Black carp A20 inhibits interferon signaling through de-ubiquitinating IKKß.

IkappaB kinase beta (IKKß) is a key member of IκB kinases and functions importantly in interferon (IFN) signaling. Phosphorylation and ubiquitination are involved in the activation of IKKß. A20 is a de-ubiquitin enzyme and functions as a suppressor in inflammation signaling, which has been reported to be phosphorylated and activated by IKKß. However, the role and relationship of IKKß and A20 in teleost remains unclear. In this study, IKKß (bcIKKß) and A20 (bcA20) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Overexpressed bcIKKß in EPC cells showed strong anti-viral ability by activating both NF-κB and IFN signaling. EPC cells stable expressing bcIKKß presented improved anti-viral activity as well. The interaction between bcA20 and bcIKKß was identified, and overexpression of bcA20 was able to suppress bcIKKß-mediated activation of NF-κB and IFN signaling. Meanwhile, knock-down of A20 increased host the antiviral ability of host cells. Importantly, it has been identified that bcA20 was able to remove K27-linked ubiquitination and decrease the phosphorylation of bcIKKß. Thus, our data conclude that bcA20 suppresses the anti-viral activity of bcIKKß and removes its K27-linked ubiquitination, which presents a new mechanism of IKKß regulation.

STAT2 negatively regulates RIG-I in the antiviral innate immunity of black carp.

The signal transducer and activator of transcription 2 (STAT2), a downstream factor of type I interferons (IFNs), is a key component of the cellular antiviral immunity response. However, the role of STAT2 in the upstream of IFN signaling, such as the regulation of pattern recognition receptors (PRRs), remains unknown. In this study, STAT2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The open reading frame (ORF) of bcSTAT2 comprises 2523 nucleotides and encodes 841 amino acids, which presents the conserved structure to that of mammalian STAT2. The dual-luciferase reporter assay and the plaque assay showed that bcSTAT2 possessed certain IFN-inducing ability and antiviral ability against both spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Interestingly, we detected the association between bcSTAT2 and bcRIG-I through co-immunoprecipitation (co-IP) assay. Moreover, when bcSTAT2 was co-expressed with bcRIG-I, bcSTAT2 obviously suppressed bcRIG-I-induced IFN expression and antiviral activity. The subsequent co-IP assay and immunoblotting (IB) assay further demonstrated that bcSTAT2 inhibited K63-linked polyubiquitination but not K48-linked polyubiquitination of bcRIG-I, however, did not affect the oligomerization of bcRIG-I. Thus, our data conclude that black carp STAT2 negatively regulates RIG-I through attenuates its K63-linked ubiquitination, which sheds a new light on the regulation of the antiviral innate immunity cascade in vertebrates.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

Black carp ATG16L1 negatively regulates STING-mediated antiviral innate immune response.

The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.

Niclosamide subacute exposure alters the immune response and microbiota of the gill and gut in black carp larvae, Mylopharyngodon piceus.

Niclosamide (NIC) is a commonly used insecticide and molluscicide in the prevention and treatment of parasitic diseases in fish. The utilization of NIC has the potential to disrupt the microbial community present on the mucosal tissue of fish, leading to localized inflammatory responses. The objective of this study was to evaluate the impact of NIC on the immune system and bacterial populations within the gill and gut of Mylopharyngodon piceus. Fish were subjected to varying concentrations of NIC, including a control group (0 µg/L), a low NIC group (15% 96 h LC50, LNG, 9.8 µg/L), and a high NIC group (80% 96 h LC50, HNG, 52.5 µg/L). Gill and gut samples were collected 28 days post-exposure for analysis. The findings revealed that the 96-h LC50 for NIC was determined to be 65.7 µg/L, and histopathological examination demonstrated that exposure to NIC resulted in gill filament subepithelial edema, exfoliation, degeneration, and a decrease in gill filament length. Furthermore, the gut exhibited apical enterocyte degeneration and leucocyte infiltration following NIC exposure. Additionally, NIC exposure led to a significant elevation in the levels of immunoglobulin M (IgM), complement component 3 (C3), and complement component 4 (C4) in both gill and gut tissues. Moreover, the activity of lysozyme (LYZ) was enhanced in the gill, while the activities of peroxidase (POD) and immunoglobulin T (IgT) were increased in gut tissue. The exposure to NIC resulted in enhanced mRNA expression of c3, c9, tnfα, il6, il8, and il11 in the gill tissue, while decreasing c3 and il8 expression in the gut tissue. Furthermore, the natural resistance-associated macrophage protein (nramp) mRNA increased, and liver-expressed antimicrobial peptide 2 (leap2) mRNA decreased in gill and gut tissues. And hepcidin (hepc) mRNA levels rose in gill but fell in gut tissue. NIC exposure also led to a decrease in gill bacterial richness and diversity, which significantly differed from the control group, although this separation was not significant in the gut tissue. In conclusion, the administration of NIC resulted in alterations in both the immune response and mucosal microbiota of fish. Furthermore, it was noted that gills displayed a heightened vulnerability to sublethal effects of NIC in comparison to gut tissues.

