biostudies-arrayexpressAndrew FirthHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14994Ribosome profiling of enterovirus infected cells was used to investigate expression of the virus polyprotein and upstream open reading frames. Samples were either pretreated with initiation inhibitor lactimidomycin (LTM) to cause ribosomes to accumulate at initiation sites, or were flash frozen with no drug pretreatment (NT) to capture elongating ribosomes.biostudies-arrayexpressSequencing - Amplicon libraries were deep sequenced using an Illumina NextSeq platform (76 cycles, single-end).Nucleic Acid Extraction - Cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a surface of dry ice-ethanol bath for 10 s, transferred to dry ice and 400 µl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TURBO DNase (Life Technologies)] dripped on. Cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 ºC, the supernatants recovered and stored in liquid nitrogen.Library Construction - The purification of RPFs was performed using Direct-zol RNA MiniPrep kit (Zymo research). The further methodologies employed were based on the original protocols (Ingolia et al., 2012; Irigoyen et al., 2016), except ribosomal RNA contamination was removed using RiboZero Gold rRNA removal kit (Illumina) and library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing. Amplicon libraries were deep sequenced using an Illumina NextSeq platform.Growth Protocol - HeLa Ohio cells were grown on 150-mm dishes to a confluency of 80% and infected with CVA-13 at an MOI of 10 to ensure synchronous infection.Sample Treatment - For lactimidomycin (LTM) treated cells, 30 min before the specified time point, cells were treated with 50 mM LTM for 30 min.Sample Collection - At 5 and 7 hpi, LTM-treated and non-treated cells were flash frozen in an ethanol/dry ice bath and lyzed in the presence of 0.36 mM cycloheximide (CHX).OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesUnknownTranscriptomicsGenomicsProteomicsNextSeq 500Enteroviruses comprise a large group of mammalian pathogens that often utilize two open reading frames (ORFs) to encode their proteins: the upstream protein (UP) and the main polyprotein. In some enteroviruses, in addition to the canonical upstream AUG (uAUG), there is another AUG that may represent an alternative upstream initiation site. An analysis of enterovirus sequences containing additional upstream AUGs identified several clusters, including strains of pathogenic Enterovirus alphacoxsackie and E. coxsackiepol . Using ribosome profiling on coxsackievirus CVA-13 ( E. coxsackiepol ), we demonstrate that both upstream AUG codons can be used for translation initiation in infected cells. Moreover, we confirm translation from both upstream AUGs using a reporter system. Mutating the additional upstream AUG in the context of CVA-13 did not result in phenotypic changes in immortalized cell lines. However, the wild-type virus outcompeted this mutant in human intestinal organoids and differentiated neuronal systems, representing an advantage in physiologically relevant infection sites. Mutation of the stop codon of the shorter upstream ORF led to dysregulated translation of the other ORFs in the reporter system, suggesting a potential role for the additional uORF in modulating the expression level of the other ORFs. These findings demonstrate the remarkable plasticity of enterovirus IRES-mediated initiation and the competitive advantage of double-upstream-AUG-containing viruses in terminally differentiated intestinal organoids and neuronal systems.Ribo-seqHomo sapiensFlexibility and modulation of translation initiation in enterovirus genomesValeria LullaRhian L O'Connor, Georgia M Cook, Jacqueline Hankinson, Ksenia Fominykh, Samantha H Cheng, Daniel A Nash, Aurelie Cenier, Komal M Nayak, Stephen C Graham, Janet E Deane, Matthias Zilbauer, Andrew E Firth, Valeria LullaAndrew FirthfalseRibosome profiling of HeLa cells infected with Enterovirus coxsackiepol (strain CVA-13/Flores)Ribosome profiling of enterovirus infected cells was used to investigate expression of the virus polyprotein and upstream open reading frames. Samples were either pretreated with initiation inhibitor lactimidomycin (LTM) to cause ribosomes to accumulate at initiation sites, or were flash frozen with no drug pretreatment (NT) to capture elongating ribosomes.2025-11-26T00:00:00Z2025-11-26T11:10:03.359Z2025-04-01T14:58:33.253ZE-MTAB-14994ERP171065EFO_0002944EFO_0004170EFO_0003789EFO_0008891EFO_0005518EFO_0004184EFO_000396910.1101/2025.03.24.645098