MARCH6 suppresses Tembusu virus replication by targeting viral NS5 protein for TOLLIP-mediated selective autophagic degradation.

The E3 ligase membrane-associated RING finger 6 (MARCH6) plays a pivotal role in various cellular processes; however, its role in viral defense remains largely unexplored. In this study, we have elucidated a novel antiviral mechanism of avian MARCH6 against duck Tembusu virus (TMUV), revealing a previously uncharacterized host defense strategy. Notably, MARCH6 expression was significantly upregulated during TMUV infection in several duck cell lines, suggesting a conserved cellular response. Functional analyses revealed that overexpression of MARCH6 effectively suppressed TMUV replication, whereas its knockdown markedly enhanced viral replication. Mechanistically, MARCH6 directly interacts with the viral non-structural protein 5 (NS5), mediating its targeted degradation through an unprecedented E3 ligase activity-independent mechanism. Moreover, MARCH6 recruits the autophagic cargo receptor TOLLIP, which facilitates the NS5-TOLLIP interaction independent of ubiquitin signaling and subsequently directs NS5 to phagophores for degradation. These findings reveal a novel antiviral mechanism that focuses on the MARCH6-NS5-TOLLIP axis and represents a critical host defense strategy against viral infections. This study not only provides insights into the antiviral functions of MARCH6 but also emphasizes the importance of selective autophagy as a fundamental mechanism to control viral infection.IMPORTANCETMUV, an emerging pathogenic flavivirus, has rapidly spread across major duck farming regions in Asia since 2010, causing substantial economic losses in the duck industry. More recently, TMUV has expanded its host range, raising concerns about its potential threat to mammals. Understanding TMUV-host interactions is essential for developing effective treatments and vaccines. Here, we uncover a previously uncharacterized role of avian MARCH6 in antiviral defense against TMUV. We demonstrate that MARCH6 restricts TMUV replication through an E3 ligase activity-independent mechanism by targeting the viral NS5 protein for degradation. Notably, MARCH6 promotes NS5 degradation via selective autophagy by recruiting the cargo receptor TOLLIP, bypassing conventional ubiquitin signaling. These findings reveal a novel host antiviral strategy centered on the MARCH6-NS5-TOLLIP axis, broadening our understanding of selective autophagy in antiviral defense.