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Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection [1]

['Mengqiong Xu', 'Guangdong Provincial Key Laboratory Of Virology', 'Institute Of Medical Microbiology', 'Jinan University', 'Guangzhou', 'Department Of Biotechnology', 'College Of Life Science', 'Technology', 'Sisi Xia', 'Department Of Biological Engineering']

Date: 2022-11 

Abstract Influenza virus has the ability to circumvent host innate immune system through regulating certain host factors for its effective propagation. However, the detailed mechanism is still not fully understood. Here, we report that a host sphingolipid metabolism-related factor, sphingosine kinase 2 (SPHK2), upregulated during influenza A virus (IAV) infection, promotes IAV infection in an enzymatic independent manner. The enhancement of the virus replication is not abolished in the catalytic-incompetent SPHK2 (G212E) overexpressing cells. Intriguingly, the sphingosine-1-phosphate (S1P) related factor HDAC1 also plays a crucial role in SPHK2-mediated IAV infection. We found that SPHK2 cannot facilitate IAV infection in HDAC1 deficient cells. More importantly, SPHK2 overexpression diminishes the IFN-β promoter activity upon IAV infection, resulting in the suppression of type I IFN signaling. Furthermore, ChIP-qPCR assay revealed that SPHK2 interacts with IFN-β promoter through the binding of demethylase TET3, but not with the other promoters regulated by TET3, such as TGF-β1 and IL6 promoters. The specific regulation of SPHK2 on IFN-β promoter through TET3 can in turn recruit HDAC1 to the IFN-β promoter, enhancing the deacetylation of IFN-β promoter, therefore leading to the inhibition of IFN-β transcription. These findings reveal an enzymatic independent mechanism on host SPHK2, which associates with TET3 and HDAC1 to negatively regulate type I IFN expression and thus facilitates IAV propagation.

Author summary Sphingosine-1-phosphate (S1P) metabolizing related proteins are important regulators in response to the infection and pathogenicity of influenza virus and may serve as potential anti-viral targets. Our previous study identifies SPHK2 as a positive regulator of IAV replication, the present study demonstrates that IAV can increase the expression of SPHK2 which in turn diminishes the type I IFN mediated antiviral responses through HDAC1 and TET3. The study highlights a subtle non-enzymatic mechanism of SPHK2 in inhibiting the transcription of type I interferon, and reveals how IAV evade host immunity and replicate in host cells with the help of SPHK2, giving us a novel insight into the interplay between IAV and host immunity.

Citation: Xu M, Xia S, Wang M, Liu X, Li X, Chen W, et al. (2022) Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection. PLoS Pathog 18(9): e1010794. https://doi.org/10.1371/journal.ppat.1010794 Editor: George A. Belov, University of Maryland, UNITED STATES Received: October 12, 2021; Accepted: August 4, 2022; Published: September 7, 2022 Copyright: © 2022 Xu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was supported by the National Natural Science Foundation of China (81730061 to JW, 32070149 to HL), Guangdong Innovative and Entrepreneurial Research Team Program (2014ZT05S136 to HL), Project for Construction of Guangzhou Key Laboratory of Virology (201705030003 to JW), Youth Program of National Natural Science Foundation of China (31500137 to JC), and Guangdong Basic and Applied Basic Research Foundation (2019A1515011742 to CX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Pandemic influenza viruses, leading to severe acute respiratory syndromes, result in immense health risks and great economic losses worldwide owing to the highest rates of morbidity and fatality to humans and animals during each influenza season [1,2]. IAV infection in certain groups are more severe than it in others because of individual variation in immune responses (susceptibility, duration, and intensity), which may be associated with host genetic factors that influence viral replication [3]. Multiple genome-wide screening approaches applied by different studies have also identified a myriad of host factors that may have pro-viral effect on IAV infection [4–7]. Our previous research has showed that poly (ADP-ribose) polymerase 1 (PARP1), a cellular protein that its interaction with hemagglutinin (HA) of influenza A virus is originally identified by mass spectrometry analysis, has been proved to facilitate viral propagation via mediating the IAV HA-induced reduction of IFNAR1 [8,9]. These host factors have been described to be necessary for virus entry, genomic replication, or negative regulation of cell-intrinsic immunity at various steps of IAV infection. However, the exact mechanisms are still lacking for most of the IAV-related host cell factors. As genetic mutation and drift of viral genome in several different strains occurs at an ever-increasing rate during each flu season, the better understanding of these factors not only informs us of the individual immune differences in response to IAV infection, but also provides alternative therapeutic strategies and ideas for anti-viral drug