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

全基因组 CRISPRa 筛选以确定细胞的抗病毒库

• 批准号: 10927987
• 负责人: Sonja Best
• 金额: $ 24.77万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10927987
• 关键词: Antiviral Therapy; Attenuated Vaccines; CRISPR-mediated transcriptional activation; Case Fatality Rates; Cell Death; Cell Line; Cell Survival; Cells; Data; Ebola virus; Genes; Genetic Transcription; Goals; Guide RNA; Human; Infection; Interferon Type I; Interferon-beta; Interferons; Life Cycle Stages; Liver; Medical; Methods; Microbe; Molecular; Mus; Organ; Proteins; Public Health; RNA Viruses; Signal Transduction; Transcription Initiation; Vaccine Design; Viral; Viral Genes; Viral Hemorrhagic Fevers; Viral Pathogenesis; Viral Proteins; Virulence; Virus; Virus Diseases; Virus Replication; Zinc Fingers; animal model development; cell killing; comparison control; genome wide screen; genome-wide; helicase; in vivo; novel; promoter; transmission process; viral outbreak

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.

该提案的目标是从功能上表征新兴 RNA 病毒的限制因素。首先，我们重点关注埃博拉病毒 (EBOV)。埃博拉病毒引起人类病毒性出血热暴发，病死率高达 60%。埃博拉病毒因其极高的毒力、人际传播以及埃博拉病毒出现的不可预测性而成为一个主要的公共卫生问题。 为了确定 EBOV 的新限制因子，对活的传染性 EBOV 进行了全基因组 CRISPRa 筛选。由于肝脏是埃博拉病毒感染期间的主要靶器官，因此产生了人类肝脏 CRISPRa 细胞系。 EBOV 选择后，超过 300 个基因显着富集。最佳候选者是锌指解旋酶 2 (Helz2)，它是一种已知的 ISG。事实证明，Helz2 是 EBOV 的一种极其有效的限制因子，与对照相比，它能阻止病毒复制 1000 倍以上。值得注意的是，这种限制水平高于在 IFNb 转录诱导的细胞中观察到的水平，但对于 Helz2 作为抗病毒蛋白几乎一无所知。我们目前正在使用 Helz2-/- C57/BL6J 小鼠确定 1) Helz2 是否与 EBOV 发病机制相关，2) Helz2 限制 EBOV 复制的机制，以及 3) 扩大 CRISPRa 筛选，以鉴定其他新兴 RNA 病毒的新限制因子。这些数据将提供对细胞抗病毒基因库的更广泛了解，并更全面地了解医学上重要的新兴 RNA 病毒的关键 ISG 的分子抗病毒机制。