TRIM25 negatively regulates IKKε-mediated interferon signaling in black carp.

IKKε plays an important role in the activation of IRF3/IRF7 and the production of interferon (IFN), however, its regulation remains obscure in human. E3 ligase TRIM25 has been reported to manipulate the K63-linked ubiquitination of RIG-I, leading to the activation of RIG-I/IFN signaling. To elucidate the role of TRIM25 in teleost, a TRIM25 homolog (bcTRIM25) was cloned and characterized from black carp (Mylopharyngodon piceus). bcTRIM25 contains 653 amino acids, possessing conservative RING, B-box and SPRY domain, which is highly expressed in muscle, spleen and skin. bcTRIM25 knock-down enhanced the antiviral ability of host cells. bcTRIM25 over-expression alone in EPC cells attenuated bcIFNa promoter transcription in the reporter assays and impeded PKR and MX1 expression in qRT-PCR. Interestingly, co-IP assays indicated that bcTRIM25 interacted with bcIKKε and the induced bcIFNa promoter transcription by bcIKKε was notably hindered by bcTRIM25. Furthermore, bcIKKε-induced expression of interferon stimulated genes (ISGs) and antiviral activity were dampened by bcTRIM25. Further exploration showed that bcTRIM25 visibly enhanced the ubiquitination of bcIKKε but significantly attenuated the phosphorylation of bcIKKε. Thus, our data demonstrate for the first time in vertebrate that TRIM25 negatively regulates IKKε through enhancing its ubiquitination, which sheds a light on the regulation of IKKε/IFN signaling.

Negatively regulation of MAVS-mediated antiviral innate immune response by E3 ligase RNF5 in black carp.

Mitochondrial antiviral signaling protein (MAVS) is as an adaptor in RIG-I like receptor (RLR) signaling, which plays the key role in interferon (IFN) production during host antiviral innate immune activation. MAVS is fine tuned to avoid excess IFN production, which have been extensively studied in human and mammals. However, the regulation of MAVS in teleost still remains obscure. In this manuscript, we cloned ring finger protein 5 (bcRNF5) of black carp (Mylopharyngodon piceus) and characterized this teleost E3 ubiquitin ligase as a negative regulator of MAVS. The coding region of bcRNF5 consists of 615 nucleotides which encode 205 amino acids, containing two trans-membrane domain (TM) and a ring-finger domain (RING). The transcription regulation of bcRNF5 varies in host cells in response to stimulations of LPS, poly (I:C), grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). bcRNF5 migrates around 22 KDa in immunoblot (IB) assay and distributes mainly in cytoplasm by immunofluorescent (IF) staining test. Moreover, bcRNF5 significantly inhibits bcMAVS-mediated IFN promoter transcription. In addition, both IF and co-immunoprecipitation assay showed that bcRNF5 interacts with bcMAVS. Furthermore, bcMAVS-mediated antiviral ability is distinctly impaired by bcRNF5. Taken together, these results conclude that bcRNF5, as a negative regulator of the MAVS-mediated IFN signaling, may play a key role in host protection upon virus infection in black carp.

ATG16L1 negatively regulates MAVS-mediated antiviral signaling in black carp Mylopharyngodon piceus.

