N⁶-methyladenosine modifications enhance enterovirus 71 ORF translation through METTL3 cytoplasmic distribution

During replication, numerous viral RNAs are modified by N⁶-methyladenosine (m⁶A), the most abundant internal RNA modification. m⁶A is believed to regulate elements of RNA metabolism, such as splicing, stability, translation, secondary structure formation, and viral replication. In this study, we assessed the occurrence of m⁶A modification of the EV71 genome in human cells and revealed a preferred, conserved modification site across diverse viral strains. A single m⁶A modification at the 5' UTR-VP4 junction was shown to perform a protranslational function. Depletion of the METTL3 methyltransferase or treatment with 3-deazaadenosine significantly reduced EV71 replication. Specifically, METTL3 colocalized with the viral dsRNA replication intermediate in the cytoplasm during EV71 infection. As a nuclear resident protein, METTL3 relies on the binding of the nuclear import protein karyopherin to its nuclear localization signal (NLS) for nuclear translocation. We observed that EV71 2A and METTL3 share nuclear import proteins. The results of this study revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, providing an increased understanding of RNA epitranscriptomics during the EV71 replication cycle.