Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-κB

Bo Chen, Chen Li, Jian Yao, Lin Shi, Wanmeng Liu, Fang Wang, Shitian Huo, Yongan Zhang, Yuanan Lu, Usama Ashraf, Jing Ye and Xueqin Liu
J Immunol April 1, 2020, 204 (7) 1881-1891; DOI: https://doi.org/10.4049/jimmunol.1900561

Key Points

  • Zebrafish NIK positively regulates antiviral response through the induction of IFN.

  • IKKα phosphorylates the NIK at the residue Thr432.

  • The IKKα–NIK complex mediates the activation of IRF3 and NF-κB.

Abstract

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB–inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.

Introduction

Innate immunity provides the first step of host defense against microbial pathogens. In response to viral infection, the induction of IFNs, especially the type I IFN, plays a decisive role in the host defense processes (1). IFN is imperative for the potent activation to IFN-stimulated genes (ISGs), which rapidly culminate in the inhibition of viral replication and spread (2, 3). IFN regulatory factors (IRFs) are transcriptional factors that perform crucial functions in several aspects of the immune response, including the response to pathogens. Among the IRFs, IRF3 and IRF7 are critical for the induction of type I IFN signaling cascades in response to the sensing of viral DNA and/or RNA motifs by the pattern recognition receptors. Upon activation, both IRF3 and IRF7 are capable of translocating from cytoplasm to the nucleus of the cell, where they trigger the expression of type I IFNs (4, 5). Albeit the expression levels of IRF3 and IRF7 are diverse in different cells, they are consistent in terms of virus-induced phosphorylation, dimerization, and nuclear translocation (5).

NF-κB activation occurs through two pathways: the canonical and noncanonical pathways. Both pathways are vital in regulating the transcription of an array of genes involved in the several processes of immune and inflammatory responses, such as cytokine receptor adhesion molecules, acute phase protein gene transcription factors, and some regulatory factors (6, 7). NF-κB is also reported to exert antiviral function by inducing the expression of IFN (1, 6). A better understanding of the mechanism of NF-κB activation is of great importance for harnessing the uncontrolled activation of immune and inflammatory responses.

In mammals, the role of NIK (also known as MAP3K14) in immunity is well established. According to previous studies, NIK activates the noncanonical NF-κB pathway, which responds to selective receptor signals that mediate adaptive immune functions, such as lymphoid organ development and B cell survival (7, 8). Furthermore, the activation of the nonclassical pathway can also induce the activation of the classical pathway because the stimulation of LTα1β2 leads to the accumulation of intracellular NIK and the upregulation of proinflammatory cytokine expression (9). NIK phosphorylates IKKα at Ser176 and Ser180 residues, and mutation in these two residues to glutamate causes the constitutive activation of NF-κB (10). Furthermore, IKKα (S176E) constitutively activates IRF3/7 and is involved in IFN-α production through TLR 7/9 signaling cascades (10). NIK interacts with the DNA pathway adapter stimulator of IFN genes (STING; also known as TMEM173/MITA) to enhance the induction of IFN (11). IFN can induce NIK expression in return (12). Although extensive studies have been conducted in mammals, the role of NIK in fish has not yet been investigated.

Aquatic diseases are the main factors in restricting the development of the aquatic industry and threatening food safety. Among the aquatic diseases caused by different pathogens, viral diseases are the most difficult to prevent and cure (13, 14). Spring viremia of carp virus (SVCV; ssRNA) (15), giant salamander iridovirus (GSIV; dsDNA) (16), and grass carp reovirus (GCRV; dsRNA) (17) are fish pathogens that cause severe diseases and significant mortalities in the affected fish, which ultimately leads to huge economic losses to the aquaculture industry. However, there is still no effective method presently available to prevent and control these viral diseases.

In this study, we demonstrated a previously undescribed function of zebrafish NIK in inducing the immune response in vitro. We found that IKKα activates NIK by directly interacting with and phosphorylating it at the residue threonine (Thr)-432. The IKKα-NIK complex mediates the activation of IRF3 and NF-κB, which in turn leads to the induction of IFN response and inhibition of viral replication. In addition, by taking advantage of the zebrafish model, we examined the role of NIK in response to SVCV infection in vivo. We elucidated that zebrafish NIK positively regulates antiviral response through the induction of IFN. These findings may provide new understanding on functions of fish NIK and facilitate the establishment of antiviral therapy to combat fish viruses.

Materials and Methods

Fish cell lines and viruses

The fathead minnow (FHM) cell line (American Type Culture Collection [ATCC] CCL-42) and epithelioma papulosum cyprini (EPC) cell line (ATCC CRL-2872) were maintained at 28°C in Medium 199 (Hyclone) or MEM (Hyclone) supplemented with 10% FBS (Life Technologies). The zebrafish embryo (ZF4) cells (ATCC CRL-2050) were maintained in DMEM/F-12 (Hyclone) with 10% FBS (Life Technologies) at 28°C. Human embryonic kidney (HEK) 293T cells were grown in DMEM (Hyclone) supplemented with 10% FBS at 37°C. SVCV (ATCC VR-1390) was propagated in FHM cells at 28°C and harvested when >80% virus-induced cytopathic effect appeared. GSIV and GCRV were propagated in EPC cells at 25°C and harvested upon appearance of extensive cytopathic effect (>80%). Following three cycles of freezing and thawing, culture media were collected from infected cultures and centrifuged at 10,000 × g for 10 min at 4°C, and recovered supernatant was aliquoted and stored at −80°C.

