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

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