Fish ubiquitin-specific protease 8 (USP8) inhibits IFN production through autophagy-lysosomal dependent degradation of IRF7.

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response.

Zebrafish TMEM47 is an effective blocker of IFN production during RNA and DNA virus infection.

IMPORTANCE: Mitochondrial antiviral signaling protein (MAVS) and stimulator of interferon (IFN) genes (STING) are key adaptor proteins required for innate immune responses to RNA and DNA virus infection. Here, we show that zebrafish transmembrane protein 47 (TMEM47) plays a critical role in regulating MAVS- and STING-triggered IFN production in a negative feedback manner. TMEM47 interacted with MAVS and STING for autophagic degradation, and ATG5 was essential for this process. These findings suggest the inhibitory function of TMEM47 on MAVS- and STING-mediated signaling responses during RNA and DNA virus infection.

The induction of ferroptosis by KLF11/NCOA4 axis: the inhibitory role in clear cell renal cell carcinoma.

Ferroptosis is a form of cell death and has great potential application in the treatment of many cancers, including clear cell renal cell carcinoma (ccRCC). Herein, we identified the essential roles of Krüppel-like factor 11 (KLF11) in suppressing the progression of ccRCC. By analyzing mRNA expression data from the Gene Expression Omnibus (GEO) database, we found that KLF11 was a significantly downregulated gene in ccRCC tissues. The results of subsequent functional assays verified that KLF11 played an antiproliferative role in ccRCC cells and xenograft tumors. Furthermore, gene set enrichment analysis indicated that ferroptosis was involved in ccRCC development, and correlation analysis revealed that KLF11 was positively related to ferroptosis drivers. We also found that KLF11 promoted ferroptosis in ccRCC by downregulating the protein expression of ferritin, system xc (-) cystine/glutamate antiporter (xCT), and glutathione peroxidase 4 (GPX4), acting as the inhibitory factors of ferroptosis and increasing the intracellular levels of lipid reactive oxygen species (ROS). As a transcriptional regulator, KLF11 significantly increased the promoter activity of nuclear receptor coactivator 4 (NCOA4), a gene significantly downregulated in ccRCC and whose low expression is associated with poor survival. The characteristics of ccRCC cells caused by KLF11 overexpression were reversed after NCOA4 silencing. In summary, the present study suggests that KLF11 suppresses the progression of ccRCC by increasing NCOA4 transcription. Therefore, the KLF11/NCOA4 axis may serve as a novel therapeutic target for human ccRCC.

Zebrafish MAP2K7 Simultaneously Enhances Host IRF7 Stability and Degrades Spring Viremia of Carp Virus P Protein via Ubiquitination Pathway.

During viral infection, host defensive proteins either enhance the host immune response or antagonize viral components directly. In this study, we report on the following two mechanisms employed by zebrafish mitogen-activated protein kinase kinase 7 (MAP2K7) to protect the host during spring viremia of carp virus (SVCV) infection: stabilization of host IRF7 and degradation of SVCV P protein. In vivo, map2k7+/- (map2k7-/- is a lethal mutation) zebrafish showed a higher lethality, more pronounced tissue damage, and more viral proteins in major immune organs than the controls. At the cellular level, overexpression of map2k7 significantly enhanced host cell antiviral capacity, and viral replication and proliferation were significantly suppressed. Additionally, MAP2K7 interacted with the C terminus of IRF7 and stabilized IRF7 by increasing K63-linked polyubiquitination. On the other hand, during MAP2K7 overexpression, SVCV P proteins were significantly decreased. Further analysis demonstrated that SVCV P protein was degraded by the ubiquitin-proteasome pathway, as the attenuation of K63-linked polyubiquitination was mediated by MAP2K7. Furthermore, the deubiquitinase USP7 was indispensable in P protein degradation. These results confirm the dual functions of MAP2K7 during viral infection. IMPORTANCE Normally, during viral infection, host antiviral factors individually modulate the host immune response or antagonize viral components to defense infection. In the present study, we report that zebrafish MAP2K7 plays a crucial positive role in the host antiviral process. According to the weaker antiviral capacity of map2k7+/- zebrafish than that of the control, we find that MAP2K7 reduces host lethality through two pathways, as follows: enhancing K63-linked polyubiquitination to promote host IRF7 stability and attenuating K63-mediated polyubiquitination to degrade the SVCV P protein. These two mechanisms of MAP2K7 reveal a special antiviral response in lower vertebrates.