Reagents

Polyinosinic-polycytidylic acid (poly[I:C]) (18, 19) and LPS (20, 21) were purchased from Invitrogen. Poly(dA:dT) was purchased from Sigma-Aldrich. Cell transfections were performed using FuGENE HD transfection reagent from Promega. Calf intestinal phosphatase (CIP) was obtained from New England Biolabs.

Quantitative real-time PCR

Total RNA was extracted using TRIzol Reagent (TAKARA) according to the instruction of the manufacturer. The reverse transcription was carried out using the ReverTra Ace qPCR RT kit (TAKARA). The relative expression of each cDNA was determined by quantitative real-time PCR (qRT-PCR) using TB Green Real-Time PCR Master Mix (TAKARA). Amplification was performed for 5 min at 95°C, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. Fluorescent signals were analyzed by a Light Cycler/Light Cycler 480 System (Roche). The relative mRNA levels were calculated using the 2−ΔΔthreshold cycle method. All the primers employed in this study are listed in Table I.

Plasmid conduction and mutation

The coding sequence of zebrafish NIK (XM_002661208.6), IRF3 (NM_001143904), or IRF7 (NM_200677.2) (https://www.ncbi.nlm.nih.gov/genbank) was cloned into pcDNA4. The coding sequence of zebrafish IKKα (NM_200317.1) was cloned into 3×Flag pCMV-14 or pEGFP-N1. The pGL3–basic firefly luciferase reporter vector harboring the IFNφ1 promoter (IFNφ1pro)–Luc or NF-kBpro-Luc and the Renilla luciferase internal control vector (pRL-TK) were provided by professor Yongan Zhang (College of Fisheries, Huazhong Agricultural University) (22). The IFN-stimulated response element (ISRE)–Luc was received from Professor Shaobo Xiao (College of Veterinary Medicine, Huazhong Agricultural University) (23).

Wild-type NIK (wtNIK) plasmid was mutated using FastPfu PCR (94°C for 5 min, followed by 25 cycles of 98°C for 10 s, 55°C for 5 s, 72°C for 6 min, and 72°C for 10 min) (TransGen). Recovered PCR product was treated with Dpn I (TAKARA) at 37°C for 30 min. Subsequently, the plasmid was extracted and stored at −20°C.

Plasmid transfection and virus infection

The recombinant plasmids were transfected using FuGENE HD (Promega) following the manufacturer’s protocol. For virus infection assays, cells were infected with SVCV (0.05 multiplicity of infection [MOI]) at 28°C or GSIV (1.0 MOI) or GCRV (1.0 MOI) at 25°C. After 1 h of virus absorption, cells were washed with PBS three times and subsequently maintained in Medium 199 supplemented with 5% FBS.

Luciferase assays

FHM cells were seeded in 12-well plates at a density of 0.5–2 × 105 cells per well. When cells were grown to 70–80% confluence, 500 ng of luciferase reporter plasmid (IFNφ1pro-Luc, ISRE-Luc, or NF-κB-Luc) and 50 ng of the Renilla luciferase construct pRL-TK (Promega), which served as an internal control, were cotransfected with empty vector or a plasmid encoding NIK protein. At 24 h posttransfection, firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system (Promega). The data are expressed as relative firefly luciferase activity normalized to the value of Renilla luciferase.

Fluorescent microscopy

FHM cells were seeded in a 20-mm dish and transfected with indicated plasmids. Following 24 h of incubation at 28°C, the cells were washed twice with PBS and fixed, then incubated with mouse anti-His Abs (1:3000 dilution; ABclonal) for 2 h, followed with Cy3 goat anti-mouse Abs (1:500 dilution; ABclonal) for 45 min. The cell nuclei were stained with DAPI and washed three times. Images were obtained using a confocal microscope (Leica).

Immunoassays

Coimmunoprecipitation (Co-IP) assay was operated using Pierce Co-IP Kit (Thermo Fisher Scientific) according to the instructions of the manufacturer. For immunoblot analysis, cells were harvested with ice-cold PBS and lysed on ice with immunoprecipitation lysis/wash buffer for 5 min. After centrifugation at 13,000 × g for 10 min, the supernatants were collected. Immunoprecipitants or whole-cell extracts were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad). The membranes were blocked in 2% albumin from bovine serum at room temperature for 1 h and subsequently incubated with mouse anti-His (1:3000 dilution; ABclonal), mouse anti-Flag (1:3000 dilution; ABclonal), rabbit anti–β-actin (1:10000 dilution; ABclonal), or anti–SVCV-G mAbs (24) for 2 h. Rabbit anti–β-actin Abs were used as internal control. After washing with TBST, the membranes were incubated with horse radish peroxidase–conjugated secondary goat anti-mouse or anti-rabbit (1:2000 dilution; ABclonal) Abs for 45 min. Finally, the reactive proteins were detected using chemical luminescence substrate (General Electric) with Amersham Imager 600 (General Electric). Protein dephosphorylation was carried out in 100-μl reaction mixtures consisting of 100 μg of cell protein and 10 U of CIP. Then the mixtures were maintained at 37°C for 45 min.

