Grass Carp (Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3

Abstract

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.

Grass Carp (Ctenopharyngodon idella) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

“…There may have several kinds of PTMs for some specific proteins, such as IRF3 and IRF7, to regulate their activities. For instance, GPATCH3, PKZ and NEK6 regulate the phosphorylation and nuclear translocalization of IRF3/IRF7 to control IFN 1 response (37,39,41); VP35 represses IFN transcription by increasing PIAS1-mediated SUMOylation of IRF7 (48); Methyltransferase NSD3 catalyzes the methylation of IRF3 at K366 to enhance antiviral immune response (49); Recently, acetylation also has been reported as an unconventional modification to regulate the activities of IRF3 and IRF7 (50,51). The various and complicated PTMs may effectively regulate immune homeostasis in cells.…”

“…CIK cells transfected with 2 µg oligomeric nucleic acids [poly(I: C) or B-DNA or Z-DNA] or 50 µl of 10 −8 TCID 50 viruses (GCRV or SVCV) were separately cultured in 6-well plates for 6, 12, 24, 48, and 72 h, as described in our previous studies (37)(38)(39).…”

Fish Paralog Proteins RNASEK-a and -b Enhance Type I Interferon Secretion and Promote Apoptosis

“…To examine whether RNASEK-a is colocalized with RNASEK-b, cells were transfected with p3×FLAG-RNASEK-a and pEGFP-RNASEK-b together. After 24 h of transfection, cells were fixed as above and permeabilized with 0.5% Triton X-100, then blocked with 5% bovine serum albumin, followed by incubation with the FLAG-tag antibody (1:5,000, Sigma, USA, F1804) ( 27 ) for 12 h (overnight) at 4°C. The next day, cells were incubated in the secondary antibody goat anti-mouse Cy3 for 1 h (1:200, BioLegend, USA, 405309) and counterstained with DAPI for 20 min without light.…”

“…Finally, the membranes were exposed using a chemiluminescence imaging system (CLINX, China). Specific antibodies used here are against grass carp type I IFN (1:800, saved in our lab) ( 28 ), phosphorylated IRF3 (p-IRF3; 1:1,000, Beyotime Biotechnology, China, AF1594) ( 27 ), basal and phosphorylated IRF7 (p-IRF7; 1:1,000, HuaBio, China, ET1610-89) ( 29 ), eIF2α (1:500, saved in our lab) ( 30 ), phosphorylated eIF2α (p-eIF2α; 1:1,000, Abcam, UK, ab32157) ( 30 ), and GAPDH (loading control; 1:10,000, saved in our lab) ( 30 ). All commercial antibodies against p-IRF3, IRF7/p-IRF7, and p-eIF2α cross-react with grass carp, which is confirmed by transfection of pEGFP-IRF3, -IRF7, and -eIF2α into grass carp cells followed by comparison of the size of the basal and GFP recombinant proteins of each gene in our previous work ( 27 , 29 , 30 ).…”

DataSheet_1.docx

“…Finally, the membranes were exposed using a chemiluminescence imaging system (CLINX, China). Specific antibodies used here are against grass carp type I IFN (1:800, saved in our lab) (28), phosphorylated IRF3 (p-IRF3; 1:1,000, Beyotime Biotechnology, China, AF1594) (27), basal and phosphorylated IRF7 (p-IRF7; 1:1,000, HuaBio, China, ET1610-89) (29), eIF2a (1:500, saved in our lab) (30), phosphorylated eIF2a (p-eIF2a; 1:1,000, Abcam, UK, ab32157) (30), and GAPDH (loading control; 1:10,000, saved in our lab) (30). All commercial antibodies against p-IRF3, IRF7/p-IRF7, and p-eIF2a cross-react with grass carp, which is confirmed by transfection of pEGFP-IRF3, -IRF7, and -eIF2a into grass carp cells followed by comparison of the size of the basal and GFP recombinant proteins of each gene in our previous work (27,29,30).…”