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Hao Feng - One of the best experts on this subject based on the ideXlab platform.

  • Multi-omics analysis revealed crucial genes and pathways associated with Black Carp antiviral innate immunity
    Fish & shellfish immunology, 2020
    Co-Authors: Ji Liu, Jun Yan, Jing Wei, Jun Xiao, Yujie Yan, Junting Wang, Yong Zeng, Hao Feng
    Abstract:

    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.

  • Black Carp RIPK1 negatively regulates MAVS-mediated antiviral signaling during the innate immune activation.
    Developmental and comparative immunology, 2020
    Co-Authors: Xinchi Xie, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Zhaoyuan Chen, Hao Feng
    Abstract:

    Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of Black Carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black Carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that Black Carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.

  • Black Carp TRAFD1 restrains MAVS-mediated antiviral signaling during the innate immune activation.
    Fish & shellfish immunology, 2020
    Co-Authors: Jun Yan, Yinyin Zhang, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Hao Feng
    Abstract:

    TRAFD1 negatively regulates TLR and RLR signaling in human and mammal; however, its role in teleost fish remains unknown. In this paper, the TRAFD1 homologue has been cloned and characterized from Black Carp (Mylopharyngodon piceus). Black Carp TRAFD1 (bcTRAFD1) consists of 567 amino acids and shows low similarity to that of mammalian TRAFD1, which has been identified as a cytosolic protein through immunofluorescence staining. When co-expressed with bcTRAFD1, the IFN promoter-inducing ability of Black Carp MAVS (bcMAVS) was obviously dampened in the luciferase reporter assay. Accordingly, bcMAVS-mediated antiviral activity against grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV) was potently repressed by bcTRAFD1 in plaque assay. And the co-immunoprecipitation assay between bcTRAFD1 and bcMAVS has identified the association between these two molecules. Thus, our data supports the conclusion that bcTRAFD1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling during the innate immune activation, which sheds a light on the regulation of MAVS in teleost.

  • Black Carp NAP1 positively regulates MDA5-mediated antiviral signaling during the innate immune activation.
    Developmental and comparative immunology, 2020
    Co-Authors: Xinchi Xie, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Zhaoyuan Chen, Hao Feng
    Abstract:

    Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of Black Carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black Carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that Black Carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.

  • Identification and characterization of IRF9 from Black Carp Mylopharyngodon piceus
    Developmental and comparative immunology, 2019
    Co-Authors: Ji Liu, Jun Yan, Hao Feng
    Abstract:

    Interferon regulatory factor 9 (IRF9) plays a crucial role in JAK-STAT signaling in human and mammal. However, the relationship between IRF9 and STAT1 in teleost fish remains largely unknown. The previous study has elucidated that two STAT1 isoforms (bcSTAT1a and bcSTAT1b) of Black Carp (Mylopharyngodon piceus) play an important role during the innate immune activation initiated by grass Carp reovirus (GCRV). In this paper, Black Carp IRF9 (bcIRF9) has been identified and characterized. bcIRF9 was distributed majorly in the nucleus and the linker domain (LD) of bcIRF9 was vital for its nuclear localization. bcIRF9 showed ISRE-inducing activity in reporter assay and presented antiviral activity against GCRV in plaque assay, in which both DNA binding domain (DBD) and LD of bcIRF9 were essential for its antiviral signaling. bcIRF9 was identified to interact with both bcSTAT1a and bcSTAT1b in the co-immunoprecipitation assay. It was interesting that bcIRF9-mediated antiviral signaling was up-regulated by bcSTAT1a; however, down-regulated by bcSTAT1b. Thus, our data support the conclusion that bcIRF9 plays an important role in the innate immune defense against GCRV, in which two STAT1 proteins function differently.

Jun Xiao - One of the best experts on this subject based on the ideXlab platform.

  • Multi-omics analysis revealed crucial genes and pathways associated with Black Carp antiviral innate immunity
    Fish & shellfish immunology, 2020
    Co-Authors: Ji Liu, Jun Yan, Jing Wei, Jun Xiao, Yujie Yan, Junting Wang, Yong Zeng, Hao Feng
    Abstract:

    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.

  • Black Carp prmt6 inhibits tbk1 irf3 7 signaling during the antiviral innate immune activation
    Fish & Shellfish Immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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 PRMT6 inhibits TBK1-IRF3/7 signaling during the antiviral innate immune activation
    Fish & shellfish immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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.

  • tak1 of Black Carp positively regulates irf7 mediated antiviral signaling in innate immune activation
    Fish & Shellfish Immunology, 2019
    Co-Authors: Chanyuan Wang, Jun Xiao, Jun Peng, Minyu Zhou, Guancheng Liao, Xiao Yang, Hao Feng
    Abstract:

    Abstract Transforming growth factor β-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.

