In vivo multiscale analyses of spring viremia of carp virus (SVCV) infection: From model organism to target species.

Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1ß. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.

Toward the Development of a Pan-Lyssavirus Vaccine.

In addition to the rabies virus (RABV), 16 more lyssavirus species have been identified worldwide, causing a disease similar to RABV. Non-rabies-related human deaths have been described, but the number of cases is unknown, and the potential of such lyssaviruses causing human disease is unpredictable. The current rabies vaccine does not protect against divergent lyssaviruses such as Mokola virus (MOKV) or Lagos bat virus (LBV). Thus, a more broad pan-lyssavirus vaccine is needed. Here, we evaluate a novel lyssavirus vaccine with an attenuated RABV vector harboring a chimeric RABV glycoprotein (G) in which the antigenic site I of MOKV replaces the authentic site of rabies virus (RABVG-cAS1). The recombinant vaccine was utilized to immunize mice and analyze the immune response compared to homologous vaccines. Our findings indicate that the vaccine RABVG-cAS1 was immunogenic and induced high antibody titers against both RABVG and MOKVG. Challenge studies with different lyssaviruses showed that replacing a single antigenic site of RABV G with the corresponding site of MOKV G provides a significant improvement over the homologous RABV vaccine and protects against RABV, Irkut virus (IRKV), and MOKV. This strategy of epitope chimerization paves the way towards a pan-lyssavirus vaccine to safely combat the diseases caused by these viruses.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model.

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

Histopathology and transcriptome profiling reveal features of immune responses in gills and intestine induced by Spring viremia of carp virus.

The immune system of bony fish closely resembles that of mammals, comprising both specific (adaptive) and non-specific (innate) components. Notably, the mucosa-associated lymphoid tissue (MALT) serves as the first line of defense within the non-specific immune system, playing a critical role in protecting these aquatic organisms against invading pathogens. MALT encompasses a network of immune cells strategically distributed throughout the gills and intestines, forming an integral part of the mucosal barrier that interfaces directly with the surrounding aquatic environment. Spring Viremia of Carp Virus（SVCV）, a highly pathogenic agent causing substantial harm to common carp populations, has been designated as a Class 2 animal disease by the Ministry of Agriculture and Rural Affairs of China. Utilizing a comprehensive array of research techniques, including Hematoxylin and Eosin (HE)、Alcian Blue Periodic Acid-Schiff (AB-PAS)、transcriptome analysis for global gene expression profiling and Reverse Transcription-Polymerase Chain Reaction (RT-qPCR), this study uncovered several key findings: SVCV is capable of compromising the mucosal architecture in the gill and intestinal tissues of carp, and stimulate the proliferation of mucous cells both in gill and intestinal tissues. Critically, the study revealed that SVCV's invasion elicits a robust response from the carp's mucosal immune system, demonstrating the organism's capacity to resist SVCV invasion despite the challenges posed by the pathogen.

SIRT6 positively regulates antiviral response in a bony fish, the Chinese perch Siniperca chuatsi.

SIRT6, a key member of the sirtuin family, plays a pivotal role in regulating a number of vital biological processes, including energy metabolism, oxidative stress, and immune system modulation. Nevertheless, the function of SIRT6 in bony fish, particularly in the context of antiviral immune response, remains largely unexplored. In this study, a sirt6 was cloned and characterized in a commercial fish, the Chinese perch (Siniperca chuatsi). The SIRT6 possesses conserved SIR2 domain with catalytic core region when compared with other vertebrates. Tissue distribution analysis indicated that sirt6 was expressed in all detected tissues, and the sirt6 was significantly induced following infection of infectious haemorrhagic syndrome virus (IHSV). The overexpression of SIRT6 resulted in significant upregulation of interferon-stimulated genes (ISGs), such as viperin, mx, isg15, irf3 and ifp35, and inhibited viral replication. It was further found that SIRT6 was located in nucleus and could enhance the expression of ISGs induced by type I and II IFNs. These findings may provide new information in relation with the function of SIRT6 in vertebrates, and with viral prevention strategy development in aquaculture.

Zebrafish mylipb attenuates antiviral innate immunity through two synergistic mechanisms targeting transcription factor irf3.

