BVDV E2 hijacks host TSG101 to facilitate transplacental transmission by regulating exosome biogenesis and release

Bovine viral diarrhea virus (BVDV) infects bovine trophoblast cells (BTCs) to induce abortion, stillbirth and other reproductive disorders, and severely threatens the global cattle industry. Exosomes serve as vital viral transmission carriers mediating immune evasion. Tumor susceptibility gene 101 (TSG101), a core ESCRT component, regulates exosome biogenesis and cargo sorting. Nevertheless, the mechanism of TSG101 in exosome-mediated BVDV transmission across BTCs remains unknown. In this study, BTCs were used as cell models to explore the role and mechanism of TSG101 in BVDV transmission. BVDV infection caused obvious cytopathic effects (CPE) in BTCs, with viral particles packaged in exosomes to facilitate immune evasion and transmission. BVDV also upregulated TSG101 and exosomal CD63, and TSG101 colocalized with BVDV E2. TSG101 knockdown suppressed BVDV proliferation and exosome-mediated transmission, and impaired CD63 trafficking into exosomes. Co-IP and immunofluorescence assays further confirmed the interaction between BVDV E2 and TSG101. Molecular docking and 100-ns MD simulation showed that E2 and TSG101 formed a stable binding complex, with key interacting residues identified at the contact surface. TSG101 mutations at Y110K, K118A, and E284A exerted distinct effects. TSG101 Y110K and K118A restricted BVDV replication by impairing TSG101 function, while the E284A mutation preserved TSG101 responsiveness, promoted viral proliferation. These findings expand understanding of BVDV E2-mediated ESCRT homeostasis regulation and provide a molecular basis for targeting the TSG101 to limit BVDV vertical transmission in BTCs.