RNA interference

To knockdown the expressions of NIK or IKKα, RNA interference assay was performed by transfecting the three pairs of either NIK- or IKKα-specific small interfering RNAs (siRNAs) (GenePharma, China). FHM cells were seeded in a 12-well plate and transfected with 100 nM of siRNA against NIK (siNIK) or negative control (NC) by using Lipofectamine 2000. Twenty-four hours later, cells were infected with SVCV (MOI = 0.05). At 24 h postinfection, total RNA were isolated and processed by qRT-PCR. The NIK-specific siRNA sequences were as follows: sequence 1 (S1): 5′-CCUGUCGUCAUCUC-UCAUUTT-3′ (forward) and 5′-AAUGAGAGAUGACAGACAGGTT-3′ (reverse); sequence 2 (S2): 5′-GCUGUCAGACGAUGGGAAATT-3′ (forward) and 5′-UUUCCCAUCGUCUGACAGCTT-3′ (reverse); and sequence 3 (S3): 5′-GGGUGAAAGUA-GGCCACAUTT-3′ (forward) and 5′-AUGUGGCCUACUUUCACCCTT-3′ (reverse).

The IKKα-specific siRNA sequences were as follows: S1: 5′-CCACAAGAUAAUCGACCUUTT-3′ (forward) and 5′-AAGGUCGAUUAUCUUGUGGTT-3′ (reverse); S2: 5′-GCAGCUGAAAG-CAAAGCUUTT-3′ (forward) and 5′-AAGCUUUGCUUUCAGCUGCTT-3′ (reverse); and S3: 5′- GGUACUAAGGGAUCUAUAUTT-3′ (forward) and 5′- AUAUAGAUCCCUUAGUACCTT-3′ (reverse).

The NC siRNA sequences were 5′-UUCUCCGAACGUGUCACGUTT-3′ (forward) and 5′-ACGUGACACGUUCGGAGAATT -3′ (reverse).

Viral plaque assay

FHM cells transfected with empty vector or a plasmid encoding NIK were infected with SVCV at an MOI of 0.05. At 6, 12, 24, and 36 h postinfection, cell supernatants were harvested, and virus titers were determined with a plaque assay. Briefly, viruses were serially diluted and inoculated onto monolayers of cells. After 1 h of absorption, cells were washed with serum-free DMEM and cultured in DMEM containing 3% FBS and 1.5% sodium carboxymethyl cellulose (Sigma-Aldrich). Visible plaques were counted, and viral titers were calculated after 3 d of incubation.

NIK gene knockdown in zebrafish embryos

Knockdown of NIK gene in zebrafish embryos was accomplished via morpholinos (MO) technology (YSY Biotech). The sequences of MO targeting NIK (NIK-MO) and STD (STD-MO), which served as an NC, were designed as follows: NIK-MO: 5′-TGCTAACGTGGAAGAAGATATCACA-3′ and STD-MO: 5′-CCTCTTACCTCAGTTACA-ATTTATA-3′. NIK-MO (4 ng) or STD-MO (8 ng) was microinjected into one-cell stage zebrafish embryos. To evaluate the knockdown efficiency, mRNA levels of NIK at different developmental stages were determined by qRT-PCR. The primers for qRT-PCR were 5′-TCCATTATTGCACAGGCGGA-3′ (forward) and 5′-GAACCCTGCTTTGCATACGG-3′ (reverse).

Statistics analysis

All statistical analyses and calculations were done using GraphPad Prism 7.0 (GraphPad Software, Inc). The significance of the variability between different treatment groups was determined by two-way ANOVA. A p value of <0.05 was considered statistically significant and marked with an asterisk (*). Data are expressed as means ± SD of results from three independent experiments.

Results

Poly(dA:dT) and poly(I:C) promote NIK expression

The coding sequence of zebrafish NIK encodes an 825-aa protein, whereas human and mouse NIK encode a protein of 947 and 942 aa, respectively. Phylogenetic analysis indicates that zebrafish NIK shares a protein sequence homology of 46 and 45% with human and mouse NIK, respectively (Supplemental Fig. 1). This low similarity of NIK sequences between zebrafish and mammals suggests that the function of NIK in zebrafish might be different from that known for mammalian species.

To investigate the role of zebrafish NIK in host defense against microbial pathogens, the mRNA expression level of NIK was determined following treatment with poly(dA:dT), poly(I:C), or LPS in FHM cells. It was observed that both poly(dA:dT) and poly(I:C), but not LPS, significantly increased the NIK mRNA expression at 12, 24, and 36 h postinfection in the cultured cells (Fig. 1A–C). As poly(I:C) and poly(dA:dT) are synthetic mimics of RNA and DNA viruses, respectively, we next assessed the effect of pathogenic fish RNA and DNA viruses on the NIK mRNA expression in different fish cell line models. Interestingly, SVCV (ssRNA virus) infection markedly enhanced the NIK mRNA expression levels in cultured FHM and ZF4 cells at 24 and 36 h postinfection (Fig. 1D, 1E). Moreover, SVCV, GCRV (dsRNA virus), or GSIV (dsDNA virus) infection of FHM cells caused a significantly upregulated pattern of NIK mRNAs in a dose-dependent manner (Fig. 1F–H). Overall, these results suggest that NIK mRNA expression is elevated upon infection with RNA or DNA viruses.