IFN regulatory factor 3 (IRF3) is the transcription factor crucial for the production of type I IFN in viral defence and inflammatory responses. The activity of IRF3 is strictly modulated by post-translational modifications (PTMs) to effectively protect the host from infection while avoiding excessive immunopathology. Here, we report that zebrafish myosin-regulated light chain interacting protein b (mylipb) inhibits virus-induced type I IFN production via two synergistic mechanisms: induction of autophagic degradation of irf3 and reduction of irf3 phosphorylation. In vivo, mylipb-null zebrafish exhibit reduced lethality and viral mRNA levels compared to controls. At the cellular level, overexpression of mylipb significantly reduces cellular antiviral capacity, and promotes viral proliferation. Mechanistically, mylipb associates with irf3 and targets Lys 352 to increase K6-linked polyubiquitination, dependent on its E3 ubiquitin ligase activity, leading to autophagic degradation of irf3. Meanwhile, mylipb acts as a decoy substrate for the phosphokinase tbk1 to attenuate irf3 phosphorylation and cellular antiviral responses independent of its enzymatic activity. These findings support a critical role for zebrafish mylipb in the limitation of antiviral innate immunity through two synergistic mechanisms targeting irf3.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.).

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Antiviral effect of miR-206 in rainbow trout (Oncorhynchus mykiss) against infectious hematopoietic necrosis virus infection.

Infectious hematopoietic necrosis (IHN), caused by IHN virus, is a highly contagious and lethal disease that seriously hampers the development of rainbow trout (Oncorhynchus mykiss) aquaculture. However, the immune response mechanism of rainbow trout underlying IHNV infection remains largely unknown. MicroRNAs act as post-transcriptional regulators of gene expression and perform a crucial role in fish immune response. Herein, the regulatory mechanism and function of miR-206 in rainbow trout resistance to IHNV were investigated by overexpression and silencing. The expression analysis showed that miR-206 and its potential target receptor-interacting serine/threonine-protein kinase 2 (RIP2) exhibited significant time-dependent changes in headkidney, spleen and rainbow trout primary liver cells infected with IHNV and their expression displayed a negative correlation. In vitro, the interaction between miR-206 and RIP2 was verified by luciferase reporter assay, and miR-206 silencing in rainbow trout primary liver cells markedly increased RIP2 and interferon (IFN) expression but significantly decreased IHNV copies, and opposite results were obtained after miR-206 overexpression or RIP2 knockdown. In vivo, overexpressed miR-206 with agomiR resulted in a decrease in the expression of RIP2 and IFN in liver, headkidney and spleen. This study revealed the key role of miR-206 in anti-IHNV, which provided potential for anti-viral drug screening in rainbow trout.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish.

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

Innate immune response of snakehead fish to Indian strain of snakehead rhabdovirus (SHRV-In) infection and the infectivity potential of the virus to other freshwater fishes.

A new virus known as snakehead rhabdovirus (SHRV-In) was discovered in South India in striped snakehead (Channa striata) that had hemorrhagic patches and cutaneous ulcerations. The virus is the most potentially harmful pathogen of snakehead because it could cause 100% mortality within 5 days. The goal of the current investigation was to evaluate the infectivity of rhabdovirus in freshwater fishes and to analyze the immune response in snakehead fish after challenge with SHRV-In. The infectivity study of SHRV-In against three freshwater fish such as tilapia, grass carp and loach showed that the virus could not induce mortality in any of them. Snakehead fish challenged with SHRV-In showed significant (p < 0.05) changes in haematological parameters such as red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), white blood cell (WBC), total platelet (PLT) counts, mean platelet volume (MPV) and immunological markers such as respiratory burst, superoxide dismutase, catalase activity and myeloperoxidase activity at 6, 12, 24 and 48 hpi. Real time PCR was executed to examine the expression profile of innate immune genes such as IRF-7, IL-8 and IL-12 in Snakehead fish at 6, 12, 24 and 48 h post SHRV-In infection. Immune gene expression of IRF-7, IL-8 and IL-12 were up-regulated in the spleen when compared to kidney at 6 and 12 hpi. However, the expression level of all the genes was down-regulated at 24 and 48 hpi. The down regulation of innate immune genes after 24 hpi in these tissues may be the result of increased multiplication of SHRV-In by interfering with the immune signaling pathway.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

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.

