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

Innate immunity is the host’s first line of defense against pathogen infection. Germline-encoded cellular pattern recognition receptors recognize conserved microbial components, such as virus-derived nucleic acids, to initiate host antimicrobial response (1, 2). Viral RNAs are mainly sensed by the retinoic acid–inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I and melanoma differentiation-associated gene 5 (MDA5), respectively (3–6). Activation of RIG-I and MDA5 leads to the recruitment of the signaling adaptor protein MAVS (also referred to as IPS-1, VISA, or Cardif) to activate the downstream protein kinases TBK1/IKKε (7–10). These kinases phosphorylate the transcription factor IRF3 and the inhibitor of NF-κB, leading to activation of IRF3 and NF-κB and induction of type I IFNs, proinflammatory cytokines, and other downstream effector genes (1, 2). Innate immunity needs to be tightly regulated to efficiently protect the host from infections and concomitantly avoid excessive immunopathology (1, 11–14).

Sirtuin 5 (SIRT5) is one of the seven members of the sirtuin family proteins (SIRT1–SIRT7), which are mammalian homologs of the yeast protein Sirt2 and NAD (NAD⁺)-dependent histone deacetylases (15, 16). Sirtuins regulate diverse cellular and biological functions, including cell metabolism, cell proliferation, gene regulation, cell division, cellular stress response, and tumor development (17, 18). In fact, among these sirtuin family proteins, the deacetylase activity of SIRT5 is barely detected (19). It is reported that SIRT5 can promote acetylation of p65 through blocking the deacetylation of p65 catalyzed by SIRT2 in a deacetylase activity-independent manner (20). Interestingly, the potent desuccinylase activity has been revealed for SIRT5 (17, 21–26). LPS decreases SIRT5 expression in macrophages and increases protein succinylation (24). SIRT5-deficient mice exhibit hypersuccinylation and increased IL-1β production (25).

Recently, by mammalian models, we show that SIRT5 negatively regulates innate immunity by catalyzing desuccinylation of Lys-7 of MAVS in response to RNA viral infection (26). Because Lys-7 of MAVS is not evolutionarily conserved in zebrafish mavs, we are interested in determining the role of zebrafish sirt5 in antiviral innate immunity. In this study, we found that zebrafish sirt5 also negatively regulated antiviral innate immunity by attenuating phosphorylation and ubiquitination of zebrafish mavs.

Zebrafish liver (ZFL) cells (American Type Culture Collection) were cultured at 28°C in a humidified incubator containing 5% CO₂ using Ham’s F12 nutrient mixture medium (Invitrogen) supplemented with 10% FBS. Epithelioma papulosum cyprini (EPC) cells (derived from skin of cyprinid fathead minnow Pimephales promelas) (American Type Culture Collection) were cultured using M199 medium (HyClone) supplemented with 10% FBS and maintained at 28°C in a humidified incubator containing 5% CO₂. HEK293T cells were cultured using DMEM (HyClone) supplemented with 10% FBS and maintained at 37°C in a humidified incubator containing 5% CO₂. Spring viremia of carp viruses (SVCVs, ssRNA viruses that cause severe diseases affecting cyprinids) were propagated in EPC cells. The culture medium containing SVCVs was collected and stored at −80°C until use.

For viral infection of EPC cells, the cells were grown overnight and transfected with indicated plasmids. After 24 h, the cells were infected with SVCV (∼2.0 × 10⁸ 50% tissue culture infective dose (TCID₅₀)/ml) for indicated times, and then the assays were conducted.

For viral infection of zebrafish larvae, zebrafish larvae (3 days post-fertilization [dpf]) were placed in a disposable 60-mm cell culture dish filled with 5 ml egg water and 2 ml SVCV (∼2.0 × 10⁸ TCID₅₀/ml) culture medium. After incubation at 28°C for 18 h, the larvae were photographed.

For viral injection of adult zebrafish, 5-mo-old zebrafish were i.p. injected with SVCV (∼2.0 × 10⁸ TCID₅₀/ml) at 10 μl/individual. An i.p. injection with cell culture medium was used as the control.