GCRV NS38 counteracts SVCV proliferation by intracellular antagonization during co-infection.

Viral co-infection has been found in animals; however, the mechanisms of co-infection are unclear. The abundance and diversity of viruses in water make fish highly susceptible to co-infection. Here, we reported a co-infection in fish, which resulted in reduced host lethality and illustrated the intracellular molecular mechanism of viral co-infection. The spring viremia of carp virus (SVCV) is a highly lethal virus that infects Cyprinidae, such as zebrafish. The mortality of SVCV infection was significantly reduced when co-infected with the grass carp reovirus (GCRV). The severity of tissue damage and viral proliferation of SVCV was also reduced in co-infection with GCRV. The transcriptome bioinformatics analysis demonstrated that the effect on the host transcripts in response to SVCV infection was significantly reduced in co-infection. After excluding the extracellular interactions of these two viruses, the intracellular mechanisms were studied. We found that the GCRV NS38 remarkably decreased SVCV infection and viral proliferation. The interaction between GCRV NS38 and SVCV nucleoprotein (N) and phosphoprotein (P) proteins was identified, and NS38 downregulated both N and P proteins. Further analysis demonstrated that the N protein was degraded by NS38 indispensable of the autophagy receptor, sequestosome 1 (p62). Meanwhile, K63-linked ubiquitination of the P protein was reduced by NS38, leading to ubiquitinated degradation of the P protein. These results reveal that the intracellular viral protein interactions are a crucial mechanism of co-infection and influence the host pathology and expand our understanding in intracellular viral interactions co-infection.

Spring Viremia of Carp Virus N Protein Negatively Regulates IFN Induction through Autophagy-Lysosome-Dependent Degradation of STING.

Fish possess a powerful IFN system to defend against aquatic virus infections. Nevertheless, spring viremia of carp virus (SVCV) causes large-scale mortality in common carp and significant economic losses to aquaculture. Therefore, it is necessary to investigate the strategies used by SVCV to escape the IFN response. In this study, we show that the SVCV nucleoprotein (N protein) negatively regulates cellular IFN production by degrading stimulator of IFN genes (STING) via the autophagy-lysosome-dependent pathway. First, overexpression of N protein inhibited the IFN promoter activation induced by polyinosinic-polycytidylic acid and STING. Second, the N protein associated with STING and experiments using a dominant-negative STING mutant demonstrated that the N-terminal transmembrane domains of STING were indispensable for this interaction. Then, the N protein degraded STING in a dose-dependent and autophagy-lysosome-dependent manner. Intriguingly, in the absence of STING, individual N proteins could not elicit host autophagic flow. Furthermore, the autophagy factor Beclin1 was found to interact with the N protein to attenuate N protein-mediated STING degradation after beclin1 knockdown. Finally, the N protein remarkably weakened STING-enhanced cellular antiviral responses. These findings reveal that SVCV uses the host autophagic process to achieve immune escape, thus broadening our understanding of aquatic virus pathogenesis.

PRAME is a useful marker for the differential diagnosis of melanocytic tumours and histological mimics.

AIMS: Although the morphological assessment of melanoma is generally straightforward, diagnosis can be especially difficult when the significant morphological and immunohistochemical results overlap with those of benign and malignant melanocytic tumours and histological mimics. This study assessed the potential diagnostic utility of measuring PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemically in naevi, melanomas and clear cell sarcomas (CCSs) in Chinese patients. METHODS: We examined the immunohistochemical expression of PRAME in 317 melanocytic naevi, 178 primary melanomas, 72 metastatic melanomas and 19 CCSs and compared the sensitivity and specificity of PRAME immunohistochemistry (IHC) in the differential diagnosis of melanocytic tumours and histological mimics. RESULTS: Of the 317 melanocytic naevi, 98.1%were completely negative for PRAME; six cases showed focal PRAME immunoreactivity in a minor population of lesional melanocytes. Diffuse nuclear immunoreactivity for PRAME was found in 89.9% of primary melanomas and 93.1% of metastatic melanomas. Regarding melanoma subtypes, PRAME was expressed in 100% of superficial spreading melanomas, 100% of melanomas arise in congenital naevus, 91.4% of nodular melanomas, 87.8% of acral lentigo melanomas, 80.0% of lentigo malignant melanomas, 60.0% of Spitz melanomas, 96.2% of mucosal melanomas and 80.0% of uveal melanomas. None of the two desmoplastic melanomas expressed PRAME. Of the 19 CCS cases, 89.5% were negative for PRAME and 10.5% showed focal weak PRAME immunoreactivity in a minor population of tumour cells. CONCLUSIONS: Our findings indicate that PRAME may be a useful marker to support a suspected diagnosis of melanoma. In addition, lack of PRAME expression is a valuable hint to CCS in a suspected case, and then molecular confirmation of the presence of EWSR1 rearrangement is necessary.