design. Sphingosine-1-phosphate (S1P), an important regulator of inflammation and immune responses, engages in the immune cells-trafficking through sharp gradient between the levels of S1P in the circulation and those in the tissues. S1P synthesized by sphingosine after phosphorylation is catalyzed by sphingosine kinase, SPHK1 and SPHK2 [10]. Both of them exert various biological and physiological functions through binding to the five tightly regulated spatial and temporal expressed sphingosine-1-phosphate receptor subfamily comprising five members (S1PR1-5), with activation of various signaling cascades [10,11]. Besides, S1P produced by cytoplasmic membrane SPHK1, a cofactor for TNF receptor-associated factor 2 (TRAF2), has important intracellular function, motivating TRAF2-mediated ubiquitylation of receptor-interacting serine/threonine protein kinase 1 (RIP1) and leading to the activation of NF-κB [12]. Therefore, it leaves us wondering whether metabolizing related proteins of S1P have significant role on restricting the infection and pathogenicity of influenza virus and may serve as potential anti-viral targets. We have found S1P lyase (SPL), which results in the irreversible cleavage of S1P, can protect cells from IAV infection through the activation of JAK/STAT pathway, while sphingosine kinases that produce S1P make the cultured cells and mice more susceptible to IAV infection [13,14]. Furthermore, we have proved that sphingosine kinase 1 (SPHK1) is served as a favorable factor for viral replication by activating the NF-κB signaling to support viral RNA synthesis and regulating the nuclear export of vRNP during IAV infection [15]. Yet, as another isoenzyme of sphingosine kinase, the specific immune regulation mechanism of SPHK2 on promotion of IAV propagation still needs to be elucidated. Although SPHK1 and SPHK2 share high homology of amino acid sequence with each other, they have overlapping but distinct functions. Studies have reported that SPHK1 mainly localizes to the cytosol and plasma membrane; in contrast, SPHK2 is found to shuttle between the nucleus, nucleoplasm and cytoplasm, but it cannot be ruled out to function in the endoplasmic reticulum [16]. In addition, SPHK1 is verified to activate TNF-α and NF-κB signaling in response to inflammation, promoting cell survival and proliferation, whereas SPHK2 has been controversially reported to stimulate cell apoptosis [16,17]. Especially, Nitai C. Hait et al. have revealed that SPHK2 is assembled in corepressor complexes containing histone deacetylase 1 (HDAC1) and the overexpression of SPHK2 leads to nuclear S1P accumulation, which inhibits HDAC1 activity, thereby causes increased acetylation of histone lysine residues and subsequently activates p21 and c-fos gene transcription [18]. Recently, it has also been reported that inhibition of SPHK2 can enhance protective T cell immune responses and gradually terminate lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) persistence in mice [19]. SPHK2 colocalizes with chikungunya virus (CHIKV) RNA and nonstructural proteins, and impairment of SPHK2 expression can significantly inhibit CHIKV infection [20]. Inhibition of SPHK2 also limits HIV-1 infection in CD4 T cells [21]. Moreover, inhibition of SPHK2 induced viral lytic gene expression with KSHV-infected endothelial cells, indicating the function of SPHK2 in maintaining viral latency [22]. Interestingly, it has been reported that SPHK2 functions differently during the infection of different genotypes of HCV. The cell-cultured adapted genotype 1 HCVs in Huh-7.5 cells can be propagated by the SPHKs inhibitor, SKI, when its concentration set to selectively inhibit SPHK2, however, a chimeric genotype 2 virus (HJ3-5/GLuc) of HCVs is not suppressed after SKI treatment [23]. In view of the complex functions of SPHK2 in response to virus infection, the role of the SPHK2 protein itself and the S1P-producing kinase activity of SPHK2 in regulation of IAV replication should be evaluated. HDAC1, as the most widely studied deacetylase, plays a significant role in epigenetic regulation of cellular genes and serves as an important regulator of innate immunity during viral infection [24–26]. Porcine epidemic diarrhea virus (PEDV) inhibits the expression of HDAC1 through the interaction of its N protein with the transcription factor Sp1, therefore facilitating the viral replication [27]. However, inhibition of HDAC1 suppresses pseudorabies virus (PRV) infection through cGAS-STING antiviral innate immunity [28]. Transient knockdown of HDAC1 also suppresses HIV-1 replication [29]. Moreover, HIV-1 integrase (IN) and cellular protein INI1/hSNF5 bind SIN3 Associated Protein 18 (SAP18) and recruit components of Sin3a-HDAC1 complex into HIV-1 virions to facilitate the viral replication [30]. Conversely, HDAC1, in complex with KAP1 (TRIM28) can deacetylate HIV-1 IN, downregulating the efficiency of HIV-1 integration [31]. These studies indicate that the functions of HDAC1 on the regulation of viral innate immunity are much more complicated. Ten-eleven translocation protein 3 (TET3), a member of dioxygenases family, induces cytosine demethylation