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.
Sirtuin 7 (SIRT7) is one of the seven members of the sirtuin family proteins (SIRT1–SIRT7), which are mammalian homologs of the yeast protein Sir2 and NAD (NAD+)-dependent histone deacetylases (1, 2). Sirtuins regulate diverse cellular and biological functions, including cell metabolism, cell proliferation, gene regulation, cell division, cellular stress response, and tumor development (3, 4). Compared with other sirtuin members, the enzymatic activity, the molecular targets, and the physiological function of SIRT7 are less well understood (5). Similar to other sirtuin members, however, the deacetylase activity of SIRT7 has been widely recognized (5, 6). Since the clear deacetylase activity of SIRT7 was first identified, in which SIRT7 deacetylates lysine 18 of histone H3 involved in the stabilization of transformed cancer cells (5), a wide variety of transcription factors, enzymes, and signaling kinases, including PAF53, ATM, USP39, FKBP51, OSX, GATA4, STRAP, fibrillarin, CDK9, and NPM, have been revealed to be deacetylated targets of SIRT7 (7–16). Apart from its deacetylase activity, its other enzymatic activities have also been reported (17, 18). The function of SIRT7 is linked to cell proliferation, DNA damage repair, metabolic homeostasis, stress resistance, aging, and tumorigenesis (6, 19, 20).
Innate immunity is the first line of defense against viral infection and is activated through the recognition of viral Ags by germline encoded pattern recognition receptors (21, 22). Retinoic acid–inducible gene I–like receptors (RLRs), including retinoic acid–inducible gene I and melanoma differentiation-associated gene 5 (MDA5), sense the viral component in the cytosol upon infection and subsequently trigger mitochondrial antiviral signaling protein (MAVS; also referred to as IPS-1 or VISA)-dependent activation of TANK-binding kinase 1 (TBK1) (23–26). These cascades eventually converge on the activation of transcription factors of the IFN regulatory factor (IRF) family, primarily IRF3 and IRF7, which are master regulators that induce type I IFN gene expression (22). Upon pathogen detection, IRF3 or IRF7 is phosphorylated at several serine and threonine residues by the upstream kinases TBK1 and inhibitor of NF-κB kinase ε. Then, phosphorylated IRF3 or IRF7 undergoes dimerization, leading to the translocation of IRF3 or IRF7 dimers from the cytoplasm to the nucleus, where IFR3 or IRF7 activates the expression of type I IFNs, a family of cytokines essential for host protection against viral infection, and subsequently IFN-stimulated genes (ISGs) (27, 28). During the process of IRF3 and IRF7 activation, each step, including phosphorylation, dimerization, and nuclear translocation, can be regulated by different mechanisms (22). Due to the critical roles of IRF3 and IRF7 in host antiviral innate immunity, the regulatory mechanisms of IRF3 or IRF7 function have undergone extensive study (29).
The function of some sirtuin family proteins in innate immunity has been reported, either dependent on or independent of their enzymatic activity (30–33). Interestingly, SIRT7 can suppress LPS-induced inflammation and apoptosis via the NF-κB signaling pathway (34). Recently, it has been reported that HIV-1 Vpr activates CRL4-DCAF1 E3 ligase to degrade SIRT7. These observations implicate a possible role of SIRT7 in innate immunity. However, the function of SIRT7 in antiviral innate immunity is still poorly understood.
Similar to mammals, zebrafish have the conserved type I IFN signaling in response to viral infection (35–38). Thus, this allows us to use a zebrafish model for investigating IRF regulation in vivo. In this study, we investigated the function of sirt7 in response to viral infection and found that zebrafish sirt7 negatively regulates antiviral response through attenuating phosphorylation of irf3 and irf7. This finding may provide insights into the function and underlying mechanisms of sirt7 in the antiviral response.
