Abstract
Bone morphogenetic protein (BMP) is a kind of classical multi-functional growth factor that plays a vital role in the formation and maintenance of bone, cartilage, muscle, blood vessels, and the regulation of energy balance. Whether BMP plays a role in antiviral immunity is unknown. Here we demonstrate that Bmp8a is a newly-identified positive regulator for antiviral immune responses. The bmp8a−/− zebrafish, when infected with the viruses of GCRV, SVCV or TSVDV, show significantly reduced antiviral immunity, increased viral load and morbidity. We also show for the first time that Bmp8a interacts with Alk6a, which promotes the phosphorylation of Tbk1 and Irf3 through p38 MAPK pathway, and induces the production of type I IFNs in response to virus infection. Upon virus infection, bmp8a expression is activated through the binding of Stat1a/Stat1b to the GAS motifs in bmp8a promoter region, enlarging the antiviral innate immune signal. Our study uncovers a previously unrecognized role of Bmp8a in regulation of antiviral immune responses and provides a new target for controlling viral infection.
Introduction
Viruses infect all groups of living things and produce a variety of diseases. Host cell detection of viral infection depends on the recognition of pathogen-associated molecular patterns (PAMPs) from viral proteins or nucleic acids by the pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs) and cytoplasmic DNA or RNA sensors1–6. The PRRs, once stimulated by their appropriate ligands, trigger distinct intracellular signaling pathways that converge on the activation of IFN regulatory factor 3 (IRF3) and/or IFN regulatory factor 7 (IRF7), which then translocate into the nucleus and activate the production of interferons (IFNs) that play important roles in inhibiting virus replication7,8.
IFNs are classified into type I IFNs that include IFNα and IFNβ; type II IFN which has only a single member IFN-γ; and type III IFNs that consist of IFN-λs9. All these three types of IFNs trigger intracellular signaling cascades generally via the Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway10. Canonically, type I IFNs initiate the signaling via binding to a heterodimeric receptor complex IFN-α/β receptor (IFNAR), inducing phosphorylation of transcription factors STAT1 and STAT2. The phosphorylated STATs form a trimolecular complex with IRF9, which translocates to the nucleus and binds to IFN-stimulated response elements (ISREs) of IFN-stimulated genes (ISGs), resulting in transcriptional activation of the target ISGs, including those encoding numerous cytokines and antiviral proteins11–16. In contrast, type II IFN binds to its unique receptor, IFN-γ receptor (IFNGR), resulting in the phosphorylation of STAT1 and the formation of STAT1 homodimers that recognize gamma-activated sequences (GASs) present in the promoter regions of IFN-γ-regulated genes9.
Bone morphogenetic proteins (BMPs) are potent growth factors belonging to the transforming growth factor beta (TGFβ) superfamily, which are intercellular signaling molecules with multiple functions in development and differentiation, such as skeletal formation and hematopoiesis, as well as lipid oxidation17–21. However, accumulating data suggests that BMPs also play important roles in the regulation of immune responses22–27. For example, BMP7 has been shown to drive Langerhans cell differentiation28. Interestingly, BMP signaling appears to have different responses in T cells: BMP4 and BMP6 were shown to promote T cell proliferation, whereas BMP2 was seen to inhibit T cell proliferation29,30. In addition, although BMPs were found to promote tumor growth by inhibiting the functions of cytotoxic T lymphocytes (CTLs) and dendritic cells (DCs), they were also shown to inhibit cancer cell proliferation and promote the activity of NK cells that typically display antitumour activity31. Therefore, it remains controversial regarding the functions of BMPs in immune responses.
In humans and mice, two closely related BMP8 genes, BMP8A and BMP8B, have been identified32,33. BMP8B has been found to play roles in the regulation of thermogenesis in mature brown adipose tissue and spermatogenesis in male germ cells, while BMP8A shown to closely correlate with the progression of spermatogenesis34,35. In contrast, only one bmp8 gene, bmp8a, has been isolated in zebrafish, and shown to be involved in the regulation of lipid metabolism36. To date, whether Bmp8a plays a role in immune responses is completely unknown. The present study was thus performed to answer this question. We demonstrated that Bmp8a was a previously unrecognized regulator functioning in antiviral immune responses of zebrafish Danio rerio, providing a new target for control of viral infection.