Zebrafish (AB strain) were raised and maintained in a recirculating water system following standard protocols. All experiments with zebrafish were approved by the Institutional Animal Care and Use Committee of Institute of Hydrobiology, Chinese Academy of Sciences under the protocol number 2020-005.

The open reading frame of zebrafish sirt5 (accession number: NM_001002605, https://www.ncbi.nlm.nih.gov/genbank/) was amplified from AB zebrafish larvae cDNAs and subcloned into pCMV-Flag and pCMV-Myc (Clontech). Similarly, zebrafish mavs (accession number: NM_001327873), tbk1 (accession number: NM_001044748), and yod1 (accession number: NM_001017716) were subcloned into pCMV-Flag, pCMV-HA, and pCMV-Myc (Clontech), respectively. All of these constructs were confirmed for accuracy by DNA sequencing.

CRISPR/Cas9 was used to knock out sirt5 in zebrafish. Sirt5 single guide RNA (sgRNA) was designed using an online CRISPR design tool (http://crispr.mit.edu). The zebrafish Codon-Optimized Cas9 plasmid was digested with XbaI, then purified, and transcribed using the T7 mMessagem Machine Kit (Ambion). pUC19-guide RNA vector was used for amplifying sirt5 sgRNA. The primers for amplifying sirt5 sgRNA template were: 5′-GTAATACGACTCACTATAGCTTCTGGAGGAAATGGCGTTTTAGAGCTAGAAATAGC-3′ and 5′-AAAAGCACCGACTCGGTGCC-3′. The Transcript Aid T7 High Yield Transcription Kit (Fermentas) was used to synthesize sgRNA. Cas9 RNA (0.75–1.25 ng/embryo) and sgRNA (0.075 ng/embryo) were mixed and injected into embryos at the one-cell stage. The mutants were initially detected using an HMA (heteroduplex mobility assay). If the HMA results were positive, the remaining embryos were raised up to adulthood as the F0 generation and then back-crossed with wild-type (WT) zebrafish (strain AB) to generate the F1 generation, which was genotyped by HMA initially and confirmed by sequencing of target sites. Heterozygous F1s were back-crossed with WT (disallowing offspring–parent mating) to generate the F2 generation. F2 adults carrying the target mutation (^+/−) were intercrossed to generate F3 offspring. The F3 generation contained WT (^+/+), heterozygous (^+/−), and homozygous (^−/−) individuals. The zebrafish sirt5 mutants were named following the zebrafish nomenclature guidelines. One mutant was obtained, namely, ihbxys5 (https://zfin.org/ZDB-ALT-210929-4).

Total protein of HEK293T cells was extracted with radioimmunoprecipitation assay buffer containing 50 mM Tris (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA (pH 8), 150 mM NaCl, 1 mM NaF, 1 mM PMSF, 1 mM Na₃VO₄, and a 1:100 dilution of protease inhibitor mixture (Sigma-Aldrich). The following Abs were used for immunoblotting: anti–c-Myc (1:1000, sc-40; Santa Cruz Biotechnology), anti-Flag (1:5000; F1804; Sigma) and anti–β-actin (1:20,000, AC026; ABclonal Technology), anti-ubiquitin (1:1000, 3936; Cell Signaling Technology), and anti-Histone H3 (1:1000, 4499; Cell Signaling Technology). Anti-sirt5 and anti-mavs Abs were generated by immunizing rabbit with bacterially expressed zebrafish sirt5 or mavs protein as indicated Ag (ABclonal Technology). The cell lysates were separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane (Millipore), blocked with 5% (w/v) nonfat milk, probed with indicated primary Abs and corresponding secondary Abs, visualized by ECL Western blotting detection reagent (Millipore), and photographed by Fuji Film LAS4000 mini-luminescent image analyzer.

Total RNA was extracted using RNAiso Plus (TaKaRa Bio., Beijing, China) following the protocol provided by the manufacturer. cDNAs were synthesized using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA). MonAmp SYBR Green qPCR Mix (high Rox) (Monad Bio., Shanghai, China) was used for quantitative real-time PCR (qRT-PCR) assays. The primers for qRT-PCR are listed in Supplemental Table I.

