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 This information is current as Ashraf, Jing Ye and Xueqin Liu of October 1, 2021. J Immunol published online 17 February 2020 http://www.jimmunol.org/content/early/2020/02/14/jimmun ol.1900561 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published February 17, 2020, doi:10.4049/jimmunol.1900561 The Journal of Immunology

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

Bo Chen,*,†,1 Chen Li,*,†,1 Jian Yao,*,† Lin Shi,*,† Wanmeng Liu,*,† Fang Wang,*,† Shitian Huo,*,† Yongan Zhang,*,† Yuanan Lu,‡ Usama Ashraf,x Jing Ye,x and Xueqin Liu*,†

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-kB–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 IKKa and confers its ability to phosphorylate p100 (also known as NF-kB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKa in the cytoplasm and that IKKa phosphorylates the NIK at the residue Thr432, which is different from k the mammals. Overexpression of NIK caused the activation of IRF3 and NF- B, which in turn led to the production of IFN and Downloaded from IFN-stimulated (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. The Journal of Immunology, 2020, http://www.jimmunol.org/ 204: 000–000.

nnate immunity provides the first step of host defense against including the response to pathogens. Among the IRFs, IRF3 and microbial pathogens. In response to viral infection, the in- IRF7 are critical for the induction of type I IFN signaling cascades I duction of IFNs, especially the type I IFN, plays a decisive in response to the sensing of viral DNA and/or RNA motifs by the role in the host defense processes (1). IFN is imperative for the pattern recognition receptors. Upon activation, both IRF3 and potent activation to IFN-stimulated genes (ISGs), which rapidly IRF7 are capable of translocating from cytoplasm to the nucleus culminate in the inhibition of viral replication and spread (2, 3). of the cell, where they trigger the expression of type I IFNs by guest on October 1, 2021 IFN regulatory factors (IRFs) are transcriptional factors that per- (4, 5). Albeit the expression levels of IRF3 and IRF7 are diverse form crucial functions in several aspects of the immune response, in different cells, they are consistent in terms of virus-induced phosphorylation, dimerization, and nuclear translocation (5). NF-kB activation occurs through two pathways: the canonical *College of Fisheries, Huazhong Agricultural University, Wuhan 430070, Hubei, and noncanonical pathways. Both pathways are vital in regulating † China; Hubei Engineering Technology Research Center for Aquatic Animal Dis- the transcription of an array of genes involved in the several eases Control and Prevention, Wuhan 430070, Hubei, China; ‡Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii 96822; and xState processes of immune and inflammatory responses, such as cyto- Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, kine receptor adhesion molecules, acute phase tran- Wuhan 430070, Hubei, China scription factors, and some regulatory factors (6, 7). NF-kB is also 1 B.C. and C.L. contributed equally to this work. reported to exert antiviral function by inducing the expression of ORCIDs: 0000-0002-3258-6224 (J.Y.); 0000-0002-0012-4084 (X.L.). IFN (1, 6). A better understanding of the mechanism of NF-kB Received for publication May 20, 2019. Accepted for publication January 9, 2020. activation is of great importance for harnessing the uncontrolled This work was supported by the National Key Research and Development Program of activation of immune and inflammatory responses. China (2018YFD0900505), the Natural Science Foundation of China (31972834), In mammals, the role of NIK (also known as MAP3K14) in and Fundamental Research Funds for the Central Universities (2662018YJ022). immunity is well established. According to previous studies, NIK Address correspondence and reprint requests to Assoc. Prof. Jing Ye or Prof. Xueqin k Liu, Huazhong Agricultural University, Wuhan, Hubei, China (X.L.). E-mail ad- activates the noncanonical NF- B pathway, which responds to dresses: [email protected] (J.Y.) or [email protected] (X.L.) selective receptor signals that mediate adaptive immune functions, The online version of this article contains supplemental material. such as lymphoid organ development and B cell survival (7, 8). Abbreviations used in this article: ATCC, American Type Culture Collection; CIP, calf Furthermore, the activation of the nonclassical pathway can also intestinal phosphatase; Co-IP, coimmunoprecipitation; EPC, epithelioma papulosum induce the activation of the classical pathway because the stimu- cyprini; FHM, fathead minnow; GCRV, grass carp reovirus; GSIV, giant salamander lation of LTa1b2 leads to the accumulation of intracellular NIK iridovirus; HEK, human embryonic kidney; IFNw1pro, IFNw1 promoter; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; and the upregulation of proinflammatory expression (9). MO, morpholino; MOI, multiplicity of infection; mutNIK, mutant of zebrafish NIK; NIK phosphorylates IKKa at Ser176 and Ser180 residues, and mutation 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; in these two residues to glutamate causes the constitutive activation siNIK, siRNA against NIK; siRNA, small interfering RNA; STD-MO, MO targeting of NF-kB (10). Furthermore, IKKa (S176E) constitutively ac- STD; STING, stimulator of IFN geneS; SVCV, spring viremia of carp virus; wtNIK, tivates IRF3/7 and is involved in IFN-a production through wild-type NIK. TLR 7/9 signaling cascades (10). NIK interacts with the DNA Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 pathway adapter stimulator of IFN genes (STING; also known as

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900561 2 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB

