N6-methyladenosine modification is not a general trait of viral RNA genomes

Despite the nuclear localization of the m 6 A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m 6 A-modified. However, these findings are mostly based on m 6 A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m 6 A in CHIKV and DENV RNAs. For this, we combine m 6 A-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 m 6 A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m 6 A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m 6 A machinery’s localization. Our results challenge the prevailing notion that m 6 A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.