Host-specific ubiquitination of prM orchestrates ESCRT recruitment to mediate efficient Japanese Encephalitis Virus assembly in vertebrates.

Mosquito-borne orthoflavivirus, such as Japanese encephalitis virus (JEV), Dengue virus (DENV), and Zika virus (ZIKV), pose a serious global health threat. As obligate intracellular parasites, they often hijack the host ubiquitin system to modify their own proteins, thereby regulating the viral life cycle, host adaptation, transmission, and pathogenesis. Despite its critical roles, the precise molecular mechanisms and functional significance of viral protein ubiquitination in orthoflavivirus infection remain incompletely understood. Here, we identify JEV prM as a novel target for host-specific ubiquitination, which occurs exclusively in vertebrate hosts but not in mosquitoes. Ubiquitin conjugation at the evolutionarily conserved lysine residues (K107/108/116) in multiple mosquito-borne orthoflaviviruses (USUV, MVEV, and WNV) confers differential adaptation between vertebrate hosts and mosquito vectors. Mechanistically, prM ubiquitination serves as a recruitment signal for the ESCRT-I subunit TSG101, an early-acting component of the ESCRT machinery, which in turn serves as an adaptor to recruit downstream ESCRT components (VPS28, CHMP2A, and CHMP4B), thereby driving viral particle budding. These findings elucidate a novel mechanism by which viral protein ubiquitination regulates JEV infection and host adaptation, and provide important insights into the adaptive evolution of orthoflaviviruses across different hosts and vectors.