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.