Materials and Methods
Cells, zebrafish, and viruses
We cultured zebrafish liver (ZFL) cells (originally obtained from the American Type Culture Collection) in Ham’s F-12 medium (HyClone) supplemented with 1.0 mM L-glutamine and 10% FBS (Biological Industries). We cultured epithelioma papulosum cyprini (EPC) cells (originally obtained from the American Type Culture Collection) in Medium 199 Earle’s Salts Base (Biological Industries) supplemented with 10% FBS. ZFL and EPC cells were maintained at 28°C in a humidified incubator containing 5% carbon dioxide (CO2). Human embryonic kidney (HEK293T) cells (originally obtained from the American Type Culture Collection) were grown in DMEM (Biological Industries) supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO2.
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 the Institute of Hydrobiology, Chinese Academy of Sciences, under protocol number 2019-015.
We propagated spring viremia of carp virus (SVCV; an ssRNA virus that causes severe diseases affecting cyprinids) and determined the titers as described previously (36).
For viral infection of EPC cells, the cells were grown overnight and transfected with the indicated plasmids. After 24 h, the cells were infected with SVCV (∼2.51 × 107 median tissue culture-infective dose [TCID50]/ml) for the indicated times, and then the assays were conducted.
For viral infection of zebrafish larvae, zebrafish larvae (3 d postfertilization [dpf]; n = 40) were placed in a disposable 60-mm cell culture dish filled with 5 ml egg water and 2 ml SVCV (∼2.51 × 107 TCID50/ml) culture medium. After incubation at 28°C for 24 h, the larvae were photographed. For gene expression assays, after the larvae were incubated for the indicated times, total RNAs were extracted from the larvae and quantitative real-time PCR (QRT-PCR) was performed.
For survival ratio assays, zebrafish larvae were placed in a 96-well plate individually, and then 100 μl egg water containing SVCV (5 ml egg water plus 2 ml SVCV [∼2.51 × 107 TCID50/ml] culture medium) was added to each well. The mortality was monitored every 4 h over a 36-h period.
The open reading frame of zebrafish sirt7 (GenBank accession number NC_007133) was amplified from AB zebrafish larvae cDNAs and subcloned into pCMV-Myc (Clontech), pCMV-hemagglutinin (HA) (Clontech), or pCMV-Flag vectors (Clontech). Similarly, zebrafish mda5 (Gene ID: 565759; Ensembl: ENSDARG00000018553), mavs (Gene ID: 562867; Ensembl: ENSDARG00000069733), irf3 (Gene ID: 564854; Ensembl: ENSDARG00000076251), irf7 (Gene ID: 393650; Ensembl: ENSDARG00000045661), and enzymatic activity–deficient mutants of sirt7 (mutant 1 [M1]: S115A; mutant 2 [M2]: H191Y) were subcloned into pCMV-Myc vector, individually. Zebrafish tbk1 (Gene ID: 692289; Ensembl: ENSDARG00000103095) was subcloned into pCMV-Myc and pCMV-Flag vectors. Zebrafish irf3 and irf7 were subcloned into pCMV-HA (Clontech) and pCMV-Flag and PM (Clontech) vectors, respectively. For promoter activity analysis, the gene promoters of zebrafish IFNφ1 (Dr-IFNφ1)-luciferase (luc) and EPC-IFN-luc were cloned and inserted into the KpnI and XhoI sites, respectively, of the pGL3-basic-luc reporter vector (Promega) as described previously (39). The IFN-sensitive response element (ISRE) luciferase reporter construct (ISRE-luc) contains five ISRE motifs in series. To verify the Abs, human IRF3 (Gene ID: 3661; Ensembl: ENSG00000126456) and IRF7 (Gene ID: 3665; Ensembl: ENSG00000185507), amplified from HEK293T cells cDNAs, and common carp irf3 (Gene ID: 109068023; Ensembl: ENSCCRG00025004), amplified from common carp liver cDNAs, were subcloned into pCMV-Myc vector, respectively. In addition, human SIRT7 (Gene ID: 51547; Ensembl: ENSG00000187531) was subcloned into pCMV-HA vector. All of these constructs were confirmed for accuracy by DNA sequencing (40–43). The plasmid and VigoFect (T001; Vigorous Biotech, Beijing, China) were transfected into cells at a ratio of 0.4 μl VigoFect per 1 μg of plasmid.