TMEM173/MITA) to enhance the induction of IFN (11). IFN can IKKa (NM_200317.1) was cloned into 33Flag pCMV-14 or pEGFP-N1. induce NIK expression in return (12). Although extensive studies The pGL3–basic firefly luciferase reporter vector harboring the IFNw1 w have been conducted in mammals, the role of NIK in fish has not promoter (IFN 1pro)–Luc or NF-kBpro-Luc and the Renilla luciferase internal control vector (pRL-TK) were provided by professor Yongan yet been investigated. Zhang (College of Fisheries, Huazhong Agricultural University) (22). The Aquatic diseases are the main factors in restricting the develop- IFN-stimulated response element (ISRE)–Luc was received from Professor ment of the aquatic industry and threatening food safety. Among the Shaobo Xiao (College of Veterinary Medicine, Huazhong Agricultural aquatic diseases caused by different pathogens, viral diseases are the University) (23). Wild-type NIK (wtNIK) plasmid was mutated using FastPfu PCR (94˚C most difficult to prevent and cure (13, 14). Spring viremia of carp for 5 min, followed by 25 cycles of 98˚C for 10 s, 55˚C for 5 s, 72˚C for virus (SVCV; ssRNA) (15), giant salamander iridovirus (GSIV; 6 min, and 72˚C for 10 min) (TransGen). Recovered PCR product was dsDNA) (16), and grass carp reovirus (GCRV; dsRNA) (17) are fish treated with Dpn I (TAKARA) at 37˚C for 30 min. Subsequently, the pathogens that cause severe diseases and significant mortalities in plasmid was extracted and stored at 220˚C. the affected fish, which ultimately leads to huge economic losses to Plasmid transfection and virus infection the aquaculture industry. However, there is still no effective method presently available to prevent and control these viral diseases. The recombinant plasmids were transfected using FuGENE HD (Promega) In this study, we demonstrated a previously undescribed following the manufacturer’s protocol. For virus infection assays, cells were infected with SVCV (0.05 multiplicity of infection [MOI]) at 28˚C or function of zebrafish NIK in inducing the immune response GSIV (1.0 MOI) or GCRV (1.0 MOI) at 25˚C. After 1 h of virus absorption, in vitro. We found that IKKa activates NIK by directly inter- cells were washed with PBS three times and subsequently maintained in acting with and phosphorylating it at the residue threonine Medium 199 supplemented with 5% FBS. (Thr)-432. The IKKa-NIK complex mediates the activation of Luciferase assays Downloaded from IRF3 and NF-kB, which in turn leads to the induction of IFN 5 response and inhibition of viral replication. In addition, by FHM cells were seeded in 12-well plates at a density of 0.5–2 3 10 cells taking advantage of the zebrafish model, we examined the role per well. When cells were grown to 70–80% confluence, 500 ng of lu- ciferase reporter plasmid (IFNw1pro-Luc, ISRE-Luc, or NF-kB-Luc) and of NIK in response to SVCV infection in vivo. We elucidated 50 ng of the Renilla luciferase construct pRL-TK (Promega), which served that zebrafish NIK positively regulates antiviral response through as an internal control, were cotransfected with empty vector or a plasmid the induction of IFN. These findings may provide new under- encoding NIK protein. At 24 h posttransfection, firefly and Renilla lucif- standing on functions of fish NIK and facilitate the establishment erase activities were measured using a dual-luciferase reporter assay sys- http://www.jimmunol.org/ tem (Promega). The data are expressed as relative firefly luciferase activity of antiviral therapy to combat fish viruses. normalized to the value of Renilla luciferase. Fluorescent microscopy Materials and Methods Fish cell lines and viruses 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 The fathead minnow (FHM) cell line (American Type Culture Collection with PBS and fixed, then incubated with mouse anti-His Abs (1:3000 di- [ATCC] CCL-42) and epithelioma papulosum cyprini (EPC) cell line lution; ABclonal) for 2 h, followed with Cy3 goat anti-mouse Abs (1:500 (ATCC CRL-2872) were maintained at 28˚C in Medium 199 (Hyclone) dilution; ABclonal) for 45 min. The cell nuclei were stained with DAPI or MEM (Hyclone) supplemented with 10% FBS (Life Technologies). and washed three times. Images were obtained using a confocal micro- by guest on October 1, 2021 The zebrafish embryo (ZF4) cells (ATCC CRL-2050) were maintained scope (Leica). in DMEM/F-12 (Hyclone) with 10% FBS (Life Technologies) at 28˚C. Human embryonic kidney (HEK) 293T cells were grown in DMEM Immunoassays (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- Coimmunoprecipitation (Co-IP) assay was operated using Pierce Co-IP induced cytopathic effect appeared. GSIV and GCRV were propagated in Kit (Thermo Fisher Scientific) according to the instructions of the EPC cells at 25˚C and harvested upon appearance of extensive cyto- manufacturer. For immunoblot analysis, cells were harvested with pathic effect (.80%). Following three cycles of freezing and thawing, ice-cold PBS and lysed on ice with immunoprecipitation lysis/wash 3 culture media were collected from infected cultures and centrifuged at buffer for 5 min. After centrifugation at 13,000 g for 10 min, the 10,000 3 g for 10 min at 4˚C, and recovered supernatant was aliquoted supernatants were collected. Immunoprecipitants or whole-cell extracts andstoredat280˚C. wereseparatedby10%SDS-PAGEand transferred onto a polyvinyli- dene difluoride membrane (Bio-Rad). The membranes were blocked in Reagents 2% albumin from bovine serum at room temperature for 1 h and sub- sequently incubated with mouse anti-His (1:3000 dilution; ABclonal), Polyinosinic-polycytidylic acid (poly[I:C]) (18, 19) and LPS (20, 21) were mouse anti-Flag (1:3000 dilution; ABclonal), rabbit anti–b-actin purchased from Invitrogen. Poly(dA:dT) was purchased from Sigma- (1:10000 dilution; ABclonal), or anti–SVCV-G mAbs (24) for 2 h. Rabbit Aldrich. Cell transfections were performed using FuGENE HD transfec- anti–b-actin Abs were used as internal control. After washing with tion reagent from Promega. Calf intestinal phosphatase (CIP) was obtained TBST, the membranes were incubated with horse radish peroxidase– from New England Biolabs. conjugated secondary goat anti-mouse or anti-rabbit (1:2000 dilution; Quantitative real-time PCR ABclonal) Abs for 45 min. Finally, the reactive were detected using chemical luminescence substrate (General Electric) with Amersham Total RNA was extracted using TRIzol Reagent (TAKARA) according to Imager 600 (General Electric). Protein dephosphorylation was carried the instruction of the manufacturer. The reverse transcription was carried out in 100-ml reaction mixtures consisting of 100 mg of cell protein and out using the ReverTra Ace qPCR RT (TAKARA). The relative ex- 10 U of CIP. Then the mixtures were maintained at 37˚C for 45 min. pression of each cDNA was determined by quantitative real-time PCR (qRT-PCR) using TB Green Real-Time PCR Master Mix (TAKARA). RNA interference Amplification was performed for 5 min at 95˚C, followed by 40 cycles of To knockdown the expressions of NIK or IKKa, RNA interference assay 95˚C for 15 s, 60˚C for 20 s, and 72˚C for 20 s. Fluorescent signals were was performed by transfecting the three pairs of either NIK- or IKKa- analyzed by a Light Cycler/Light Cycler 480 System (Roche). The relative 2DDthreshold cycle specific small interfering RNAs (siRNAs) (GenePharma, China). FHM mRNA levels were calculated using the 2 method. All the cells were seeded in a 12-well plate and transfected with 100 nM primers employed in this study are listed in Table I. of siRNA against NIK (siNIK) or negative control (NC) by using Plasmid conduction and mutation Lipofectamine 2000. Twenty-four hours later, cells were infected with SVCV (MOI = 0.05). At 24 h postinfection, total RNA were isolated The coding sequence of zebrafish NIK (XM_002661208.6), IRF3 andprocessedbyqRT-PCR.TheNIK-specific siRNA sequences were (NM_001143904), or IRF7 (NM_200677.2) (https://www.ncbi.nlm.nih.gov/ as follows: sequence 1 (S1): 59-CCUGUCGUCAUCUC-UCAUUTT-39 genbank) was cloned into pcDNA4. The coding sequence of zebrafish (forward) and 59-AAUGAGAGAUGACAGACAGGTT-39 (reverse); sequence The Journal of Immunology 3