  • Molecular cloning and characterization of TANK of Black Carp Mylopharyngodon piceus.
    Fish & shellfish immunology, 2018
    Co-Authors: Chaoliang Feng, Yinyin Zhang, Ji Liu, Jun Xiao, Hao Feng
    Abstract:

    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.

Xuejiao Song - One of the best experts on this subject based on the ideXlab platform.

  • Black Carp prmt6 inhibits tbk1 irf3 7 signaling during the antiviral innate immune activation
    Fish & Shellfish Immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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 PRMT6 inhibits TBK1-IRF3/7 signaling during the antiviral innate immune activation
    Fish & shellfish immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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.

  • NLRX1 of Black Carp suppresses MAVS-mediated antiviral signaling through its NACHT domain.
    Developmental and comparative immunology, 2019
    Co-Authors: Xuejiao Song, Jing Wei, Xinchi Xie, Ziqi Zou, Hao Feng
    Abstract:

    Abstract NOD-like receptor (NLR) family member X1 (NLRX1) of human localizes on mitochondria and serves as a negative regulator of antiviral signaling. However, the function of NLRX1 in teleost fish still remains elusive. To explore its role in the innate immunity of teleost fish, NLRX1 homologue has been cloned and characterized from Black Carp (Mylopharyngodon piceus). Black Carp NLRX1 (bcNLRX1) consists of 1008 amino acids, which includes a N-terminal mitochondrial targeting sequence, a central NACHT domain and a C-terminal leucine-rich repeat (LRR) domain. bcNLRX1 was identified as a cytosolic protein locating on mitochondria through immunofluorescence (IF) staining. The overlapped subcellular distribution of bcNLRX1 and Black Carp MAVS (bcMAVS) was detected in IF staining, and the direct interaction between these two molecules in vitro was identified through co-immunoprecipitation assay. When co-expressed with bcMAVS, bcNLRX1 fiercely reduced bcMAVS-mediated IFN induction in reporter assay. Accordingly, the antiviral activity of bcMAVS against both grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV) was forcefully repressed by bcNLRX1 in plaque assay. Mutagenic analyses further revealed that the NACHT domain of bcNLRX1 was essential for it to interact with bcMAVS and to suppress bcMAVS-mediated antiviral signaling. Taken together, our data support the conclusion that bcNLRX1 negatively regulates bcMAVS-mediated antiviral signaling through its NACHT domain during host innate immune activation.

  • TRAF3 enhances STING-mediated antiviral signaling during the innate immune activation of Black Carp.
    Developmental and comparative immunology, 2018
    Co-Authors: Xu Wang, Xinchi Xie, Xuejiao Song, Song Chen, Hao Feng
    Abstract:

    Abstract Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a main regulator of antiviral and anti-inflammatory pathways in mammals, which is considered to induce type I interferon (IFN) activation and negatively regulate the activation of the canonical and non-canonical NF-κB pathways. To elucidate its function in teleost fish, TRAF3 homologue of Black Carp ( Mylopharyngodon piceus ) has been cloned and characterized in this study. The open reading frame (ORF) of Black Carp TRAF3 (bcTRAF3) consists of 1722 nucleotides and bcTRAF3 contains 574 amino acids. bcTRAF3 protein migrated around 65 KDa in immunoblot analysis of both EPC and HEK293T cells. bcTRAF3 was identified as a cytosolic protein and suggested to form aggregates or be associated with vesicles scattering in the cytoplasm. It was interesting that both NF-κB and IFN transcription was activated by bcTRAF3 in reporter assay. When co-expressed with Black Carp STING (bcSTING), bcTRAF3 was redistributed in the cytoplasm and its subcellular location overlapped with that of bcSTING no matter what the cells was infected with GCRV or not, which suggested the association between these two molecules. bcSTING-mediated IFN production was up-regulated by bcTRAF3 in a dose dependent manner in reporter assay. Accordingly, EPC cells transfected with both bcSTING and bcTRAF3 showed enhanced antiviral activity comparing EPC cells expressing bcSTING alone. Taken together, the data generated in this paper supported the conclusion that bcTRAF3 was recruited into host innate immune activation and positively regulated bcSTING-mediated antiviral signaling.