Total RNA was extracted from cells or embryos using RNAiso Plus (Takara Biomedical Technology, Beijing, China), following the manufacturer’s instruction. In brief, cells or zebrafish larvae were homogenized by adding appropriate amounts of RNAiso Plus. Then, the homogenate was kept at room temperature for 5 min. Next, the homogenate was centrifuged at 12,000 × g for 5 min at 4°C. The supernatant was transferred to a new centrifuge tube and chloroform of 0.2 vol of RNAiso Plus was added. This mixture was kept at room temperature for 5 min after vortexing vigorously. Then, centrifugation was performed at 12,000 × g for 15 min at 4°C, and the upper layer was transferred to a new centrifuge tube. Subsequently, isopropanol with 0.5–1.0 vol of RNAiso Plus was added, and then the mixture was kept at room temperature for 10 min. Next, centrifugation was performed at 12,000 × g for 10 min at 4°C. RNA was washed with an equivalent amount of 75% ethanol. Centrifugation was then performed at 7,500 × g for 5 min at 4°C. The supernatant was discarded, and the precipitate was kept for drying in air. Finally, the transparent precipitate was dissolved with an appropriate amount of diethyl pyrocarbonate–treated water. Extracted RNA was measured for its concentration and for the OD260/OD280 ratio. The OD260/OD280 ratio for all RNAs was in the range of 1.7–2.1.
Equivalent amounts of total RNA (2000 ng) were used for cDNA synthesis with the Revert Aid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) in a 20-µl reaction volume. The synthesized cDNA was used as a template for QRT-PCR analysis. QRT-PCR was performed using the CFX Connect Real-Time PCR System (Bio-Rad Laboratories) with MonAmp SYBR Green qPCR Mix (HIGH Rox; Monad Biotech Co., Shanghai, China) under the following conditions: 95°C for 5 min, followed by 50 cycles at 95°C for 3 s and at 60°C for 15 s. The instrument’s default dissolution curve acquisition program was used to draw the cycle threshold value (44). The changes of gene expression were calculated as the relative fold changes by the comparative cycle threshold method, and the β-actin of each species was used as an internal control gene for normalization. The results were obtained from three independent experiments, and each was performed in triplicate. The primers used are listed in Supplemental Table I.
Luciferase reporter assays
We grew EPC cells in 24-well plates and transfected them with the indicated amounts of vectors, including Renilla as an internal control, by VigoFect (Vigorous Biotech). pER-luc vector was purchased from Stratagene. Luciferase activity was assayed 16–28 h after transfection. The luciferase activity in cell extracts was determined by a dual-luciferase reporter assay system (Promega) according to the protocol supplied by the manufacturer. The relative light units were measured using a luminometer (Sirius; Zylux Corp., Oak Ridge, TN). Data were normalized to Renilla luciferase. Data are reported as means ± SD of three repeated experiments. The statistical analysis was performed using GraphPad Prism 7.0 software (unpaired t tests).
Western blot and immunoprecipitation analyses
Western blot and coimmunoprecipitation analyses were performed as described previously (36). Anti-HA and anti-Flag Ab–conjugated agarose beads were purchased from Sigma-Aldrich. We used the FUJIFILM LAS 4000 mini luminescent image analyzer to image the blots.