2(S2):59-GCUGUCAGACGAUGGGAAATT-39 (forward) and 59-U- stage zebrafish embryos. To evaluate the knockdown efficiency, mRNA UUCCCAUCGUCUGACAGCTT-39 (reverse); and sequence 3 (S3): levels of NIK at different developmental stages were determined by qRT- 59-GGGUGAAAGUA-GGCCACAUTT-39 (forward) and 59-AUGUGG- PCR. The primers for qRT-PCR were 59-TCCATTATTGCACAGGCGGA-39 CCUACUUUCACCCTT-39 (reverse). (forward) and 59-GAACCCTGCTTTGCATACGG-39 (reverse). The IKKa-specific siRNA sequences were as follows: S1: 59-CCAC- AAGAUAAUCGACCUUTT-39 (forward) and 59-AAGGUCGAUUAUC- Statistics analysis UUGUGGTT-39 (reverse); S2: 59-GCAGCUGAAAG-CAAAGCUUTT- 9 9 9 All statistical analyses and calculations were done using GraphPad 3 (forward) and 5 -AAGCUUUGCUUUCAGCUGCTT-3 (reverse); and S3: Prism 7.0 (GraphPad Software, Inc). The significance of the variability 59- GGUACUAAGGGAUCUAUAUTT-39 (forward) and 59- AUAUA- 9 between different treatment groups was determined by two-way GAUCCCUUAGUACCTT-3 (reverse). , 9 ANOVA. A p value of 0.05 was considered statistically significant The NC siRNA sequences were 5 -UUCUCCGAACGUGUCACGUTT- 6 9 9 9 and marked with an asterisk (*). Data are expressed as means SD of 3 (forward) and 5 -ACGUGACACGUUCGGAGAATT -3 (reverse). results from three independent experiments. Viral plaque assay Results 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 post- Poly(dA:dT) and poly(I:C) promote NIK expression infection, cell supernatants were harvested, and virus titers were de- The coding sequence of zebrafish NIK encodes an 825-aa protein, termined with a plaque assay. Briefly, viruses were serially diluted and whereas human and mouse NIK encode a protein of 947 and 942 aa, inoculated onto monolayers of cells. After 1 h of absorption, cells were washed with serum-free DMEM and cultured in DMEM containing 3% FBS respectively. Phylogenetic analysis indicates that zebrafish NIK and 1.5% sodium carboxymethyl cellulose (Sigma-Aldrich). Visible plaques shares a protein of 46 and 45% with human were counted, and viral titers were calculated after 3 d of incubation. and mouse NIK, respectively (Supplemental Fig. 1). This low similarity of NIK sequences between zebrafish and mammals Downloaded from NIK gene knockdown in zebrafish embryos suggests that the function of NIK in zebrafish might be different Knockdown of NIK gene in zebrafish embryos was accomplished via from that known for mammalian species. morpholinos (MO) technology (YSY Biotech). The sequences of MO To investigate the role of zebrafish NIK in host defense against targeting NIK (NIK-MO) and STD (STD-MO), which served as an NC, were designed as follows: NIK-MO: 59-TGCTAACGTGGAAGAAGA- microbial pathogens, the mRNA expression level of NIK was TATCACA-39 and STD-MO: 59-CCTCTTACCTCAGTTACA-ATTTATA- determined following treatment with poly(dA:dT), poly(I:C), or

