The autophagy-related degradation of MDA5 by Tembusu virus nonstructural 2B disrupts IFNβ production

Duck Tembusu virus (TMUV) is a serious avian pathogen causing a decline in egg production, but the mechanism of the virus that breaks through the innate immune system is poorly understood. Here, we show that TMUV inhibits poly(I:C)-induced interferon (IFN) production. Because poly(I:C) transfection can specifically activate the MDA5 pathway in duck primary cells, we found that infection with TMUV can specifically target MDA5 and lead to its degradation. MDA5 downregulation could be blocked by the autophagy inhibitor 3-methyladenine (3-MA) but not a proteasome inhibitor, strongly implicating MDA5 degradation as an autophagy-related degradation pathway. Pretreatment with 3-MA enhanced the expression of MDA5 and inhibited TMUV replication. To screen TMUV proteins that degraded MDA5, the TMUV replicon and MDA5-Flag were cotransfected into cells, and the western blot analysis showed that nonstructural 2B (NS2B) can degrade MDA5 in a dose-dependent manner. Dual-luciferase assays indicate that NS2B alone inhibits MDA5- or poly(I:C)-mediated IFN production. NS2B binds MDA5 in the presence of 3-MA. The deletion of the amino acids of NS2B from residues 51 to 92 (hydrophilic area) restored the expression of MDA5 and relieved the MDA5-mediated IFNβ production inhibition by NS2B, indicating that the hydrophilic area of NS2B is important for its interaction with host innate immunity.