Abs and reagents
The Abs used were as follows: anti-sirt7 Ab (Frdbio), anti-Myc Ab (9E10; Santa Cruz Biotechnology), anti-Flag Ab (Sigma-Aldrich), anti-HA Ab (Covance), anti-GAPDH Ab (Santa Cruz Biotechnology), anti–β-actin Ab (AC026; Abclonal), anti-irf3 Ab (A11921; Abclonal), and anti-acetylated lysine Ab (9441S; Cell Signaling Technology). Anti-irf7 was raised by immunization of rabbit with prokaryotically expressed crucian carp IRF7 DNA binding domain, as described previously (45). Polyinosinic-polycytidylic acid (poly I:C) was purchased from InvivoGen (San Diego, CA).
Cytopathic effect assays
EPC cells were transfected with 0.5 μg of Myc-tagged sirt7 or Myc empty vector. After 24 h, cells were infected with SVCV for 48 h at the dose indicated. Then, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Visible plaques were used to monitor viral infection. If the cytopathic effect (CPE) appeared later, the anti-SVCV ability in cells was stronger.
Virus titer determination
EPC cells were seeded into 6-well plates overnight and transfected with Myc-empty (2 μg) or Myc- sirt7 (2 μg), respectively. After 24 h, the cells were infected with SVCV (multiplicity of infection [MOI], 10) for 14 h, and the culture supernatant (containing virus) was collected and stored at −80°C until use. EPC cells were cultured in 96-well plates. The culture supernatant (containing virus) was diluted in serial dilutions (10−1 to 10−11) in sterile 1.5-ml tubes using M-199 medium. Subsequently, the diluted viruses were added to EPC cells seeded into 96-well plates. After 4 d, the plates were observed under a microscope, and the detached cells >50% in one well were counted as positive. The titers for SVCV infection were calculated using the Spearman-Kärber method and represented as TCID50/ml. The experiments were repeated three times for statistical analysis.
Generation of sirt7-null zebrafish
sirt7 ihblqs7/ihblqs7 was generated by CRISPR/Cas9-mediated mutagenesis. We designed sirt7 single-guide RNA (sgRNA) using the CRISPR design tool provided (http://crispr.mit.edu). The zebrafish codon-optimized Cas9 plasmid was digested with XbaI and purified and transcribed using the T7 mMessage Machine Kit (Ambion). pUC19-gRNA vector was used for amplifying the sgRNA template. The primers used for amplifying the gRNA template were as follows: 5'-GTAATACGACTCACTATAGGAGGAGCGGTCGATGCTGCGTTTTAGAGCTAGAAATAGC-3' and 5'-AAAAGCACCGACTCGGTGCC-3'. We used the Transcript Aid T7 High Yield Transcription Kit (Fermentas) to synthesize sgRNA. We mixed Cas9 RNA (0.75–1.25 ng/embryo) with sgRNA (0.075 ng/embryo) and injected it into embryos at the one-cell stage. The sirt7 ihblqs7 allele (40-bp deletion in exon 2) was identified by heteroduplex mobility assay (HMA) using the primers listed in Supplemental Table I (36). If the HMA results were positive, the remaining embryos were raised to adulthood as the F0 generation and were then backcrossed with the wild-type (WT) zebrafish (strain AB) to generate the F1 generation, which was genotyped by HMA initially and confirmed by sequencing of targeting sites. Heterozygous F1s were backcrossed with the WT zebrafish (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 (+/+), heterozygote (+/−), and homozygote (−/−) individuals. The zebrafish sirt7 mutant was named following zebrafish nomenclature guidelines (https://zfin.atlassian.net/wiki/spaces/general/pages/1818394635/ZFIN+Zebrafish+Nomenclature+Conventions). We obtained a mutant: sirt7ihblqs7/ihblqs7 (https://zfin.org/ZDB-ALT-210308-2).
EPC cells were harvested and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and protease inhibitor), and 20 μg total protein was applied for native PAGE after centrifugation at 11,950 × g. Native PAGE was performed with 10% gel without SDS. The gel was prerun with the running buffer (25 mM Tris-HCl, pH 8.4, 192 mM glycine, with or without 0.2% deoxycholate in the cathode and anode buffers, respectively) at 40 V for 30 min. The samples were electrophoresed at 80 V for 3 h in a cold temperature (4°C), followed by transferring onto a membrane for immunoblot analysis.
