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. Proposed role of EV71 2A in regulating the nucleocytoplasmic localization of METTL3 and the dynamics of EV71 RNA m6A modification in regulating viral protein translation in cells. Upon entering the cell, the genomic ORF of EV71 is translated, and cleaved 2A can interact with the nuclear import chaperone karyopherin alpha to retain METTL3 in the cytosol and modify EV71 RNA, leading to subsequent enhancement of translation. [Display omitted] •The most prevalent causative enterovirus of hand, foot and mouth disease, EV71 RNA genome is m6A modified in human cells.•Enriched EV71 m6A modification peaks in human cells similar to those found in a monkey-derived cell cultured virus.•METTL3 methyltransferase binds and modify EV71 RNA, and enhanced EV71 replication.•2A share the same karyopherin alpha subunits interaction with METTL3.