Results
Bmp8a inhibited RNA virus replication in vitro
To explore the function of zebrafish Bmp8a in the antiviral immune response, we infected both the wild-type zebrafish liver (ZFL) cells and ZFL cells over-expressing bmp8a as well as the epithelioma papulosum cyprini (EPC) cells and EPC cells over-expressing bmp8a with Grass carp reovirus (GCRV), a dsRNA virus, and then monitored the cytopathic effect(CPE)and the viral titers of the supernatants. In response to GCRV challenge, an apparent CPE was observed in the control cells, while the bmp8a-overexpressing ZFL cells had much less CPE (Fig. 1a). In accordance with the CPE results, a significant decrease of the viral titers was observed in the supernatants of bmp8a-overexpressing ZFL cells (Fig. 1b). Similarly, the GCRV-induced CPE and GCRV yields of the supernatants were also markedly reduced in the bmp8a-overexpressing EPC cells (Fig. 1c, d). These suggested that Bmp8a suppressed the replication of the RNA virus in the cells.
Bmp8a was involved in antiviral responses in vivo
To test the function of Bmp8a in host defense against virus infection in vivo, we generated bmp8a deficient (bmp8a−/−) zebrafish using the TALEN approach (Supplementary Fig. 1a), which caused 7 nucleotides deletion in the exon 4 (Supplementary Fig. 1b, c). We challenged both the wild-type and bmp8a−/− mutant zebrafish by intraperitoneal injection with GCRV. Compared with wild-type zebrafish, the levels of GCRV RNA in the liver, kidney, intestine, and spleen of bmp8a−/− zebrafish were considerably increased (Fig. 1e). Accordingly, bmp8a−/− zebrafish exhibited significantly reduced survival rate than wild-type zebrafish upon GCRV infection (Fig. 1f). When the wild-type and bmp8a−/− zebrafish were similarly challenged with spring viremia of carp virus (SVCV), a negative ssRNA virus, the survival rate of bmp8a−/− zebrafish was also remarkably lower than that of wild-type zebrafish (Fig. 1g). Moreover, when the wild-type and bmp8a−/− zebrafish were infected with turbot skin verruca disease virus (TSVDV) isolated from skin (Supplementary Fig. 2), mortality occurred in bmp8a−/− zebrafish at 108 h postinfection, and all the fish died at 156 h post infection, while none of the wild type zebrafish were dead during the period (Fig. 1h). These data indicated that bmp8a−/− zebrafish were more susceptible to virus infection than wild-type zebrafish, suggesting involvement of Bmp8a in antiviral immune responses in vivo.
Bmp8a promoted expression of antiviral genes
In zebrafish, type I IFNs contained 4 members ifnφ1, ifnφ2, ifnφ3, and ifnφ4, among which ifnφ1 and ifnφ3 were shown to be involved in the antiviral response. In contrast, in carp, only one type I IFN was identified to fulfill antiviral role in EPC cell37,38. To pinpoint the role of Bmp8a in the antiviral immune response, both the wild-type and bmp8a−/− zebrafish were injected intraperitoneally with GCRV, and the expression of type I IFN genes and the antiviral protein gene mx were detected using quantitative reverse transcription PCR (qRT-PCR). We found that the expression of both ifnφ1 and ifnφ3 as well as mx was all significantly down-regulated in the liver, kidney, intestine, and spleen of bmp8a−/− zebrafish, compared with those of wild-type fish (Fig. 2a-d). We then over-expressed bmp8a in ZFL cells and found that ZFL cells with over-expressed Bmp8a showed markedly higher expression of ifnφ1, ifnφ3, and mx than that of control cells, infected with or without GCRV (Fig. 2e-h). In addition, bmp8a knockdown caused remarkably decreased expression of ifnφ1, ifnφ3, and mx in ZFL cells (Fig. 2i-l). Similarly, the expression of ifn and mx in EPC cells with over-expressed Bmp8a was also remarkably up-regulated than that in control cells (Fig. 2m-p). Moreover, EPC cells cotransfected with bmp8a expressing plasmid and IFNφ1, IFNφ3, or EPC IFN promoter-driven luciferase plasmid, followed by infection with or without GCRV, had markedly increased intracellular IFNφ1, IFNφ3, or EPC IFN promoter-driven luciferase activities (Fig. 2q-s). All these data denoted that Bmp8a promoted the expression of both type I IFN and mx genes.