by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) at target gene promoters, thereby creating a global and local dynamic methylation landscape in epigenetic expression under specific physiological conditions [32,33]. For example, TET3 in B cells is needed to repress CD86 and prevent autoimmunity [34]. TET3 increases IL6 expression by up-regulation of 5hmC in IL6 promoter in chronic hypoxia induced atherosclerosis in offspring rats [35]. TET3 and TGF-β1 form a positive feedback loop to promote TGF-β signaling and subsequently profibrotic gene expression in hepatic stellate cells (HSCs) [36]. TET3 also demethylates the gene promoter of miR-30d precursor to block TGF-β1-induced epithelial-mesenchymal transition (EMT), thus can act as a suppressor of ovarian cancer [37]. In this study, we explored the potential molecular mechanisms of SPHK2 in influenza A virus infection. We substantiated that, IAV manipulates SPHK2 to subvert host type I IFN responses to gain a better replication. However, SPHK2 catalytic-inactive mutant does not lose the ability to enhance the IAV replication, suggesting the pro-viral function of SPHK2 is independent of its enzymatic activity. Furthermore, we proved that the inhibitory effect on IAV-induced type I IFN transcription by SPHK2 is dependent on HDAC1 and the specific IFN-β promoter binding demethylase TET3 [38]. The study reveals a subtle non enzymatic mechanism of SPHK2 in downregulating the expression of type I interferon upon IAV infection.

Discussion In this study, we demonstrate that SPHK2, an IAV-induced and pro-viral host factor, depends on the histone deacetylase HDAC1, negatively regulates type I IFN response, and thus facilitates IAV infection. Unexpectedly, the core enzymatic activity of SPHK2 that inhibits HDAC1 by producing S1P is unrelated to the increased IAV propagation. More interestingly, SPHK2 using the substrate binding domain to interact with TET3, an inhibitor of IFN-β production with DNA binding activity, is recruited to the IFN-β promoter. Therefore, the IFN-β transcription is inhibited by the SPHK2-interacting cooperator HDAC1, and IAV replication was increased within host cells (Fig 9). PPT PowerPoint slide

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TIFF original image Download: Fig 9. SPHK2 interacts with HDAC1 and TET3 to inhibit IFN-β transcription. The expression of SPHK2 in IAV infected cells is upregulated and then the location of SPHK2 is shifted from cytoplasm to nucleus. Afterwards, SPHK2 accumulated in the nucleus interacts with HDAC1 and its substrate binding domain also interacts with TET3 that bands to IFN-β promoter. Therefore, HDAC1 is recruited to IFN-β promoter and inhibited IFN-β transcription by enhancing the deacetylation of IFN-β promoter. https://doi.org/10.1371/journal.ppat.1010794.g009 We found that SPHK2 was obviously upregulated in IAV infected A549 cells, and its location was shifted from cytoplasm to nucleus, suggesting SPHK2 may play a role in influenza virus infection. SPHK1, homologous to SPHK2, has been reported to possess a pro-viral property. Inhibition of SPHK1 suppresses virus-induced NF-κB activation [15,48]. Therefore, SPHK2 may also have a similar character with SPHK1 during IAV infection. However, different from SPHK1 which upregulates NF-κB activity, we found that SPHK2 downregulates type I IFN responses during IAV infection. SPHK2, essential for sphingolipid metabolism, catalyzes the phosphorylation of sphingosine which is derived from the N-deacetylation of ceramide by ceramidase, brings it into generating S1P, and activates the S1P receptor signaling [49]. However, we found that the catalytic-incompetent SPHK2 mutant has the same ability to support IAV replication with the WT SPHK2 (Fig 5). Thus, we believe that the pro-viral function of SPHK2 is independent of its enzymatic activity. It has been reported that endogenous lipid peroxidation restricts the replication of some HCV strains, leading to the increased viral persistence [23], SPHK2 maintains KSHV latency in virus infected endothelial cells and promotes CHIKV replication in vitro [20,22]. Moreover, the sphingosine precursor ceramide inhibits IAV infection in vitro [50], S1P receptor signaling protects mice from pathogenic influenza [51], while S1P lyase (SPL) enhances type I IFN production to restrict IAV replication [52,53], indicating that sphingolipid metabolism occupies an important place in influenza virus infection. However, as one member of sphingolipid metabolism, SPHK2 facilitates IAV infection independent of its enzymatic activity, suggesting the sphingolipid metabolism function is not involved in SPHK2 enhanced IAV replication but by another novel mechanism. Besides, genetic deletion of SPHK2 impairs dengue virus (DENV) infection, but inhibition of SPHK2 activity does not affect DENV replication, which also suggests SPHK2 activity may not be required for some viruses such as DENV and IAV [54]. Therefore, we wonder to know whether the SPHK2 protein itself or there might be some unknown proteins involved in the regulation and promotion of IAV infection. HDACs play multiple roles during viral infection. For instance, HDAC11 and