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.