Bmp8a activated Tbk1-Irf3-Ifn antiviral signaling via p38 MAPK pathway
To putatively examine the molecular mechanism by which Bmp8a stimulates ifn and mx expression, we carried out transcriptome analysis of the livers of bmp8a−/− mutant and wild-type zebrafish, that both had been intraperitoneally injected with GCRV. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that, compared with wild-type zebrafish, the down-regulated genes in the mutant were remarkably enriched in the RIG-I-like receptor signaling pathway, NF-kappa B signaling pathway, TNF signaling pathway, and protein digestion and absorption that function in the antiviral immune process (Supplementary Fig. 3). In agreement with transcriptome analysis, we found that the expressions of tbk1, irf3, and irf7 were significantly upregulated in bmp8a-overexpressing ZFL cells than control cells, infected with or without GCRV (Fig. 3a-d). Similar expression patterns of tbk1, irf3, and irf7 were also observed in EPC cells, infected with or without GCRV (Fig. 3e-h). In addition, bmp8a knockdown resulted in considerably reduced expression of tbk1, irf3, and irf7 in ZFL cells than control cells, infected with or without GCRV (Fig. 3i-l). We then evaluated the activation of Tbk1 and Irf3 by immunoblot assays in both bmp8a-overexpressing and wild-type ZFL cells as well as EPC cells, infected with or without GCRV. It was found that phosphorylation levels of Tbk1 and Irf3 were significantly increased in both types of the cells with overexpressed Bmp8a (Fig. 3m-p). Moreover, bmp8a knockdown markedly reduced the phosphorylation levels of Tbk1 and Irf3 in ZFL cells, infected with or without GCRV (Fig. 3q, r). Furthermore, in luciferase activity assays, transfection of dominant negative mutations of tbk1 (tbk1-K38M), irf3 (irf3DN), or irf7 (irf7DN) in EPC cells induced a significant loss in the ability of Bmp8a to activate the IFN promoter (Fig. 3s-u). This was further supported by the observations in vivo that the expression of tbk1, irf3, and irf7 in the liver, kidney, intestine, and spleen was significantly downregulated in bmp8a−/− mutant than that in wild-type zebrafish (Fig. 3v-y). These data together revealed that Bmp8a induced ifn and mx expression via promoting Tbk1-Irf3-Ifn antiviral signaling.
BMPs transmit signals through smad1/5/8, smad2/3, ERK, JNK, or p38 MAPK pathways39–41. Thus, the effects of p38 MAPK inhibitor SB203580, JNK inhibitor SP600125, MEK1/2 inhibitor U0126, SMAD1/5/8 inhibitor DMH1, and smad2/3 inhibitor TP0427736 HCl on the expression of ifn was tested in bmp8a-overexpressing ZFL cells and EPC cells. We have shown above that Bmp8a overexpression increased the expression of ifn (Fig. 2). Here we found that only p38 MAPK inhibitor SB203580 significantly reduced the expression of ifnφ1 and ifnφ3 in ZFL cells and the expression of ifn in EPC cells (Fig. 4a-c). Furthermore, the IFN promoter-driven luciferase assays revealed that the luciferase activities were markedly reduced in EPC cells upon treatment with p38 MAPK inhibitor SB203580 (Fig. 4d-f), consistent with the observation that Bmp8a induced the expression of ifn through p38 MAPK pathway. Taken the previous report that the MAPK signaling increased the phosphorylation of TBK1 and IRF3 upon viral infection into consideration42, we suggested that Bmp8a activated Tbk1-Irf3-Ifn antiviral signaling via p38 MAPK pathway.
BMP type I receptor Alk6a interacted with Bmp8a and participated in antiviral immunity
BMPs exert their biological effects through the sequential activation of two types of transmembrane receptors, namely BMP receptor type I and BMP receptor type II, that both possess intrinsic serine/threonine kinase activity43,44. To detect which receptor is involved in the regulation of the antiviral immune responses, the expression of both type I receptor genes (alk2, alk3, alk6a) and type II receptor genes (bmpr2a, bmpr2b, actr2a, actr2b) in ZFL cells transfected with poly(I:C) or infected with GCRV were measured. Among these receptor genes, alk6a showed the highest expression upon poly(I:C) or virus challenge (Fig. 5a, b). We also overexpressed all these receptors in ZFL and EPC cells, and examined the expression of ifn in these cells. The upregulation of ifnφ1 was only found in the alk6a-overexpressing ZFL cells (Fig. 5c). Moreover, the overexpression of alk6a or alk2 resulted in significantly increased expression of ifnφ3 in ZFL cells and ifn in EPC cells, with overexpressed alk6a being more effective (Fig. 5d, e). Based on these findings, the function of Alk6a was selected for further test.
Compared to wild-type ZFL cells, the expressions of antiviral protein genes ifnφ1, ifnφ3 and mx as well as the antiviral signaling genes tbk1, irf3, and irf7 were all markedly upregulated in alk6a-overexpressed ZFL cells with or without GCRV infection (Fig. 5f, g). The similar results were also observed in the alk6a-overexpressed EPC cells (Fig. 5h, i). Because the phosphorylation of Gly-Ser (GS) domain of the type I receptor is required for its activation, we thus constructed dominant-negative mutant of alk6a (alk6a-ΔGS) plasmid (Fig. 5j), and then transferred it into both ZFL and EPC cells. It revealed that the dominant-negative mutation of alk6a significantly reduced the expression of mx, ifn (ifnφ1 and ifnφ3 in ZFL cells), tbk1, irf3, and irf7 in ZFL and EPC cells, with or without GCRV infection (Fig. 5k-n). The data suggested that Alk6a was apparently involved in the Tbk1-Irf3/7-Ifn antiviral signaling.