Mammalian one-hybridization assay
Zebrafish irf3 and irf7 were cloned into pM vector (Clontech), in which irf3 or irf7 is fused with the yeast GAL4 DNA binding domain contained in pM vector. The luciferase reporter plasmid pFR-luc (Stratagene), which contains the Photinus pyralis luciferase gene under the control of five tandem repeats of the GAL4 binding site, was cotransfected with pM-irf3 or pM-irf7 together with sirt7 in HEK293T cells. After 24 h, the cell lysates were subjected to luciferase assays.
Nuclear cytoplasmic fractionation
Nuclear cytoplasmic fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833; Thermo Fisher Scientific) following the manufacturer’s instructions. Briefly, EPC cells were seeded into 6-well plates overnight and transfected with Myc- sirt7 or Myc empty vector (2 μg each), respectively. After 24 h, poly I:C was transfected into cells for 12 h. Subsequently, the cells were washed with ice-cold PBS, lysed in 200 μl of cytoplasmic extraction reagent I, and incubated on ice for 10 min, then 11 μl of cytoplasmic extraction reagent II was added. The lysate was incubated on ice for 1 min and immediately centrifuged at 16,000 × g for 5 min at 4°C. The supernatants were treated as cytoplasmic components. A 100 μl nuclear extraction reagent was added per deposit, and the lysate was incubated on ice for 40 min and then immediately centrifuged at 16,000 × g for 10 min at 4°C. The pellets were treated as nuclei. The endogenous proteins were immunoblotted using the indicated Abs. Anti–α-tubulin Ab was used to monitor cytoplasmic protein, and anti-histone H3 Ab was used to monitor nuclear protein.
The statistical analysis was performed using an unpaired t test in GraphPad Prism 7.00 (GraphPad Software). Data are representative of at least three independent experiments with similar results, and error bars indicate the mean with SD. A p value <0.05 was considered significant. Statistical significance is represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001.
Zebrafish sirt7 is induced upon SVCV infection
In ZFL cells, upon SVCV infection, sirt7 was steadily increased over time (6, 12, and 24 h), similar to the key antiviral genes ifn1, lta, mxc, and pkz (Fig. 1A–(1E) (36). Subsequently, we challenged zebrafish larvae (3 dpf) with SVCV, and we found that sirt7 was also induced (Fig. 1F). Immunoblot analysis indicated that the sirt7 protein level was indeed induced in response to SVCV infection, even though its relative protein level decreased after 12 h of viral infection (Fig. 1G and (1H). These data suggest that zebrafish sirt7 is increased upon viral infection and thus may be involved in the antiviral responses.
Zebrafish sirt7 represses SVCV- or poly I:C–induced IFN activation
To determine the function of sirt7 in response to SVCV infection or poly I:C treatment, we performed promoter assays to evaluate the effect of sirt7 overexpression on SVCV or poly I:C–induced IFN activation. Overexpression of Myc-tagged sirt7 (Myc-sirt7) suppressed ISRE and Dr-IFNφ1 (36) reporter activity induced by SVCV infection or poly I:C transfection in EPC cells (Fig. 2A–(2D).
Subsequently, we examined expression of ifn, a typical irf3/irf7 downstream gene, and two typical ISGs (isg15 and viperin) in EPC cells after challenge with SVCV via QRT-PCR assays (36). Overexpression of sirt7 reduced expression of ifn, isg15, and viperin in a dose-dependent manner, consistent with the promoter assays (Fig. 2E–(2G). Overexpression of sirt7 was confirmed by Western blot assay (Fig. 2H). Similarly, overexpression of sirt7 also suppressed expression of ifn, isg15, and viperin in EPC cells upon poly I:C treatment (Fig. 2I–(2K). These data suggest that IFN activation induced by SVCV or poly I:C is suppressed by overexpression of sirt7.
