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In mammals, the signaling adaptor mitochondrial antiviral signaling protein (MAVS) is a critical determinant in antiviral innate immunity. However, because of the lack of in vivo data, the physiological function of zebrafish mavs in response to viral infection is still not determined. In this study, we demonstrate that the long splicing isoform of zebrafish mavs promotes IFN regulatory factor 3 signaling and NF-κB signaling. Overexpression of this isoform of mavs enhances cellular antiviral responses. Disruption of mavs in zebrafish attenuates survival ratio on challenge with spring viremia of carp virus. Consistently, the antiviral-responsive genes and inflammatory genes are significantly reduced, and the replication of spring viremia of carp virus is increased in mavs-null zebrafish. Therefore, we provide in vivo evidence to support that zebrafish mavs is essential for antiviral innate immunity, similar to mammalian MAVS.

The signaling adaptor MAVS is a critical determinant in retinoic acid–inducible gene 1–like receptor signaling, and its activation is tightly controlled by multiple mechanisms in response to viral infection, including phosphorylation and ubiquitination. In this article, we demonstrate that zebrafish sirt5, one of the sirtuin family proteins, negatively regulates mavs-mediated antiviral innate immunity. Sirt5 is induced by spring viremia of carp virus (SVCV) infection and binds to mavs, resulting in attenuating phosphorylation and ubiquitination of mavs. Disruption of sirt5 in zebrafish promotes survival ratio after challenge with SVCV. Consistently, the antiviral responsive genes are enhanced, and the replication of SVCV is diminished in sirt5-dificient zebrafish. Therefore, we reveal a function of zebrafish sirt5 in the negative regulation of antiviral innate immunity by targeting mavs.

Sirt7 is one member of the sirtuin family proteins with NAD (NAD+)-dependent histone deacetylase activity. In this study, we report that zebrafish sirt7 is induced upon viral infection, and overexpression of sirt7 suppresses cellular antiviral responses. Disruption of sirt7 in zebrafish increases the survival rate upon spring viremia of carp virus infection. Further assays indicate that sirt7 interacts with irf3 and irf7 and attenuates phosphorylation of irf3 and irf7 by preventing tbk1 binding to irf3 and irf7. In addition, the enzymatic activity of sirt7 is not required for sirt7 to repress IFN-1 activation. To our knowledge, this study provides novel insights into sirt7 function and sheds new light on the regulation of irf3 and irf7 by attenuating phosphorylation.

TNFR-associated factor 6 (TRAF6) not only recruits TBK1/IKKε to MAVS upon virus infection but also catalyzes K63-linked polyubiquitination on substrate or itself, which is critical for NEMO-dependent and -independent TBK1/IKKε activation, leading to the production of type I IFNs. The regulation at the TRAF6 level could affect the activation of antiviral innate immunity. In this study, we demonstrate that zebrafish prmt2, a type I arginine methyltransferase, attenuates traf6-mediated antiviral response. Prmt2 binds to the C terminus of traf6 to catalyze arginine asymmetric dimethylation of traf6 at arginine 100, preventing its K63-linked autoubiquitination, which results in the suppression of traf6 activation. In addition, it seems that the N terminus of prmt2 competes with mavs for traf6 binding and prevents the recruitment of tbk1/ikkε to mavs. By zebrafish model, we show that loss of prmt2 promotes the survival ratio of zebrafish larvae after challenge with spring viremia of carp virus. Therefore, we reveal, to our knowledge, a novel function of prmt2 in the negative regulation of antiviral innate immunity by targeting traf6.

Ovarian tumor domain-containing 6B (OTUD6B) belongs to the OTU deubiquitylating enzyme family. In this study, we report that zebrafish otud6b is induced upon viral infection, and overexpression of otud6b suppresses cellular antiviral response. Disruption of otud6b in zebrafish increases the survival rate upon spring viremia of carp virus and grass carp reovirus exposure. Further assays indicate that otud6b interacts with irf3 and irf7 and diminishes traf6-mediated K63-linked polyubiquitination of irf3 and irf7. In addition, the OTU domain is required for otud6b to repress IFN-1 activation and K63-linked polyubiquitination of irf3 and irf7. Moreover, otud6b also attenuates tbk1 to bind to irf3 and irf7, resulting in the impairment of irf3 and irf7 phosphorylation. This study provides, to our knowledge, novel insights into otud6b function and sheds new lights on the regulation of irf3 and irf7 by deubiquitination in IFN-1 signaling.

