The Molecular Mechanism Of SVCV N Protein Negatively Regulates Interferon Through Autophagy-Lysosome-Dependent Degradation Of STING

As a major aquaculture country,China accounts for more than 60%of the world’s total aquaculture production..However,with the increase of aquaculture volume,the economic losses caused by aquatic animal diseases,especially viral diseases,are becoming more and more serious.Spring viremia of carp（SVCV）caused by Spring viremia of carp virus（SVCV）has caused significant economic losses in carp fish culture.The interferon（IFN）system is the host’s first line of defense against viral invasion.Upon virus infection of host cells,host pattern recognition receptors（PRRs）recognize viral nucleic acids,activate relevant signal transduction responses,and then initiate IFN expression,ultimately inducing downstream interferon-stimulated gene activation for virus clearance.However,viruses have evolved multiple immune escape strategies to evade the host IFN system during their long-term evolution with the host cells,so it is crucial to study the immune escape mechanism of SVCV to prevent fish disease outbreaks and save the aquaculture economy.In this study,we found that the SVCV viral N protein negatively regulates host IFN production through the autophagy-lysosome-dependent pathway of degradation of STING（Stimulator of interferon genes）.First,overexpression of N protein inhibits the activity of the viral mimic polyinosinic-polycytidylic acid（poly I:C）/SVCV-induced IFN promoter.Second,N protein inhibited poly I:C/SVCV-induced expression of ifn as well as IFN-stimulated genes at the m RNA level.Preliminary results suggest that the SVCV virus N protein is able to inhibit the cellular IFN response.The signaling pathway mediated by RIG-I Retinoic acid-induced gene I-like receptors（RLRs）is one of the typical pattern recognition receptors signaling pathways for host-induced IFN.Based on this,we investigated the effect of SVCV N protein on IFN induced by RLR signaling pathway molecules.Luciferase experiments showed that the N protein significantly inhibited STING-induced IFN promoter activity.Subsequently,the interaction between N protein and RLR pathway molecules was investigated,and immunoprecipitation experiments showed that N protein binds to STING and that the N-terminal structural domain（TM）of STING is essential for this binding.Next,the degradation mechanism of STING by N protein was explored,and treatment with different drugs revealed that the degradation of STING by N protein was restored by the autophagy inhibitors 3-MA and NH₄Cl,indicating that N protein degrades STING in an autophagic lysosome-dependent manner with a dose-dependent effect.Further investigation of the molecular mechanism of STING degradation by N protein using autophagy system revealed that N protein bound to autophagy factor Beclin1,and knockdown of Beclin1 attenuated N protein-mediated STING degradation.In addition,N protein significantly attenuated the STING-mediated cellular antiviral response and affected the host cell function.Taken together,our study shows that SVCV N protein degrades STING through an autophagy-lysosome-dependent pathway to suppress host IFN expression,thereby inhibiting the IFN response to achieve immune escape.This study enriches the understanding of the pathogenesis of SVCV,and also provides new ideas for the prevention and treatment of viral diseases in fish.