Zebrafish sirt7 negatively regulates cellular antiviral responses
To determine the effect of zebrafish sirt7 on cellular antiviral response, we performed CPE assays. Overexpression of sirt7 in EPC cells caused enhanced CPE compared with the empty vector control upon challenge with different titers of SVCV (from an MOI of 1 to an MOI of 100) (Fig. 3A). Consistently, the titer of SVCV was significantly increased in the supernatant of sirt7-overexpressed EPC cells as determined by plaque assays (Fig. 3B). The copy numbers of SVCV genes, including N gene, P gene, and G gene, were dramatically increased in sirt7-overexpressed EPC cells as revealed by QRT-PCR assays (Fig. 3C–(3E). These data suggest that zebrafish sirt7 negatively regulates cellular antiviral response by facilitating the replication of SVCV.
Disruption of sirt7 in zebrafish facilitates antiviral responses
Sirt7 is evolutionarily conserved among human, mouse, and zebrafish (Supplemental Fig. 1A). Its predicted catalytic residues are identical from zebrafish to human (S111 and H187 in human; S115 and H191 in zebrafish) (Supplemental Fig. 1A) (5). To determine the physiological function of sirt7 in vivo, we disrupted sirt7 in zebrafish via CRISPR/Cas9 (Supplemental Fig. 1B–1E). In the mutant line, a 40-bp nucleotide was deleted in exon 2 of sirt7, resulting in a reading frame shift to generate a mutated peptide with 75 aa instead of 405 aa encoded by WT sirt7 (Supplemental Fig. 1C). To exclude off-targeting effects, we backcrossed sirt7+/− to WT zebrafish (sirt7+/+) (disallowing offspring–parent mating), and then sirt7+/− zebrafish were intercrossed to obtain sirt7+/+, sirt7+/−, and sirt7−/−. The offspring with sirt7+/+, sirt7+/−, and sirt7−/− genetic backgrounds were born at a Mendelian ratio (1:2:1), and no obvious defects in growth rate and reproductive capability were detected in sirt7−/− zebrafish under normal conditions. Sirt7 mRNA was reduced in sirt7−/− zebrafish compared to sirt7+/+ zebrafish, and no sirt7 protein was detected in sirt7−/− zebrafish, indicating that sirt7 was completely disrupted in sirt7-null zebrafish (Supplemental Fig. 1F and 1G). The specificity of Abs against zebrafish irf3, irf7, and sirt7, respectively, was validated by Western blot assay (Supplemental Fig. 2A–2D).
We challenged sirt7-null larvae (n = 120 in total) and the WT larvae (having WT allele of sirt7; n = 120 in total) with high-titer SVCV and counted the number of dead larvae at different time points. The dead larvae exhibited no movement, no blood circulation, and a degenerated body (Fig. 4A, circled by red rectangles). As shown in (Fig. 4A and (4B, sirt7-null larvae had a higher survival rate than the WT larvae. Consistently, the key antiviral genes, including ifn1, mxc, lta, and pkz were enhanced in sirt7-null larvae compared with the WT larvae after challenge with SVCV (Fig. 4C–(4F) (35, 36).
These data suggest that zebrafish sirt7 negatively regulates the antiviral responses in vivo.
Zebrafish sirt7 negatively regulates antiviral responses mainly through suppressing irf3 and irf7 transactivity
To determine the mechanisms of zebrafish sirt7 in negative regulation of antiviral immunity, we examined the activity of EPC-IFN-luc in EPC cells stimulated by overexpression of cascade factors in the RLR signaling pathway, including mda5, mavs, tbk1, irf3, or irf7, together with empty Myc empty vector or Myc-tagged sirt7. Overexpression of the cascade factors in the RLR signaling pathway significantly activated the activity of EPC-IFN-luc (Fig. 5A). However, coexpression of sirt7 suppressed the activity induced by overexpression of all these factors, from upstream factors to downstream factors. Thus, sirt7 might negatively regulate antiviral immunity by suppressing the transactivity of irf3 and irf7 (36).
