O-GlcNAcylation of AMFR stabilizes TSPAN4 to regulate migrasome formation for viral release.

Migrasomes are migration-dependent organelles, serving as delivery packets to mediate release of cytoplasmic contents. Tetraspanin 4 (TSPAN4) acts as a marker for migrasomes and is essential for their formation. However, the regulatory mechanism(s) underlying TSPAN4-mediated migrasome biogenesis and its physiological functions remain to be elucidated. Here, we identified AMFR, an ER-resident E3 ligase, regulates migrasome formation through catalyzing the K48-linked polyubiquitination of TSPAN4 for degradation. Further, O-GlcNAcylation of AMFR by OGT at threonine 643 disrupts AMFR-TSPAN4 interactions, thereby stabilizing TSPAN4 and promoting migrasome formation. Additionally, viruses dynamically regulate migrasome formation by modulating AMFR O-GlcNAcylation and TSPAN4 ubiquitination. During the early stages of VSV or HSV-1 infection, viruses enhance migrasome formation and exploit these structures to spread among neighboring cells, whereas abolish migrasome formation during the late stages of infection. Our findings reveal a negatively regulatory mechanism governing migrasome biogenesis, and highlight how VSV and HSV-1 manipulate this process to facilitate their release.