Cross-Neutralization Activities of Antibodies against 18 Lyssavirus Glycoproteins.

Some lyssaviruses, including the rabies virus (RABV), cause lethal neurological symptoms in humans. However, the efficacy of commercial vaccines has only been evaluated against RABV. To assess cross-reactivity among lyssaviruses, including RABV, sera from rabbits inoculated with human and animal RABV vaccines and polyclonal antibodies from rabbits immunized with expression plasmids of the glycoproteins of all 18 lyssaviruses were prepared, and cross-reactivity was evaluated via virus-neutralization tests using Duvenhage lyssavirus (DUVV), European bat lyssavirus-1 (EBLV-1), Mokola lyssavirus (MOKV), Lagos bat lyssavirus (LBV), and RABV. The sera from rabbits inoculated with RABV vaccines showed cross-reactivity with EBLV-1 and DUVV, both belonging to phylogroup I. However, reactivity with MOKV and LBV in phylogroup II was notably limited or below the detection level. Next, we compared the cross-reactivity of the polyclonal antibodies against all lyssavirus glycoproteins. Polyclonal antibodies had high virus-neutralization titers against the same phylogroup but not different phylogroups. Our findings indicate that a new vaccine should be developed for pre- and post-exposure prophylaxis against lyssaviral infections.

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.

Expression characteristics of non-virion protein of Hirame novirhabdovirus and its transfection induced response in hirame natural embryo cells.

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process.

Cytokine Receptor-Like Factor 3 Negatively Regulates Antiviral Immunity by Promoting the Degradation of TBK1 in Teleost Fish.

The cytokine receptor-like factor 3 (Crlf3) belongs to the orphan class I cytokine receptors and is identified as a neuroprotective erythropoietin receptor. In previous studies of Crlf3, few focused on its role in innate immunity. Therefore, this study explored the regulatory role of Crlf3 in innate immunity. TANK-binding kinase 1 (TBK1) is a vital adaptor protein for the activation of the RLRs-MVAS-IRF3 antiviral signaling axis; thus, its expression and activity must be tightly regulated to maintain immune homeostasis and avoid undesirable effects. Here, we report that Crlf3 is a negative regulator of type I interferon production. The expression of Crlf3 is induced by poly(I·C) or Siniperca chuatsi rhabdovirus (SCRV) treatment. Silencing of Crlf3 enhanced poly(I·C)- and SCRV-induced type I interferon production, whereas overexpression of Crlf3 suppressed type I interferon production. Mechanistically, Crlf3 interacted with TBK1 via its N domain and then inhibited type I interferon production by promoting TBK1 proteasomal degradation through K48-linked polyubiquitination. Our study shows that Crlf3 is a key factor for viral escape from innate antiviral immunity in fish and provides a new perspective on mammalian resistance to viral invasion. IMPORTANCE The expression of Crlf3 was upregulated with SCRV invasion, which proved that Crlf3 was involved in the regulation of the antiviral immune response. In this study, we found that the existence of Crlf3 promoted the replication of SCRV. Therefore, it is reasonable to believe that SCRV evades innate immune attack with the assistance of Crlf3. In addition, we report that Crlf3 negatively regulates interferon (IFN) induction by promoting the degradation of TBK1 in fish. We showed that Crlf3 is evenly distributed in the cytoplasm and interacts with TBK1. Further studies showed that Crlf3 specifically mediates K48-linked ubiquitination of TBK1 and promotes TBK1 degradation, resulting in a marked inhibition of retinoic acid-inducible gene I (RIG-I) downstream signaling.

IRF2 Cooperates with Phosphoprotein of Spring Viremia of Carp Virus to Suppress Antiviral Response in Zebrafish.