EPC cells were grown in 24-well plates and transfected with various amounts of plasmids by VigoFect (Vigorous Biotech., Beijing, China), as well as with CMV-Renilla used as a control. The ratio between the luciferase reporters (ISRE-Luc, Dr-IFNφ1-Luc, or EPC-IFN-Luc) and CMV-Renilla is 10:1, and pCMV-Myc empty was used to ensure equivalent amounts of total DNA in each well. After the cells were transfected for 24 h, SVCV infection was performed as indicated for 24 h, and then the cells were harvested and the luciferase activity was determined by the Dual-Luciferase Reporter Assay System (Promega). Data were normalized to Renilla luciferase. Data are representative of three independent experiments (mean ± SD).

For analysis of the ubiquitination of mavs in HEK293T cells, HEK293T cells were transfected with plasmids expressing Flag-sirt5, Myc-mavs, and His-ubiquitin for 24 h and then lysed by denatured buffer (6 M guanidine-HCl, 0.1 M Na₂HPO₄/NaH₂PO₄, 10 mM imidazole), followed by nickel bead purification and immunoblotting with the indicated Abs. Ubiquitination assays with His-ubiquitin were performed by affinity purification using Ni²⁺-NTA resin (Novogen) as described previously (27) and Western blot analysis with the indicated Abs.

For ubiquitination assay in sirt5-deficient or WT zebrafish (sirt5^−/− or sirt5^+/+), zebrafish larvae (7 dpf) were infected with SVCV for 18 h, then collected and lysed with lysis buffer (100 μl). The supernatants were denatured at 95°C for 5 min in the presence of 1% SDS. The denatured lysates were diluted with lysis buffer to reduce the concentration of SDS (<0.1%). Immunoprecipitation (denature-IP) was conducted using anti-mavs Ab and then subjected to immunoblotting with anti-ubiquitin Ab.

Unpaired Student t test and log-rank (Mantel–Cox) test were used for statistical analysis with GraphPad Prism 6 Software. All data are representative of three independent experiments performed in triplicates. A p value <0.05 was considered significant: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

We have previously shown that mammalian SIRT5 acts as a desuccinylase to negatively regulate RLR signaling through desuccinylating MAVS at Lys-7 (26). Given that SIRT5 is evolutionarily conserved between mammal and zebrafish (Supplemental Fig. 1), but the succinylated lysine residue is not conserved between mammal and zebrafish (Supplemental Fig. 2), we were interested in determining whether zebrafish sirt5 has an impact similar to that of mammalian SIRT5 on RLR signaling and whether zebrafish sirt5 acts its function on RLR signaling through a mechanism other than that of mammalian SIRT5 exhibited. Initially, we examined sirt5 expression in response to SVCV infection and polyriboinosinic:polyribocytidylic acid [poly (I:C)] treatment. As shown in (Fig. 1, SVCV infection and poly(I:C) treatment in ZFL cells caused sirt5 expression to steadily increase, even though it was not as dramatic as those of typical antiviral responsive genes (Fig. 1). These data implicate that zebrafish sirt5 may be involved in RLR signaling.

To determine the impact of zebrafish sirt5 in RLR signaling, we employed promoter assays. In EPC cells, overexpression of zebrafish sirt5 suppressed ISRE, Dr-IFNφ1, and EPC-IFN (28–30) reporter activity induced by SVCV infection (Fig. 2A–2C). Consistently, overexpression of zebrafish sirt5 reduced expression of ifn, a typical irf3/irf7 downstream gene, and two typical IFN-stimulated genes (viperin and isg15) in EPC cells after challenge with SVCV via qRT-PCR assays (28) (Fig. 2D–2F). These data suggest that zebrafish sirt5 negatively regulates RLR signaling.