Considering we have revealed that Bmp8a activated Tbk1-Irf3-Ifn antiviral signaling via p38 MAPK pathway, we then examined whether the the levels of phosphorylated p38 MAPK protein were regulated by Alk6a. Clearly, compared to wild-type ZFL or EPC cells, phosphorylation levels of p38 MAPK were significantly increased in alk6a-overexpressed both types of the cells with or without GCRV infection (Fig. 6a-d). Moreover, dominant-negative mutation of alk6a significantly reduced the phosphorylation levels of p38 MAPK in ZFL or EPC cells, with or without GCRV infection (Fig. 6e-h). Thus, Alk6a was shown to be involved in the phosphorylation of p38 MAPK. Next, we investigated whether the antiviral immune responses of Bmp8a were mediated by Alk6a. In the IFN promoter-driven luciferase assays, it was found that the ability of Bmp8a to activate the IFN promoter was markedly blocked after the dominant-negative mutation of alk6a in EPC cells (Fig. 6i-k). Importantly, co-immunoprecipitation (Co-IP) experiments confirmed that Bmp8a interacted with Alk6a (Fig. 6l). Collectively, our data demonstrated that BMP type I receptor Alk6a interacted with Bmp8a, and participated in the antiviral immunity. To our best knowledge, Alk6a is the first BMP receptor identified thus far which is being directly required for antiviral immune responses.
Virus induced expression of bmp8a
Next, we measured the expression of bmp8a in ZFL cells upon virus infection. GCRV significantly induced the expression of bmp8a at 12 h post-stimulation, and reached a peak at 48 h (Fig. 7a). We also detected the expression of bmp8a upon infection with GCRV or poly(I:C) in vivo. The expression of bmp8a in the liver, kidney, intestine, and spleen of zebrafish infected with GCRV or poly(I:C) was all significantly elevated (Fig. 7b-i). This indicated that the expression of bmp8a was inducible by infection with virus or its mimic poly(I:C).
Binding of Stat1 to GAS motifs activated bmp8a expression
To explore how virus induces expression of bmp8a, we searched for the transcription factor binding sites in bmp8a promoter region at the web http://jaspar.genereg.net/. Two gamma-activated sites (GAS), 5‘-ATTCCGGGAAA-3’ (P1) and 5‘-TTTACTAGAAC-3’ (P2), in bmp8a promoter region were identified (Supplementary Fig. 4). In mammals, the transcription factor STAT1 homodimer was known to bind to the GAS motif in the promoter region of IFN-γ triggered downstream genes11,16. We thus wonder if Stat1 can bind to the bmp8a promoter region to activate the expression of bmp8a. In zebrafish, there exist two Stat1 factors, Stat1a (GenBank accession number NM_131480.1) and Stat1b (GenBank accession number NM_200091.2). Thus we constructed dominant-negative mutant plasmids of stat1a-ΔC and stat1b-ΔC, that were transferred into ZFL cells, respectively. It was found that the expression of bmp8a was significantly blocked in both cases (Fig. 8a, b). By contrast, the overexpression of either stat1a or stat1b markedly promoted bmp8a promoter-driven luciferase activities (Fig. 8c). In addition, when one of the GAS motifs in bmp8a promoter region was deleted, bmp8a promoter-driven luciferase activities were significantly reduced; when both of the GAS motifs were deleted, bmp8a promoter-driven luciferase activities were reduced even more significantly (Fig. 8d, e). These suggested that both Stat1a and Stat1b may interact with the GAS motifs.
To verity the binding of Stat1a/Stat1b to the GAS motifs in bmp8a promoter region, the recombinant proteins of both Stat1a and Stat1b were expressed and purified (Fig. 8f, g), and used for electrophoretic mobility shift assay (EMSA). As shown in Fig. 8h-k, a remarkable band of protein-DNA complex was observed (lane 2). The protein-DNA complex band was obviously weaker in the presence of an excess unlabeled competitor probe, compared to that of biotin labeled probe group (lane 3), but the intensity of the band showed little change in the presence of GAS mutant probes (lane 4). To detect the specific binding, a supershift experiment with the antibody against His tag was performed. It revealed that His tag antibody caused a specific supershift of slower migrating protein-DNA-antibody complex (Fig. 8h-k, lane 5). There was no protein-DNA complex observed in the negative control group (Fig. 8h-k, lane 1). These demonstrated that both Stat1a and Stat1b could directly bind to the GAS motifs in bmp8a promoter region, suggesting that bmp8a expression was subjected to the regulation by binding of Stat1a/Stat1b to the GAS motifs.