39. NIK-MO (4 ng) or STD-MO (8 ng) was microinjected into one-cell LPS in FHM cells. It was observed that both poly(dA:dT) and http://www.jimmunol.org/ by guest on October 1, 2021

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 mg/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)FHMcellswereinfectedwithGSIV,GCRV,orSVCVatanMOIof0.1,1,or10foraperiodof24h. 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. 4 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB poly(I:C), but not LPS, significantly increased the NIK mRNA positively regulates the production of IFN and ISGs during viral expression at 12, 24, and 36 h postinfection in the cultured cells infection. (Fig. 1A–C). As poly(I:C) and poly(dA:dT) are synthetic mimics NIK interacts with IKKa of RNA and DNA viruses, respectively, we next assessed the effect of pathogenic fish RNA and DNA viruses on the NIK mRNA Human NIK is reported to interact with several cellular factors, expression in different fish cell line models. Interestingly, SVCV including TRAF3, IRF3, Pellino3, GRB10, ARRB1, and ARRB2 (ssRNAvirus)infectionmarkedlyenhancedtheNIKmRNA (11, 27–30). Human NIK is also known to physically assemble expression levels in cultured FHM and ZF4 cells at 24 and 36 h with IKKa (31). As a key regulatory factor of the NF-kB pathway, postinfection (Fig. 1D, 1E). Moreover, SVCV, GCRV (dsRNA IKKa plays an important role in triggering the innate immune virus), or GSIV (dsDNA virus) infection of FHM cells caused a response (31). To verify whether the zebrafish NIK can interact significantly upregulated pattern of NIK mRNAs in a dose- with IKKa, a Co-IP assay was carried out by cotransfecting the dependent manner (Fig. 1F–H). Overall, these results suggest NIK-His and IKKa-Flag plasmids in HEK 293T cells. Using either that NIK mRNA expression is elevated upon infection with a captured anti-Flag or anti-His Ab, NIK-His and IKKa-Flag RNA or DNA viruses. were found to be coprecipitated in the lysates of transfected cells (Fig. 3A, 3B). These data demonstrate that the zebrafish NIK induces IFN and ISG production NIK interacts with IKKa. Based on the data presented above, we reasoned that NIK might be To further visualize the subcellular localization of NIK and associated with the host response to viral infection. IFN is one of IKKa in FHM cells, a laser confocal microscopy was employed. the most critical of the host in providing protection Plasmids expressing NIK-His and IKKa-pEGFP or pEGFP were against the invading pathogens. To determine whether NIK is in- cotransfected in the cultured cells. By using a laser confocal mi- Downloaded from volved in inducing the host IFN response, we first examined the croscopy, a similar distribution pattern of NIK (red) and IKKa effect of NIK overexpression on the production of IFN and ISGs. (green) was observed in the cytoplasm (Fig. 3C), which may Our data revealed that overexpression of NIK significantly in- support the interaction of NIK and IKKa as demonstrated by the creased the mRNA expression levels of IFNw1 (25, 26) and ISGs Co-IP assay. (PKR, viperin, and ISG15) in FHM cells (Fig. 2A, 2B). Moreover, a 432 ectopic expression of NIK augmented the promoter activities of IKK phosphorylates NIK at Thr http://www.jimmunol.org/ IFNw1 and ISRE, as assessed by luciferase assay (Fig. 2C, 2D). To In our Co-IP assay, NIK exhibited an obvious shift in its m.w., substantiate that NIK is indeed involved in the induction of IFNw1 whereas IKKa had no such effect (Fig. 3A, 3B). Because phos- and ISGs, cultured FHM cells were transfected with siNIK or control phorylation is known to induce the shift of m.w. of proteins, we siRNAs and subsequently infected with SVCV. The specificity and surmised that the observed shift in the NIK weight may have silencing efficiency of siRNAs for NIK was confirmed (Fig. 2E). occurred owing to NIK phosphorylation. To this end, plasmids Compared with nonsilenced cells, silencing of NIK reduced the encoding IKKa-Flag and NIK-His were cotransfected in HEK mRNA levels of IFNw1, PKR, viperin, and ISG15 in SVCV- 293T cells, and a significant shift of NIK protein was observed. infected cells (Fig. 2F–I). Thus, these data demonstrate that NIK However, the shift of NIK m.w. disappeared upon treatment with by guest on October 1, 2021

