复制型 HIV 指导的 CRISPR 筛选鉴定出抗病毒因子，包括辅助蛋白 Nef 的靶标。
Replication competent HIV-guided CRISPR screen identifies antiviral factors including targets of the accessory protein Nef.
机构信息
Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.
Laboratory for Functional Genome Analysis Gene Center, LMU Munich, 81377, Munich, Germany.
出版信息
Nat Commun. 2024 May 7;15(1):3813. doi: 10.1038/s41467-024-48228-x.
Innate antiviral factors are essential for effective defense against viral pathogens. However, the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here, we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4 T cells robustly enriched HIV-1 encoding sgRNAs against GRN, CIITA, EHMT2, CEACAM3, CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene, we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4 T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry, transcription, release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle.
先天抗病毒因子对于有效抵御病毒病原体至关重要。然而，主要的限制机制仍不明确。目前发现抗病毒因子的方法通常集中在病毒复制的初始步骤，并且仅限于单次感染。在这里，我们构建了超过 1500 个具有复制能力的 HIV-1 文库，每个文库都表达一个 gRNA，以靶向超过 500 个细胞基因，用于通过病毒驱动的方式发现抗病毒因子。在 CD4 T 细胞中传代可使编码 sgRNA 的 HIV-1 针对 GRN、CIITA、EHMT2、CEACAM3、CC2D1B 和 RHOA 的富集程度提高 50 多倍。使用缺乏辅助 nef 基因的 HIV-1 文库，我们鉴定了 IFI16 是 Nef 的靶标。在细胞系和原代 CD4 T 细胞中的功能分析表明，HIV 驱动的 CRISPR 筛选鉴定了针对病毒进入、转录、释放和感染力的限制因子。我们的 HIV 指导的 CRISPR 技术能够在整个病毒复制周期中灵敏地发现生理相关的细胞防御因子。
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