Designing and building artificial nanodevices and nanoarchitectures in living systems are extremely intriguing subjects in nanotechnology and synthetic biology. Taking advantage of cellular machinery and endogenous biomacromolecules, such as proteins, is key to achieving the precise and sophisticated manipulation of nanoarchitectures. In this study, we proposed a protein-mediated DNA self-assembly strategy in a molecular crowding environment. By carefully controlling the surface charge of basic nuclear proteins in a crowding environment that mimicked the intracellular environment, we demonstrated that highly positively charged protamine can assemble individual DNA strands into defined structures similar to a catalytic process manner. Successful self-assembly required an optimized protamine surface charge and a crowding environment; otherwise, this self-assembly was impossible. Polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), and dynamic light scattering (DLS) results showed that tile-based DNA tubular structures, tetrahedra, and two dimensional DNA origami structures were successfully assembled. We inferred that the assembly process occurred between the arginine-rich domain of protamine and DNA strands that repeatedly interacted with each other in the viscous system. The current study provides a potential strategy to construct nanodevices in living systems and presents an alternative protein-DNA interaction for the potential fabrication of protein-DNA hybrid nanomaterials.

Arginine methylation catalyzed by protein arginine methyltransferases (PRMT) is a common post-translational modification in histone and nonhistone proteins, which regulates many cellular functions. Protein arginine methyltransferase 3 (prmt3), a type I arginine methyltransferase, has been shown to carry out the formation of stable monomethylarginine as an intermediate before the establishment of asymmetric dimethylarginine. To date, however, the role of PRMT3 in antiviral innate immunity has not been elucidated. This study showed that zebrafish prmt3 was upregulated by virus infection and that the overexpression of prmt3 suppressed cellular antiviral response. The PRMT3 inhibitor, SGC707, enhanced antiviral capability. Consistently, prmt3-null zebrafish were more resistant to Spring Viremia of Carp Virus (SVCV) and Grass Carp Reovirus (GCRV) infection. Further assays showed that the overexpression of prmt3 diminished the phosphorylation of irf3 and prmt3 interacted with rig-i. In addition, both zinc-finger domain and catalytic domain of prmt3 were required for the suppressive function of prmt3 on IFN activation. Our findings suggested that zebrafish prmt3 negatively regulated the antiviral responses, implicating the vital role of prmt3-or even arginine methylation-in antiviral innate immunity.

Transcriptional programs regulated by the NF-κB family are essential for the inflammatory response as well as for innate and adaptive immunity. NF-κB activation occurs via two major signaling pathways: the canonical and the noncanonical. The canonical NF-κB pathway responds to diverse immune stimulations and leads to rapid but transient activation. As a member of the canonical NF-κB family, p65 is thought to be a key regulator of viral infection. Because of the embryonic lethality of p65-null mice, the physiological role of p65 in the antiviral immune response is still unclear. In this study, we generated p65-null zebrafish, which were viable and indistinguishable from their wildtype (WT) siblings under normal conditions. However, p65-null zebrafish were more sensitive to spring viremia of carp virus infection than their WT siblings. Further assays indicated that proinflammatory and antiviral genes, including IFN, were downregulated in p65-null zebrafish after spring viremia of carp virus infection compared with their WT siblings. Our results thus suggested that p65 is required for the antiviral response, activating not only proinflammatory genes but also antiviral genes (including IFN).

TNFR-associated factor 6 (TRAF6) not only recruits TBK1/IKKε to MAVS upon virus infection but also catalyzes K63-linked polyubiquitination on substrate or itself, which is critical for NEMO-dependent and -independent TBK1/IKKε activation, leading to the production of type I IFNs. The regulation at the TRAF6 level could affect the activation of antiviral innate immunity. In this study, we demonstrate that zebrafish

Retinoic acid-inducible-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and cyclic GMP-AMP synthase (cGAS) genes encode essential cytosolic receptors mediating antiviral immunity against viruses. Here, we show that OTUD3 has opposing role in response to RNA and DNA virus infection by removing distinct types of RIG-I/MDA5 and cGAS polyubiquitination. OTUD3 binds to RIG-I and MDA5 and removes K63-linked ubiquitination. This serves to reduce the binding of RIG-I and MDA5 to viral RNA and the downstream adaptor MAVS, leading to the suppression of the RNA virus-triggered innate antiviral responses. Meanwhile, OTUD3 associates with cGAS and targets at Lys279 to deubiquitinate K48-linked ubiquitination, resulting in the enhancement of cGAS protein stability and DNA-binding ability. As a result, Otud3-deficient mice and zebrafish are more resistant to RNA virus infection but are more susceptible to DNA virus infection. These findings demonstrate that OTUD3 limits RNA virus-triggered innate immunity but promotes DNA virus-triggered innate immunity.