FIGURE 1.
FIGURE 1.

The effects of poly(dA:dT), poly(I:C), LPS, and different viruses on NIK expression. (A–C) FHM cells were treated with 1 μg/ml poly(dA:dT), poly(I:C), or LPS for the indicated time points (6, 12, 24, 36 h). (D and E) FHM and ZF4 cells were infected with SVCV (0.05 MOI) for the indicated time points (6, 12, 24, 36 h). (F–H) FHM cells were infected with GSIV, GCRV, or SVCV at an MOI of 0.1, 1, or 10 for a period of 24 h. The NIK mRNA expression levels were quantified by qRT-PCR. All data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

NIK induces IFN and ISG production

Based on the data presented above, we reasoned that NIK might be associated with the host response to viral infection. IFN is one of the most critical cytokines of the host in providing protection against the invading pathogens. To determine whether NIK is involved in inducing the host IFN response, we first examined the effect of NIK overexpression on the production of IFN and ISGs. Our data revealed that overexpression of NIK significantly increased the mRNA expression levels of IFNφ1 (25, 26) and ISGs (PKR, viperin, and ISG15) in FHM cells (Fig. 2A, 2B). Moreover, ectopic expression of NIK augmented the promoter activities of IFNφ1 and ISRE, as assessed by luciferase assay (Fig. 2C, 2D). To substantiate that NIK is indeed involved in the induction of IFNφ1 and ISGs, cultured FHM cells were transfected with siNIK or control siRNAs and subsequently infected with SVCV. The specificity and silencing efficiency of siRNAs for NIK was confirmed (Fig. 2E). Compared with nonsilenced cells, silencing of NIK reduced the mRNA levels of IFNφ1, PKR, viperin, and ISG15 in SVCV-infected cells (Fig. 2F–I). Thus, these data demonstrate that NIK positively regulates the production of IFN and ISGs during viral infection.

FIGURE 2.
FIGURE 2.

NIK enhances the expression and promoter activities of IFN and ISGs. (A and B) NIK induces IFNφ1 and ISG mRNA production. FHM cells were transfected with NIK-His or empty vector (2 μg each per well) for 24 h. The mRNA expression levels of IFN and ISGs (PKR, Viperin, and ISG15) were detected by qRT-PCR. (C and D) NIK enhances the IFNφ1pro-luc and ISREpro-luc promoter activities. FHM cells were transfected with 500 ng of NIK plasmid or empty plasmid along with 500 ng of IFNφ1pro-luc or ISREpro-luc (pGL3-basic empty plasmid as NC) and 50 ng of pRL-TK. At 24 h posttransfection, luciferase activities were measured. (E) RNA interference effect on the expression of endogenous NIK. FHM cells were transfected with NIK-specific siRNAs (S1, S2, and S3) or control siRNA (NC) for 24 h. The efficiency of siRNAs to inhibit the NIK expression was detected by qRT-PCR. S2 siRNA showed the best silencing efficiency. (F–H) Effects of NIK silencing on the SVCV-induced IFN and ISG transcription. FHM cells were transfected with S2 NIK-specific siRNA or control siRNA (NC) for 24 h, followed by SVCV infection for a period of 24 h. The mRNA expression levels of IFNφ1 and ISGs (PKR, Viperin, and ISG15) were measured by qRT-PCR. All data are representative of three independent experiments. ***p < 0.001, ****p < 0.0001.

NIK interacts with IKKα

Human NIK is reported to interact with several cellular factors, including TRAF3, IRF3, Pellino3, GRB10, ARRB1, and ARRB2 (11, 27–30). Human NIK is also known to physically assemble with IKKα (31). As a key regulatory factor of the NF-κB pathway, IKKα plays an important role in triggering the innate immune response (31). To verify whether the zebrafish NIK can interact with IKKα, a Co-IP assay was carried out by cotransfecting the NIK-His and IKKα-Flag plasmids in HEK 293T cells. Using either a captured anti-Flag or anti-His Ab, NIK-His and IKKα-Flag were found to be coprecipitated in the lysates of transfected cells (Fig. 3A, 3B). These data demonstrate that the zebrafish NIK interacts with IKKα.

FIGURE 3.
FIGURE 3.

NIK interacts and colocalizes with IKKα. (A and B) NIK interacts with IKKα. HEK 293T cells were seeded in T25 cell flasks and transfected with 5 μg of NIK-His or IKKα-Flag plasmids for 24 h. Cell lysates were precipitated with anti-Flag or anti-His beads, followed by analysis with immunoblot (IB) assay. (C) Colocalization of NIK and IKKα. FHM cells were cotransfected with NIK-His and IKKα-EGFP or EGFP plasmid for 24 h. Subsequently, cells were fixed and subjected to laser scanning confocal microscopy. Green and red signals indicate the IKKα and NIK proteins, respectively. Blue signals represent cellular nuclei. The yellow staining in the merged picture indicates the colocalization of NIK and IKKα. All data are the representative of three independent experiments.