HDAC4 are involved in the modulation of HBV replication [55], HDAC8 is required for influenza A virus entry [56], HDAC6 is crucial for IAV-induced apoptosis of infected cells [57], HDAC2 is a key factor of host anti-influenza virus response [58]. Endogenous SPHK2 associates with HDAC1 and interacts with histone H3. SPHK2-generated S1P specially binds to HDAC1 to inhibit their enzymatic activities, enhancing p21 and c-fos transcription [18]. It has been reported that HDAC1 inhibits IAV infection [41], but intriguingly, others have also reported that HDAC1 facilitates IAV infection by supporting the nuclear preservation of NP and negatively regulating TBK1-IRF3 pathway [39]. Thus, it is attractive to determine whether HDAC1 participates in SPHK2 facilitated IAV infection. As previously reported, TET3 exerts a methylation epigenetic regulation of the corresponding promoters under various physiological conditions [35–38]. Specially, TET3 can function in a DNA demethylation-independent manner and binds to the IFN-β promoter and leads to the recruitment of HDAC1 to the IFN-β promoter, consequently inhibiting IFN-β transcription under viral infection [38]. However, no evidence has been found to support a direct interaction between TET3 and HDAC1. Of note, our study has proved that SPHK2 is a negative regulator of IFN-β transcription and HDAC1 is known to be a binding partner of SPHK2, not SPHK1 [18], thus we speculate that TET3 binds to HDAC1 through SPHK2, leading to the recruitment of HDAC1 and the inhibition of IFN-β transcription. This assumption has been verified in our study (Figs 7 and S9). We have found SPHK2 serves as an adaptor protein between TET3 and HDAC1. Of note, in Fig 7D, endogenous SPHK2 could precipitate HDAC1 and TET3 in IAV infected A549 cells, but could not precipitate TET3 in mock infected cells. Our explanation is that, compared to uninfected A549 cells, IAV infection induces a large amount of accumulation of SPHK2 in the nucleus, and perhaps some modifications on SPHK2 proteins, leading to the interaction with HDAC1 and TET3. This may be a way of protecting cells from non-physiological activation of IFN-β. According to the previous reports and the results of Fig 2A, we speculate that the necessary and small amount of SPHK2 present in nucleus could interact with HDAC1 to regulate other cellular progress in normal conditions [18]. Upon IAV infection, some modifications or changes were created in both SPHK2 and TET3, leading to SPHK2’s accumulation in the nucleus and the interaction of SPHK2-HDAC1 complex with TET3, subsequently the specific binding of the IFN-β promoter recruited by TET3 in response to viral infection. However, it’s still confusing that this process is unrelated with the enzymatic activity of SPHK2, but depends on its substrate binding domain, suggesting a mechanism distinct from SPHK2 and HDAC1 regulated p21 and c-fos transcription. Finally, we have found TET3 binds to the substrate binding domain of SPHK2, which inhibits the enzymatic activity of SPHK2 and releases the deacetylation function of HDAC1 to the IFN-β promoter, resulting in IFN-β transcriptional inhibition. Furthermore, the result is in accordance with our previously findings that inhibition of SPHK2 in IAV infected mice by ABC294640 which competitively binds to the substrate binding domain of SPHK2 increases IFN-β production, thus protecting mice from lethal influenza A virus infection [14]. In summary, our study determines that SPHK2 displays a pro-IAV function which is dependent on HDAC1, and the regulation of IAV replication is independent of the enzymatic activity of SPHK2. Furthermore, SPHK2 negatively regulates type I IFN signaling during IAV infection. Most importantly, SPHK2 interacts with TET3 through its substrate binding domain and recruits HDAC1 to the IFN-β promoter to inhibit IFN-β transcription by increasing the deacetylation of IFN-β promoter. Since the two TET3 regulated TGF-β1 and IL6 promoters could not be enriched by SPHK2 in IAV infected cells, we speculate that the SPHK2-mediated mechanism which specially modulate the IFN-β promoter is dependent both on the binding properties of TET3 and SPHK2 induced by IAV infection. However, the mechanism of how IAV induces the accumulation of SPHK2 to the nucleus is not extensively elucidated. There are reports that protein kinase D mediated phosphorylation regulates the nuclear export of SPHK2 [59], and serum deprivation can also induce SPHK2’s translocation in the cells [24]. Our findings demonstrate that the accumulation of SPHK2 in the nucleus was specific to influenza A virus infection, indicating that there may be existing other unknow factors related to IAV infection that make the difference on the cellular location of SPHK2 in response to IAV infection, and the mechanism needs to be further investigated in the future work. Anyway, our findings further broaden our horizon regarding the multiple immune regulation functions of SPHK2 and the special role of SPHK2 in promotion of IAV infection. The new understanding of the interplay between IAV and SPHKs may promote the development of alternative or next generation of anti-IAV strategies.

