m⁶A modification associated with YTHDF1 is involved in Japanese encephalitis virus infection

N6-methyladenosine (m⁶A), the most common modification in mammalian mRNA and viral RNA, regulates mRNA structure, stability, translation, and nuclear export. The Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus causing severe neurologic disease in humans. To date, the role of m⁶A modification in JEV infection remains unclear. Herein, we aimed to determine the impact of m⁶A methylation modification on JEV replication in vitro and in vivo. Our results demonstrated that the overexpression of the m⁶A reader protein YTHDF1 in vitro significantly inhibits JEV proliferation. Additionally, YTHDF1 negatively regulates JEV proliferation in YTHDF1 knockdown cells and YTHDF1 knockout mice. MeRIP-seq analysis indicated that YTHDF1 interacts with several interferon-stimulated genes (ISGs), especially in IFIT3. Overall, our data showed that YTHDF1 played a vital role in inhibiting JEV replication. These findings bring novel insights into the specific mechanisms involved in the innate immune response to infection with JEV. They can be used in the development of novel therapeutics for controlling JEV infection.