Research progress on medical image dataset expansion methods / 生物医学工程学杂志

Computer-aided diagnosis (CAD) systems play a very important role in modern medical diagnosis and treatment systems, but their performance is limited by training samples. However, the training samples are affected by factors such as imaging cost, labeling cost and involving patient privacy, resulting in insufficient diversity of training images and difficulty in data obtaining. Therefore, how to efficiently and cost-effectively augment existing medical image datasets has become a research hotspot. In this paper, the research progress on medical image dataset expansion methods is reviewed based on relevant literatures at home and abroad. First, the expansion methods based on geometric transformation and generative adversarial networks are compared and analyzed, and then improvement of the augmentation methods based on generative adversarial networks are emphasized. Finally, some urgent problems in the field of medical image dataset expansion are discussed and the future development trend is prospected.

A survey of loss function of medical image segmentation algorithms / 生物医学工程学杂志

Medical image segmentation based on deep learning has become a powerful tool in the field of medical image processing. Due to the special nature of medical images, image segmentation algorithms based on deep learning face problems such as sample imbalance, edge blur, false positive, false negative, etc. In view of these problems, researchers mostly improve the network structure, but rarely improve from the unstructured aspect. The loss function is an important part of the segmentation method based on deep learning. The improvement of the loss function can improve the segmentation effect of the network from the root, and the loss function is independent of the network structure, which can be used in various network models and segmentation tasks in plug and play. Starting from the difficulties in medical image segmentation, this paper first introduces the loss function and improvement strategies to solve the problems of sample imbalance, edge blur, false positive and false negative. Then the difficulties encountered in the improvement of the current loss function are analyzed. Finally, the future research directions are prospected. This paper provides a reference for the reasonable selection, improvement or innovation of loss function, and guides the direction for the follow-up research of loss function.

Macrophage heterogeneity role in NAFLD and NASH disease progression / 中华肝脏病杂志

Nonalcoholic fatty liver disease (NAFLD) is a type of metabolic stress liver injury that is closely associated with insulin resistance and genetic susceptibility. The continuum of liver injury in NAFLD can range from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) and even lead to cirrhosis and liver cancer. The pathogenesis of NAFLD is complicated. Pro-inflammatory cytokines, lipotoxicity, and gut bacterial metabolites play a key role in activating liver-resident macrophages (Kupffer cells, KCs) and recruiting circulating monocyte-derived macrophages (MoDMacs) to deposit fat in the liver. With the application of single-cell RNA-sequencing, significant heterogeneity in hepatic macrophages has been revealed, suggesting that KCs and MoDMacs located in the liver exert distinct functions in regulating liver inflammation and NASH progression. This study focuses on the role of macrophage heterogeneity in the development and occurrence of NAFLD and NASH, in view of the fact that innate immunity plays a key role in the development of NAFLD.

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 (CaHV) 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.

Zebrafish CERKL Enhances Host TBK1 Stability and Simultaneously Degrades Viral Protein via Ubiquitination Modulation.

In the viral infection process, host gene function is usually reported as either defending the host or assaulting the virus. In this study, we demonstrated that zebrafish ceramide kinase-like (CERKL) mediates protection against viral infection via two distinct mechanisms: stabilization of TANK-binding kinase 1 (TBK1) through impairing K48-linked ubiquitination and degradation of spring viremia of carp virus (SVCV) P protein by dampening K63-linked ubiquitination, resulting in an improvement of the host immune response and a decline in viral activity in epithelioma papulosum cyprini (EPC) cells. On SVCV infection, ifnφ1 expression was increased or blunted by CERKL overexpression or knockdown, respectively. Subsequently, we found that CERKL localized in the cytoplasm, where it interacted with TBK1 and enhanced its stability by impeding the K48-linked polyubiquitination; meanwhile, the antiviral capacity of TBK1 was significantly potentiated by CERKL. In contrast, CERKL also interacted with and degraded SVCV P protein to disrupt its function in viral proliferation. Further mechanism analysis revealed K63-linked deubiquitination is the primary means of CERKL-mediated SVCV P protein degradation. Taken together, our study reveals a novel mechanism of fish defense against viral infection: the single gene cerkl is both a shield for the host and a spear against the virus, which strengthens resistance.