Materials and methods Cells and viruses Human embryonic kidney 293 (ATCC), Madin-Darby canine kidney (MDCK) epithelial cells (ATCC), human lung epithelial lung cell line A549 (ATCC), and the related knock-down A549 cell lines were grown at 37°C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with high glucose, GlutaMax, pyruvate (Gibco), 10% fetal bovine serum (FBS, Lonsera), and 1% P/S (100 units/ml penicillin, 100 μg/ml streptomycin; Gibco). Influenza virus A/PR/8/34 H1N1 (a gift from Dr. Wenbao Qi at the Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture) and A/WSN/34 H1N1 (a gift from Dr. Wenjun Song at Guangzhou Medical University) strains were propagated and titrated on MDCK cells, and then stored at -80°C. EV71 (Xiangyang-Hubei-09) and ZIKV (z16006) strains were kindly provided by Dr. Zhen Luo at Jinan University. For viral infection, the cell monolayers were washed twice with PBS, the virus inoculum was diluted in infection media, i.e., serum-free DMEM containing 0.3% BSA (Sigma-Aldrich), 1% P/S and 1 μg/ml TPCK-trypsin (Sigma-Aldrich). Next, cells were incubated with virus at 37°C for 1 h. After removing the virus inoculum, cells were washed twice with PBS, subsequently replaced with infection media. Generally, cells were infected at the multiplicity of infection (MOI) of 0.01–5.0 plaque-forming units (PFUs) per cell. Regents and antibodies HDAC inhibitor Trichostatin A (TSA) was purchased from Sigma (T1952). SPHK1 inhibitor SK1-I was purchased from MCE (HY-119016). Trypan blue solution was purchased from Gibco (15250061). NE-PER Nuclear and Cytoplasmic Extraction Reagents was purchased from Thermo Fisher (78833). NBD sphingosine was purchased from Avanti (810205). ATP was purchased from Invitrogen (PV3227). Antibodies against influenza A virus NP, human GAPDH, Lamin B1 and β-Actin were purchased from Abcam (ab128193, ab181602, ab16048, ab6276). Anti-human Myc and Flag tagged antibodies were purchased from Sigma (M4439, F1804). The antibodies against human HDAC1, STAT1 and pSTAT1 were obtained from CST (34589, 9172S, 9167S). The antibodies against influenza A virus M1, NS1 and Enterovirus 71 VP1 antibody were obtained from GeneTex (GTX125928, GTX25990, GTX132338). Anti-human TET3 antibody was purchased from Affinity (DF13335). Anti-human SPHK2 polyclonal antibody was purchased from Proteintech (17096-1-AP). Anti-human SPHK2 monoclonal antibody and Zika virus NS1 monoclonal antibody was purchased from Invitrogen (9C5E1, EA88). FITC-Goat polyclonal to influenza A virus (ab20388), Goat Anti-Rabbit IgG H&L (Alexa Flour 647, ab150079) and Goat Anti-Mouse IgG H&L (Alexa Flour 555, ab150114, Alexa Flour 568, ab175473) were purchased from Abcam. DAPI (C1002) was purchased from Beyotime. Plasmids design and construction The constructs generated in our study are presented in Table 1. The pGL3-IFN-β-promoter and pRL-TK plasmids were obtained as previously described [60]. The site-directed mutagenesis of SPHK2 (G212E) was generated using the mutation primers by Fast Mutagenesis system (Transgen) according to the manufacturer’s protocol. The mutation primers are as follows: G212E-F: 5’-TGATCTCTGAAGCTGGGCTGTCCTTC-3’, G212E-R: 5’-CCAGCTTCAGAGATCATGGGAAGCA-3’. PPT PowerPoint slide

