biostudies-arrayexpressJernej UleHomo sapiensDoradohttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15097The 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.biostudies-arrayexpressLibrary Construction - The total RNA was purified with the Monarch Spin RNA Cleanup Kit (NEB). The 5’ RACE was performed from 500 ng purified RNA using the SMARTer RACE 5’/3’ Kit and according to the manufacturer’s instructions. 5’ RACE sequencing librairies were prepared using the Native Barcoding Kit 24 V14 (Oxford Nanopore Technologies).Nucleic Acid Extraction - Total RNA was extracted using the Direct-zol RNA Miniprep Plus KitSequencing - Reads were sequenced on PromethION with R10.4.1 flowcells.Sample Collection - Cells were infected at MOI=3. The medium was replaced 18 hours (h) post inoculation. Cell lysates and supernatants were collected at 48 hours post-infection. Supernatants were spun down at 300 ×g for five minutes and analysed by TZM-bl assay and immunoblotting.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsPromethIONRNA-seq of coding RNAHomo sapiensJernej UleGrega Gimpeljfalse5′ RACE-seq of HeLa cells treated with siRNA control, siZAP or siXRN1 cells and infected with wild type HIV-1 or CpG-recoded HIV-1The 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.2025-09-30T00:00:00Z2025-09-30T01:04:50.864Z2025-04-28T15:39:38.678ZE-MTAB-15097ERP172036EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184