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"Lu, Long-Feng"
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Spring viraemia of carp virus modulates p53 expression using two distinct mechanisms
p53, which regulates cell-cycle arrest and apoptosis, is a crucial target for viruses to release cells from cell-cycle checkpoints or to protect cells from apoptosis for their own benefit. Viral evasion mechanisms of aquatic viruses remain mysterious. Here, we report the spring viremia of carp virus (SVCV) degrading and stabilizing p53 in the ubiquitin-proteasome pathway by the N and P proteins, respectively. Early in an SVCV infection, significant induction was observed in the S phase and p53 was decreased in the protein level. Further experiments demonstrated that p53 interacted with SVCV N protein and was degraded by suppressing the K63-linked ubiquitination. However, the increase of p53 was observed late in the infection and experiments suggested that p53 was bound to SVCV P protein and stabilized by enhancing the K63-linked ubiquitination. Finally, lysine residue 358 was the key site for p53 K63-linked ubiquitination by the N and P proteins. Thus, our findings suggest that fish p53 is modulated by SVCV N and P protein in two distinct mechanisms, which uncovers the strategy for the subversion of p53-mediated host innate immune responses by aquatic viruses.
Journal Article
Fish TOLLIP manipulates ATG5 for autophagic degradation of STING to attenuate antiviral interferon responses
While robust interferon (IFN) responses in fish are critical for viral clearance, dysregulated signalling can trigger detrimental hyperinflammation, necessitating precise immunoregulatory mechanisms. This study identified Toll-interacting protein (TOLLIP) as a pivotal negative regulator of IFN production in grass carp (Ctenopharyngodon idella) . Upon grass carp reovirus (GCRV) infection, TOLLIP expression increases significantly in tissues and cells. Furthermore, TOLLIP overexpression reduced GCRV- and polyinosinic-polycytidylic acid (poly I:C)-induced IFN expression, whereas tollip knockdown increased the cellular IFN production capacity. TOLLIP subsequently binds and degrades STING. Further mechanistic studies revealed that TOLLIP degrades STING in a dose-dependent manner via an autophagy-lysosome-dependent pathway. Interestingly, autophagy-related protein 5 (ATG5) was found to interact with TOLLIP and reduce TOLLIP-mediated STING degradation after atg5 knockdown. In addition, TOLLIP attenuated STING-driven IFN activation and compromised antiviral efficacy. These findings demonstrate that fish TOLLIP plays a specialized regulatory role in antiviral innate immunity, balancing immune defence with homeostasis maintenance.
Journal Article
Fish female-biased gene cyp19a1a leads to female antiviral response attenuation between sexes by autophagic degradation of MITA
From insects to mammals, both innate and adaptive immune response are usually higher in females than in males, with the sex chromosome and hormonal differences considered the main reasons. Here, we report that zebrafish cyp19a1a (cytochrome P450, family 19, subfamily A, polypeptide 1a), an autosomal gene with female-biased expression, causes female fish to exhibit a lower antiviral response. First, we successfully constructed an infection model by intraperitoneal injection of spring viremia of carp virus (SVCV) into zebrafish ( Danio rerio ) and Carassius auratus herpesvirus ( Ca HV) in gibel carp ( Carassius gibelio ). Specifically, female fish were more vulnerable to viral infection than males, accompanied by a significantly weaker interferon (IFN) expression. After screening several candidates, cyp19a1a , which was highly expressed in female fish tissues, was selected for further analysis. The IFN expression and antiviral response were significantly higher in cyp19a1a -/- than in cyp19a1a +/+ . Further investigation of the molecular mechanism revealed that Cyp19a1a targets mediator of IRF3 activation (MITA) for autophagic degradation. Interestingly, in the absence of MITA, Cyp19a1a alone could not elicit an autophagic response. Furthermore, the autophagy factor ATG14 (autophagy-related 14) was found interacted with Cyp19a1a to either promote or attenuate Cyp19a1a-mediated MITA degradation by either being overexpressed or knocked down, respectively. At the cellular level, both the normal and MITA-enhanced cellular antiviral responses were diminished by Cyp19a1a. These findings demonstrated a sex difference in the antiviral response based on a regulation mechanism controlled by a female-biased gene besides sex chromosome and hormonal differences, supplying the current understanding of sex differences in fish.