  • Black Carp STING functions importantly in innate immune defense against RNA virus
    Fish & shellfish immunology, 2017
    Co-Authors: Xu Wang, Jun Xiao, Xuejiao Song, Xinchi Xie, Ziqi Zou, Song Chen, Hao Feng
    Abstract:

    Abstract Stimulator of interferon genes (STING) is a central and multifaceted mediator in the innate immune response of higher vertebrates. To explore its role in teleost fish, the STING homolog of Black Carp ( Mylopharyngodon piceus ) (bcSTING) has been cloned and characterized in this paper. bcSTING transcription in Mylopharyngodon piceus fin (MPF) cells increased remarkably in response to GCRV and SVCV infection, or poly (I:C) stimulation. bcSTING migrated around 42 KDa in immunoblot assay and was identified as a cytosolic protein locating on ER majorly through immunofluorescence staining. Under condition of SVCV/GCRV infection or poly (I:C) stimulation, the subcellular distribution of bcSTING majorly displayed on mitochondria, which overlapped with that of bcMAVS. HA-bcSTING instead of bcSTING-HA presented strong IFN-inducing activity in reporter assay and antiviral ability against both SVCV and GCRV in plaque assay. Site mutation of serine (S) on C-terminus of bcSTING demonstrated that both S371 and S379 were crucial for its mediated signaling. Taken together, our study support the conclusion that bcSTING plays an important role in host innate immune defense against RNA virus such as SVCV and GCRV, in which its C-terminus functions crucially.

Ji Liu - One of the best experts on this subject based on the ideXlab platform.

  • Multi-omics analysis revealed crucial genes and pathways associated with Black Carp antiviral innate immunity
    Fish & shellfish immunology, 2020
    Co-Authors: Ji Liu, Jun Yan, Jing Wei, Jun Xiao, Yujie Yan, Junting Wang, Yong Zeng, Hao Feng
    Abstract:

    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.

  • Identification and characterization of IRF9 from Black Carp Mylopharyngodon piceus
    Developmental and comparative immunology, 2019
    Co-Authors: Ji Liu, Jun Yan, Hao Feng
    Abstract:

    Interferon regulatory factor 9 (IRF9) plays a crucial role in JAK-STAT signaling in human and mammal. However, the relationship between IRF9 and STAT1 in teleost fish remains largely unknown. The previous study has elucidated that two STAT1 isoforms (bcSTAT1a and bcSTAT1b) of Black Carp (Mylopharyngodon piceus) play an important role during the innate immune activation initiated by grass Carp reovirus (GCRV). In this paper, Black Carp IRF9 (bcIRF9) has been identified and characterized. bcIRF9 was distributed majorly in the nucleus and the linker domain (LD) of bcIRF9 was vital for its nuclear localization. bcIRF9 showed ISRE-inducing activity in reporter assay and presented antiviral activity against GCRV in plaque assay, in which both DNA binding domain (DBD) and LD of bcIRF9 were essential for its antiviral signaling. bcIRF9 was identified to interact with both bcSTAT1a and bcSTAT1b in the co-immunoprecipitation assay. It was interesting that bcIRF9-mediated antiviral signaling was up-regulated by bcSTAT1a; however, down-regulated by bcSTAT1b. Thus, our data support the conclusion that bcIRF9 plays an important role in the innate immune defense against GCRV, in which two STAT1 proteins function differently.

  • Black Carp IRF5 interacts with TBK1 to trigger cell death following viral infection.
    Developmental and comparative immunology, 2019
    Co-Authors: Can Yang, Ji Liu, Liqun Liu, Ping-hui Feng, Hao Feng
    Abstract:

    Interferon regulated factor 5 (IRF5) is a key regulator of inflammatory responses in human and mammals; however, its role in teleost remains largely unknown. In this study, IRF5 homologue of Black Carp (Mylopharyngodon piceus) has been cloned and characterized, which possesses conservation in structure and sequence to its mammalian counterparts. Black Carp IRF5 (bcIRF5) was characterized as a predominantly cytosolic protein by immunofluorescent staining and showed little IFN promoter-inducing ability in reporter assay. The direct association between bcIRF5 and Black Carp TBK1 (bcTBK1) were identified through co-immunoprecipitation assay, and co-expressed bcIRF5 in EPC cells suppressed bcTBK1-mediated IFN promoter transcription in reporter assay. Surprisingly, the titer of grass Carp reovirus (GCRV) in the media of EPC cells co-expressing bcIRF5 and bcTBK1 was obviously lower than that of EPC cells expressing bcTBK1 alone. It was interesting that expression of bcIRF5 and/or bcTBK1 in EPC cells showed little effect on cell growth; however, the survival ratio of EPC cells co-expressing bcTBK1 and bcIRF5 post GCRV infection was much lower than that of EPC cells expressing bcIRF5 or bcTBK1 alone. These results indicate that bcIRF5 negatively regulates bcTBK1-mediated IFN signaling in healthy cells; however, it correlates with bcTBK1 and triggers cell death to inhibit the virus replication during the innate immune activation.