Discussion
BMPs, canonical multi-functional growth factors, are suggested to play roles in regulation of immune responses, but it remains controversial over their functions in immunity. We show here for the first time that Bmp8a is a previously unrecognized factor involved in the regulation of antiviral immune responses in zebrafish. Evidences supporting this nature of Bmp8a include: Bmp8a inhibits RNA viruses replication in vitro and in vivo; Bmp8a promotes the expression of antiviral protein genes including type I IFNs and mx; the expression of bmp8a is induced by CGRV or poly(I:C), and Bmp8a activates TBK1-IRF3-IFN signaling. Bmp8a is a member of the TGF-β superfamily, which includes more than 30 genes encoding TGFβ, BMPs and activins. TGFβ has been shown to have both pro- and anti-inflammatory effects depending on the context it is acting in the immune system45. Like TGFβ, BMP members have also been shown to play different immunoregulatory roles46. BMP2, 4 and 7 deficiency or knockdown results in partial loss of the thymic capsule, reduced size of thymus and increased Helicobacter pylori-induced inflammation, whereas BMP6 inhibits macrophage growth by inducing cell cycle arrest in vitro27,47–50. The finding that Bmp8a is a positive regulator in antiviral immune responses provides a new angle for our understanding of the functions of TGF-β superfamily members including BMPs.
On one hand, it is generally regarded that the recognition of virus by PRRs elicits the activation of IRF3 and NF-κB, and induces the production of IFNα/β, that then activate JAK-STAT pathway, eventually leading to the production of ISGs such as cytokines and antiviral proteins. On the other hand, BMPs are known to transmit signals through both Smad-dependent pathways (smad1/5/8 and smad2/3 pathways) and Smad-independent pathways (ERK, JNK, and p38 MAPK pathways). Here we clearly demonstrate that Bmp8a promotes the expression of endogenous type I IFNs independent of Smad signaling pathways. Moreover, it is p38 MAPK pathway, rather than ERK and JNK pathways, that is involved in the antiviral responses induced by Bmp8a. Furthermore, we show that Alk6a participates in Tbk1-Irf3/7-Ifn antiviral signaling via interaction with Bmp8a. In addition, Alk6a was found to be required to induce the phosphorylation of p38 MAPK, which is consistent with the findings in mouse that Alk6 knockdown suppresses p38 MAPK phosphorylation51. Previous studies have shown that MAPK signaling pathway is crucial in the phosphorylation of TBK1 and IRF3 upon viral infection42. Thus, we probably discover a new pathway of Bmp8a signaling in antiviral immune responses that Bmp8a acts as a positive regulator through the promotion of phosphorylation of Tbk1 via p38 MAPK pathway, i.e., Bmp8a binds to Alk6a, which induces p38 MAPK phosphorylation, and in turn enhances Tbk1 and Irf3 phosphorylation, eventually resulting in increased synthesis of type I IFN (Fig. 9).
Our study also show that the challenge with GCRV and poly(I:C) both significantly promotes the expression of zebrafish bmp8a. Searching for the binding sites of transcript factors in bmp8a promoter region reveals that two IFN-γ activation sites (GAS) that may interact with STAT1 are identified. Functionally, we demonstrate that Stat1a and Stat1b, orthologues of human STAT1, can directly bind the GAS sites in bmp8a promoter, resulting in activation of bmp8a expression. It has been reported that in black carp, Stat1a and Stat1b can both form homodimer and heterodimer in vivo like their mammalian counterpart52. It is thus highly likely that in zebrafish, Stat1a and Stat1b can form homodimer or heterodimer, which then binds to the GAS sites in bmp8a promoter region. In zebrafish, IFN-γ has been found to be able to induce the expression of both stat1a and stat1b, and the GAS motif is required for Ifn-γ activation53,54. It is widely known that the JAK-STAT pathway is one of the most important candidate pathways through which IFN-γ works. Activation of JAK-STAT signaling leads to transcription of various downstream ISGs for antiviral activity55. We thus think that Bmp8a is another important antiviral factor which can be activated through Ifn-γ-Jak-Stat1 pathway (Fig. 9).
In summary, we demonstrate for the first time that Bmp8a is a positive regulator in antiviral immune responses, which functions through promotion of phosphorylation of Tbk1 via p38 MAPK pathway. Upon virus infection, the expression of bmp8a is activated by Stat1a/Stat1b directly. Bmp8a knockdown leads to significantly decreased phosphorylation of Tbk1 and Irf3. As a consequence, bmp8a-deficient zebrafish produces significantly lowered type I IFNs in response to RNA virus infection, exhibits significantly reduced immune responses, and increases significantly higher viral load and morbidity in vivo. Our study is significant to understand the regulatory mechanism of BMP involving in antiviral immune response and provides a new target for controlling viral infection.