Many aerobic organisms have developed molecular mechanism to tolerate hypoxia, but the specifics of these mechanisms remain poorly understood. It is important to develop genetic methods that confer increased hypoxia tolerance to intensively farmed aquatic species, as these are maintained in environments with limited available oxygen. As an asparaginyl hydroxylase of hypoxia-inducible factors (HIFs), factor inhibiting HIF (FIH) inhibits transcriptional activation of hypoxia-inducible genes by blocking the association of HIFs with the transcriptional coactivators CREB-binding protein (CBP) and p300. Therefore, here we sought to test whether fih is involved in regulating hypoxia tolerance in the commonly used zebrafish model. Overexpressing the zebrafish fih gene in epithelioma papulosum cyprini (EPC) cells and embryos, we found that fih inhibits the transcriptional activation of zebrafish HIF-α proteins. Using CRISPR/Cas9 to obtain fih-null zebrafish mutants, we noted that the fih deletion makes zebrafish more tolerant of hypoxic conditions than their WT siblings, but does not result in oxygen consumption rates that significantly differ from those of WT fish. Of note, we identified fewer apoptotic cells in adult fih-null zebrafish brains and in fih-null embryos, possibly explaining why the fih-null mutant had greater hypoxia tolerance than the WT. Moreover, the fih deletion up-regulated several hypoxia-inducible genes in fih-null zebrafish exposed to hypoxia. The findings of our study suggest that fih plays a role in hypoxia tolerance by affecting the rate of cellular apoptosis in zebrafish.

Summary Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that arginine monomethylation precisely regulates the mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. Protein arginine methyltransferase 7 (PRMT7) forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled in a timely manner due to automethylation at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression of MAVS activation. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo but also uncover a mechanism by which PRMT7 is tightly controlled to ensure the timely activation of antiviral defense.

Accurate control of innate immune responses is required to eliminate invading pathogens and simultaneously avoid autoinflammation and autoimmune diseases. Here, we demonstrate that the arginine monomethylation, a relative barely studied protein modification in innate immunity, precisely regulates mitochondrial antiviral-signaling protein (MAVS)-mediated antiviral response. PRMT7 forms aggregates to catalyze MAVS monomethylation at arginine residue 52 (R52), attenuating its binding to TRIM31 and RIG-I, which leads to the suppression of MAVS aggregation, and subsequent activation. Upon virus infection, aggregated PRMT7 is disabled timely due to automethylated at arginine residue 32 (R32), and SMURF1 is recruited to PRMT7 by MAVS to induce proteasomal degradation of PRMT7, resulting in the relief of PRMT7 suppression on MAVS activation. PRMT7 inhibitor SGC3027 can protect mice from virus infection by blocking enzymatic activity of PRMT7. Therefore, we not only reveal that arginine monomethylation by PRMT7 negatively regulates MAVS-mediated antiviral signaling in vitro and in vivo, but also uncover a mechanism by which PRMT7 is tightly controlled to ensure timely activation of antiviral defense.

