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.

Lysine 39 of IKKε of black carp is crucial for its regulation on IRF7-mediated antiviral signaling.

Interferon regulatory factor 7 (IRF7) plays a crucial role in the interferon (IFN) signaling in mammals, in which it is activated by the TBK1/IKKε complex during host antiviral innate immune response. There are few reports about the relation between IRF7 and IKKε in teleost fishes. In this study, the IRF7 homologue (bcIRF7) of black carp (Mylopharyngodon Piceus) has been cloned and characterized. The transcription of bcIRF7 gene increased in host cells in response to the stimulation of LPS, poly (I:C) and viral infection. bcIRF7 migrated around 56 KDa in immunoblot assay and was identified as a predominantly cytosolic protein by immunofluorescent staining. bcIRF7 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF7 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). IKKε of black carp (bcIKKε) was found to be recruited into host innate immune response initiated by SVCV and GCRV in the previous work; in this paper, the kinase dead mutant of bcIKKε, bcIKKε-K39A was constructed and showed no IFN-inducing activity. The data of reporter assay and plaque assay demonstrated that bcIKKε but not bcIKKε-K39A obviously enhanced bcIRF7-mediated IFN production and antiviral activity. Our data support the conclusion that bcIKKε upregulates bcIRF7-mediated antiviral signaling, which most likely depends on its kinase activity.

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.

TRAF2 of black carp upregulates MAVS-mediated antiviral signaling during innate immune response.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of the tumor necrosis factor (TNF) mediated signaling of higher vertebrates. To elucidate its function in teleost fish, TRAF2 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. The open reading frame (ORF) of black carp TRAF2 (bcTRAF2) consists of 1611 nucleotides and bcTRAF2 contains 536 amino acids. bcTRAF2 protein migrated around 65 KDa in immunoblot analysis of both EPC and HEK293T cells. bcTRAF2 was identified as a cytosolic protein and suggested to be associated with vesicles scattering in the cytoplasm. NF-κB transcription instead of IFN transcription was activated by bcTRAF2 in reporter assay. It was interesting that bcMAVS-mediated IFN production was up-regulated by bcTRAF2 in a dose dependent manner in reporter assay. Accordingly, EPC cells transfected with both bcMAVS and bcTRAF2 showed enhanced antiviral activity comparing EPC cells only expressing bcMAVS. When co-expressed with bcMAVS, bcTRAF2 was redistributed in the cytoplasm and its subcellular location overlapped with the subcellular location of bcMAVS, which suggested the association between these two molecules. Taken together, the data generated in this paper supported the conclusion that bcTRAF2 was recruited into host innate immune response and positively regulated bcMAVS-mediated antiviral signaling.

TBK1 of black carp plays an important role in host innate immune response against SVCV and GCRV.

Tank-binding kinase 1 (TBK1) plays a pivotal role in the induction of type I IFNs in higher vertebrates. To explore the function of TBK1 in teleost, TBK1 of black carp (Mylopharyngodon Piceus) was cloned and characterized in this paper. The full-length cDNA of black carp TBK1 (bcTBK1) consists of 2857 nucleotides and the predicted bcTBK1 protein contains 727 amino acids, which includes an N-terminal kinase domain (KD), an ubiquitin-like domain (ULD) and two C-terminal coiled-coils. The transcription of bcTBK1 was constitutively detected in all the selected tissues and bcTBK1 mRNA level was increased in all selected tissues in response to SVCV or GCRV infection except that in muscle post GCRV invasion. The transcription of bcTBK1 in Mylopharyngodon Piceus fin (MPF) cells was up-regulated by the stimulation of SVCV, GCRV or poly (I:C) but not by LPS treatment. bcTBK1 migrated around 80 kDa in immunoblot assay and was identified as a cytosolic protein by immunofluorescence staining. bcTBK1 showed strong IFN-inducing ability in reporter assay and presented strong antiviral activity against both GCRV and SVCV in EPC cells. The reporter assay demonstrated that TRAF6 of black carp (bcTRAF6) up-regulated bcTBK1-induced IFN expression and the subcellular distribution of bcTBK1 overlapped with that of bcTRAF6 when these two proteins were co-expressed in EPC cells. Taken together, our study support the conclusion that bcTBK1 plays an important role in the antiviral innate immune response of black carp against SVCV and GCRV, in which its activity was positively regulated by bcTRAF6.

Characterization of the black carp TRAF6 signaling molecule in innate immune defense.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a vital role in the innate immune response of higher vertebrates. To elucidate its function in teleost fish, TRAF6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp TRAF6 (bcTRAF6) transcription in Mylopharyngodon piceus fin (MPF) cells was up-regulated in response to both poly (I:C) treatment and viral infection, but was suppressed by LPS stimulation. bcTRAF6 migrated around 72 KDa in immunoblot analysis and was identified as a cytosolic protein suggested to be associated with vesicles scattering in the cytoplasm. Reporter assay demonstrated that NF-κB instead of IFN was activated by bcTRAF6; and EPC cells expressing bcTRAF6 presented the same cytopathic effect (CPE) ratio to that of control cells. When co-expressed with bcMAVS, bcTRAF6 was redistributed and overlapped with the subcellular location of bcMAVS. It was interesting that bcMAVS mediated the IFN induction was up-regulated by low input of bcTRAF6 but down-regulated by high input of bcTRAF6. Taken together, the data generated in this paper supported the conclusion that bcTRAF6 associated with bcMAVS and was recruited into bcMAVS mediated signaling during host innate immune response.