FIGURE 2. NIK enhances the expression and promoter activities of IFN and ISGs. (A and B) NIK induces IFNw1 and ISG mRNA production. FHM cells were transfected with NIK-His or empty vector (2 mg 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 IFNw1pro-luc and ISREpro-luc promoter activities. FHM cells were transfected with 500 ng of NIK plasmid or empty plasmid along with 500 ng of IFNw1pro-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 IFNw1 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. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 3. NIK interacts and colocalizes with IKKa.(A and B) NIK interacts with IKKa. HEK 293T cells were seeded in T25 cell flasks and transfected with 5 mg of NIK-His or IKKa-Flag plasmids for 24 h. Cell lysates were precipitated with anti-Flag or anti-His beads, followed by analysis with im- munoblot (IB) assay. (C) Colocalization of NIK and IKKa. FHM cells were cotransfected with NIK-His and IKKa-EGFP or EGFP plasmid for 24 h. Subsequently, cells were fixed and subjected to laser scanning confocal microscopy. Green and red signals indicate the IKKa and NIK proteins, re- spectively. Blue signals represent cellular nuclei. The yellow staining in the merged picture indicates the colocalization of NIK and IKKa. All data are the representative of three independent experiments.

CIP (Fig. 4A), suggesting that the shift of NIK was caused by of wtNIK-expressing samples with CIP (Fig. 4D). To further phosphorylation. confirm the role of IKKa in phosphorylating the NIK protein, To identify the NIK phosphorylated site, multiple sequence IKKa was subjected to siRNA-mediated silencing in FHM cells. alignment of human, mouse, common carp, and zebrafish NIK Reduced mRNA levels of IKKa in siIKKa-treated cells confirmed proteins was performed. A highly conserved sequence, ranging the specificity and silencing efficiency of siRNAs (Fig. 4E). Inhi- from 560 to 570 aa, was detected in these proteins (Supplemental bition of endogenous IKKa caused a reduction in the phosphory- Fig. 1B). NIK is previously reported to undergo phosphoryla- lating state of exogenous NIK in the treated cells (Fig. 4F). tion in mammals (Thr561 in mouse and Thr559 in human) (11), Moreover, endogenous IKKa was found to be upregulated in re- and the phosphorylation sites are located within the conserved sponse to SVCV infection of FHM cells (Supplemental Fig. 2). protein motifs. To investigate whether the phosphorylation site Taken together, these data demonstrate that IKKa phosphorylates ofzebrafishNIKproteinalsoliesintheseconservedaaresi- NIK at the residue Thr432. dues, a mutant of zebrafish NIK (mutNIK) was generated by k substituting the Thr with alanine at position 432 (T432A) of the NIK regulates IRF3 and NF- B activation wtNIK (Fig. 4B). Immunoblot analysis of the lysates of HEK To elucidate how NIK positively regulates the IFN production, 293T cells overexpressed with wtNIK or mutNIK in the absence the effect of NIK on the transcriptional factors of IFN, IRF3/7, or presence of IKKa revealed that wtNIK, but not the mutNIK, and NF-kB was assessed in this study. To illustrate any regu- undergoes shifting of m.w. when coexpressed with IKKa (Fig. 4C). latory effect of NIK on IRF3 and IRF7 at the protein level, the Furthermore, different molecular weights of these two proteins plasmids of NIK-His or IKKa-Flag were cotransfected with either were detected in wtNIK- and mutNIK-expressing FHM cells. No IRF3-His or IRF7-His in HEK 293T cells. Immunoblot analysis detectable shift of wtNIK m.w. was observed following treatment revealed no obvious effect on the protein levels of both IRF3 and 6 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB Downloaded from

FIGURE 4. IKKa phosphorylates NIK at the residue Thr432.(A) HEK 293T cells were cotransfected with NIK-His and IKKa-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 a D cotransfected with NIK-His or mutNIK-His with IKK -Flag or empty vector for 24 h. Later, cell lysates were prepared and subjected to IB analysis. ( ) http://www.jimmunol.org/ 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 IKKa-specific siRNAs on the endogenous expression of IKKa. FHM cells were transfected with IKKa-specific siRNAs (S1, S2, and S3) or control siRNAs (NC) for 24 h. The efficiency of siRNAs to inhibit the IKKa expression was detected by qRT-PCR. S1 siRNA showed the highest silencing efficiency. (F) Effect of IKKa silencing on the phos- phorylation state of NIK. FHM cells were transfected with S1 IKKa-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.

