Lyssavirus Nucleoprotein Induces GPAT4‐Mediated CYB5R1 Depalmitoylation to Suppress Ferroptosis and Promote Viral Replication

Rabies virus (RABV) and other lyssaviruses exploit lipid droplet (LD) formation to evade host defenses, but the underlying mechanisms remain unclear. Here, we demonstrate that lyssavirus N proteins induce LD biogenesis by upregulating glycerol‐3‐phosphate acyltransferase 4 (GPAT4) expression and promoting its translocation to the LD surface, a conserved mechanism across the lyssavirus genus. Mechanistically, GPAT4‐mediated LD formation sequesters free fatty acids, leading to acyl‐protein thioesterase 1 (APT1)‐dependent depalmitoylation of NADH‐cytochrome B5 reductase 1 (CYB5R1) at Cys208 and Cys278. This post‐translational modification triggers autophagic degradation of CYB5R1, thereby impairing its ability to induce ferroptosis via two complementary pathways: nuclear receptor co‐activator 4 (NCOA4)‐mediated ferritinophagy and H 2 O 2 production. Conversely, diacylglycerol O‐acyltransferase (DGAT) inhibitors or GPAT4 knockdown restores CYB5R1 palmitoylation and stability, reinstates ferroptosis, and suppresses RABV infection. Our findings reveal a novel “ lyssavirus N‐GPAT4‐LD‐CYB5R1 palmitoylation” axis that modulates ferroptosis susceptibility, highlighting protein palmitoylation as a critical regulatory node in virus‐host interactions and identifying GPAT4 as a potential antiviral target.