5' RACE-seq of HeLa cells treated with siRNA control, siZAP or siXRN1 cells and infected with wild type HIV-1 or CpG-recoded HIV-1

The zinc finger antiviral protein (ZAP, also known as PARP13 or ZC3HAV1) is an antiviral factor that restricts the replication of a wide range of RNA and DNA viruses. It exerts its antiviral function primarily by binding specific sequences known as ZAP response elements (ZREs) within single-stranded RNA, promoting RNA degradation or inhibiting its translation. The ZAP RNA-binding domain shows a high affinity for binding to CpG dinucleotides, which are generally depleted in vertebrate RNA viruses. There are two major isoforms of ZAP, the long isoform (ZAP-L) and the short isoform (ZAP-S). ZAP has no enzymatic activity and requires cofactors to effectively restrict viral replication. We aimed to characterise the mechanisms underlying ZAP-mediated RNA decay and, in particular, to investigate how ZAP cofactors influence its binding to viral RNA and subsequent RNA degradation. We used an HIV-1 model that was sensitised to ZAP activity by introducing 36 additional CpG dinucleotides through silent mutations into the env gene. The role of ZAP cofactors TRIM25 and KHNYN as well as the ZAP-L and ZAP-S isoforms was investigated by CRISPR-mediated depletion. ZAP interactions with viral RNA was analysed using iCLIP assays performed in TRIM25, KHNYN, ZAP-L and ZAP-S knockout cell lines. In addition, we used 3' and 5' RACE-seq in combination with Oxford Nanopore Technologies sequencing technology to accurately identify KHNYN-mediated cleavage sites on viral RNA.

锌指抗病毒蛋白（zinc finger antiviral protein, ZAP，又称PARP13或ZC3HAV1）是一类可广谱限制RNA与DNA病毒复制的抗病毒因子。其抗病毒功能主要通过结合单链RNA内的ZAP应答元件（ZAP response elements, ZREs）这一特定序列实现，可促进RNA降解或抑制其翻译过程。ZAP的RNA结合结构域对CpG二核苷酸具有高亲和力，而脊椎动物RNA病毒的基因组中通常存在CpG二核苷酸缺失现象。ZAP存在两种主要亚型：长亚型（ZAP-L）与短亚型（ZAP-S）。ZAP本身不具备酶活性，需依赖辅助因子才能有效限制病毒复制。本研究旨在解析ZAP介导的RNA降解机制，重点探究ZAP辅助因子如何影响其与病毒RNA的结合及后续的RNA降解过程。我们构建了对ZAP活性敏感的HIV-1模型：通过对env基因引入沉默突变，添加36个额外的CpG二核苷酸位点。通过CRISPR介导的基因敲除技术，我们探究了ZAP辅助因子TRIM25、KHNYN以及ZAP-L、ZAP-S亚型的功能。在TRIM25、KHNYN、ZAP-L与ZAP-S敲除细胞系中开展iCLIP实验，分析ZAP与病毒RNA的相互作用。此外，我们结合3'与5' RACE-seq技术及牛津纳米孔技术（Oxford Nanopore Technologies）测序平台，精准鉴定病毒RNA上KHNYN介导的切割位点。