Grass Carp (Ctenopharyngodon Idella) NLK2 Phosphorylation Of MAVS Negatively Regulates Type Ⅰ Interferon Expression

MAVS(mitochondrial antiviral signaling),as a connector protein anchored on the mitochondrial membrane surface,is a key protein in RLR mediated antiviral innate immunity signal transduction.When stimulated by incoming RNA viruses,RIG-1(retinoic acid inducible gene I)receptor proteins are activated and their spatial structure changes.The CARD domain(caspase recruitment domain)is exposed and binds to the CARD domain of MAVS,resulting in the formation of a virus-like aggregate of MAVS and the activation of the innate antiviral immune response as an upstream platform protein.NLK2(Nemo-like kinase 2)is a vertebrate homology of the Nemo gene,similar to MAPK1/ERK2,and phosphorylates serine and threonine residues prior to protein proline residues.NLK2 plays an important role in Wnt/β-catenin signaling pathway and Notch signaling pathway.In mammals,the molecular mechanism of tight regulation of MAVS has been studied,but little has been reported in grass carp.In order to study the molecular mechanism of NLK2(Nemo-like kinase 2)regulating MAVS in grass carp,we amplified and identified the NLK2 gene by homologous cloning.The complete CDS sequence of the grass carp NLK2 was 1281 bp and encodes a polypeptide containing 427 amino acids.Phylogenetic analysis showed that Ci NLK2 was more closely related to carp.When grass carp were stimulated with LPS,GCRV and poly I:C,IT was found that NLK2 did not participate in LPS-induced signal transduction,but could respond to GCRV and Poly I:C stimulation.Moreover,overexpression and interference experiments showed that Ci NLK2 could inhibit IFN Ⅰ(interferon I)expression in grass carp.To study the role of NLK2 in innate immunity,it is necessary to understand its distribution in cells.It was observed by confocal microscopy that NLK2 existed in both cytoplasm and nucleus,mainly in the cytoplasm.In order to investigate the specific molecular mechanism of NLK2 inhibition of type I interferon,NLK2 was overexpressed or interfered in CO cells(Grass carp overy cells),and it was found that NLK2 inhibited IRF3(interferon regulatory factor 3)phosphorylation.This suggests that NLK2 may inhibit interferon signal transduction by acting upstream of IRF3.Then MAVS and NLK2 were co-transformed in CO cells,and co-IP experiment proved that Ci NLK2 and Ci MAVS could interact.To clarify the mechanism,WE transfected NLK and MAVS into CIK cells(Grass carp kidney cells)individually or jointly.The results showed that the overexpression of MAVS and IFN Ⅰ was significantly increased.NLK2 overexpression,IFN Ⅰ expression decreased.IFN Ⅰ expression increased after NLK2 and MAVS cotransformation compared with NLK2 overexpression alone.This suggests that overexpression of MAVS prevents NLK2 induced IFN Ⅰ downregulation.After interfering with MAVS,there was no change in IFN Ⅰ expression compared with the control group,whether or not NLK2 was overexpressed.These results suggest that NLK2 inhibits the innate immunity of grass carp by acting on MAVS.To investigate how NLK inhibits innate immunity through MAVS and overexpresses NLK2,it was found that MAVS degrades and NLK2 phosphorylates MAVS.In conclusion,the formation of NLK2 complex with MAVS will phosphorylate MAVS,thus affecting the stability of MAVS and leading to degradation of MAVS.