To further determine whether irf3 and irf7 activity was influenced by sirt7 directly, we examined their protein–protein interactions. As shown in (Fig. 5B–(5E, coimmunoprecipitation assays indicated that sirt7 associated with irf3 or irf7 (Fig. 5B–(5E). It seemed that sirt7 had no effect on the relative mRNA levels or the protein levels of irf3 and irf7 (Fig. 5B–(5E; Supplemental Fig. S3A–S3D). Moreover, we confirmed that overexpression of sirt7 suppressed the transactivity of irf3 and irf7 by mammalian one-hybridization assay (Fig. 5F and (5G) (46).
These data suggest that zebrafish sirt7 might negatively regulate the antiviral response through binding to irf3 and irf7 and suppressing irf3 and irf7 transactivity.
Zebrafish sirt7 attenuates phosphorylation of orthographic irf3 and orthographic irf7 by preventing orthographic tbk1 binding to orthographic irf3 and orthographic irf7
To get insights into the mechanisms of sirt7 in suppressing the transactivity of irf3 and irf7, we examined whether sirt7 had impacts on irf3 and irf7 phosphorylation and subsequent dimerization. As shown in (Fig. 6A, poly I:C treatment, overexpression of sirt7 attenuated phosphorylation of endogenous irf3 and irf7. Consistently, dimerization of irf7 was also reduced when sirt7 was overexpressed (Fig. 6A). Due to the lack of suitable anti-irf3 Ab that can readily detect endogenous zebrafish irf3, we could not detect the status of endogenous irf3 dimerization.
Subsequently, we found that overexpression of sirt7 attenuated irf7 translocation from cytoplasm to nucleus in response to poly I:C treatment (Fig. 6B). Moreover, we detected that overexpression of sirt7 prevented tbk1 binding to irf3 and irf7 (Fig. 6C and (6D).
These data suggest that zebrafish sirt7 might negatively regulate antiviral responses through attenuating irf3 and irf7 phosphorylation, dimerization, and nuclear translocation. In addition, it seems that through competitive binding, sirt7 prevents tbk1 binding to irf3 and irf7, resulting in the attenuation of irf3 and irf7 phosphorylation.
Zebrafish sirt7 negatively regulates antiviral responses independent of its enzymatic activity
As one of the putative NAD+-dependent deacetylases, the catalytic residues of SIRT7 have been identified (5, 47). To determine whether the negative regulation of antiviral responses by sirt7 is dependent on sirt7 enzymatic activity, we made two enzymatic inactive mutants of zebrafish sirt7: sirt7-S115A (corresponding to human SIRT7-S111A) and sirt7-H191Y (corresponding to human SIRT7-H187Y). Initially, we examined whether these two mutants really lost their enzymatic activity. As shown in (Fig. 7A and (7B, the deacetylation activity of sirt7-S115A and sirt7-H191Y was indeed less than that of WT sirt7. Subsequently, we compared their effect on RLR signaling with that of WT sirt7 (sirt7-WT). By promoter assays, the two mutants had effects similar to those of WT sirt7 on IFN activation in response to both poly I:C treatment and SVCV infection (Fig. 7C–(7F). Overexpression of sirt7-WT, sirt7-S115A, and sirt7-H191Y was confirmed by Western blot assay (Fig. 7G).
These data suggest that zebrafish sirt7 negatively regulates antiviral responses independent of its enzymatic activity.
Sirtuin proteins have barely been detected as being involved in antiviral innate immunity, particularly through their NAD+-dependent deacetylase activity (30, 31). In this study, we identified (to our knowledge) that sirt7 is a virus-responsive gene that negatively regulates antiviral innate immunity by attenuating phosphorylation of irf3 and irf7 independent of its enzymatic activity, uncovering a novel function of sirt7 in antiviral responses by an in vivo model.