Burkholderia pseudomallei is a zoonotic pathogen that usually affects patients' lungs and causes serious melioidosis. The interaction of B. pseudomallei with its hosts is complex, and cellular response to B. pseudomallei infection in humans still remains to be elucidated. In this study, transcriptomic profiling of B. pseudomallei-infected human lung epithelial A549 cells was performed to characterize the cellular response dynamics during the early infection (EI) stage. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed by using the online databases DAVID 6.8 and KOBAS 3.0. Real-time quantitative PCR and western blot were used for validation experiments. Compared with the negative control group (NC), a set of 36 common genes varied over time with a cut-off level of 1.5-fold change, and a P-value < 0.05 was identified. Bioinformatics analysis indicated that the PERK-mediated unfolded protein response (UPR) was enriched as the most noteworthy biological process category, which was enriched as a branch of UPR in the signaling pathway of protein processing in the endoplasmic reticulum. Other categories, such as inflammatory responses, cell migration, and apoptosis, were also focused. The molecular chaperone Bip (GRP78), PERK, and PERK sensor-dependent phosphorylation of eIF2α (p-eIF2α) and ATF4 were verified to be increasing over time during the EI stage, suggesting that B. pseudomallei infection activated the PERK-mediated UPR in A549 cells. Collectively, these results provide important initial insights into the intimate interaction between B. pseudomallei and lung epithelial cells, which can be further explored toward the elucidation of the cellular mechanisms of B. pseudomallei infections in humans.

There were errors in [Development (2019) 146, dev179572][1] ([doi:10.1242/dev.179572][2]). In the supplementary information, the primer sequences for sox9a in Table S1 were not correct. Corrected: sox9a forward primer (5′→3′), GTCTGATGCACCCAGTCCG; sox9a reverse primer (5′→3′)

RLR‐mediated type I IFN production plays a pivotal role in innate antiviral immune responses, where the signaling adaptor MAVS is a critical determinant. Here, we show that MAVS is a physiological substrate of SIRT5. Moreover, MAVS is succinylated upon viral challenge, and SIRT5 catalyzes desuccinylation of MAVS. Mass spectrometric analysis indicated that Lysine 7 of MAVS is succinylated. SIRT5‐catalyzed desuccinylation of MAVS at Lysine 7 diminishes the formation of MAVS aggregation after viral infection, resulting in the inhibition of MAVS activation and leading to the impairment of type I IFN production and antiviral gene expression. However, the enzyme‐deficient mutant of SIRT5 (SIRT5‐H158Y) loses its suppressive role on MAVS activation. Furthermore, we show that Sirt5‐deficient mice are resistant to viral infection. Our study reveals the critical role of SIRT5 in limiting RLR signaling through desuccinylating MAVS.

The hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) are master regulators of the cellular response to O2. In addition to HIF-1α and HIF-2α, HIF-3α is another identified member of the HIF-α gene family. Even though whether some HIF-3α isoforms have transcriptional activity or repressive activity is still under debate, it is evident that the full length of HIF-3α acts as a transcription factor. However, its function in hypoxia signaling is largely unknown. Here, we showed that loss of hif-3α in zebrafish reduced hypoxia tolerance. Further assays indicated that erythrocyte number was decreased because red blood cell maturation was impeded by hif-3α disruption. We found that gata-1 expression was downregulated in hif-3α-null zebrafish, as were several hematopoietic marker genes, including alas2, band3, hbae1, hbae3 and hbbe1. hif-3α recognized the hypoxia response element (HRE) located in the promoter of gata-1 and directly bound to the promoter to transactivate gata-1 expression. Our results suggested that hif-3α facilities hypoxia tolerance by modulating erythropoiesis via gata-1 regulation.

The hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) are master regulators of the cellular response to O

FOXO3a, a member of the forkhead homeobox type O (FOXO) family of transcriptional factors, regulates cell survival in response to DNA damage, caloric restriction, and oxidative stress. The von Hippel-Lindau (VHL) tumor suppressor gene encodes a component of the E3 ubiquitin ligase complex that mediates hypoxia-inducible factor α degradation under aerobic conditions, thus acting as one of the key regulators of hypoxia signaling. However, whether FOXO3a impacts cellular hypoxia stress remains unknown. Here we show that FOXO3a directly binds to the VHL promoter and up-regulates VHL expression. Using a zebrafish model, we confirmed the up-regulation of vhl by foxo3b, an ortholog of mammalian FOXO3a. Furthermore, by employing the clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease Cas9 (CRISPR/Cas9) technology, we deleted foxo3b in zebrafish and determined that expression of hypoxia-inducible genes was affected under hypoxia. Moreover, foxo3b-null zebrafish exhibited impaired acute hypoxic tolerance, resulting in death. In conclusion, our findings suggest that, by modulating hypoxia-inducible factor activity via up-regulation of VHL, FOXO3a (foxo3b) plays an important role in survival in response to hypoxic stress.

