Arginine monomethylation by PRMT7 controls MAVS-mediated antiviral innate immunity.

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.

Dual modifying of MAVS at lysine 7 by SIRT3-catalyzed deacetylation and SIRT5-catalyzed desuccinylation orchestrates antiviral innate immunity.

To effectively protect the host from viral infection while avoiding excessive immunopathology, the innate immune response must be tightly controlled. However, the precise regulation of antiviral innate immunity and the underlying mechanisms remain unclear. Here, we find that sirtuin3 (SIRT3) interacts with mitochondrial antiviral signaling protein (MAVS) to catalyze MAVS deacetylation at lysine residue 7 (K7), which promotes MAVS aggregation, as well as TANK-binding kinase I and IRF3 phosphorylation, resulting in increased MAVS activation and enhanced type I interferon signaling. Consistent with these findings, loss of Sirt3 in mice and zebrafish renders them more susceptible to viral infection compared to their wild-type (WT) siblings. However, Sirt3 and Sirt5 double-deficient mice exhibit the same viral susceptibility as their WT littermates, suggesting that loss of Sirt5 in Sirt3-deficient mice may counteract the increased viral susceptibility displayed in Sirt3-deficient mice. Thus, we not only demonstrate that SIRT3 positively regulates antiviral immunity in vitro and in vivo, likely via MAVS, but also uncover a previously unrecognized mechanism by which SIRT3 acts as an accelerator and SIRT5 as a brake to orchestrate antiviral innate immunity.

Asymmetric arginine dimethylation of cytosolic RNA and DNA sensors by PRMT3 attenuates antiviral innate immunity.

The cytosolic RNA and DNA sensors initiate type I interferon signaling when binding to RNA or DNA. To effectively protect the host against virus infection and concomitantly avoid excessive interferonopathy at resting states, these sensors must be tightly regulated. However, the key molecular mechanisms regulating these sensors' activation remain elusive. Here, we identify PRMT3, a type I protein arginine methyltransferase, as a negative regulator of cytosolic RNA and DNA sensors. PRMT3 interacts with RIG-I, MDA5, and cGAS and catalyzes asymmetric dimethylation of R730 on RIG-I, R822 on MDA5, and R111 on cGAS. These modifications reduce RNA-binding ability of RIG-I and MDA5 as well as DNA-binding ability and oligomerization of cGAS, leading to the inhibition of downstream type I interferon production. Furthermore, mice with loss of one copy of Prmt3 or in vivo treatment of the PRMT3 inhibitor, SGC707, are more resistant to RNA and DNA virus infection. Our findings reveal an essential role of PRMT3 in the regulation of antiviral innate immunity and give insights into the molecular regulation of cytosolic RNA and DNA sensors' activation.

USP38 promotes deubiquitination of K11-linked polyubiquitination of HIF1α at Lys769 to enhance hypoxia signaling.

HIF1α is one of the master regulators of the hypoxia signaling pathway and its activation is regulated by multiple post-translational modifications (PTMs). Deubiquitination mediated by deubiquitylating enzymes (DUBs) is an essential PTM that mainly modulates the stability of target proteins. USP38 belongs to the ubiquitin-specific proteases (USPs). However, whether USP38 can affect hypoxia signaling is still unknown. In this study, we used quantitative real-time PCR assays to identify USPs that can influence hypoxia-responsive gene expression. We found that overexpression of USP38 increased hypoxia-responsive gene expression, but knockout of USP38 suppressed hypoxia-responsive gene expression under hypoxia. Mechanistically, USP38 interacts with HIF1α to deubiquitinate K11-linked polyubiquitination of HIF1α at Lys769, resulting in stabilization and subsequent activation of HIF1α. In addition, we show that USP38 attenuates cellular ROS and suppresses cell apoptosis under hypoxia. Thus, we reveal a novel role for USP38 in the regulation of hypoxia signaling.

Zebrafish mavs Is Essential for Antiviral Innate Immunity.

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.

SIRT5 impairs aggregation and activation of the signaling adaptor MAVS through catalyzing lysine desuccinylation.

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.

Zebrafish sirt5 Negatively Regulates Antiviral Innate Immunity by Attenuating Phosphorylation and Ubiquitination of 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.

Zebrafish Hif3α modulates erythropoiesis via regulation of gata1 to facilitate hypoxia tolerance.

The hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) are master regulators of the cellular response to O2. In addition to HIF1α and HIF2α, HIF3α is another identified member of the HIFα family. Even though the question of whether some HIF3α isoforms have transcriptional activity or repressive activity is still under debate, it is evident that the full length of HIF3α acts as a transcription factor. However, its function in hypoxia signaling is largely unknown. Here, we show that loss of hif3a in zebrafish reduced hypoxia tolerance. Further assays indicated that erythrocyte number was decreased because red blood cell maturation was impeded by hif3a disruption. We found that gata1 expression was downregulated in hif3a null zebrafish, as were several hematopoietic marker genes, including alas2, band3, hbae1, hbae3 and hbbe1 Hif3α recognized the hypoxia response element located in the promoter of gata1 and directly bound to the promoter to transactivate gata1 expression. Our results suggested that hif3a facilities hypoxia tolerance by modulating erythropoiesis via gata1 regulation.

Zebrafish prmt2 Attenuates Antiviral Innate Immunity by Targeting traf6.

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.

Zebrafish otud6b Negatively Regulates Antiviral Responses by Suppressing K63-Linked Ubiquitination of irf3 and irf7.

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.

Zebrafish sirt7 Negatively Regulates Antiviral Responses by Attenuating Phosphorylation of irf3 and irf7 Independent of Its Enzymatic Activity.

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.

Zebrafish prmt5 arginine methyltransferase is essential for germ cell development.

Protein arginine methyltransferase 5 (Prmt5), a type II arginine methyltransferase, symmetrically dimethylates arginine in nuclear and cytoplasmic proteins. Prmt5 is involved in a variety of cellular processes, including ribosome biogenesis, cellular differentiation, germ cell development and tumorigenesis. However, the mechanisms by which prmt5 influences cellular processes have remained unclear. Here, prmt5 loss in zebrafish led to the expression of an infertile male phenotype due to a reduction in germ cell number, an increase in germ cell apoptosis and the failure of gonads to differentiate into normal testes or ovaries. Moreover, arginine methylation of the germ cell-specific proteins Zili and Vasa, as well as histones H3 (H3R8me2s) and H4 (H4R3me2s), was reduced in the gonads of prmt5-null zebrafish. This resulted in the downregulation of several Piwi pathway proteins, including Zili, and Vasa. In addition, various genes related to meiosis, gonad development and sexual differentiation were dysregulated in the gonads of prmt5-null zebrafish. Our results revealed a novel mechanism associated with prmt5, i.e. prmt5 apparently controls germ cell development in vertebrates by catalyzing arginine methylation of the germline-specific proteins Zili and Vasa.

Zebrafish NF-κB/p65 Is Required for Antiviral Responses.

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).

Zebrafish prmt7 negatively regulates antiviral responses by suppressing the retinoic acid-inducible gene-I-like receptor signaling.

Arginine methylation is a post-translational modification in histone and nonhistone proteins that can affect numerous cellular activities. Protein arginine methyltransferase 7 (Prmt7), a type III arginine methyltransferase, catalyzes the formation of stable monomethylarginines of histones. The role of PRMT7 in virus-induced innate immunity signaling, however, remains largely unknown. We demonstrate that zebrafish prmt7 could be inhibited by spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV) infection. The overexpression of prmt7 suppresses cellular antiviral responses that are partially dependent on the arginine methyltransferase activity of prmt7. Consistently, prmt7-null zebrafish were more resistant to SVCV or GCRV infection, exhibiting enhanced expression of key antiviral genes and fewer necrotic cells in the liver and kidney upon viral infection. Furthermore, we established a zebrafish model to investigate grass carp hemorrhagic disease. Our findings suggest that by suppressing the RIG-I-like receptors signaling, zebrafish prmt7 negatively regulates antiviral responses, indicating the vital role of prmt7 and its arginine methyltransferase activity in innate immunity.

Zebrafish prmt3 negatively regulates antiviral responses.

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.

Deletion of the fih gene encoding an inhibitor of hypoxia-inducible factors increases hypoxia tolerance in zebrafish.

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.

Model construction of Niemann-Pick type C disease in zebrafish.

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.

Zebrafish foxo3b Negatively Regulates Antiviral Response through Suppressing the Transactivity of irf3 and irf7.

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.

Forkhead Transcription Factor 3a (FOXO3a) Modulates Hypoxia Signaling via Up-regulation of the von Hippel-Lindau Gene (VHL).

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.