Fibrillarin-mediated 2'-O-methylation serves as a translation brake on uncapped enterovirus RNA.

2'-O-methylation (Nm) is a key epitranscriptomic mark across diverse RNAs, yet its role in antiviral defense remains largely unexplored. To systematically investigate the landscape and functional significance of internal Nm modifications on enterovirus RNAs, a sensitive sequencing method termed RACE-Nm was established, in combination with the machine learning-based predictor NanoNm, to map Nm sites on low-abundance RNAs at single-nucleotide resolution. A conserved Nm site was identified at nucleotide 307C within the viral internal ribosome entry site (IRES), which was catalyzed by the methyltransferase FBL. The 2'-O-methylation at 307C caused steric hindrance that disrupted PCBP2 recruitment to the IRES and repressed cap-independent translation. Consequently, FBL-mediated suppression of IRES activity limited the production of the viral RNA polymerase 3D, which in turn prevented the upregulation of a set of pro-viral host genes, including FOSL1, ITGB3, and KCNQ4. The physiological importance of this defense mechanism was further demonstrated in vivo, as EV71 mutant lacking the 307C site showed markedly enhanced replication, aggravated tissue damage, and increased lethality in mouse model. Our study establishes FBL-mediated internal Nm modification as a novel host defense pathway that directly targets cap-independent translation to restrict enteroviral infection, highlighting the epitranscriptomic regulation of host-virus interactions.