biostudies-arrayexpressHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15060The 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-arrayexpressNucleic Acid Extraction - Total RNA was extracted using the RNeasy Mini Kit (Qiagen).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.Sequencing - The sequencing libraries were multiplexed and loaded on the flowcell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument.Library Construction - RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR.MINSEQE ScoreAssays and DataorganisationMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensE-MTAB-15056Jernej UleGrega GimpeljfalseRNA-seq of HeLa CRISPR KHNYN knockout cells reconstituted with GFP or GFP-tagged KHNYN infected with 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.2025-09-30T00:00:00Z2025-09-30T01:04:50.844Z2025-04-17T16:24:08.191ZE-MTAB-15060ERP171783E-MTAB-15061E-MTAB-15056EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184