Upon pathogen detection, IRF3 and IRF7 are activated through a successive process, including phosphorylation, dimerization, and subsequent nuclear translocation (22). So, the modulation of IRF phosphorylation usually occurs in the cytoplasm mediated by the disruption of TBK1 function or association with IRF. In this study, we identified that sirt7 prevented tbk1 binding to irf3 and irf7, revealing a mechanism for the role of sirt7 in antiviral immunity. As a histone deacetylase, SIRT7 is mainly distributed in the nucleus (3), but its cytoplasmic pool has also been identified (48). Given that sirt7 attenuates IRF phosphorylation independent of its enzymatic activity, it appears that sirt7 affects IRF function mostly in the cytoplasm instead of in the nucleus. Due to lack of suitable anti-irf3 Ab for readily detecting endogenous zebrafish irf3, we cannot provide evidence to show that overexpression of sirt7 could also diminish dimerization of irf3. Considering a successive process of IRF activation, sirt7 may also affect dimerization of irf3 as well as that of irf7.
Of note, the induction of sirt7 by viral infection is similar to that of antiviral genes, which appear to have a completely opposite function in antiviral immune responses. So, the viruses might escape from host innate immunity through hijacking host sirt7 to promote its expression, resulting in inhibition of antiviral immunity. Insight into the underlying mechanism of sirt7 in antiviral innate immunity will help us to fully understand the process of viral infection and host responses during viral infection.
As a deacetylase, sirt7 acts in its role in antiviral innate immunity independent of its enzymatic activity. Mechanistic studies suggest that sirt7 prevents tbk1 binding to irf3 and irf7 through competitive association, resulting in attenuating irf3/irf7 phosphorylation, dimerization, and nuclear translocation, leading to suppression of antiviral immunity eventually. However, we still do not know the process and the underlying mechanisms of sirt7 in preventing tbk1 binding, and we still cannot rule out the possibility that sirt7 works as an enzyme other than a deacetylase or targets other molecules to act in its role in affecting RLR signaling. To further address these questions, future studies will shed new light on the action of sirt7 in antiviral innate immunity.
Viral infection in fish causes tremendous losses in the aquaculture industry (49–51). In this study, we found that sirt7-deficient zebrafish are more resistant to SVCV infection but do not have any defects in development, growth rate, and reproduction ability. These data suggest that sirt7 might be a good candidate gene for cultivating antiviral fish strains by gene-targeting technology, greatly benefiting the aquaculture industry.
In this study, we mainly used a zebrafish cell line and a zebrafish model to demonstrate the function of sirt7 in antiviral innate immunity. Given the evolutionarily conserved activity of SIRT7 between zebrafish and mammals, mammalian SIRT7 might have the same function as that of zebrafish sirt7 in response to viral infection.
We thank Hong-Bing Shu and Yibing Zhang for providing the reagents.
This work was supported by Strategic Priority Research Program of the Chinese Academy of Sciences Grant XDA24010308, National Natural Science Foundation of China Grants 31721005 and 31830101, and National Key Research and Development Program of China Grant 2018YFD0900602.
Mutant sirt7 data presented in this article have been submitted to the Zebrafish Information Network under accession number ZDB-ALT-210308-2.
The online version of this article contains supplemental material.
Abbreviations used in this article
epithelioma papulosum cyprini
human embryonic kidney
heteroduplex mobility assay
IFN regulatory factor
IFN-sensitive response element
mitochondrial antiviral signaling protein
melanoma differentiation-associated gene 5
- poly I:C, polyinosinic-polycytidylic acid; QRT-PCR
quantitative real-time PCR
retinoic acid–inducible gene I–like receptor
- SIRT, sirtuin; SVCV
spring viremia of carp virus
TANK-binding kinase 1
median tissue culture-infective dose
The authors have no financial conflicts of interest.