{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":[null],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15071"],"description":["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."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - The nitrocellulose membrane strips were digested with 10μl proteinase K in 200μl proteinase K SDS buffer (10mM Tris-HCl pH 7.4, 100mM NaCl, 1mM EDTA, 0.2% SDS) for 1 hour at 50oC. The supernatant was transferred to a phase lock gel heavy tube. The RNA extracted were then phenol/chloroform extracted from the supernatant by adding 200μl of Phenol:Chloroform:Isoamyl Alcohol 25:24:1, incubating shaking for 5 min at 30°C, and centrifuged to separate the aqueous and organic phases. The aqueous phase was further extracted with 0.8ml of chloroform, centrifuged, and the upper phase containing the RNA is transferred to a new tube, and then precipitated with 20μl 3M NaAc, 0.75μl glycoblue and 0.5ml EtOH overnight at −20°C. The RNA is pelleted for 30 minutes at 4°C at 13000G.","Sequencing - RNA-seq libraries were sequenced as paired-end 150 bp reads on Illumina Novaseq 6000.","Sample Collection - Cells were infected at MOI=3 and media was replaced 18 h post-inoculation with DMEM supplemented with 2% fetal bovine serum. At 48hpi, cells were washed with ice-cold PBS, irradiated once with 150 mJ/cm2 in a Stratalinker 2400 at 254 nm, harvested by scrapping in PBS, pelleted by centrifugation then resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; protease inhibitor).","Library Construction - Custom individual cross-linking and immunoprecipitation (iCLIP) library construction protocol."],"figure_sub":["MINSEQE Score","Assays and Data","organisation","MAGE-TAB Files"],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"additional_accession":["E-MTAB-15056"],"pubmed_authors":["Jernej Ule","Grega Gimpelj"]},"is_claimable":false,"name":"CLIP-seq of ZAP in HeLa CRISPR control and TRIM25 knockout cells infected with wild type HIV-1 or CpG-recoded HIV-1","description":"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.","dates":{"release":"2025-09-30T00:00:00Z","modification":"2025-09-30T01:04:50.834Z","creation":"2025-04-23T13:34:34.265Z"},"accession":"E-MTAB-15071","cross_references":{"ENA":["ERP171895"],"Biostudies":["E-MTAB-15064","E-MTAB-15077","E-MTAB-15065","E-MTAB-15095","E-MTAB-15072","E-MTAB-15056"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005518","EFO_0003738","EFO_0004184"]}}