IRF7 (Fig. 5A). Interestingly, cotransfection of NIK-His with the SVCV-induced IFN production was investigated by qRT-PCR by guest on October 1, 2021 IRF3-His or IRF7-His in the presence of IKKa-Flag did not and indicated that NIK significantly upregulated the IFN expres- cause any change on the IRF7 level (Fig. 5A); however, a shifted sion upon SVCV infection (Fig. 6E). strip appeared above the IRF3-His level (Fig. 5B). To determine To consolidate our above findings, the impact of mutNIK on the whether the shifted strip represented the phosphorylated zebra- SVCV replication was examined. As expected, mutNIK caused no fish IRF3, samples were treated with CIP and subjected to influence on the SVCV mRNAs (G, M, and N) and G protein immunoblot analysis. It was found that IRF3 shifted strip dis- (Fig. 7A, 7B) expression levels. However, the comparison of appeared upon treatment with CIP, whereas nontreated samples viral tiers between wtNIK (Fig. 6D) and mutNIK (Fig. 7C) exhibited no effect on the phosphorylated IRF3 (Fig. 5C), revealed that the latter had obviously less-strong influence on the suggesting that NIK activates IRF3, but not IRF7, by regulating SVCV particle production. To further confirm whether NIK is the IRF3 phosphorylation. Furthermore, we determined the ef- indeed involved in restricting the viral infection, FHM cells were fect of zebrafish NIK on the promoter activity of NF-kB. As transfected with siNIK or control siRNAs (NC) and subsequently assessed by luciferase assay, NF-kB–pro activity was significantly infected with SVCV. When compared with nonsilenced cells, stimulated upon expression with NIK (Fig. 5D). Overall, these knockdown of NIK markedly promoted the SVCV mRNA ex- findings demonstrate that zebrafish NIK regulates IRF3 and NF-kB pression (Fig. 7D) and viral titers (Fig. 7E) in the cultured cells. activation. In short, these data indicate a critical role of NIK in controlling the viral infection. NIK restricts virus infection in vitro To evaluate the role of NIK in response to virus infection, NIK was Knockdown of NIK in zebrafish inhibits antiviral response overexpressed in EPC cells for 24 h, followed by infection with To determine the physiological role of NIK in response to viral GSIV, GCRV, or SVCV. The mRNA expression levels of these infection, the zebrafish embryos injected with NIK-MO or STD- viruses were found to be inhibited (Fig. 6A). We next determined MO and the mRNA levels of NIK were determined by qRT-PCR the effect of NIK on SVCV replication in detail by conducting a at different developmental stages, which showed that the NIK series of experiments. Ectopic expression of NIK significantly gene was significantly knocked down in NIK-MO–injected decreased the level of SVCV mRNA (G, M, and N) and G protein embryos (Fig. 8A). The NIK-MO–injected (n = 100) or STD-MO– measured at different infection time points by qRT-PCR (Fig. 6B, injected (n = 100) embryos were infected with SVCV at 48 h Supplemental Fig. 3A, 3B) and immunoblot assays, respectively postinjection, and the numbers of dead larvae were counted at (Fig. 6C). The production of infectious SVCV particles in the su- different time points. The NIK knockdown embryos showed a pernatants of NIK-overexpressed cells was also found to be signif- reduced survival rate compared with the control embryos after icantly reduced at different infection time points, as assessed by SVCV infection (Fig. 8B). To further evaluate the role of NIK plaque formation assay (Fig. 6D). In addition, the effect of NIK on on antiviral response of zebrafish, the expressions of IFNw1and The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 5. NIK regulates IRF3 phosphorylation and NF-kB activation. (A and B) Effect of NIK and IKKa on IRF7 and IRF3 phosphorylation. HEK 293T cells were transfected with the indicated plasmids (1 mg 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 mg 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-kB promoter activity. FHM cells were transfected with 500 ng of NIK plasmid or empty plasmid along with 500 ng of NF-kBpro-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. by guest on October 1, 2021

ISGs, such as PKR, viperin, and ISG15, in response to viral the subcellular localization in HEK 293T cells (11). NIK has been infection were measured. As shown in Fig. 8C–F, SVCV in- confirmed to interact with several proteins in mammals. For fection elicited the expression of IFNw1, PKR, viperin, and instance, NIK interacts with RIG-I and MAVS to activate the ISG15, whereas NIK knockdown resulted in decreased ex- expression of inflammatory genes via the NF-kB pathway (42). pression of IFNw1, PKR, viperin, and ISG15 in the embryos NIK interacts with STING to mediate the IRF3 activation, which after SVCV infection. Consistently, the copy numbers of G, M, contributes to curb a DNA virus infection (11). The interaction of and N genes of SVCV indicated by RNA level were signifi- NIK with IKKa has been documented in mammalian species, cantly increased in NIK knockdown embryos compared with wherein NIK activates IKKa by inducing the phosphorylation of that in control embryos (Fig. 8G). Taken together, these data the IKKa residue Ser176 (10, 31). In agreement with the pre- further confirm the positive regulation role of NIK on antiviral vious studies conducted in mammals, our study demonstrates response in zebrafish (Table I). that NIK interacts and colocalizes with IKKa in the cultured fish cells. However, in contrast to mammals, IKKa was found Discussion to activate NIK by phosphorylating it at the site Thr432 in Viral infection triggers the innate immune responses and promotes zebrafish. Owing to the limited availability of fish-specific Abs, it the induction of IFNs. Four zebrafish IFNs, namely, IFNw1to remained impractical to test and determine the phosphorylation IFNw4, have been classified into two distinct subsets: group I and with respective Abs directly in this study. The development of group II. IFNw1 and IFNw4 belong to group I, whereas IFNw2 and Abs recognizing the fish proteins will facilitate further research in IFNw3 belong to group II. All of the IFNs, except IFNw4, have this area. been reported to induce strong antiviral activity in adult zebrafish Transcriptional factors IRF3 and IRF7 are known to play pivotal (32–34). In mammals and fish, IFNs play a critical role in the roles in inducing the production of IFN during viral infections. host innate immune system and trigger a massive expression of During the early stages of infection in mammals, IRF3 has been ISGs that can interfere with viral transcription, translation, as- found to express abundantly, whereas IRF7 expression remained at sembly, and release (26, 32, 35). IFN activation is dependent on lower levels. A higher amount of IRF3 induces the IFN production, the regulation of transcriptional factors that include IRF3, IRF7, which subsequently leads to an increased expression of IRF7 and and NF-kB (36–41). However, the mechanisms of these path- thereby regulates a variety of IFN-related genes to initiate a ways are separately or differentially modulated and remain cascade amplification effect for the increased secretion of IFNs largely uncharacterized. (5). IKKa is critically involved in IRF7 activation and IFN-a This study revealed that NIK was distributed in the cytoplasm production in TLR 7/9 signaling cascades. NIK and IKKa at the periphery of the nuclei in FHM cells, which is similar to are known to induce the IRF3 and IRF7 phosphorylation 8 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB Downloaded from