Conserved function of crucian carp cGAS in the MITA-mediated interferon signaling.

Mammalian cyclic GMP-AMP synthase (cGAS) is pivotal for cytosolic DNA-triggered interferon (IFN) response. However, the function of cGAS in fish IFN response remains unclear. Our recent study has reported that cGAS from crucian and grass carps downregulates the IFN response by attenuating the K63-linked ubiquitination of retinoic acid-inducible gene-I (RIG-I) and its interaction with mitochondrial antiviral signaling protein (MAVS). Here, the function of crucian carp cGAS was further investigated. We found that crucian carp cGAS directly binds to poly deoxyadenylic-deoxythymidylic acid (poly (dA:dT)) and exhibits mediator of IFN regulatory factor 3 (IRF3) activation (MITA)-dependent activation of the IFN response, indicating a conserved function of crucian carp cGAS in the MITA-mediated IFN signaling. However, crucian carp cGAS could suppress the IFN activation stimulated by polyinosinic: polycytidylic acid (poly (I:C)) in time- and dose-dependent manners. These data collectively suggest complicated functions of crucian carp cGAS in the IFN antiviral response.

MicroRNA miR-155 inhibits cyprinid herpesvirus 3 replication via regulating AMPK-MAVS-IFN axis.

Since emerged in the late 1990s, cyprinid herpesvirus 3 (CyHV-3) has caused huge economic losses in common and koi carp culture worldwide. Accumulating evidences suggest that teleost fish microRNA (miRNA), a class of non-coding RNA of ∼22 nucleotides, can participate in many cellular processes, especially in host antiviral defenses. However, the roles of miRNAs in CyHV-3 infection are still unclear. Here, using high-throughput miRNA sequencing and quantitative real-time PCR (qRT-PCR) verification, we found that miR-155 was significantly upregulated in common carp brain (CCB) cells upon CyHV-3 infection. Overexpression of miR-155 effectively inhibited CyHV-3 replication in CCB cells and promoted type I interferon (IFN-I) expression. Further study revealed that miR-155 targeted the 3' untranslated region (UTR) of the mRNA of 5'AMP-activated protein kinase (AMPK), and that AMPK could interact with and degrade the mitochondrial antiviral signaling protein (MAVS), resulting in the reduction of interferon (IFN) expression. Collectively, our results show that miR-155, induced by CyHV-3 infection, exhibits anti-CyHV-3 activity via regulating AMPK-MAVS-IFN axis, which will help design anti-CyHV-3 drugs.

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 CgSHP1-A (encoded by the Cgptpn6-A gene) and CgSHP1-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 CaTBK1-induced phosphorylation of CaMITA shared by CgSHP1-A and CgSHP1-B, and the autophagic degradation of CaMITA exclusively by CgSHP1-A. Meanwhile, the data support that CgSHP1-A and CgSHP1-B have sub-functionalized and that CgSHP1-A overwhelmingly dominates CgSHP1-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.

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 CgViperin-A and CgViperin-B were found to interact with crucian carp (C. auratus) herpesvirus (CaHV) 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, CgViperin-B also mediated ORF46R degradation through autophagosome pathway, which was absent in CgViperin-A. Moreover, we found that the N-terminal α-helix domain was necessary for the localization of CgViperin-A and CgViperin-B at the endoplasmic reticulum (ER), and the C-terminal domain of CgViperin-A and CgViperin-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.

Clinicopathological and prognostic features of hepatitis B virus-associated diffuse large B-cell lymphoma: a single-center retrospective study in China.

