m6A epigenetic modification of viral RNA is critical for the transmission cycle of mosquito-borne flaviviruses

Arboviruses maintain alternative transmission routes between arthropods and vertebrates through precise dual-host adaptation of their viral genome. However, how epigenetic modifications impact flavivirus transmission cycle remains obscure. Depleting the m6A modification complex revealed that m6A modifications preferentially facilitate mosquito-borne flaviviruses (MBFs) propagation in vertebrates but not in mosquitoes. Stem loop I (SLI) deletion in the MBF 3' UTR decreased m6A expression and viral propagation in vertebrates but not in mosquitoes, indicating viral genome component-epigenetic modification interplay. Mechanistically, m6A modification in stress granules was found to be driven by dynamic SLI-cellular G3BP1 interaction. The m6A-methylated viral genome is ejected by G3BP1 and undergoes viral assembly. Chemical perturbation of m6A modification via METTL3 inhibition potently blocked MBF proliferation in mice and abolished mouse-mosquito viral transmission. Overall, m6A modification is indispensable for efficient arthropod-to-vertebrate transmission of MBF, highlighting its potential as a target for antiviral and transmission-control strategies.