To further visualize the subcellular localization of NIK and IKKα in FHM cells, a laser confocal microscopy was employed. Plasmids expressing NIK-His and IKKα-pEGFP or pEGFP were cotransfected in the cultured cells. By using a laser confocal microscopy, a similar distribution pattern of NIK (red) and IKKα (green) was observed in the cytoplasm (Fig. 3C), which may support the interaction of NIK and IKKα as demonstrated by the Co-IP assay.

IKKα phosphorylates NIK at Thr432

In our Co-IP assay, NIK exhibited an obvious shift in its m.w., whereas IKKα had no such effect (Fig. 3A, 3B). Because phosphorylation is known to induce the shift of m.w. of proteins, we surmised that the observed shift in the NIK weight may have occurred owing to NIK phosphorylation. To this end, plasmids encoding IKKα-Flag and NIK-His were cotransfected in HEK 293T cells, and a significant shift of NIK protein was observed. However, the shift of NIK m.w. disappeared upon treatment with CIP (Fig. 4A), suggesting that the shift of NIK was caused by phosphorylation.

FIGURE 4.
FIGURE 4.

IKKα phosphorylates NIK at the residue Thr432. (A) HEK 293T cells were cotransfected with NIK-His and IKKα-Flag or empty vector. At 24 h posttransfection, cell lysates were treated with or without CIP, followed by immunoblot (IB) analysis. (B) Sequence comparison of zebrafish NIK with other indicated species. The mutNIK was generated by inducing a mutation T432A in the wtNIK plasmid using FastPfu PCR. (C) HEK 293T cells were cotransfected with NIK-His or mutNIK-His with IKKα-Flag or empty vector for 24 h. Later, cell lysates were prepared and subjected to IB analysis. (D) NIK undergoes phosphorylation in FHM cells. Cultured cells were transfected with NIK-His or mutNIK-His plasmids for 24 h. Subsequently, cell lysates were prepared, treated with or without CIP for a period of 40 min at 37°C, and analyzed by IB assay. (E) Effect of IKKα-specific siRNAs on the endogenous expression of IKKα. FHM cells were transfected with IKKα-specific siRNAs (S1, S2, and S3) or control siRNAs (NC) for 24 h. The efficiency of siRNAs to inhibit the IKKα expression was detected by qRT-PCR. S1 siRNA showed the highest silencing efficiency. (F) Effect of IKKα silencing on the phosphorylation state of NIK. FHM cells were transfected with S1 IKKα-specific siRNA or control siRNA (NC). After 24 h transfection, cell lysates were prepared and used to perform IB analysis. Data are representative of three independent experiments. ***p < 0.001.

To identify the NIK phosphorylated site, multiple sequence alignment of human, mouse, common carp, and zebrafish NIK proteins was performed. A highly conserved sequence, ranging from 560 to 570 aa, was detected in these proteins (Supplemental Fig. 1B). NIK is previously reported to undergo phosphorylation in mammals (Thr561 in mouse and Thr559 in human) (11), and the phosphorylation sites are located within the conserved protein motifs. To investigate whether the phosphorylation site of zebrafish NIK protein also lies in these conserved aa residues, a mutant of zebrafish NIK (mutNIK) was generated by substituting the Thr with alanine at position 432 (T432A) of the wtNIK (Fig. 4B). Immunoblot analysis of the lysates of HEK 293T cells overexpressed with wtNIK or mutNIK in the absence or presence of IKKα revealed that wtNIK, but not the mutNIK, undergoes shifting of m.w. when coexpressed with IKKα (Fig. 4C). Furthermore, different molecular weights of these two proteins were detected in wtNIK- and mutNIK-expressing FHM cells. No detectable shift of wtNIK m.w. was observed following treatment of wtNIK-expressing samples with CIP (Fig. 4D). To further confirm the role of IKKα in phosphorylating the NIK protein, IKKα was subjected to siRNA-mediated silencing in FHM cells. Reduced mRNA levels of IKKα in siIKKα-treated cells confirmed the specificity and silencing efficiency of siRNAs (Fig. 4E). Inhibition of endogenous IKKα caused a reduction in the phosphorylating state of exogenous NIK in the treated cells (Fig. 4F). Moreover, endogenous IKKα was found to be upregulated in response to SVCV infection of FHM cells (Supplemental Fig. 2). Taken together, these data demonstrate that IKKα phosphorylates NIK at the residue Thr432.

