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

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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.