  • Molecular cloning and characterization of TANK of Black Carp Mylopharyngodon piceus.
    Fish & shellfish immunology, 2018
    Co-Authors: Chaoliang Feng, Yinyin Zhang, Ji Liu, Jun Xiao, Hao Feng
    Abstract:

    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.
    Fish & Shellfish Immunology, 2018
    Co-Authors: Yu Tian, Ji Liu, Chaoliang Feng, Chanyuan Wang, Hao Feng
    Abstract:

    Interferon regulatory factor 7 (IRF7) plays a crucial role in the interferon (IFN) signaling in mammals, in which it is activated by the TBK1/IKKe complex during host antiviral innate immune response. There are few reports about the relation between IRF7 and IKKe 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). IKKe of Black Carp (bcIKKe) 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 bcIKKe, bcIKKe-K39A was constructed and showed no IFN-inducing activity. The data of reporter assay and plaque assay demonstrated that bcIKKe but not bcIKKe-K39A obviously enhanced bcIRF7-mediated IFN production and antiviral activity. Our data support the conclusion that bcIKKe upregulates bcIRF7-mediated antiviral signaling, which most likely depends on its kinase activity.

Yingyi Cao - One of the best experts on this subject based on the ideXlab platform.

  • Black Carp RIPK1 negatively regulates MAVS-mediated antiviral signaling during the innate immune activation.
    Developmental and comparative immunology, 2020
    Co-Authors: Xinchi Xie, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Zhaoyuan Chen, Hao Feng
    Abstract:

    Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of Black Carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black Carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that Black Carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.

  • Black Carp TRAFD1 restrains MAVS-mediated antiviral signaling during the innate immune activation.
    Fish & shellfish immunology, 2020
    Co-Authors: Jun Yan, Yinyin Zhang, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Hao Feng
    Abstract:

    TRAFD1 negatively regulates TLR and RLR signaling in human and mammal; however, its role in teleost fish remains unknown. In this paper, the TRAFD1 homologue has been cloned and characterized from Black Carp (Mylopharyngodon piceus). Black Carp TRAFD1 (bcTRAFD1) consists of 567 amino acids and shows low similarity to that of mammalian TRAFD1, which has been identified as a cytosolic protein through immunofluorescence staining. When co-expressed with bcTRAFD1, the IFN promoter-inducing ability of Black Carp MAVS (bcMAVS) was obviously dampened in the luciferase reporter assay. Accordingly, bcMAVS-mediated antiviral activity against grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV) was potently repressed by bcTRAFD1 in plaque assay. And the co-immunoprecipitation assay between bcTRAFD1 and bcMAVS has identified the association between these two molecules. Thus, our data supports the conclusion that bcTRAFD1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling during the innate immune activation, which sheds a light on the regulation of MAVS in teleost.

  • Black Carp NAP1 positively regulates MDA5-mediated antiviral signaling during the innate immune activation.
    Developmental and comparative immunology, 2020
    Co-Authors: Xinchi Xie, Yaqi Tan, Yuhan Dai, Jing Wei, Yingyi Cao, Zhaoyuan Chen, Hao Feng
    Abstract:

    Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important regulator of necroptosis and involved in innate immune response in human and mammal; however, its function in teleost fish mains largely unknown. In this paper, the RIPK1 homologue of Black Carp (Mylopharyngodon piceus) has been cloned and characterized to explore its role in immunity. Black Carp RIPK1 (bcRIPK1) possesses the similar structure to its mammalian counterpart, which has been identified as a cytosolic protein by immunofluorescence staining. Overexpressed bcRIPK1 in host cells led to the decreased transcription of interferon (IFN) and interferon stimulated genes, and exogenous bcRIPK1 in EPC cells led to the decreased transcription of interferon promoters in reporter assay. Our previous study has identified that Black Carp MAVS (bcMAVS) functions as an antiviral adaptor protein against both grass Carp reovirus (GCRV) and spring viremia of Carp virus (SVCV). The reporter assay showed that the IFN-inducing ability of bcMAVS was dampened by bcRIPK1 and the plaque assay demonstrated that the antiviral activity of bcMAVS was inhibited by bcRIPK1. The immunofluorescent staining and co-immunoprecipitation identified the interaction between these two molecules. Thus, the data generated in this paper support the conclusion that bcRIPK1 interacts with bcMAVS and negatively regulates bcMAVS-mediated antiviral signaling.

  • Black Carp prmt6 inhibits tbk1 irf3 7 signaling during the antiviral innate immune activation
    Fish & Shellfish Immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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 PRMT6 inhibits TBK1-IRF3/7 signaling during the antiviral innate immune activation
    Fish & shellfish immunology, 2019
    Co-Authors: Yuanyuan Jiang, Yingyi Cao, Jun Xiao, Liqun Liu, Xuejiao Song, Shisi Yang, Hao Feng
    Abstract:

    Abstract 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.