Functional characterization of EcPSMB10: Suppression of RGNNV replication via inhibiting autophagy in vitro

Red-spotted grouper nervous necrosis virus (RGNNV) is a major pathogen, causing high mortality in grouper aquaculture. During infection, the viral capsid protein (CP) induces host cell autophagy to promote viral replication. Thus, modulating the host autophagy pathway represents a promising therapeutic strategy against RGNNV. In this study, we cloned the proteasome subunit PSMB10 from orange-spotted grouper (Epinephelus coioides) and performed mechanistic investigation in grouper spleen (GS) cells. Functional assays revealed that EcPSMB10 acts as a host restriction factor and strongly suppresses RGNNV replication. Mechanistically, EcPSMB10 inhibits CP-induced autophagy by acting on viral CP. Specifically, EcPSMB10 reverses CP-mediated inhibition of AKT and mTOR phosphorylation, thereby blocking autophagy initiation. Furthermore, EcPSMB10 downregulates CP-enhanced expression of ATG5, Beclin1, and LC3-II, and suppresses the formation of LC3-II-positive autophagosomes. EcPSMB10 also specifically interacts with the host protein EcATG5, which downregulates ATG5 protein expression and disrupts assembly of the ATG5-ATG12-ATG16L complex, thereby inhibiting autophagy. Collectively, our in vitro results indicate that EcPSMB10 restricts RGNNV replication by inhibiting cellular autophagy. These findings provide new insights into the antiviral regulatory mechanism of immunoproteasome.