NIK regulates IRF3 and NF-κB activation

To elucidate how NIK positively regulates the IFN production, the effect of NIK on the transcriptional factors of IFN, IRF3/7, and NF-κB was assessed in this study. To illustrate any regulatory effect of NIK on IRF3 and IRF7 at the protein level, the plasmids of NIK-His or IKKα-Flag were cotransfected with either IRF3-His or IRF7-His in HEK 293T cells. Immunoblot analysis revealed no obvious effect on the protein levels of both IRF3 and IRF7 (Fig. 5A). Interestingly, cotransfection of NIK-His with IRF3-His or IRF7-His in the presence of IKKα-Flag did not cause any change on the IRF7 level (Fig. 5A); however, a shifted strip appeared above the IRF3-His level (Fig. 5B). To determine whether the shifted strip represented the phosphorylated zebrafish IRF3, samples were treated with CIP and subjected to immunoblot analysis. It was found that IRF3 shifted strip disappeared upon treatment with CIP, whereas nontreated samples exhibited no effect on the phosphorylated IRF3 (Fig. 5C), suggesting that NIK activates IRF3, but not IRF7, by regulating the IRF3 phosphorylation. Furthermore, we determined the effect of zebrafish NIK on the promoter activity of NF-κB. As assessed by luciferase assay, NF-κB–pro activity was significantly stimulated upon expression with NIK (Fig. 5D). Overall, these findings demonstrate that zebrafish NIK regulates IRF3 and NF-κB activation.

FIGURE 5.
FIGURE 5.

NIK regulates IRF3 phosphorylation and NF-κB activation. (A and B) Effect of NIK and IKKα on IRF7 and IRF3 phosphorylation. HEK 293T cells were transfected with the indicated plasmids (1 μg each) for 24 h. Later, the cell lysates were collected and analyzed by immunoblot (IB). (C) HEK 293T cells were transfected with the indicated plasmids (1 μg each). After 24 h of transfection, cell lysates were treated with or without CIP for 40 min at 37°C and were assessed by IB assay. (D) NIK regulates the NF-κB promoter activity. FHM cells were transfected with 500 ng of NIK plasmid or empty plasmid along with 500 ng of NF-κBpro-luc (where pGL3-basic empty plasmid is NC) and 50 ng of pRL-TK. After 24 h of stimulation, luciferase activities were monitored. Data are the representative of three independent experiments. ****p < 0.0001.

NIK restricts virus infection in vitro

To evaluate the role of NIK in response to virus infection, NIK was overexpressed in EPC cells for 24 h, followed by infection with GSIV, GCRV, or SVCV. The mRNA expression levels of these viruses were found to be inhibited (Fig. 6A). We next determined the effect of NIK on SVCV replication in detail by conducting a series of experiments. Ectopic expression of NIK significantly decreased the level of SVCV mRNA (G, M, and N) and G protein measured at different infection time points by qRT-PCR (Fig. 6B, Supplemental Fig. 3A, 3B) and immunoblot assays, respectively (Fig. 6C). The production of infectious SVCV particles in the supernatants of NIK-overexpressed cells was also found to be significantly reduced at different infection time points, as assessed by plaque formation assay (Fig. 6D). In addition, the effect of NIK on the SVCV-induced IFN production was investigated by qRT-PCR and indicated that NIK significantly upregulated the IFN expression upon SVCV infection (Fig. 6E).

FIGURE 6.
FIGURE 6.

NIK restricts viral infection in vitro. (A) Effects of NIK on the expression of viral mRNAs. EPC cells were transfected with NIK plasmid or empty vector (2 μg each) for 24 h and then infected with GCRV (1.0 MOI), GSIV (1.0 MOI), or SVCV (0.05 MOI) for 24 h. The GCRV-NS4, GSIV-MCP, and SVCV-N mRNA expressions were determined by qRT-PCR. (B–D) Effect of NIK on the SVCV replication. FHM cells were transfected with NIK plasmid and empty vector (2 μg each) for a period of 24 h, followed by infection with SVCV at MOI of 0.05. Cell samples and supernatants were collected at the indicated time points and were analyzed by qRT-PCR (B), Western blot (C), or plaque formation assay (D). (E) NIK enhances the SVCV-induced IFNφ1 expression. FHM cells were transfected with NIK plasmid or empty vector (2 μg each) for 24 h and then infected with SVCV (0.05 MOI) for 24 h. The relative IFNφ1 mRNA expression was quantified by qRT-PCR. All data are representative of three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001.

To consolidate our above findings, the impact of mutNIK on the SVCV replication was examined. As expected, mutNIK caused no influence on the SVCV mRNAs (G, M, and N) and G protein (Fig. 7A, 7B) expression levels. However, the comparison of viral tiers between wtNIK (Fig. 6D) and mutNIK (Fig. 7C) revealed that the latter had obviously less-strong influence on the SVCV particle production. To further confirm whether NIK is indeed involved in restricting the viral infection, FHM cells were transfected with siNIK or control siRNAs (NC) and subsequently infected with SVCV. When compared with nonsilenced cells, knockdown of NIK markedly promoted the SVCV mRNA expression (Fig. 7D) and viral titers (Fig. 7E) in the cultured cells. In short, these data indicate a critical role of NIK in controlling the viral infection.

FIGURE 7.
FIGURE 7.