Methods
Cells and viruses
The epithelioma papulosum cyprini cells (EPC), zebrafish liver cells (ZFL) were acquired from the China Zebrafish Resource Center (CZRC) and cultured according to the instructions from the CZRC. EPC and ZFL cells were grown in a 28 °C incubator supplied with 5% CO2. The flounder gill cells (FG) were grown in a 22 °C incubator supplied. The EPC or FG cells were cultured in MEM media supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin. The ZFL cells were maintained in DMEM/F-12 media supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Grass carp reovirus (GCRV), a dsRNA virus, and spring viremia of carp virus (SVCV), a negative ssRNA virus, were kindly provided by Yibing Zhang (Institute of Hydrobiology, Chinese Academy of Sciences), and tittered according to the method of Reed and Muench56, by a 50% tissue culture infective dose (TCID50) assay on EPC cells. The turbot skin verruca disease virus (TSVDV) was isolated from the focal site of turbot skin in our lab.
Zebrafish
The animals used in the experiment followed the ethical guidelines established by the Institutional Animal Care and Use Committee of the Ocean University of China (permit number, SD2007695). The zebrafish bmp8a knockout mutant lines (bmp8a−/−) were established from the zebrafish AB line using TALENs technology. The TALENs for zebrafish bmp8a were assembled using the Unit Assembly (UA) method as previously described57. All the UA method starting vectors were the gifts from laboratories of Bo Zhang at Peking University and of Shuo Lin at University of California Los Angeles. TALENs mRNAs were transcribed using the mMESSAGE mMACHINE SP6 kit (Invitrogen, #AM1340) and purifified using the RNeasy Mini kit (QIAGEN, #74106). To generate zebrafish mutant lines, TALEN mRNAs (50–200 pg) were microinjected into 1-cell stage zebrafish embryos. Two days after injection, genomic DNA was isolated from 8 to 10 pooled larvae. The target genomic regions were amplified by nested PCR and subcloned into the pGEM-T vector. Single colonies were genotyped by sequencing. To obtain germline mutations, the TALEN-injected embryos were raised to adulthood and outcrossed with wild-type (WT) fish. The F1 progeny were genotyped by sequencing. To obtain homozygous mutants, heterozygous mutant of the same mutation were obtained and self-crossed. The primers used in this study were listed in Supplemental Table 1.
Plasmid construction
The open reading frames (ORF) of zebrafish bmp8a, stat1a, stat1b, alk2, alk3, alk6a, actr2a, actr2b, bmpr2a and bmpr2b were cloned into the pcDNA3.1 expression vector for the eukaryotic expression. The ORF of zebrafish stat1a and stat1b were cloned into the pET-28a expression vector for the expression of recombinant proteins. The promoter regions of bmp8a, bmp8a-ΔP1 (delete the GAS motif of 5’-attccgggaaa-3’ in the bmp8a promoter), bmp8a-ΔP2 (delete another GAS motif of 5’-tttactagaac-3’ in the bmp8a promoter), bmp8a-ΔP1and ΔP2 (delete both the two GAS motifs), IFNφ1, IFNφ3, and EPC IFN were cloned into the pGL3-basic vector. Dominant negative mutant plasmids including tbk1-K38M, stat1a-ΔC, stat1b-ΔC, irf3DN, and irf7DN were described previously58,59. Dominant negative mutant plasmids alk6a-ΔGS (delete the sequence of 5’-agctccggttctggctcagga-3’ encoding the GS domain) was cloned into the pcDNA3.1 expression vector. The deletions and mutations were created using Mut Express II Fast Mutagenesis Kit V2 (Vazyme, #C214-01). The primers used in this study were listed in Supplemental Table 2.
Transcriptome Sequencing
The zebrafish were fasted for 24 h prior to tissue collection. Three mutant (bmp8a−/−) and three wild type (WT) adult zebrafish (0.4 g ± 0.05 g) were randomly selected from each group and were injected intraperitoneally with 50 μl of GCRV (108TCID50 per ml) per fish. Seventy-two hours after the injection, the livers from three fish each group were pooled together for total RNA extraction. Three independent samples from each group were prepared for transcriptome sequencing. mRNAs were enriched by magnetic beads combining the oligo (dT). cDNA fragments were subjected to the end repair process by the addition of a single ‘A’ base and the ligation of Illumina sequencing adapters. The ligation products were selected by agarose gel electrophoresis, amplified by PCR, and sequenced using Illumina HiSeq 4000 by Annoroad Technology (Beijing, China).
