Chicken Anemia Virus Negatively Regulates STING Via NOD-like Receptor X1 To Affect IFN-β Expression

NOD-like receptor X1（NLRX1）in mitochondria is a member of the nucleotide-binding oligomerization domain like receptors（NLRs）family,whichcan play a negative regulatory role in inflammatory response,autophagy and viral infection.Stimulator of interferon genes（STING）is an important natural immune molecule that induces type I interferon and protects against viral infections.Previous studies in our laboratory have shown that chicken anemia virus（CAV）can inhibit the production of IFN-βby suppressing the c GAS-STING signalling pathway in cells,thereby escaping immunity.However,it is not clear whether CAV negatively regulates the STING signaling pathway through NLRX1,thus affecting IFN-βproduction and the mechanism.In this study,based on the clarification of the mitochondrial localization sequence（MT）of chicken NLRX1（chNLRX1）,we analyzed whether CAV negatively regulates IFN-βthrough chNLRX1 at the molecular and cellular levels and the possible pathways of action.The main elements are as follows:In this study,the gene sequence of the predicted chNLRX1 mitochondrial localization MT sequence（N1-53aa）was first cloned by PCR,inserted into the eukaryotic recombinant expression vector pEGFP-N1 to construct a MT recombinant plasmid,transfected into HEK293T and the chicken macrophage cell line（HD11）via liposome-mediated transfection,and marked with a mitochondrial fluorescent probe（red）to mark the intracytoplasmic mitochondrial location,and observed the co-localization of MT with mitochondria by laser confocal microscopy;In order to clarify the effect of CAV infection on the expression of chNLRX1 in cells,the chicken lymphocyte line（MDCC-MSB1）was infected with 10^-3.525/0.1 mL TCID₅₀ of CAV,The transcript levels of the chNLRX1 gene were measured by SYBR Green I relative fluorescence PCR at 0h before and 12 h,24 h,36h and 48 h after infection.The effect of chNLRX1 overexpression on CAV proliferation in MSB1 cells was detected by applying Taq Man fluorescence quantitative PCR;Finally,to investigate the effect of chNLRX1 on STING-regulated IFN-βproduction,chNLRX1 was overexpressed with STING and then the interaction between chNLRX1 and STING was analyzed by subcellular co-localization,Co-IP and Western blot.Different doses of chNLRX1 were also co-expressed with STING and TBK1,the junction molecule of STING.Co-IP was applied to detect the competitive binding of chNLRX1 and TBK1 to STING,and the effect of chNLRX1 on STING-induced IFN-βpromoter activity was analyzed by a dual luciferase reporter gene assay system.The results showed that after overexpression of the recombinant plasmid pEGFP-N1-MT in HEK293T cells and HD11 cells,the chNLRX1 N-terminal MT-fused GFP-tagged protein co-localized with the mitochondria,indicating that,as predicted,the MT sequence was present in the N-terminal 1-53aa of chNLRX1.SYBR Green I relative fluorescence quantification results showed that after CAV infection with MSB1,chNLRX1mRNA transcript levels were significantly higher at 12 h,24 h and 36 h post-infection compared to the 0 h control（p<0.05）,and were not significantly different from the control by 48 h.This suggests that chNLRX1 may be involved in the viral immune response induced by CAV infection.Taq Man absolute fluorescence quantification of CAV copy number in cells overexpressing chNLRX1 showed a significant increase in CAV copy number in both cell culture supernatant and lysate（p<0.05）.This suggests that chNLRX1promotes CAV replication.Overexpression of chNLRX1 with STING in HEK293T cells and HD11 cells showed subcellular co-localization,whichwas further confirmed by Co-IP assay,and the junction molecule TBK1,whichinteracts with STING,was significantly reduced with increasing chNLRX1 dose,suggesting that chNLRX1 can bind competitively with TBK1.Subsequently,overexpression of chNLRX1 was found to significantly attenuate STING-induced IFN-βpromoter activity in a dual luciferase reporter gene assay system（p<0.05）,suggesting that chNLRX1 could inhibit the STING-related signalling pathway to produce IFN-βexpression,thus exerting a negative regulatory effect.In summary,this study firstly confirmed that chNLRX1 N-terminal 1-53aa has a mitochondrial localization role,and secondly confirmed that chNLRX1 can promote CAV infection and inhibit IFN-βexpression,thus playing a role in assisting virus proliferation;it further clarified that chNLRX1 negatively regulates IFN-βexpression through STING.