Genome-wide CRISPRa screen to determine the antiviral repertoire of the cell

该提案的目标是功能性地表征新兴RNA病毒的限制因子。首先，我们重点关注埃博拉病毒（EBOV）。EBOV引起人类病毒性出血热暴发，病死率高达60%。EBOV是一个主要的公共卫生问题，因为它的极端毒力，它的人际传播，以及EBOV出现的不可预测性。 为了鉴定EBOV的新限制因子，用活的感染性EBOV进行全基因组CRISPRa筛选。产生了人肝脏CRISPRa细胞系，因为肝脏是EBOV感染期间的主要靶器官。 在EBOV选择后，超过300个基因显著富集。最佳候选物是具有锌指2的解旋酶（Helz 2），其是已知的ISG。Helz 2被证明是一种非常有效的EBOV限制因子，与对照相比，它可以阻断病毒复制超过1000倍。值得注意的是，这种限制水平大于在IFNb转录诱导的细胞中观察到的限制水平，但关于Helz 2作为抗病毒蛋白几乎一无所知。我们目前正在确定1）使用Helz 2-/- C57/BL 6 J小鼠在体内Helz 2是否与EBOV发病机制相关，2）Helz 2限制EBOV复制的机制，以及3）扩展CRISPRa筛选以鉴定具有其他新兴RNA病毒的新型限制因子。这些数据将提供对细胞的抗病毒基因库的扩展理解，以及对医学上重要的新兴RNA病毒的关键ISG的分子抗病毒机制的更透彻理解。

The goal for this proposal is to functionally characterize restriction factors for emerging RNA viruses. As a starting point, we have focused on Ebola virus (EBOV). EBOV causes outbreaks of viral hemorrhagic fever in humans with a case-fatality rate as high as 60%. EBOV is a major public health concern because of its extreme virulence, its human-to-human transmission, and the unpredictability of EBOV emergence. To identify new restriction factors for EBOV, a genome-wide CRISPRa screen was performed with live, infectious EBOV. A human liver CRISPRa cell line was generated because the liver is a major target organ during infection with EBOV. Over 300 genes were significantly enriched after EBOV selection. The top candidate was helicase with zinc finger 2 (Helz2), which is a known ISG. Helz2 proved to be an incredibly potent restriction factor for EBOV by blocking virus replication by more than 1000-fold compared to the control. Remarkably, this level of restriction was greater than observed in cells that were transcriptionally induced for IFNb, but almost nothing is known about Helz2 as an antiviral protein. We are currently determining 1) whether Helz2 is relevant in vivo to EBOV pathogenesis using Helz2-/- C57/BL6J mice, 2) the mechanism of Helz2 restriction of EBOV replication, and 3) expanding the CRISPRa screen to identify novel restriction factors with other emerging RNA viruses. These data will provide an expanded understanding of the antiviral gene repertoire of the cell and a more thorough understanding of molecular antiviral mechanisms of key ISGs for medically important emerging RNA viruses.