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TIFF original image Download: Table 1. Plasmid constructs used in the study. https://doi.org/10.1371/journal.ppat.1010794.t001 Generation of CRISPR-Cas9 knock out cell lines and RNA-i transduced stable cell lines SPHK2 knock out A549 cells were constructed using the sgRNA lentivirus packaging system as previously described [61]. HDAC1 knock down A549 cells were constructed using the shRNA lentivirus packaging system as previously described [42]. The KO efficiency of SPHK2 or the knock down efficiency of HDAC1 in A549 cells was confirmed by qPCR and Western blotting assay. The sequences of SPHK2 sgRNA and HDAC1 shRNA used in this study are as follows: sgSPHK2-1-F: 5’-CACCGTTAAGCCATAGTAGGCTGGT-3’, sgSPHK2-1-R: 5’-AAACACCAGCCTACTATGGCTTAAC-3’; sgSPHK2-2-F: 5’-CACCGCTGTTACATCAACGGGACCC-3’, sgSPHK2-2-R: 5’-AAACGGGTCCCGTTGATGTAACAGC-3’; sgSPHK2-3-F: 5’-GGTCTTAAGCCATAGTAGGC-3’, sgSPHK2-3-R: 5’-GCCTACTATGGCTTAAGACC-3’. The 3’-UTR targeting sequence for shHDAC1 was: 5’-CGTTCTTAACTTTGAACCAATA-3’ [42]. Interfering RNA design and transfection siRNA and control siRNA were designed and synthesized by Sangon Biotech. For silencing, A549 cells were seeded in 12 well plates (2×105 cells/well), at 80–90% confluence, cells were transfected with the indicated siRNA at 100 nM with 3μL RNAiMax reagent (Invitrogen) according to the indicated protocol. The sequences of siRNA used in this study are as follows: SPHK2-siRNA-1-F: 5’-GGGAGGAAGCUGUGAAGAUTT-3’, SPHK2-siRNA-1-R: 5’-AUCUUCACAGCUUCCUCCCTT-3’; SPHK2-siRNA-2-F: 5’-AGACAGAACGACAGAACCATT-3’, SPHK2-siRNA-2-R: 5’-UGGUUCUGUCGUUCUGUCUTT-3’; TET3-siRNA: 5’-AGGCCAAGCUCUACGGGAA-3’ [62]. For the overexpression of SPHK2, SPHK2-G212E, HDAC1 or other fragments, the Myc-SPHK2, Myc-SPHK2-F1(Δ1–69), Myc-SPHK2-F2(Δ391–514), Myc-SPHK2-F3(Δ245–248), Myc-SPHK2-G212E or Myc-HDAC1 expression vector was transfected into A549 or 293T cells. The plasmid DNA was transfected with LipoD293 (Signagen) as recommended protocol provided by the manufacturer. Cell viability assay A549 cells were seeded in 96 well plates (1×104 cells/well), at 90% confluence, cells were then treated with TSA at concentration of 5, 10, 20 or 50 nM (experiment groups), wells just only supplemented with complete medium were blank groups, and cells with no treatment were control groups. After 72 h, supernatants were discarded, fresh complete medium and CCK8 reagent (Beyotime, C0038) was added to the groups. After shaking, 96 well plates were incubated at 37°C for 25 to 60 min. Finally, the absorbances were measured at 450 nm on automatic microplate reader (Biotek). Cell growth rate analysis A549 cells or sgSPHK2-1 A549 cells were seeded in 12 well plates (1×105 cells/well). A549 cells were overexpressed with Myc-tagged SPHK2 or SPHK2-G212E by using reverse transfection. In next 3 days, cells were digested and counted every 24 h by using trypan blue on blood counting chamber. The growth curves of cells with different treatment were obtained by GraphPad Prism 6 software. Fluorescence-based SPHK2 activity assay A549 cells or sgSPHK2-1 A549 cells were seeded in 6 well plates (5×105 cells/well). At 80–90% confluence, A549 cells were then transfected with pCMV-Myc, Myc-SPHK2 or Myc-G212E plasmid. After 24 h, cells were collected and transferred to lysis buffer (20 mM Tris, pH 7.4, 20% glycerol, 1 mM 2-mercaptoethanol, 1 mM EDTA, 5 mM sodium orthovanadate, 40 mM glycerophosphate, 15 mM NaF, 10 μg/ml leupeptin, aprotinin, and soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 0.5 mM 4-deoxypyridoxine), then repeated the freeze-thawing cycle in this lysis buffer for 3 times (10 min/time) [63]. Cell lysates were centrifuged at 14,000 ×g for 30 min to remove cellular debris. Supernatants were collected for subsequent SPHK2 activity assay. Supernatants (10–15 μL) was incubated with NBD-sphingosine (dissolved