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 mg 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 m

plasmid and empty vector (2 g each) for a period of 24 h, followed by infection with SVCV at MOI of 0.05. Cell samples and supernatants were collected http://www.jimmunol.org/ 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 IFNw1 expression. FHM cells were transfected with NIK plasmid or empty vector (2 mg each) for 24 h and then infected with SVCV (0.05 MOI) for 24 h. The relative IFNw1 mRNA expression was quantified by qRT-PCR. All data are representative of three independent experiments. *p , 0.05, ***p , 0.001, ****p , 0.0001. and antiviral gene expression in human cultured cells (10). be involved in the activation of IRF3, but not IRF7, by enhancing Moreover, in the DNA pathway, mammalian NIK associates IRF3 phosphorylation. The augmented IRF3 phosphorylation was with STING, which in turn bridges TBK1 with IRF3 to promote proved to be responsible for IFNw1 induction. k the phosphorylation-dependent IRF3 activation (11, 43). IRF3 is As an NF- B–induced kinase, NIK functions as an inde- by guest on October 1, 2021 also reported to interact with NIK, which results in an elevated pendent factor in triggering the noncanonical NF-kBpathway. level of IFN-b and the formation of STING-IRF3 aggregates Activated noncanonical NF-kB pathway can also induce the (11). Our data from this study are partly consistent with the stimulation of the canonical NF-kB pathway (9). These find- above-mentioned findings in mammals. Zebrafish NIK was found to ings reveal the critical role of NIK in the cross-talk between the

FIGURE 7. (A–C) Effects of mutNIK on SVCV replication. FHM cells were transfected with mutNIK plasmid or empty vector (2 mg 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. The Journal of Immunology 9 Downloaded from

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 3 106 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 IFNw1, 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. http://www.jimmunol.org/ canonical and noncanonical NF-kB pathways. In alternative NF-kB unrestrained, competent transcription factor complex (44–46). In signaling, NIK phosphorylates IKKa that leads to the activation of this study, NIK was found to enhance the NF-kB transcriptional its kinase function and confers its ability to phosphorylate p100 activity in cultured fish cells, which is consistent with the finding (also known as NF-kB2) to ultimately yield the formation of an in mammals. However, to conclude whether the NIK-mediated