To determine the physiological function of sirt5 in vivo, we disrupted sirt5 in zebrafish via CRISPR/Cas9 and obtained one mutant line (Supplemental Fig. 3A–3D). By crossing sirt5^+/− (♀) × sirt5^+/− (♂), the offspring with sirt5^+/+, sirt5^+/−, and sirt5^−/− genetic backgrounds were born at a mendelian ratio (1:2:1), and no obvious defects in growth rate and reproduction capability were detected in sirt5^−/− zebrafish under normal conditions. We challenged sirt5^−/− larvae and sirt5^+/+ larvae (having WT allele of sirt5) with high-titer SVCV and photographed the larvae after 18 h (Fig. 3A). The dead larvae exhibited no movement, no blood circulation, and a degenerated body (Fig. 3A). Subsequently, we i.p. injected SVCV, as well as using cell culture medium as control, into sirt5^+/+ and sirt5^−/− adult zebrafish and then observed their phenotype. Compared with SVCV-injected sirt5^+/+ adult zebrafish (5 months post-fertilization [mpf]), SVCV-injected sirt5^−/− adult zebrafish (5 mpf) exhibited less swelling and fewer hemorrhagic symptoms in the abdomen (Fig. 3B). At different time points after SVCV injection, we counted dead zebrafish and made a survival curve. As shown in (Fig. 3C, after challenge with SVCV, sirt5^−/− zebrafish displayed a higher survival rate compared with sirt5^+/+ zebrafish.

Subsequently, we examined gene expression in different tissues of these infected zebrafish. In agreement, the key antiviral genes, including ifn1, lta (lymphotoxin α), and mxc (myxovirus resistance c), were enhanced, and the copy number of the P, G, and N genes of SVCV were decreased in sirt5^−/− zebrafish liver compared with that of sirt5^+/+ zebrafish (Fig. 4A–4F). Similarly, expression levels of ifn1, lta, and mxc were also enhanced in sirt5^−/− zebrafish spleen and kidney compared with those of sirt5^+/+ zebrafish (Figs. 4G–4L).

Taken together, these data suggest that zebrafish sirt5 negatively regulates antiviral response in vivo.

As reported previously, both RIG-I and MDA5 can recognize the synthetic viral dsRNA analogue poly(I:C). However, MDA5 recognizes the high m.w. (HMW) poly(I:C), whereas RIG-I recognizes the low m.w. (LMW) poly(I:C) and dsRNA with the 5′-triphosphate group (31). We used poly(I:C)-HMW and poly(I:C)-LMW to stimulate EPC cells, respectively, and then examined the effect of sirt5 on IFN activation by checking expression of key antiviral genes, including ifn, viperin, and isg15, via qRT-PCR assays. In fact, overexpression of sirt5 could suppress IFN activation induced by both poly(I:C)-HMW and poly(I:C)-LMW (Fig. 5A–5F). These data further suggest that zebrafish sirt5 regulates both rig-I– and mad5-mediated RLR signaling.

Notably, in mammalian cells, MAVS is the target of SIRT5 to act its role in RLR signaling; thus, zebrafish sirt5 might also affect RLR signaling by targeting zebrafish mavs. However, Lys-7 in MAVS desuccinylated by SIRT5 is not evolutionarily conserved in zebrafish (Supplemental Fig. 2). Therefore, zebrafish sirt5 might affect mavs activation through other mechanisms instead of desuccinylating Lys-7 of mavs. Initially, we examined whether zebrafish sirt5 interacts with mavs by ectopic expression of sirt5 and mavs in HEK293T cells. IP assays showed that sirt5 interacts with mavs when overexpressed (Fig. 6A, 6B). Moreover, overexpression of sirt5 suppressed mavs-activated ISRE, Dr-IFNφ1, and EPC-IFN promoter activity. These data further support that zebrafish sirt5 targets mavs in RLR signaling.

It is well defined that MAVS could be directly phosphorylated by TBK1, which facilitates IRF3 phosphorylation and activation (8). Therefore, we tested whether sirt5 had an impact on tbk1-catalyzed mavs phosphorylation. Overexpression of sirt5 attenuated phosphorylation of mavs stimulated by tbk1 (Fig. 7A, 7B). In addition, it is reported that K63-linked polyubiquitination of MAVS increases MAVS activation and downstream antiviral signaling (32, 33). Subsequently, we examined whether sirt5 affected mavs ubiquitination. Ubiquitination assays indicated that overexpression of sirt5 diminished polyubiquitination of mavs (Fig. 7C–7E). YOD1, a highly conserved deubiquitinating enzyme of the ovarian tumor family, was reported to be recruited to mitochondria on RNA virus infection to cleave the K63-linked ubiquitination of MAVS and then suppress phosphorylation of MAVS by TBK1 (34). To understand the mechanisms of sirt5 in affecting mavs polyubiquitination, we examined the effect of sirt5 on interaction between mavs and yod1. Overexpression of sirt5 enhanced yod1 binding to mavs (Fig. 7F, 7G), which might be one of the explanations about how sirt5 attenuates mavs ubiquitination and subsequent phosphorylation.

