TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.

Nucleophosmin 1a translocated from nucleus to cytoplasm and facilitate GCRV replication.

To decipher the functional characterization of Nucleophosmin 1a (NPM1a) from grass carp (Ctenopharyngodon idellus) (CiNPM1a), its cDNA was cloned and bioinformatic analysis were conducted. The full-length cDNA sequence of CiNPM1a is 1732 bp, which encodes 307 amino acids. CiNPM1a contains conserved domains of Nucleoplasmin domain, NPM1-C terminal domain, as well as nuclear localization signals, nuclear export signal (NES) and acid patches. There are 52 and 20 consensus amino acids exist in the Nucleoplasmin domain and the NPM1-C terminal domain of all blasted species. In addition, the immune function of CiNPM1a were analyzed. The Ciirf7, Ciifn1 and Ciifn2 transcription was inhibited, whereas the vp2 and vp7 expressions were enhanced in CiNPM1a overexpressing cells after GCRV infection (P < 0.05). Moreover, the Ciirf7, Ciifn1 and Ciifn2 mRNA levels were significantly up-regulated, but the vp2 and vp7 expressions were significantly down-regulated in CiNPM1a knockdown cells after infection. This indicated that CiNPM1a played negative roles in the induction of Type I IFN reaction and thus the GCRV replication. Finally, the NES domain that affect the nucleous-cytoplasm shuttle and the replication of GCRV were investigated. The deletion of NES1 and NES(1 + 2+3) absolutely limited the transloacation of CiNPM1a△NES1 protein and CiNPM1a △NES(1 + 2+3) protein to cytoplasm after infection, and the deletion of NES2 resulted in partially limitation of protein shuttle. In general, Ciirf3, Ciirf7, Ciifn1 and Ciifn2 expressions were enhanced in the CiNPM1a△NES1, CiNPM1a△NES2 and CiNPM1a△NES3 overexpression groups, and the deletion of functional domains in CiNPM1a led to significantly reduction of the vp2 and vp7 replication. The results indicated that CiNPM1a may be a target molecular for GCRV infection curation, and a candidate molecular for resistance strain breeding of grass carp.

Zebrafish MARCH7 negatively regulates IFN antiviral response by degrading TBK1.

Membrane-associated RING-CH-type finger (MARCH) proteins have been reported to regulate type I IFN production during host antiviral innate immunity. The present study reported the zebrafish MARCH family member, MARCH7, as a negative regulator in virus-triggered type I IFN induction via targeting TANK-binding kinase 1 (TBK1) for degradation. As an IFN-stimulated gene (ISG), we discovered that MARCH7 was significantly induced by spring viremia of carp virus (SVCV) or poly(I:C) stimulation. Ectopic expression of MARCH7 reduced the activity of IFN promoter and dampened the cellular antiviral responses triggered by SVCV and grass carp reovirus (GCRV), which concomitantly accelerated the viral replication. Accordingly, the knockdown of MARCH7 by siRNA transfection significantly promoted the transcription of ISG genes and inhibited SVCV replication. Mechanistically, we found that MARCH7 interacted with TBK1 and degraded it via K48-linked ubiquitination. Further characterization of truncated mutants of MARCH7 and TBK1 confirmed that the C-terminal RING of MARCH7 is essential in the MARCH7-mediated degradation of TBK1 and the negative regulation of IFN antiviral response. This study reveals a molecular mechanism by which zebrafish MARCH7 negatively regulates the IFN response by targeting TBK1 for protein degradation, providing new insights into the essential role of MARCH7 in antiviral innate immunity.