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

Abstract

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, etc., have been reported to modulate the activities of IRF3 and IRF7. In this study, we found an acetyltransferase KAT8 in grass carp (CiKAT8, MW286472) that acetylated IRF3/IRF7 and then resulted in inhibition of IFN 1 response. CiKAT8 expression was up-regulated in the cells under poly I:C, B-DNA or Z-DNA stimulation as well as GCRV(strain 873) or SVCV infection. The acetyltransferase domain (MYST domain) of KAT8 promoted the acetylation of IRF3 and IRF7 through the direct interaction with them. So, the domain is essential for KAT8 function. Expectedly, KAT8 without MYST domain (KAT8-△264-487) was granularly aggregated in the nucleus and failed to down-regulate IFN 1 expression. Subcellular localization analysis showed that KAT8 protein was evenly distributed in the nucleus. In addition, we found that KAT8 inhibited the recruitment of IRF3 and IRF7 to ISRE response element. Taken together, our findings revealed that grass carp KAT8 blocked the activities of IRF3 and IRF7 by acetylating them, resulting in a low affinity interaction of ISRE response element with IRF3 and IRF7, and then inhibiting nucleic acids-induced innate immune response.

Keywords: IFN 1 signaling pathway; IRF3 and IRF7; KAT8; acetylation; fish.

Figure 1

Nucleic acids increase the expression of CiKAT8. CIK cells seeded in 6-well plates were transfected with (poly I:C, B-DNA, and Z-DNA) (A–C) or infected with viruses (GCRV-873, SVCV) (D, E) for different time (0 h, 6 h, 12 h, 24 h, 48 h, and 72 h). qRT-PCR analyzed the mRNA level of KAT8 in CIK cells. β-actin was served as an internal reference. The group of 0 h was a control. * represented significant (p < 0.05) and ** represented highly significant (p < 0.01).

Figure 2

CiKAT8 suppresses the production of IFN 1. CO cells were transfected with 2 µg of pcDNA3.1-KAT8 or empty vector (pcDNA3.1-basic). (A) Dual-luciferase analyzed the activities of IFN 1, ISG15 promoters and ISRE element. (B) qRT-PCR was used to detect IFN 1 and ISG15 expression. (C) Western bolt analyzed the expression of IFN 1 and KAT8 in CO cells. GAPDH served as an internal reference. (D) CO cells were transfected with pcDNA3.1-KAT8 at three different dosages (1, 1.5, or 2 µg), qRT-PCR analyzed the mRNA level of IFN 1. (E, F) CIK cells were transfected with control siRNA (N.C/siCtrl) or specific siRNA targeting KAT8 (siKAT8) for 36 h, and the levels of IFN 1, ISG15 mRNA and IFN 1 protein were analyzed by qRT-PCR and Western blot, respectively. * (p < 0.05), ** (p < 0.01). Data are representative of three independent experiments (C, F). The histograms exhibit the relative expression levels, which are quantified using ImageJ software.

Figure 3

CiKAT8 enhances virus replication in GCRV873-infected CIK cells. CIK cells were transfected with 2 μg of pcDNA3.1-KAT8 for 24 h. Then cells were infected with 50 µl 10^-8 TCID₅₀ GCRV-873 for 5 day. The cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet (A). (B–D) CIK cells were transfected with 2 μg of pcDNA3.1-KAT8 and then infected with GCRV-873 (10^-8 TCID₅₀). After 5 day, total RNA were extracted for detecting the mRNA levels of the vp5, vp6, and vp7 of GCRV-873 by qRT-PCR analysis ** (p < 0.01).

Figure 4

CiKAT8 blocks IRF3/IRF7-induced IFN 1 expression. CO cells seeded in (6-well or 24-well) plates were single-transfected or co-transfected with 2 µg of pcDNA3.1-KAT8 and pcDNA3.1-IRF3/IRF7. pcDNA3.1-basic was used as a control group. 36 h later, cells were harvested for detection of the mRNA level of IFN 1 (A) or luciferase activities of IFN 1 promoter and ISRE element (B), or the protein level of IFN 1 (C). * (p < 0.05) and ** (p < 0.01). Data are representative of three independent experiments (C). The histograms exhibit the relative expression levels, which are quantified using ImageJ software.