Autophagy related 16 like 1 (ATG16L1) is a crucial component of autophagy that regulates the formation of the autophagosome. In mammals, ATG16L1 also performs important roles in immunity, including controlling viral replication and regulating innate immune signaling; however, investigation on the role of piscine ATG16L1 in immunity is rare. In this report, the ATG16L1 homolog of black carp Mylopharyngodon piceus (bcATG16L1) was cloned and identified, and its negative regulatory role in mitochondrial antiviral signaling protein (MAVS)-mediated antiviral signaling was described. The coding region of bcATG16L1 consists of 1830 nucleotides and encodes 609 amino acids, including one coiled-coil domain at the N-terminus, three low complexity region domains in the middle, and seven WD40 domains at the C-terminus. By immunofluorescence assay and immunoblotting, we found that bcATG16L1 is a cytosolic protein with a molecular weight of ∼74 kDa. In addition, over-expression of bcATG16L1 suppressed bcMAVS-mediated bcIFNa and DrIFNφ1 promoters transcriptional activity and inhibited bcMAVS-mediated antiviral activity. We further confirmed the co-localization of bcATG16L1 and bcMAVS by immunofluorescence assay and verified the protein interaction between bcATG16L1 and bcMAVS by immunoprecipitation assay. Our results report for the first time that black carp ATG16L1 suppresses MAVS-mediated antiviral signaling in teleost fish.

Black carp LGP2 suppresses RIG-I mediated IFN signaling during the antiviral innate immunity.

Laboratory of genetics and physiology 2 (LGP2), a member of retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), has been reported to play different roles in IFN signaling in both mammals and teleost fish. In our previous study, black carp (Mylopharyngodon piceus) LGP2 (bcLGP2) has been characterized to positively regulate melanoma differentiation-associated gene 5 (MDA5). In this study, knockdown of bcLGP2 decreased the expression of host genes, including bcIFNb, bcPKR, bcMx1, and bcViperin, and also attenuated the antiviral capability of host cells. The relationship between bcLGP2 and black carp RIG-Ib (bcRIG-Ib) has been explored. Dual-luciferase reporter assay and qRT-PCR assay indicated that bcLGP2 dampened bcRIG-Ib induced transcription of type I interferons (IFNs) and interferon-stimulated genes (ISGs), including PKR, ISG15, and Viperin. Consistently, the plaque assay identified that bcLGP2 attenuated bcRIG-Ib mediated antiviral ability against spring viremia of carp virus (SVCV). Co-immunoprecipitation assay identified the interaction between bcLGP2 and bcRIG-Ib, as well as bcLGP2 and bcRIG-Ib-CARD. And bcRIG-Ib-CARD mediated antiviral ability was also attenuated by bcLGP2. Truncation mutation analysis showed DExD/H-box Helicase domain of bcLGP2 possessed a similar inhibitory effect on bcRIG-Ib to that of bcLGP2, while the C-terminus repressor domain (CTD) presented little impact on bcRIG-Ib. Furthermore, bcLGP2 enhanced the K48-linked ubiquitination of bcRIG-Ib, promoting proteasome-dependent degradation of bcRIG-Ib. Thus, our data supported the conclusion that bcLGP2 interacted with and induced degradation of bcRIG-Ib through proteasome, leading to the dampened antiviral signaling mediated by bcRIG-Ib.

RIPK3 collaborates with RIPK1 to inhibit MAVS-mediated signaling during black carp antiviral innate immunity.

Both the activation and attenuation of MAVS/IFN signaling are critical for host defensing against viral infection and thus lead to an elaborate regulation of MAVS-mediated signaling. However, the regulatory mechanisms concerning MAVS/IFN signaling in teleost fish are not well understood. RIPK3 has been identified as a key regulator of necroptosis, apoptosis, and inflammatory signaling in human and mammals. Here we report the identification of the RIPK3 homologue from black carp Mylopharyngodon piceus (bcRIPK3) and describe its role in regulating MAVS/IFN signaling. qPCR results demonstrated that bcRIPK3 was transcriptionally activated in response to poly (I:C) or LPS stimulation. Immunoblot assay and immunofluorescent staining assay showed that bcRIPK3 was a cytosolic protein with molecular weights of 47 kDa. Like its mammalian counterparts, bcRIPK3 exhibited a conserved function in inducing cell death. The reporter assay and plaque assay showed that overexpression of bcRIPK3 restricted bcMAVS-activated transcription of the interferon promoters of black carp and zebrafish, and suppressed bcMAVS-mediated antiviral activity. Notably, EPC cells co-expressing bcRIPK3, bcRIPK1 and bcMAVS presented much attenuated antiviral activity than the cells co-expressing bcRIPK3 and bcMAVS; and the subsequent co-IP assay identified the interaction between bcRIPK3 and bcRIPK1. Our findings collectively elucidate for the first time in teleost that black carp RIPK3 interacts with RIPK1 to inhibit MAVS-mediated antiviral signaling.

Black carp IKKε collaborates with IRF3 in the antiviral signaling.