(A–C) Effects of mutNIK on SVCV replication. FHM cells were transfected with mutNIK plasmid or empty vector (2 μg each) for 24 h and then infected with SVCV (0.05 MOI). After 24 h of infection, samples were collected and subjected to qRT-PCR (A), Western blot (B), or plaque assay (C) analysis. (D and E) Effects of NIK silencing on the SVCV replication. FHM cells were transfected with siNIK or control siRNAs for 24 h, following SVCV infection (0.05 MOI). At 24 h postinfection, samples were collected and analyzed by qRT-PCR (D) and plaque assay (E). All data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Knockdown of NIK in zebrafish inhibits antiviral response

To determine the physiological role of NIK in response to viral infection, the zebrafish embryos injected with NIK-MO or STD-MO and the mRNA levels of NIK were determined by qRT-PCR at different developmental stages, which showed that the NIK gene was significantly knocked down in NIK-MO–injected embryos (Fig. 8A). The NIK-MO–injected (n = 100) or STD-MO–injected (n = 100) embryos were infected with SVCV at 48 h postinjection, and the numbers of dead larvae were counted at different time points. The NIK knockdown embryos showed a reduced survival rate compared with the control embryos after SVCV infection (Fig. 8B). To further evaluate the role of NIK on antiviral response of zebrafish, the expressions of IFNφ1 and ISGs, such as PKR, viperin, and ISG15, in response to viral infection were measured. As shown in Fig. 8C–F, SVCV infection elicited the expression of IFNφ1, PKR, viperin, and ISG15, whereas NIK knockdown resulted in decreased expression of IFNφ1, PKR, viperin, and ISG15 in the embryos after SVCV infection. Consistently, the copy numbers of G, M, and N genes of SVCV indicated by RNA level were significantly increased in NIK knockdown embryos compared with that in control embryos (Fig. 8G). Taken together, these data further confirm the positive regulation role of NIK on antiviral response in zebrafish (Table I).

FIGURE 8.
FIGURE 8.

Knockdown of NIK in zebrafish inhibits antiviral response against SVCV. NIK-MO (4 ng/individual embryo) or STD (STD-MO, 8 ng/individual embryo), which was used as control, was injected into one-cell zebrafish embryos. The levels of NIK expression were detected by qRT-PCR at 12 h, 24 h, 48 h, and 5 d postinjection (A). The zebrafish were then treated with SVCV (∼2 × 106 PFU/ml) in water, and the numbers of dead larvae were counted at 6, 12, 18, and 24 h postinfection (B). The mRNA levels of IFNφ1, PKR, viperin, and ISG15 and the copy number of viral genes were determined by qRT-PCR at 24 h postinfection (C–G). All data are representative of three independent experiments. ****p < 0.0001.

View this table:
Table I. Primers of this study

Discussion

Viral infection triggers the innate immune responses and promotes the induction of IFNs. Four zebrafish IFNs, namely, IFNφ1 to IFNφ4, have been classified into two distinct subsets: group I and group II. IFNφ1 and IFNφ4 belong to group I, whereas IFNφ2 and IFNφ3 belong to group II. All of the IFNs, except IFNφ4, have been reported to induce strong antiviral activity in adult zebrafish (32–34). In mammals and fish, IFNs play a critical role in the host innate immune system and trigger a massive expression of ISGs that can interfere with viral transcription, translation, assembly, and release (26, 32, 35). IFN activation is dependent on the regulation of transcriptional factors that include IRF3, IRF7, and NF-κB (36–41). However, the mechanisms of these pathways are separately or differentially modulated and remain largely uncharacterized.

This study revealed that NIK was distributed in the cytoplasm at the periphery of the nuclei in FHM cells, which is similar to the subcellular localization in HEK 293T cells (11). NIK has been confirmed to interact with several proteins in mammals. For instance, NIK interacts with RIG-I and MAVS to activate the expression of inflammatory genes via the NF-κB pathway (42). NIK interacts with STING to mediate the IRF3 activation, which contributes to curb a DNA virus infection (11). The interaction of NIK with IKKα has been documented in mammalian species, wherein NIK activates IKKα by inducing the phosphorylation of the IKKα residue Ser176 (10, 31). In agreement with the previous studies conducted in mammals, our study demonstrates that NIK interacts and colocalizes with IKKα in the cultured fish cells. However, in contrast to mammals, IKKα was found to activate NIK by phosphorylating it at the site Thr432 in zebrafish. Owing to the limited availability of fish-specific Abs, it remained impractical to test and determine the phosphorylation with respective Abs directly in this study. The development of Abs recognizing the fish proteins will facilitate further research in this area.

Transcriptional factors IRF3 and IRF7 are known to play pivotal roles in inducing the production of IFN during viral infections. During the early stages of infection in mammals, IRF3 has been found to express abundantly, whereas IRF7 expression remained at lower levels. A higher amount of IRF3 induces the IFN production, which subsequently leads to an increased expression of IRF7 and thereby regulates a variety of IFN-related genes to initiate a cascade amplification effect for the increased secretion of IFNs (5). IKKα is critically involved in IRF7 activation and IFN-α production in TLR 7/9 signaling cascades. NIK and IKKα are known to induce the IRF3 and IRF7 phosphorylation and antiviral gene expression in human cultured cells (10). Moreover, in the DNA pathway, mammalian NIK associates with STING, which in turn bridges TBK1 with IRF3 to promote the phosphorylation-dependent IRF3 activation (11, 43). IRF3 is also reported to interact with NIK, which results in an elevated level of IFN-β and the formation of STING-IRF3 aggregates (11). Our data from this study are partly consistent with the above-mentioned findings in mammals. Zebrafish NIK was found to be involved in the activation of IRF3, but not IRF7, by enhancing IRF3 phosphorylation. The augmented IRF3 phosphorylation was proved to be responsible for IFNφ1 induction.