Figure 5

Subcellular localization of CiKAT8. (A) Structural diagram of wild-type and mutants KAT8. (B–E) CIK cells seeded on microscopy dishes were separately transfected with 2 µg of KAT8-GFP or KAT8-(△1-151)-GFP or KAT8-(△151-264)-GFP or KAT8-(△264-487)-GFP. 24 hours later, the cells were fixed and examined using a confocal microscopy (gray, Brightfield/BF; green, pEGFP-KAT8 or mutants pEGFP-KAT8; blue, DAPI). Scale bar is 75 µm.

Figure 6

CiKAT8 interacts with IRF3 and IRF7 via the MYST domain. (A–D) CO cells seeded in 60mm dishes were co-transfected with 1.5 µg of KAT8-Flag and 1.5 µg of (IRF3-GFP or IRF7-GFP) plasmids, 36 h later, cell lysates were immunoprecipitated with anti-Flag Ab (A, C) or anti-GFP Ab (B, D), then Western blot analyzed the interaction of KAT8 with IRF3/7. (E, F) CO cells seeded in 60 mm dishes were transfected with 1.5 µg KAT8-Flag or Flag-tagged mutant KAT8 (△1-151, △151-264, △264-487) plus (IRF3-GFP or IRF7-GFP), 36 h later, cell lysates were immunoprecipitated with anti-Flag Ab, Western blot analyzed the interaction of three KAT8 truncations with IRF3/IRF7. “*” refers to IRF3-GFP or IRF7-GFP. The molecular masses of KAT8-Flag, KAT8-(△1-151)-Flag, KAT8-(△151-264)-Flag, KAT8-(△264-487)-Flag, IRF3-GFP, IRF7-GFP are 60, 42, 46, 34, 85, and 82 kD, respectively. Data are representative of three independent experiments.

Figure 7

CiKAT8 acetylates IRF3/7 via its MYST domain. (A, B) CO cells seeded in 60 mm dishes were co-transfected with 1.5 µg of cmv-Flag or KAT8-Flag and 1.5 µg of IRF3-GFP plasmids (A), or 1.5 µg of cmv-Flag or KAT8-Flag and IRF7-GFP plasmids (B). 36 h later, the cell lysates were added with the deacetylase inhibitors cocktail and then co-immunoprecipitated with an anti-GFP antibody. Eventually, Western blot was performed with the indicated antibodies. (C, D) CO cells were transfected with control siRNA (N.C) or specific siRNA targeting KAT8 (siKAT8) for 12 h, then transfected with IRF3-GFP (C) or IRF7-GFP (D) plasmids for other 24 h, respectively. The subsequent experiment was conducted similarly to (A, B, E, F) Effect of KAT8 and its truncated mutants on the acetylation of IRF3/IRF7, CO cells were transfected with the indicated plasmids for 36 h, and the subsequent experiment was conducted similarly to A, (B) The molecular masses of KAT8-Flag, KAT8-(△1-151)-Flag, KAT8-(△151-264)-Flag, KAT8-(△264-487)-Flag, IRF3-GFP, IRF7-GFP are 60, 42, 46, 34, 85, and 82 kD, respectively. (G–I) CO cells seeded in plates (6-well or 24-well) were transfected with 2 µg of pcDNA3.1-KAT8-WT or pcDNA3.1-KAT8-(△1-151) or pcDNA3.1-KAT8-(△151-264) or pcDNA3.1-KAT8-(△264-487). 36 h later, cells were harvested for detection of the mRNA level of IFN 1 (G) or the protein level of IFN 1 (H) or the luciferase activities of IFN 1 and ISG15 promoters (I) * (p < 0.05) and ** (p < 0.01). Data are representative of three independent experiments (A–F, H). The histograms show relative expression levels quantified using the Image J software. "ns" means no significance.

Figure 8

CiKAT8 inhibits the combination of IRF3/IRF7 with ISRE response element. (A, B) CO cells seeded in 60 mm dishes and transfected with 1.5 µg of cmv-FLAG or KAT8-FLAG puls 1.5 µg of IRF3-GFP or IRF7-GFP. 36 h later, the cell extracts were incubated with immobilized biotin-labeled ISRE or No-biotin labeled ISRE. 12 h later, the beads were washed with lysis buffer three times, eluted with 2 × SDS sample buffer and boiled at 95°C for 10 min. Western blot was used to detect the affinity of IRF3/IRF7 with ISRE response element. Data are representative of three independent experiments.