Table I. Primers of this study

Application Prime Name Sequence (59–39) by guest on October 1, 2021 qRT-PCR qTBP-F 59-TTACCCACCAGCAGTTTAG-39 qRT-PCR qTBP-R 59-ACCTTGGCACCTGTGAGTA-39 qRT-PCR qCc40S-F 59-CCGTGGGTGACATCGTTACA-39 qRT-PCR qCc40S-R 59-TCAGGACATTGAACCTCACTGTCT-39 qRT-PCR qNIK-F 59-CAAAAAGACTGAACGAGA-39 qRT-PCR qNIK-R 59-AGGACTGACTGACCAACG-39 qRT-PCR SVCV-G-F 59-CGACCTGGATTAGACTTG-39 qRT-PCR SVCV-G-R 59-AATGTTCCGTTTCTCACT-39 qRT-PCR SVCV-M-F 59-TACTCCTCCCACTTACGA-39 qRT-PCR SVCV-M-R 59-CAAGAGTCCGAGAAGGTC-39 qRT-PCR SVCV-N-F 59-GCGGTTTTCTGTATGTGTCTC-39 qRT-PCR SVCV-N-R 59-CTCTGCCAAATCACCATACTC-39 qRT-PCR qIFN1-F 59-AACGCAGCACAATGGAAC-39 qRT-PCR qIFN1-R 59-TGATGGATGGTGGTATCG-39 qRT-PCR qISG15-F 59-TAATGCCACAGTCGGTGAA-39 qRT-PCR qISG15-R 59-AGGTCCAGTGTTAGTGATGAGC-39 qRT-PCR qPKR-F 59-ACCTGAAGCCTCCAAACATA-39 qRT-PCR qPKR-R 59-GCATTCGCTCATCATTGTC-39 qRT-PCR qViperin-F 59-GCAAAGCGAGGGTTACGAC-39 qRT-PCR qViperin -R 59-CTGCCATTACTAACGATGCTGAC-39 qRT-PCR qGSIV-MCP-F 59-GACTTGGCCACTTATGAC-39 qRT-PCR qGSIV-MCP-R 59-GTCTCTGGAGAAGAAGAA-39 qRT-PCR qGCRV-NS4-F 59-CCTTCGTCTAACATGAAC-39 qRT-PCR qGCRV-NS4-R 59-GAAGGTGGGAATTTGAAG-39 Plasmid construction NIK-His-F 59-CCGGGTACCATGCAGGTGCAAAGAATTTG-39 Plasmid construction NIK-His -R 59-TGCTCTAGAGTTATCTCTGGTCTCCAGAAG-39 Plasmid construction IKKa-Flag -F 59-CGGGGTACCATGGAGAAACCCCCTTTCAG-39 Plasmid construction IKKa-Flag-R 59-CCGCTCGAGCAAACGCGCTGATTTAGCAA-39 Plasmid construction IKKa-GFP-F 59-CCGCTCGAGATGGAGAAACCCCCTTTCAG-39 Plasmid construction IKKa-GFP-R 59-CGGAATTCGCAAACGCGCTGATTTAGCAA-39 Plasmid construction IRF3-His-F 59-CGGGGTACCATGACTCAAGCAAAACCGCT-39 Plasmid construction IRF3-His-R 59-CCGCTCGAGGCAGAGCTCCATCATTTGCTC-39 Plasmid construction IRF7-His-F 59-GGGGTACCATGATTGCCTAGAATTCAAGCTAT-39 Plasmid construction IRF7-His-R 59-CCGCTCGAGTCGAATATGGGAAAAAGTTGATGT-39 Plasmid mutation mutNIK-His-F 59-GTTATTGTCAGGGATTGAAAGGCGCAGAGACTCATATGGCACCTGAGG-39 Plasmid mutation mutNIK-His-R 59-CGCCTTTCAATCCCTGACAATAACTAAGCCCTTGTTTGTCCAGTC-39 10 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB activation of NF-kB is dependent on IKKa or not needs to be 4. Smith, E. J., I. Marie´, A. Prakash, A. Garcı´a-Sastre, and D. E. Levy. 2001. IRF3 and IRF7 phosphorylation in virus-infected cells does not require double- further investigated. stranded RNA-dependent protein kinase R or Ikappa B kinase but is blocked The absence of mammalian NIK has been associated with an by Vaccinia virus E3L protein. J. Biol. Chem. 276: 8951–8957. impaired IFN response and increased replication of HSV in vivo 5. Fitzgerald, K. A., D. C. Rowe, B. J. Barnes, D. R. Caffrey, A. Visintin, E. Latz, B. Monks, P. M. Pitha, and D. T. Golenbock. 2003. LPS-TLR4 signaling to IRF- (11). Furthermore, mammalian NIK has been shown to coop- 3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. [Published er- erate with IKKa and induce the antiviral gene expression, which ratum appears in 2003 J. Exp. Med. 198: following 1450.] J. Exp. Med. 198: augmented the cellular antiviral response and enhanced defense 1043–1055. 6. Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the against vesicular stomatitis virus infection (10). Findings from control of NF-[kappa]B activity. Annu. Rev. Immunol. 18: 621–663. our study indicate that NIK responds to RNA and DNA viral 7. Sun, S.-C. 2017. The non-canonical NF-kB pathway in immunity and inflam- infections in the cultured fish cells, and overexpression of NIK mation. Nat. Rev. Immunol. 17: 545–558. 8. Sun, S.-C. 2012. The noncanonical NF-kBpathway.Immunol. Rev. 246: strengthened the resistance to the viral infections. As an im- 125–140. portant regulatory protein, the function of NIK is not limited to 9. Zarnegar, B., S. Yamazaki, J. Q. He, and G. Cheng. 2008. Control of canonical innate immunity. NIK is also reported to be involved in tissue NF-kappaB activation through the NIK-IKK complex pathway. Proc. Natl. Acad. Sci. USA 105: 3503–3508. injury (47) and tumor development (48, 49). Therefore, results 10. Wang, R. P., M. Zhang, Y. Li, F. C. Diao, D. Chen, Z. Zhai, and H. B. Shu. 2008. presented in this study may facilitate more in-depth studies in Differential regulation of IKK alpha-mediated activation of IRF3/7 by NIK. Mol. the future for a comprehensive understanding of the possible Immunol. 45: 1926–1934. 11. Parvatiyar, K., J. Pindado, A. Dev, S. R. Aliyari, S. A. Zaver, H. Gerami, function of NIK in fish. M. Chapon, A. A. Ghaffari, A. Dhingra, and G. Cheng. 2018. A TRAF3-NIK To date, zebrafish have been widely employed to investigate the module differentially regulates DNA vs RNA pathways in innate immune sig- function of genes involved in innate immunity. Thus, zebrafish could naling. Nat. Commun. 9: 2770.

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