Identification of DEGs
The RPKM (reads per kb per million reads) method was used to calculate and normalize the abundances of genes60. DEGs across groups were identified by the edgeR package (http://www.r-project.org/). Genes with a fold change ≥ 2 and a false discovery rate (FDR) < 0.05 in a comparison were regarded as significantly expressed. DEGs were then subjected to enrichment analysis of KEGG pathways.
Viral infection in vitro
EPC, FG, or ZFL cells were plated 24 h before infection. Cells were infected with GCRV (5 × 104TCID50 per ml) or TSVDV (crude virus extracts filtered by a 0.45 μm microporous membrane) for 1 h in medium without FBS. After infection, cells were washed with PBS and then medium was added with FBS. The cells and cell-free supernatants were harvested at the indicated times.
Viral infection in vivo
For in vivo viral infection, the adult WT and bmp8a−/− zebrafish were infected via intraperitoneal injection using 50 μL of SVCV (108TCID50 per ml), GCRV (108TCID50 per ml), or TSVDV (crude virus extracts filtered by a 0.45 μm microporous membrane) per fish. Total RNA was extracted and viral RNA expression in the liver, kidney, intestine, and spleen were examined by qRT-PCR. For the survival experiments, zebrafish were monitored for survival after SVCV, GCRV, or TSVDV infection.
Crystal violet staining
EPC or ZFL cells were transfected with bmp8a or empty vector plasmid. At 24 h post transfection, cells were treated with GCRV (5 × 104TCID50 per ml). At 72 h after virus challenge, cells were fixed with 4% formaldehyde for 1 h and stained with 0.5% crystal violet for 2 h. After washing with tap water, visible plaques were photographed.
Luciferase assays
EPC cells were seeded in 24-well plates overnight and cotransfected with various plasmids using FuGENE HD Transfection Reagent (Promega, #E2311) following the manufacturer’s instruction. If necessary, the cells were infected with GCRV (5 × 104TCID50 per ml). At the indicated time points, luciferase activities were measured with the Luc-Pair Duo-Luciferase Assay Kits 2.0 (iGene Biotechnology, #LF002). Data were normalized by calculating the ratio between firefly luciferase activity and Renilla luciferase activity. The primers used for cloning the promoter are shown in Supplemental Table 2.
RNA interference
Small interfering RNA (siRNA) of bmp8a and si-NC (negative control) were purchased from RiboBio. The oligonucleotide sequences (siRNA) specific targeting bmp8a mRNA was as follows: 5’-GTTCTTCAGAGCTAGTCAA-3’. Transfection was performed with Lipofectamine RNAiMAX (Invitrogen, #13778075) according to the manufacturer’s protocols. Total RNA was extracted for qRT-PCR analysis.
Drug treatment and analysis
SMAD1/5/8 inhibitor DMH1(Selleck, #S7146), smad2/3 inhibitor TP0427736 HCl (Selleck, #S8700), p38 MAPK inhibitor SB203580 (Beyotime, #S1863), JNK inhibitor SP600125 (Beyotime, #S1876), and MEK1/2 inhibitor U0126 (Beyotime, #S1901) were dissolved in dimethyl ulfoxide (DMSO). ZFL or EPC cells were treated with each inhibitor, which were diluted with culture medium at concentrations of 10 μM for 24 h. Total cellular RNA was extracted for qRT-PCR analysis.
RNA quantification
Total RNA was isolated using the miRNeasy Mini Kit (QIAGEN, #217004) according to the manufacturer’s instructions. RNA treated with DNase and the cDNAs were synthesized with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa, #RP047A). Samples without reverse transcriptase were also added as control. Gene expression was determined by amplifying the cDNA with ChamQ SYBR Color qPCR Master Mix(Vazyme, #Q431-02) by an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). The expression of zebrafish actb1 or EPC actin was used as an internal control. The 2−ΔΔCt method was used to calculate relative expression changes. All qRT-PCR experiments were performed in triplicate and repeated three times. Primers used are listed in Supplementary Table 1.