IFN regulatory factor (IRF) 2 belongs to the IRF1 subfamily, and its functions are not yet fully understood. In this study, we showed that IRF2a was a negative regulator of the interferon (IFN) response induced by spring viremia of carp virus (SVCV). Irf2a-/- knockout zebrafish were less susceptible to SVCV than wild-type fish. Transcriptomic analysis reveals that differentially expressed genes (DEGs) in the irf2a-/- and irf2a+/+ cells derived caudal fins were mainly involved in cytokine-cytokine receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and transforming growth factor-beta (TGF-beta) signaling pathway. Interestingly, the basal expression levels of interferon stimulating genes (ISGs), including pkz, mx, apol, and stat1 were higher in the irf2a-/- cells than irf2a+/+ cells, suggesting that they may contribute to the increased viral resistance of the irf2a-/- cells. Overexpression of IRF2a inhibited the activation of ifnφ1 and ifnφ3 induced by SVCV and poly(I:C) in the epithelioma papulosum cyprini (EPC) cells. Further, it was found that SVCV phosphoprotein (SVCV-P) could interact with IRF2a to promote IRF2a nuclear translocation and protein stability via suppressing K48-linked ubiquitination of IRF2a. Both IRF2a and SVCV-P not only destabilized STAT1a but reduced its translocation into the nucleus. Our work demonstrates that IRF2a cooperates with SVCV-P to suppress host antiviral response against viral infection in zebrafish. IMPORTANCE Interferon regulatory factors (IRFs) are central in the regulation of interferon-mediated antiviral immunity. Here, we reported that IRF2a suppressed interferon response and promoted virus replication in zebrafish. The suppressive effects were enhanced by the phosphoprotein of the spring viremia of carp virus (SVCV) via inhibition of K48-linked ubiquitination of IRF2a. IRF2a and SVCV phosphoprotein cooperated to degrade STAT1 and block its nuclear translocation. Our work demonstrated that IRFs and STATs were targeted by the virus through posttranslational modifications to repress interferon-mediated antiviral response in lower vertebrates.

Negative Regulatory Role of the Spring Viremia of Carp Virus Matrix Protein in the Host Interferon Response by Targeting the MAVS/TRAF3 Signaling Axis.

Spring viremia of carp virus (SVCV) is a severe infectious pathogen that causes high rates of mortality in cyprinids and other fish species. Despite numerous investigations of SVCV infection, the underlying molecular mechanisms remain poorly understood. In this study, we found that the SVCV matrix protein (SVCV-M) played an inhibitory role in the host interferon (IFN) response by targeting the MAVS/TRAF3 signaling axis, thereby uncovering a previously unrecognized mechanism of SVCV escape from host innate antiviral immunity. Mechanistically, SVCV-M was located at the mitochondria independent of MAVS, which allowed SVCV-M to build an arena for competition with the MAVS platform. A microscale thermophoresis assay showed that SVCV-M had a high affinity for TRAF3, as indicated by a lower equilibrium dissociation constant (KD) value than that of MAVS with TRAF3. Therefore, the association of MAVS with TRAF3 was competitively impaired by SVCV-M in a dose-dependent manner. Accordingly, SVCV-M showed a potent ability to inhibit the K63-linked polyubiquitination of TRAF3. This inhibition was accompanied by the impairment of the IFN response, as shown by the marked decline in IFN-φ1-promoter (pro) luciferase reporter activity. By constructing truncated TRAF3 and SVCV-M proteins, the RING finger, zinc finger, and coiled-coil domains of TRAF3 and the hydrophobic-pocket-like structure formed by the α2-, α3-, and α4-helices of SVCV-M may be the major target and antagonistic modules responsible for the protein-protein interaction between the TRAF3 and SVCV-M proteins. These findings highlighted the intervention of SVCV-M in host innate immunity, thereby providing new insights into the extensive participation of viral matrix proteins in multiple biological activities. IMPORTANCE The matrix protein of SVCV (SVCV-M) is an indispensable structural element for nucleocapsid condensation and virion formation during viral morphogenesis, and it connects the core nucleocapsid particle to the outer membrane within the mature virus. Previous studies have emphasized the architectural role of SVCV-M in viral construction; however, the potential nonstructural functions of SVCV-M in viral replication and virus-host interactions remain poorly understood. In this study, we identified the inhibitory role of the SVCV-M protein in host IFN production by competitively recruiting TRAF3 from the MAVS signaling complex and impairing TRAF3 activation via inhibition of K63-linked polyubiquitination. This finding provided new insights into the regulatory role of SVCV-M in host innate immunity, which highlighted the broader functionality of rhabdovirus matrix protein apart from being a structural protein. This study also revealed a previously unrecognized mechanism underlying SVCV immune evasion by inhibiting the IFN response by targeting the MAVS/TRAF3 signaling axis.