Interferon regulatory factor 3 (IRF3) is activated by IκB kinase ε (IKKε) and Tank-binding kinase 1 (TBK1), which plays a crucial role in the interferon signaling in vertebrates. However, the regulation of teleost IRF3 by IKKε remains largely unknown. In this study, the IRF3 homologue (bcIRF3) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcIRF3 was detected to increase in host cells in response to different stimuli. bcIRF3 distributed predominantly in the cytosolic area; however, translocated into nuclei after virus infection. bcIRF3 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF3 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). Moreover, knockdown of bcIRF3 reduced the antiviral ability of the host cells, and the transcription of antiviral-related cytokines was obviously lower in bcIRF3-deficient host cells than that of control cells. The data of reporter assay and plaque assay demonstrated that bcIKKε obviously enhanced bcIRF3-mediated IFN production and antiviral activity. Immunofluorescent staining and co-immunoprecipitation assay revealed that bcIKKε interacted with bcIRF3. It was interesting that the nuclear translocation of bcIRF3 and bcIKKε was enhanced by each other when these two molecules were co-expressed in the cells, however, the protein levels of bcIRF3 and bcIKKε were decreased mutually. Thus, our data support the conclusion that bcIKKε interacts with bcIRF3 and enhances bcIRF3-mediated antiviral signaling during host innate immune activation.

Black carp TRADD suppresses MAVS/IFN signaling during the innate immune activation.

Tumor necrosis factor receptor 1 (TNFR1) associated death domain protein (TRADD) is a pivotal adaptor in TNF signaling pathway and up-regulates MAVS/IFN signaling pathway in human and mammal. However, the role of TRADD in teleost fish remains obscure. To reveal the function of teleost TRADD in the innate immune response, the TRADD homologue (bcTRADD) of black carp (Mylopharyngodon piceus) has been cloned and the function of bcTRADD is investigated in this study, which shares similar functional domain to its mammalian counterpart. bcTRADD mRNA expression level increased in response to different stimuli, including LPS, poly (I:C) and virus infection in host cells. bcTRADD activated the transcriptional activity of NF-κB promoter in the reporter assay; however, showed hardly any effect on the transcriptional activity of IFN promoter. It was interesting that black carp mitochondria antiviral signaling protein (bcMAVS)-activated IFN promoter transcription were dramatically depressed by bcTRADD and the C-terminal death domain of bcTRADD was indispensable for its regulation of bcMAVS. Accordingly, the plaque assay result showed that EPC cells co-expressing bcMAVS and bcTRADD presented much attenuated antiviral activity than EPC cells expressing bcMAVS alone. Knockdown of bcTRADD slightly promoted the antiviral ability of the host cells against SVCV. The current data support the conclusion that bcTRADD suppresses MAVS-mediated antiviral signaling, which is different to its mammalian counterpart.

Multi-omics analysis revealed crucial genes and pathways associated with black carp antiviral innate immunity.

Multi-omics strategy contributes as an indispensable and efficient approach for the investigation of the innate immunity in vertebrates. To explore the crucial genes and pathways involved in the antiviral innate immunity of black carp (Mylopharyngodon piceus), the comparative phosphoproteomics and transcriptomics of Mylopharyngodon piceus kidney (MPK) cells with/without GCRV infection were performed in this manuscript. In phosphoproteomics analysis, 2637 phosphosites corresponding to 1532 proteins were identified and quantified, in which 1372 proteins were identified as differentially expressed proteins (DEPs) with 683 upregulated and 689 downregulated in GCRV infected cells. Functional annotation, enrichment analysis and pathway analysis highlighted that a large number of DEPs were enriched in immune related pathways including TLR pathway and NLR pathway. In transcriptomics analysis, a total of 2936 genes were identified as differentially expressed genes (DEGs), in which 2290 and 646 genes were upregulated and downregulated respectively after GCRV infection. As expected, pathway analysis based on DEGs also showed that a large proportion of DEGs were enriched in immune related pathways including TLR and RLR pathway. A combined list of DEPs and DEGs that enriched in above pathways were imported in Cytoscape for network analysis, reconstruction and visualization. The integrative study suggested that several significant DEPs and DEGs, such as MAP3K7 (TAK1), JUN, MAP2K2, CASP8, IL8 and IRF7 might be functionally crucial in host antiviral innate immunity. Thus, this study contributes as an indispensable reference map for the further investigation of the innate immune system of black carp.

Virus susceptibility of a new cell line derived from the fin of black carp Mylopharyngodon piceus.

A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.