BACKGROUND: While the epidemiologic association between hepatitis B virus (HBV) infection and diffuse large B-cell lymphoma (DLBCL) is established, little is known about the pathological characteristics and outcome of DLBCL arising in patients with HBV infection. METHODS: We retrospectively studied a cohort of 420 patients with DLBCL for the incidence of HBV infection, and the clinicopathologic features and prognostic factors in HBsAg-positive DLBCL patients in China, a hepatitis B endemic area. RESULTS: In our study, 127 (30.2%) patients were HBsAg-positive. HBsAg-positive DLBCL displayed a younger median onset age (50 vs. 54 years, P = 0.002), more frequent involvement of the spleen (19.7% vs. 6.1%, P < 0.001), less frequent involvement of the small and large intestine (2.3% vs. 11.2%, P = 0.003), more advanced disease (stage III/IV: 56.7% vs. 45.1%, P = 0.028), and lower expression rate of MYC (49.1% vs. 66.7%, P = 0.026). The median follow-up time was 61.9 months. Univariate analysis showed that there was no significant difference in overall survival (OS) between HBsAg-negative and -positive DLBCL (P = 0.577). In the HBsAg-positive DLBCL subgroup, age older than 60 years, advanced disease, elevated lactate dehydrogenase (LDH), spleen involvement, B symptoms (fever, night sweats, weight loss), and double expressers of MYC and BCL2 had a significantly worse outcome, and patients treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) had a better prognosis. Multivariate analysis further confirmed that spleen involvement and rituximab use were independent prognostic factors in HBsAg-positive DLBCL patients. CONCLUSIONS: Our study indicates that HBsAg-positive DLBCL has unique clinicopathological features and independent prognostic factors. Moreover, under antiviral prophylaxis, the survival of DLBCL patients with HBV infections was comparable to that of HBV-negative patients, and the use of rituximab significantly improved OS in HBsAg-positive DLBCL patients.

cGAS Is a Negative Regulator of RIG-I-Mediated IFN Response in Cyprinid Fish.

In mammals, cyclic GMP-AMP synthase (cGAS) recognizes cytosolic dsDNA to induce the type I IFN response. However, the functional role of cGAS in the IFN response of fish remains unclear or controversial. In this study, we report that cGAS orthologs from crucian carp Carassius auratus (CacGAS) and grass carp Ctenopharyngodon idellus (CicGAS) target the dsRNA sensor retinoic acid-inducible gene I (RIG-I) for negative regulation of the IFN response. First, poly(deoxyadenylic-deoxythymidylic) acid-, polyinosinic-polycytidylic acid-, and spring viremia of carp virus-induced IFN responses were impaired by overexpression of CacGAS and CicGAS. Then, CacGAS and CicGAS interacted with CiRIG-I and CiMAVS and inhibited CiRIG-I- and CiMAVS-mediated IFN induction. Moreover, the K63-linked ubiquitination of CiRIG-I and the interaction between CiRIG-I and CiMAVS were attenuated by CacGAS and CicGAS. Finally, CacGAS and CicGAS decreased CiRIG-I-mediated the cellular antiviral response and facilitated viral replication. Taken together, data in this study identify CacGAS and CicGAS as negative regulators in RIG-I-like receptor signaling, which extends the current knowledge regarding the role of fish cGAS in the innate antiviral response.

Zebrafish Uba1 Degrades IRF3 through K48-Linked Ubiquitination to Inhibit IFN Production.

Fish IFN regulatory factor 3 (IRF3) is a crucial transcription factor in the IFN activation signaling pathway, which leads to IFN production and a positive cycle. Unrestricted IFN expression results in hyperimmune responses and therefore, IFN must be tightly regulated. In the current study, we found that zebrafish Ub-activating enzyme (Uba1) negatively regulated IRF3 via the K-48 ubiquitin proteasome degradation of IRF3. First, ifn expression stimulated by spring viraemia of carp virus infection was blunted by the overexpression of Uba1 and enhanced by Uba1 knockdown. Afterward, we found that Uba1 was localized in the cytoplasm, where it interacted with and degraded IRF3. Functional domains analysis revealed that the C-terminal ubiquitin-fold domain was necessary for IRF3 degradation by Uba1 and the N-terminal DNA-binding domain of IRF3 was indispensable for the degradation by Uba1.The degradation of IRF3 was subsequently impaired by treatment with MG132, a ubiquitin proteasome inhibitor. Further mechanism analysis revealed that Uba1 induced the K48-linked Ub-proteasomal degradation of IRF3. Finally, the antiviral capacity of IRF3 was significantly attenuated by Uba1. Taken together, our study reveals that zebrafish Uba1 interacts with and activates the ubiquitinated degradation of IRF3, providing evidence of the IFN immune balance mechanism in fish.