Lipidomics reveals the pro-viral roles of ceramides during fish nodavirus infection

Red-spotted grouper nervous necrosis virus (RGNNV) is one of the most lethal viral pathogens with high mortality in the fry and juvenile stage of marine fish. Our previous studies demonstrated that RGNNV infection induced the remodeling of intracellular membrane and exploited cellular fatty acid synthesis for infection. However, the roles of lipid metabolism during infection still remained largely uncertain. Here, the global lipidomic profiles of RGNNV in grouper cells were analyzed, and the crucial roles of ceramides during viral infection were investigated. RGNNV significantly altered lipid homeostasis <i>in vitro</i>. Of note, almost all the detected ceramides were elevated in RGNNV-infected cells. Consistently, RGNNV infection induced a significant increase in ceramides accumulation and the mRNA expression levels of ceramide synthesis-related genes. Interestingly, virus-induced ceramides were colocalized with RGNNV coat protein (CP), but not RNA-dependent RNA-polymerase (RdRp). Furthermore, ectopic expression of CP alone also induced a significant increase in the levels of ceramide species, suggesting that RGNNV manipulated the ceramide metabolism partially via CP protein. Furthermore, disruption of ceramide synthesis pathways using pharmacological inhibitors or knockdown technology markedly suppressed RGNNV infection, and the inhibitory effects were rescued by the exogenous C16-ceramide (d18:1/16:0). In addition, C16-ceramide enhanced RGNNV-induced autophagy and reversed the antiviral action of an autophagy inhibitor chloroquine upon RGNNV infection, suggesting that ceramides might promote RGNNV replication via autophagy. Together, our findings firstly demonstrated that RGNNV altered sphingolipid metabolism and ceramide flux was one of pro-viral mediators for RGNNV replication, providing potential approaches for preventing fish nodaviral infection.IMPORTANCECeramides, as central intermediates in sphingolipid metabolism, are involved in regulating cell viability, differentiation, cycle autophagy, and immune response. Viruses can regulate ceramide synthesis by driving different ceramide synthesis pathways, resulting in differential utilization of ceramides during virus infection. In this study, we demonstrated that RGNNV infection and CP overexpression altered lipid homeostasis, especially by inducing sphingolipid metabolism. Three major ceramide synthesis pathways<i>-de novo</i> biosynthesis, salvage, and sphingomyelin degradation-were all required for RGNNV infection. Moreover, the addition of C16-ceramide (d18:1/16:0) significantly promoted RGNNV replication via increasing RGNNV-induced autophagy. Our findings not only contribute greatly to understanding the mechanism underlying fish nodavirus pathogenesis, but also shed new light on the potential of ceramides as an ideal candidate for prevention and treatment for viral nervous necrosis (VNN).