TAK1 of black carp positively regulates IRF7-mediated antiviral signaling in innate immune activation.

Transforming growth factor ß-activated kinase 1 (TAK1) plays a vital role in IL-1-mediated NF-κB, JNK, and p38 activation in human and mammals. However, the function of TAK1 in teleost fish still remains largely unknown. To explore the role of TAK1 during the antiviral innate immune response of teleost fish, TAK1 of black carp (Mylopharyngodon piceus) was cloned and characterized in this paper. The open reading frame (ORF) of black carp TAK1 (bcTAK1) consists of 1626 nucleotides and the predicted bcTAK1 protein contains 541 amino acids, which includes a N-terminal Serine/Threonine protein kinases (S/TKc) and a C-terminal coiled-coil region. bcTAK1 migrated around 75 kDa in immunoblotting assay and was identified as a cytosolic protein by immunofluorescence staining. bcTAK1 transcription in Mylopharyngodon piceus kidney (MPK) cells varied in response to the stimulation of poly (I:C), LPS, grass carp reovirus (GCRV), and spring viremia of carp virus (SVCV). bcTAK1 showed deficient IFN-inducing ability in reporter assay and feeble antiviral activity against GCRV and SVCV in plaque assay. However, when co-expressed with bcIRF7 in EPC cells, bcTAK1 obviously enhanced bcIRF7-mediated IFN promoter induction in reporter assay. Accordingly, the data of plaque assay demonstrated that the antiviral activity of bcIRF7 against both GCRV and SVCV was unregulated by bcTAK1. Thus, the data generated in this study support the conclusion that bcTAK1 up-regulates bcIRF7-mediated antiviral signaling during host innate immune activation, which is reported for the first time in vertebrates.

STAT1a and STAT1b of black carp play important roles in the innate immune defense against GCRV.

Signal transducer and activator of transcription 1 (STAT1) plays an important role in the Janus kinase (JAK)-STAT signaling of human and mammals; however, the mechanism of STAT1 in innate immune activation of teleost fishes remains largely unknown. In this study, two STAT1 homologues (bcSTAT1a and bcSTAT1b) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Both bcSTAT1a and bcSTAT1b transcription in host cells was obviously increased in response to the stimulation of poly (I:C), lipopolysaccharide (LPS), grass carp reovirus (GCRV) and interferon (IFN); however, the increase rate of bcSTAT1b transcription post stimulation was obviously higher than that of bcSTAT1a. bcSTAT1a and bcSTAT1b were distributed in both cytoplasm and nucleus in the immunofluorescence staining assay. Self-association of bcSTAT1a and bcSTAT1b, and the interaction between bcSTAT1a and bcSTAT1b have been detected through co-immunoprecipitation (co-IP) assay; and the data of native polyacrylamide gel electrophoresis (PAGE) implied that bcSTAT1a and bcSTAT1b might form homodimer and heterodimer in vivo like their mammalian counterparts. Both bcSTAT1a and bcSTAT1b presented IFN-inducing ability in report assay, and both bcSTAT1a and bcSTAT1b showed antiviral activities against GCRV in EPC cells. Our data support the conclusion that both bcSTAT1a and bcSTAT1b play important roles in host antiviral innate immune activation initiated by GCRV.

Black carp PRMT6 inhibits TBK1-IRF3/7 signaling during the antiviral innate immune activation.

Protein arginine methylation is a prevalent posttranslational modification and protein arginine methyltransferases 6 (PRMT6) has been identified as a suppressor of TBK1/IRF3 in human and mammals. To explore the role of PRMT6 in teleost fish, PRMT6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp PRMT6 (bcPRMT6) transcription in host cells varies in response to different stimuli and bcPRMT6 migrates around 43 kDa in the immunoblot assay. Like its mammalian counterpart, bcPRMT6 has been identified to distribute majorly in the nucleus through the immunofluorescent staining assay. bcPRMT6 shows little interferon (IFN) promoter-inducing activity in the reporter assay and bcPRMT6 shows no antiviral activity against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV) in plaque assay. When co-expressed with bcPRMT6, the IFN promoter-inducing abilities of black carp TBK1 (bcTBK1) and IRF3/7 (bcIRF3/7) are fiercely attenuated. Accordingly, bcTBK1-mediated antiviral activity in EPC cells is obviously dampened by bcPRMT6. The interaction between bcPRMT6 and bcIRF3/7 has been identified by co-immunoprecipitation assay; however, no direct association between bcPRMT6 and bcTBK1 has been detected. Taken together, our data elucidates for the first time in teleost fish that PRMT6 suppresses TBK1-IRF3/7 signaling during host antiviral innate immune activation.

