SUMOylation of the m6A reader YTHDF2 by PIAS1 promotes viral RNA decay to restrict EBV replication.

YTHDF2 is a member of the YTH protein family that binds to N6-methyladenosine (m6A)-modified RNA, regulating RNA stability and restricting viral replication, including Epstein-Barr virus (EBV). PIAS1 is an E3 SUMO ligase known as an EBV restriction factor, but its role in YTHDF2 SUMOylation remains unclear. In this study, we investigated the functional regulation of YTHDF2 by PIAS1. We found that PIAS1 promotes the SUMOylation of YTHDF2 at three specific lysine residues (K281, K571, and K572). Importantly, PIAS1 enhances the antiviral activity of YTHDF2, and SUMOylation-deficient YTHDF2 shows reduced anti-EBV activity. Mechanistically, YTHDF2 lacking SUMOylation exhibits reduced binding to EBV transcripts, leading to increased viral mRNA stability. Furthermore, PIAS1 mediates SUMOylation of YTHDF2's paralogs, YTHDF1 and YTHDF3. These results collectively uncover a unique mechanism whereby YTHDF2 controls EBV replication through PIAS1-mediated SUMOylation, highlighting the significance of SUMOylation in regulating viral mRNA stability and EBV replication.