Journal Article
A novel role of Zebrafish TMEM33 in negative regulation of interferon production by two distinct mechanisms
The transmembrane protein 33 (TMEM33) was originally identified as an endoplasmic reticulum (ER) protein that influences the tubular structure of the ER and modulates intracellular calcium homeostasis. However, the role of TMEM33 in antiviral immunity in vertebrates has not been elucidated. In this article, we demonstrate that zebrafish TMEM33 is a negative regulator of virus-triggered interferon (IFN) induction via two mechanisms: mitochondrial antiviral signaling protein (MAVS) ubiquitination and a decrease in the kinase activity of TANK binding kinase 1 (TBK1). Upon stimulation with viral components, tmem33 was remarkably upregulated in the zebrafish liver cell line. The IFNφ1 promoter (IFNφ1pro) activity and mRNA level induced by retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) were significantly inhibited by TMEM33. Knockdown of TMEM33 increased host ifn transcription. Subsequently, we found that TMEM33 was colocalized in the ER and interacted with the RLR cascades, whereas MAVS was degraded by TMEM33 during the K48-linked ubiquitination. On the other hand, TMEM33 reduced the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA)/IRF3 by acting as a decoy substrate of TBK1, which was also phosphorylated. A functional domain assay revealed that the N-terminal transmembrane domain 1 (TM1) and TM2 regions of TMEM33 were necessary for IFN suppression. Finally, TMEM33 significantly attenuated the host cellular antiviral capacity by blocking the IFN response. Taken together, our findings provide insight into the different mechanisms employed by TMEM33 in cellular IFN-mediated antiviral process.
Journal Article
Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production
Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.
Journal Article
Grass Carp Reovirus (GCRV) Giving Its All to Suppress IFN Production by Countering MAVS Signaling Transduction
Viruses typically target host RIG-I-like receptors (RLRs), a group of key factors involved in interferon (IFN) production, to enhance viral infection. To date, though immune evasion methods to contradict IFN production have been characterized for a series of terrestrial viruses, the strategies employed by fish viruses remain unclear. Here, we report that all grass carp reovirus (GCRV) proteins encoded by segments S1 to S11 suppress mitochondrial antiviral signaling protein (MAVS)-mediated IFN expression. First, the GCRV viral proteins blunted the MAVS-induced expression of IFN, and impair MAVS antiviral capacity significantly. Interestingly, subsequent co-immunoprecipitation experiments demonstrated that all GCRV viral proteins interacted with several RLR cascades, especially with TANK-binding kinase 1 (TBK1) which was the downstream factor of MAVS. To further illustrate the mechanisms of these interactions between GCRV viral proteins and host RLRs, two of the viral proteins, NS79 (S4) and VP3 (S3), were selected as representative proteins for two distinguished mechanisms. The obtained data demonstrated that NS79 was phosphorylated by gcTBK1, leading to the reduction of host substrate gcIRF3/7 phosphorylation. On the other hand, VP3 degraded gcMAVS and the degradation was significantly reversed by 3-MA. The biological effects of both NS79 and VP3 were consistently found to be related to the suppression of IFN expression and the promotion of viral evasion. Our findings shed light on the special evasion mechanism utilized by fish virus through IFN regulation, which might differ between fish and mammals.