Black carp TAB1 up-regulates TAK1/IRF7/IFN signaling during the antiviral innate immune activation.

TAK1-binding protein 1 (TAB1) forms the protein complex with TAK1 and enhances its kinase activity in human and mammals. To elucidate the role of TAB1 in the innate immunity of teleost sfih, the TAB1 homologue of black carp (Mylopharyngodon piceus) (bcTAB1) has been cloned and characterized in this paper. bcTAB1 is composed of 498 amino acids and contains a typical PP2Cc domain like its mammalian counterpart. The transcription of bcTAB1 gene in vivo and ex vivo varied in response to different stimuli; and the immunofluorescence staining showed that bcTAB1 was distributed in both cytoplasm and nucleus of host cell. The reporter assay showed that neither bcTAB1-expression alone nor co-expression of bcTAB1 and bcTAK1 could activate the transcription of IFN in EPC cells. Accordingly, EPC cells expressing bcTAB1 or co-expressing bcTAB1 and bcTAK1 showed no improved antiviral activity against grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). However, EPC cells co-expressing bcTAB1, bcTAK1 and bcIRF7 showed fiercely increased IFN-inducing ability in reporter assay and obviously improved antiviral activity in plaque assay compared with EPC cells co-expressing bcTAK1 and bcIRF7. The subsequent co-immunoprecipitation assay identified that bcTAB1 associated with bcTAK1 but not interacted with bcIRF7. Based on our previous finding that bcTAK1 up-regulates bcIRF7-mediated IFN signaling during host innate immune activation, the data generated in this study support the conclusion that bcTAB1 interacts with bcTAK1 and boosts bcTAK1-activated bcIRF7/IFN signaling during host antiviral innate immune response against GCRV and SVCV.

IFNb of black carp functions importantly in host innate immune response as an antiviral cytokine.

Type I interferons (IFN-Is) play an important role in the antiviral immune response in teleost fishes. In this study, one type I interferon (bcIFNb) from black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of bcIFNb gene consists of 806 nucleotides and the predicted bcIFNb protein contains 188 amino acids. Basing on the cysteine number and evolutionary position, bcIFNb was classified into group II type I IFN. q-PCR analysis demonstrated that bcIFNb mRNA level varied in vivo and ex vivo in response to different stimuli. bcIFNb was detected in both the whole cell lysate and the supernatant media of HEK293T cells or EPC cells transfected with bcIFNb through immunoblot assay. IFN stimulated genes (ISGs) were greatly upregulated when the host cells were treated with the bcIFNb-containing conditioned media. EPC cells showed greatly enhanced antiviral ability when the cells were transfected with bcIFNb or treated with the bcIFNb-containing conditioned media before GCRV or SVCV infection. Glycosidase digestion analysis determined that bcIFNb was modified with N-linked glycosylation, which occurred on the Asn (N) of 92 site of this cytokine. The un-glycosylated mutant bcIFNb-N92Q presented the similar antiviral ability as that of wild type bcIFNb, which demonstrated that N-linked glycosylation did not contribute directly to the antiviral property of this fish cytokine.

Molecular cloning and characterization of TANK of black carp Mylopharyngodon piceus.

The TRAF family member-associated NF-κB activator (TANK) is linked to the regulation of the transcription of NF-κB in mammals; however, its role in interferon induction is unclear. To elucidate the roles of TANK in teleost, the TANK homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this paper. The open reading frame (ORF) of black carp TANK (bcTANK) comprises 1050 nucleotides and the predicted bcTANK protein contains 350 amino acids. The transcription of bcTANK in host cells increased in response to the stimulation of LPS, poly (I:C), SVCV and GCRV. bcTANK migrated around 50 KDa in immunoblot assay and was identified as a cytosolic protein by immunofluorescent staining in both EPC and HeLa cells. bcTANK could not induce the activity of IFN promoter in luciferase reporter assay in EPC cells; however, the IFN-activation ability of bcTANK was obviously enhanced when the cells were treated with LPS, poly (I:C) or virus. Both CPE ratio and virus titer in the media of EPC cells expressing bcTANK were obviously lower than those of the control cells, which were examined by violet crystal staining and plaque assay separately. Taken together, our data support the conclusion that bcTANK plays an important role in the antiviral innate immune activation of black carp.