Summary Deregulation of MYC plays an essential role in T cell acute lymphoblastic leukemia (T-ALL), yet the mechanisms underlying its deregulation remain elusive. Herein, we identify a molecular mechanism responsible for reciprocal activation between Aurora B kinase (AURKB) and MYC. AURKB directly phosphorylates MYC at serine 67, counteracting GSK3β-directed threonine 58 phosphorylation and subsequent FBXW7-mediated proteasomal degradation. Stabilized MYC, in concert with T cell acute lymphoblastic leukemia 1 (TAL1), directly activates AURKB transcription, constituting a positive feedforward loop that reinforces MYC-regulated oncogenic programs. Therefore, inhibitors of AURKB induce prominent MYC degradation concomitant with robust leukemia cell death. These findings reveal an AURKB-MYC regulatory circuit that underlies T cell leukemogenesis, and provide a rationale for therapeutic targeting of oncogenic MYC via AURKB inhibition.

Niemann-Pick type C disease (NPC) is a rare human disease, with limited effective treatment options. Most cases of NPC disease are associated with inactivating mutations of the NPC1 gene. However, cellular and molecular mechanisms responsible for the NPC1 pathogenesis remain poorly defined. This is partly due to the lack of a suitable animal model to monitor the disease progression. In this study, we used CRISPR to construct an NPC1 −/− zebrafish model, which faithfully reproduced the cardinal pathological features of this disease. In contrast to the wild type (WT), the deletion of NPC1 alone caused significant hepatosplenomegaly, ataxia, Purkinje cell death, increased lipid storage, infertility and reduced body length and life span. Most of the NPC1 −/− zebrafish died within the first month post fertilization, while the remaining specimens developed slower than the WT and died before reaching 8 months of age. Filipin-stained hepatocytes of the NPC1 −/− zebrafish were clear, indicating abnormal accumulation of unesterified cholesterol. Lipid profiling showed a significant difference between NPC1 −/− and WT zebrafish. An obvious accumulation of seven sphingolipids was detected in livers of NPC1 −/− zebrafish. In summary, our results provide a valuable model system that could identify promising therapeutic targets and treatments for the NPC disease.

Forkhead box O (FOXO)3, a member of the FOXO family of transcription factors, plays key roles in various cellular processes, including development, longevity, reproduction, and metabolism. Recently, FOXO3 has also been shown to be involved in modulating the immune response. However, how FOXO3 regulates immunity and the underlying mechanisms are still largely unknown. In this study, we show that zebrafish (Danio rerio) foxo3b, an ortholog of mammalian FOXO3, is induced by polyinosinic-polycytidylic acid stimulation and spring viremia of carp virus (SVCV) infection. We found that foxo3b interacted with irf3 and irf7 to inhibit ifr3/irf7 transcriptional activity, thus resulting in suppression of SVCV or polyinosinic-polycytidylic acid–induced IFN activation. By suppressing expression of key antiviral genes, foxo3b negatively regulated the cellular antiviral response. Furthermore, upon SVCV infection, the expression of the key antiviral genes was significantly enhanced in foxo3b-null zebrafish larvae compared with wild-type larvae. Additionally, the replication of SVCV was inhibited in foxo3b-null zebrafish larvae, leading to a higher survival rate. Our findings suggest that by suppressing irf3/irf7 activity, zebrafish foxo3b negatively regulates the antiviral response, implicating the vital role of the FOXO gene family in innate immunity.

Delay Tolerant Network (DTN) was developed as a proposal for Interplanetary Network (IPN) communication at first. Its primary objective is to support the interconnection and intercommunication in highly stressed network environments including intermittent connectivity, large and/or variable delays, heterogeneous transmission rate, and high bit error rates. DTN researchers have relied heavily on simulation for research. The research object of this paper is to construct a DTN testbed, offer a real experiment environment for research like routing algorithm and DTN application development. This research construct a multiple nodes DTN experiment environment among Linux machines by using the DTN2 implement public released by DTNRG (DTN Research Group), and successfully accomplish different transmission experiments. The experiment simulates intermittent connective network of three-endpoint, we successfully accomplish one transmission experiment base on this DTN testbed. In addition, we selected HBSD as an external routing protocol, and added it to the experimental platform. Using the external routing protocol we successfully accomplish another transmission experiment. The DTN constructed using DTN2 implement is basically finish all BP functions.