Baquero Pérez, BelindaYonchev, Ivaylo D.Delgado-Tejedor, AnnaMedina, RebecaPuig-Torrents, MireiaSudbery, IanBegik, OguzhanWilson, Stuart A.Novoa, Eva MariaDíez Antón, Juana, 1962-2024-03-202024-03-202024Baquero-Pérez B, Yonchev ID, Delgado-Tejedor A, Medina R, Puig-Torrents M, Sudbery I, et al. N6-methyladenosine modification is not a general trait of viral RNA genomes. Nat Commun. 2024 Mar 11;15(1):1964. DOI: 10.1038/s41467-024-46278-92041-1723http://hdl.handle.net/10230/59509Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery's localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.application/pdfeng© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-024-46278-9AlphavirusesMicrobiologyVirologyinfo:eu-repo/semantics/openAccess