Preparation of recombinant protein
The recombinant protein of rStat1a or rStat1b was expressed and purified as previous description61. Briefly, the recombinant plasmid (pET-28a-stat1a or pET-28a-stat1b) was transformed into TransB (DE3) chemically competent cell. The positive transformants were incubated in liquid LB medium containing 50 μg/mL kanamycin at 37 °C by shaking at 160 rpm. When the cells grow to OD600 = 0.5, isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and incubated at 19 °C with shaking at 120 rpm for 14 h. After incubation, the cells were harvested by centrifugation, re-suspended in PBS, and broken by ultrasound. The supernatant was centrifuged at 12,000 g for 20 min, and the protein was purified by Ni-NTA Sepharose column. The concentration of purified protein was determined by BCA (bicinchonininc acid) method. The purified recombinant proteins were stored at −80 °C for subsequent experiment.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay (EMSA) was performed according to the instruction of Chemiluminescent EMSA Kit (Beyotime, #GS009). Oligonucleotides (Supplementary Table 1) were synthesized and biotinylated by Sangon Biotech Company as required and the complementary oligonucleotides were annealed to form the double DNA probe. The recombinant protein was pre-incubated in EMSA/Gel-Shift binding buffer with poly (dI-dC) at 25 °C for 25 min in the presence or absence of Non-Biotin probe (0.5 μM). Then, all samples were mixed with biotinylated probes (0.5 μM) and incubated at 25°C for 30 min. The binding reaction without recombinant protein was set as the negative control. For supershift analysis, anti-His tag antibody was incubated with the reaction mixture for another 30 min after the labeled probe was added. After separation in a native 6% polyacrylamide gel, the free probes and DNA-protein compound were transferred to a Hybond-N+ nylon membrane, followed by crosslinking under UV light. After immersion in the blocking buffer, membranes were incubated with streptavidin conjugated HRP for 20 min with shaking and then washed three times with washing buffer. Membranes were exposed briefly in Western Lightning-ECL and then exposure to Automatic X-ray Film Processor (Smpicgg).
Immunoblot analysis and co-immunoprecipitation
For Immunoblot analysis, cells were lysed in NP-40 buffer (150 mM NaCl, 0.5% EDTA, 50 mM Tris, 1% NP40, proteinase inhibitors, and protein phosphatase inhibitors) for 30 min on ice. For co-immunoprecipitation (co-IP), whole-cell extracts were collected 48 h after transfection and lysed in NP-40 buffer on ice for 30 min. After centrifugation for 15 min at 12,000 × g, 4 °C, supernatants were collected and incubated with Protein A/G PLUS-Agarose (Santa Cruz, #sc-2003) coupled to specific antibodies for overnight with rotation at 4°C. Beads were washed 3 × times with NP-40 buffer. Bound proteins were eluted by boiling 7min with 2 x SDS sample buffer. For immunoblot analysis, immunoprecipitates or whole-cell lysates were separated by 12.5% SDS–PAGE gels, electro-transferred to PVDF membranes and blocked for 4 h with 5% no-fat milk solution, followed by blotting with the appropriate antibodies and detection by enhanced chemiluminiscence (ECL). Antibodies from Cell Signaling Technology: TBK1 (1:1000, #3504T), p-TBK1(Ser172) (1:1000, #5483T). Antibodies from Bioss: IRF3 (1:1000, #bs-2993R), p-IRF3 (Ser386) (1:1000, #bsm-52170R), p38MAPK (1:1000, #bs-0637R), p-p38MAPK (Thr180 + Tyr182) (1:1000, #bs-2210R), Actin (1:2000, #bs-0061R). Antibodies from Beyotime: HA tag (1:1000, #AH158) and Flag tag (1:1000, #AF519). Antibodies from CWBIO: His tag (1:5000, #CW0285) and goat anti-rabbit IgG HRP secondary antibody (1:8000, #CW0103S). Antibodies from Abcam: VeriBlot for IP Detection (1:5000, #ab131366).
Statistical analysis and reproducibility
Representative experiments have been repeated at least two to three times. All statistical analysis was performed using Graphpad Prism 8.0.1. Data are presented as mean ± SEM. Statistical significance was assessed using the unpaired, two-tailed Student’s t-test. Survival analyses were performed using the Kaplan-Meier method and assessed using the log-rank (Mantel-Cox) test. *p < 0.05, **p < 0.01, *** p< 0.001; ns, not significant, p > 0.05.
Data availability
All data are available from the corresponding author upon reasonable request and that a Source Data file is available for this article.
Author contributions
Z.L.and S.-C.Z. conceived and coordinated the project. Z.L., G.J., and S.Z. designed the experiments. S.Z., H.L. and Y.-S.W. performed experiments. H.-Y.L. and Y.W. contributed to establish the bmp8a knockout mutant lines (bmp8a−/−) using TALENs technology. S.Z. performed the statistical analysis. Z.L., S.-C.Z. and S.Z. wrote the manuscript, with input from the other authors. All authors reviewed the manuscript and approved the final version.
Conflict of Interest
The work is under a patent in China “A method to improve the antiviral immunity of fish (Application No. 201910454499.6)”.
Acknowledgements
This work was supported by the grants of National Natural Science Foundation of China (31572259) and National Key Research and Development Project of the Ministry of Science and Technology(2018YFD0900505).