Taken together, these data suggest that zebrafish sirt5 attenuates phosphorylation and ubiquitination to suppress RLR signaling.

Similar to what we observed in mammalian models, zebrafish sirt5 also negatively regulated antiviral innate immunity by targeting mavs, indicating a conserved function of sirt5 in antiviral response. However, the specific desuccinylated residue catalyzed by SIRT5 identified in mammalian MAVS does not exist in zebrafish mavs, suggesting a different mechanism might account for zebrafish sirt5 in acting its role in antiviral innate immunity. Given that aggregation, phosphorylation, and ubiquitination are not only successive connected events during the process of MAVS activation but also prerequired for MAVS activation (2, 8, 35, 36), mammalian and zebrafish sirt5 might affect all these aspects of mavs in response to viral infection. In this article, we show that zebrafish sirt5 could attenuate phosphorylation and ubiquitination of mavs. Due to the lack of an anti–p-mavs Ab, we were unable to directly detect phosphorylation of endogenous mavs. Alternatively, we examined the effect of sirt5 on mavs phosphorylation by cotransfection experiment. Probably because of the relative low sensitivity of this method, we could not detect phosphorylation of mavs in sirt5^−/− and sirt5^+/+ zebrafish mutants by this method. Furthermore, we found that zebrafish sirt5 enhanced yod1 binding to mavs, which might provide an explanation about how sirt5 diminishes mavs ubiquitination and subsequent phosphorylation. However, we have no data to show how zebrafish sirt5 directly affects mavs, but it cannot be ruled out that zebrafish sirt5 desuccinylates zebrafish mavs at other lysine residue(s). In addition, the specific phosphorylation residue catalyzed by TBK1 in mammalian MAVS (S442 in human) is not identified in zebrafish mavs. But zebrafish tbk1 does induce phosphorylation of mavs; therefore, other phosphorylation site(s) by zebrafish tbk1 may exist in zebrafish mavs, which needs to be further explored. Alternatively, zebrafish might affect mavs function rather than acting on its desuccinylase activity. Further determining the process and the underlying mechanism of zebrafish sirt5 in negatively regulating antiviral innate immunity will give us a full picture about the function of SIRT5 in innate antiviral immune response.

Multiple lines of evidence support that the functions of most of the genes are evolutionarily conserved between mammal and zebrafish (37). This is actually the basis for developing human disease models using zebrafish (38, 39). However, divergent functions or even complete opposite functions have also been revealed between mammalian and zebrafish genes. For example, disruption of p65 in mice causes embryonic mortality (40), but p65-dificient zebrafish grow and reproduce normally (41). In addition, zebrafish IκB kinase 1 negatively regulates NF-κB activity, in contrast with mammalian IKK1 (42). In this study, we find that zebrafish sirt5 and mammalian SIRT5 have similar function in antiviral innate immunity, but they might act their functions through different mechanisms, further highlighting that we must be aware of the difference of function or mechanism between mammalian and zebrafish genes. Particularly, when we use zebrafish models to investigate human diseases, as well as screen therapeutic drugs, we should always be concerned with this difference. Furthermore, it is always needed to keep in mind whether the biochemical observations in the human context exactly mimic what is going on in the natural zebrafish context.

Virus infection in fish causes tremendous losses in the aquaculture industry (43–45). In this study, we found that sirt5-deficient zebrafish were more resistant to virus infection, but their growth, development, and reproduction were compatible with their WT siblings. Therefore, fish sirt5 might be a good candidate for cultivating disease-resistant fish strains. Through disrupting sirt5 in aquaculture fish by CRISPR/Cas9 techniques, we might obtain fish strains with virus-resistant ability. This application will greatly benefit the aquaculture industry.

We thank Dr. Hongbing Shu for providing reagents.

The authors have no financial conflicts of interest.