Journal Article
Divergent Antiviral Mechanisms of Two Viperin Homeologs in a Recurrent Polyploid Fish
Polyploidy and subsequent diploidization provide genomic opportunities for evolutionary innovations and adaptation. The researches on duplicated gene evolutionary fates in recurrent polyploids have seriously lagged behind that in paleopolyploids with diploidized genomes. Moreover, the antiviral mechanisms of Viperin remain largely unclear in fish. Here, we elaborate the distinct antiviral mechanisms of two viperin homeologs ( Cgviperin-A and Cgviperin-B ) in auto-allo-hexaploid gibel carp ( Carassius gibelio ). First, Cgviperin-A and Cgviperin-B showed differential and biased expression patterns in gibel carp adult tissues. Subsequently, using co-immunoprecipitation (Co-IP) screening analysis, both Cg Viperin-A and Cg Viperin-B were found to interact with crucian carp ( C. auratus ) herpesvirus ( Ca HV) open reading frame 46 right (ORF46R) protein, a negative herpesvirus regulator of host interferon (IFN) production, and to promote the proteasomal degradation of ORF46R via decreasing K63-linked ubiquitination. Additionally, Cg Viperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in Cg Viperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of Cg Viperin-A and Cg Viperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of Cg Viperin-A and Cg Viperin-B was indispensable for the interaction with degradation of ORF46R. Therefore, the current findings clarify the divergent antiviral mechanisms of the duplicated viperin homeologs in a recurrent polyploid fish, which will shed light on the evolution of teleost duplicated genes.
Journal Article
Interferon Regulatory Factors 1 and 2 Play Different Roles in MHC II Expression Mediated by CIITA in Grass Carp, Ctenopharyngodon idella
Expression of major histocompatibility complex class II (MHC II) molecules, which determines both the immune repertoire during development and subsequent triggering of immune responses, is always under the control of a unique (MHC class II) transactivator, CIITA. The IFN-γ-inducible MHC II expression has been extensively and thoroughly studied in humans, but not in bony fish. In this study, the characterization of CIITA was identified and its functional domains were analyzed in grass carp. The absence of GAS and E-box in the promoter region of grass carp CIITA, might imply that the cooperative interaction between STAT1 and USF1 to active the CIITA expression, found in mammals, is not present in bony fish. After the transfection of IFN-γ or IFN-γ rel, only IFN-γ could induce MHC II expression mediated by CIITA. Moreover, interferon regulatory factor (IRF) 2, which cooperates with IRF1 to active the CIITA promoter IV expression in mammals, played an antagonistic role to IRF1 in the activation of grass carp CIITA. These data suggested that grass carp, compared with mammals, has both conservative and unique mechanisms in the regulation of MHC II expression.
Journal Article
Two Duplicated Ptpn6 Homeologs Cooperatively and Negatively Regulate RLR-Mediated IFN Response in Hexaploid Gibel Carp
Src homology region 2 domain-containing phosphatase 1 (SHP1), encoded by the protein tyrosine phosphatase nonreceptor type 6 ( ptpn6 ) gene, belongs to the family of protein tyrosine phosphatases (PTPs) and participates in multiple signaling pathways of immune cells. However, the mechanism of SHP1 in regulating fish immunity is largely unknown. In this study, we first identified two gibel carp ( Carassius gibelio ) ptpn6 homeologs ( Cgptpn6-A and Cgptpn6-B ), each of which had three alleles with high identities. Then, relative to Cgptpn6-B , dominant expression in adult tissues and higher upregulated expression of Cgptpn6-A induced by polyinosinic-polycytidylic acid (poly I:C), poly deoxyadenylic-deoxythymidylic (dA:dT) acid and spring viremia of carp virus (SVCV) were uncovered. Finally, we demonstrated that Cg SHP1-A (encoded by the Cgptpn6-A gene) and Cg SHP1-B (encoded by the Cgptpn6-B gene) act as negative regulators of the RIG-I-like receptor (RLR)-mediated interferon (IFN) response via two mechanisms: the inhibition of Ca TBK1-induced phosphorylation of Ca MITA shared by Cg SHP1-A and Cg SHP1-B, and the autophagic degradation of Ca MITA exclusively by Cg SHP1-A. Meanwhile, the data support that Cg SHP1-A and Cg SHP1-B have sub-functionalized and that Cg SHP1-A overwhelmingly dominates Cg SHP1-B in the process of RLR-mediated IFN response. The current study not only sheds light on the regulative mechanism of SHP1 in fish immunity, but also provides a typical case of duplicated gene evolutionary fates.
Journal Article