As an NF-κB–induced kinase, NIK functions as an independent factor in triggering the noncanonical NF-κB pathway. Activated noncanonical NF-κB pathway can also induce the stimulation of the canonical NF-κB pathway (9). These findings reveal the critical role of NIK in the cross-talk between the canonical and noncanonical NF-κB pathways. In alternative NF-κB signaling, NIK phosphorylates IKKα that leads to the activation of its kinase function and confers its ability to phosphorylate p100 (also known as NF-κB2) to ultimately yield the formation of an unrestrained, competent transcription factor complex (44–46). In this study, NIK was found to enhance the NF-κB transcriptional activity in cultured fish cells, which is consistent with the finding in mammals. However, to conclude whether the NIK-mediated activation of NF-κB is dependent on IKKα or not needs to be further investigated.

The absence of mammalian NIK has been associated with an impaired IFN response and increased replication of HSV in vivo (11). Furthermore, mammalian NIK has been shown to cooperate with IKKα and induce the antiviral gene expression, which augmented the cellular antiviral response and enhanced defense against vesicular stomatitis virus infection (10). Findings from our study indicate that NIK responds to RNA and DNA viral infections in the cultured fish cells, and overexpression of NIK strengthened the resistance to the viral infections. As an important regulatory protein, the function of NIK is not limited to innate immunity. NIK is also reported to be involved in tissue injury (47) and tumor development (48, 49). Therefore, results presented in this study may facilitate more in-depth studies in the future for a comprehensive understanding of the possible function of NIK in fish.

To date, zebrafish have been widely employed to investigate the function of genes involved in innate immunity. Thus, zebrafish could be a nice model to determine the role of NIK on antiviral response in vivo. MO-mediated gene silencing is a common technique for gene knockout in zebrafish. However, most phenotypes of MO-treated zebrafish are identified in 3 d postfertilization, and gene silencing is commonly effective within 5 d. Therefore, zebrafish larvae injected with NIK-MO were used as an in vivo model in this study. The results reveal that NIK, which has been expressed in early zebrafish larvae, contributes to antiviral responses via the induction of IFN.

In summary, our study demonstrated that the expressions of zebrafish NIK and IKKα are induced upon virus infection and that NIK protein undergoes phosphorylation by IKKα in vitro. Together with IKKα, NIK induces the activation of IRF3 and NF-κB, which subsequently lead to the induction of IFN and ISG expression. The NIK-mediated elevated antiviral response provided increased resistance to GSIV, GCRV, and SVCV infections. And in this study, we took advantage of a zebrafish model and demonstrated that NIK positively regulated antiviral responses via the induction of IFN response in vivo. The data presented in this study unveiled, for the first time (to our knowledge), the role of NIK in providing protection against the viral infections in lower vertebrates. These findings may provide new insights into the understanding of the fish innate immune response and may facilitate the establishment of antiviral therapy to combat fish viruses. Future studies are needed to consider more host immune–related proteins to reveal the fish innate immune network deeply.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Prof. Zhen Xu (College of Fisheries, Huazhong Agricultural University) for providing virus (GSIV) and Prof. Shaobo Xiao (College of Veterinary Medicine, Huazhong Agricultural University) for providing plasmid (ISREpro-luc).

Footnotes

  • This work was supported by the National Key Research and Development Program of China (2018YFD0900505), the Natural Science Foundation of China (31972834), and Fundamental Research Funds for the Central Universities (2662018YJ022).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    ATCC
    American Type Culture Collection
    CIP
    calf intestinal phosphatase
    Co-IP
    coimmunoprecipitation
    EPC
    epithelioma papulosum cyprini
    FHM
    fathead minnow
    GCRV
    grass carp reovirus
    GSIV
    giant salamander iridovirus
    HEK
    human embryonic kidney
    IFNφ1pro
    IFNφ1 promoter
    IRF
    IFN regulatory factor
    ISG
    IFN-stimulated gene
    ISRE
    IFN-stimulated response element
    MO
    morpholino
    MOI
    multiplicity of infection
    mutNIK
    mutant of zebrafish NIK
    NC
    negative control
    NIK-MO
    MO targeting NIK
    poly(I:C)
    polyinosinic-polycytidylic acid
    qRT-PCR
    quantitative real-time PCR
    S1
    sequence 1
    S2
    sequence 2
    S3
    sequence 3
    siNIK
    siRNA against NIK
    siRNA
    small interfering RNA
    STD-MO
    MO targeting STD
    STING
    stimulator of IFN geneS
    SVCV
    spring viremia of carp virus
    wtNIK
    wild-type NIK.

  • Received May 20, 2019.
  • Accepted January 9, 2020.

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