基因。