in 5% Triton X-100) and ATP in SPHK2 reaction buffer (30 mM Tris-HCl, pH 7.4, 0.05% Triton X-100, 200 mM KCl, and 10% glycerol), 10 mM NaF and semicarbazide were added to prevent potential degradation of formed NBD-S1P. After incubation for 30 min at 30°C, 100 μL 1 M potassium phosphate buffer, pH 8.5, was added followed by 500 μL CHCl3/MeOH 2:1 [64]. After several vortex, phases were separate by centrifugation at 15,000 ×g for 30 s. An aliquot of the upper aqueous layer was transferred into 96-well plate, following by 100 μL of dimethylformamide. Fluorescence intensity was measured in automatic microplate reader (BioTek) with excitation at 485 nm and emission at 538 nm. The reaction mixture containing no SPHK2 served as blank, The SPHK2 activity in samples are normalized to control groups. Cytoplasmic and nuclear protein extraction 10×106 cells were transferred to 1.5 mL microcentrifuge tube and centrifuged at 500 ×g for 3 min, then the supernatant was discarded. 200 μL ice-cold Cytoplasmic Extraction Reagent I (CER I) was added to the cell pellet. Afterwards, the tube was vortexed vigorously for 15 seconds and incubated on ice for 10 min. 11 μL ice-cold Cytoplasmic Extraction Reagent II (CER II) was added to the tube. The tube was vortexed vigorously for 5 seconds and incubated on ice for 1 min. The tube was vortexed vigorously for 5 seconds again and centrifuged at 16,000 ×g for 5 min. The supernatant obtained from the centrifugation was cytoplasmic extract. After transferring the supernatant, 100 μL ice-cold Nuclear Extraction Reagent (NER) was added to the cell pellet. The tube was vortexed vigorously for 15 seconds and incubated on ice for 40 min, a continue vortex for 15 seconds every 10 min was needed during the incubation. The tube was centrifuged at 16,000 ×g for 10 min. The supernatant obtained from the centrifugation was nuclear extract. The extracts were stored at -80°C until use. Co-immunoprecipitation Cells were transfected with Myc-SPHK2, Flag-TET3 or Myc-HDAC1. After 24 h transfection, the cells were washed and then lysed for 30 min at ice with Triton X-100 lysis buffer (20mM Tris-HCl pH = 7.5, 100 mM NaCl, 0.5 mM EDTA, 5% glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (Beyotime). After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking. Subsequently, the beads were centrifuged and washed three times with ice-cold PBS, boiled in 6×SDS loading buffer for 8 min, and analyzed by Western blotting with the indicated antibodies (anti-Myc, anti-SPHK2 (R, 17096-1-AP), anti-HDAC1 (R, 34589), anti-Flag or anti-TET3 (R, DF13335) antibody). Western blotting analysis Cells were lysed in TritonX-100 lysis buffer with protease inhibitor cocktail (100×; Thermo Fisher) for 30 min on ice and then centrifuged at 4°C, 16,000 ×g for 10 min. The supernatants with 6×SDS loading buffer were boiled for 8 min and stored at -80°C. The samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked and shook with 5% non-fat milk (Biorad) in 1×TBST for 1 h at room temperature. Then the membrane was incubated with the indicated primary antibodies followed by HRP-conjuncted secondary antibodies. The expression of protein was detected by Odyssey Fc (Li-COR), and data were analyzed by Image Studio V5.2 (Li-COR). RNA Extraction and real-time q-PCR Total RNA was obtained from the cultured cells with RNA fast 200 kit (Fastagen Biotech, Shanghai, China) according to the indicated instructions. cDNA was synthesized with commercial PrimeScript RT Master Mix (Takara, Tokyo, Japan). The quantitative real-time PCR (qPCR) reaction was performed on a LightCycler 96 System (Roche, Hong Kong, China). The data represent the mRNA expression levels of experimental genes normalized to the reference gene GAPDH. Relative expression values were calculated by using the 2ˆ-ΔΔCt method [65,66]. The primers used in the qPCR are as follows: qGAPDH-F: 5’-AGCCTCAAGATCATCAGCAATGCC, qGAPDH-R: 5’-TGTGGTCATGAGTCCTTCCACGAT-3’; qIFIT1-F: 5’-AGAAGCAGGCAATCACAGAAAA-3’, qIFIT1-R: 5’-CTGAAACCGACCATAGTGGAAAT-3’; qISG15-F: 5’-CGCAGATCACCCAGAAGATCG-3’, qISG15-R: 5’-TTCGTCGCATTTGTCCACCA-3’; qIFNb-F: 5’-CGCCGCATTGACCATCTA-3’, qIFNb-R: 5’-GACATTAGCCAGGAGGTTCTC-3’; qSPHK2-F: 5’-ATGGACACCTTGAAGCAGAG-3’, qSPHK2-F: 5’-TGACCAATAGAAGCAACCGG-3’; qHDAC1-F: 5’-GGATACGGAGATCCCTAATG-3’, qHDAC1-R: 5’-CGTGTTCTGGTTAGTCATATTG-3’; qTET3-F: 5’-TGCGATTGCGTCGAACA-3’, qTET3-R: 5’-TGCGGATCACCCACTTTG-3’. qTNF-α-F: 5’-GCCTCTTCTCCTTCCTGATCGT-3’, qTNF-α-R: 5’- TGAGGGTTTGCTACAACATGGG-3’ [67]; qIL6-F: 5’- AGTGAGGAACAAGCCAGAGC-3’, qIL6-R: 5’- GTCAGGGGTGGTTATTGCAT-3’ [68]. Luciferase report assay 100 ng pGL3-IFNβ-promoter, pGL3-NF-κB-promoter or the indicated plasmids were co-transfected with 20 ng pRL-TK vector into cells in 24 well plates using LipoD293 [69]. After 24 h, the samples were obtained according to the manufacturer’s instructions (Beyotime, Shanghai, China), and Firefly and Renilla luciferase activities were detected using the Dual Luciferase Reporter Assay kit (Beyotime) according to the indicated protocol. Immunofluorescence assay Cells in confocal dishes were washed with ice-cold PBS, and then fixed with 4% paraformaldehyde for 20 min at room temperature. The cells were then wash with ice-cold PBS for three times (5min/time) again and permeabilized with 1×PBS-T containing 0.2% TritonX-100 for 10 min. Subsequently, cells were blocked with 1×PBS-B containing 0.4% BSA for 30 min at 37°C after washed with PBS. Cells were incubated with the indicated primary antibodies (anti-SPHK2 (R, 17096-1-AP), anti-IAV NP (M, ab128193)) (1:100) overnight at 4°C, followed by Alexa Flour 555, Alexa Flour 647 conjugated secondary antibodies (1:1000) for 1 h. And then DAPI was used for nuclear staining (5 min), 50% glycerin was used for sealing. Images were obtained using a Leica SP8 TCP confocal microscope. Quantitative analyses of IF images were obtained using a 63×objective with LAS X software from three representative images. ChIP assay A549 cells in 6cm plates were fixed with 1% formaldehyde and quenched with 1.25 M glycine. Subsequently, the cells were washed, collected and lysed in 0.5% SDS lysis buffer at 4°C for 30 min. After the cell lysis, the supernatants were obtained and disrupted by Ultrasonic Processor (Scientz). The supernatants were then collected, diluted and incubated with protein A/G magnetic beads (Millipore) at 4°C for 1 h. The indicated antibodies were added for overnight incubation at 4°C. Then protein A/G magnetic beads were added for another two hours, followed by washing (low salt wash buffer, high salt wash buffer, LiCl wash buffer and TE buffer), eluting (Elution buffer: 10% SDS, 1 M NaHCO 3 ), and reverse crosslinking processes (incubated with 5 M NaCl at 65°C overnight). The DNA was finally purified and quantified by qRT-PCR. The relative enrichments of promoters are normalized to IgG (-) group, and 2ˆ-ΔΔCt method was used in the calculation. The primers of ChIP-qPCR are as follows: ChIP-qPCR-IFN-β-F: 5’- TTCCTTTGCTTTCTCCCAAGTC-3’, ChIP-qPCR-IFN-β-R: 5’- CAGAGGAATTTCCCACTTTCACTT-3’ [69]; ChIP-qPCR-TGF-β1-F: 5’-CCTGCCGACCCAGCC-3’, ChIP-qPCR-TGF-β1-R: 5’- CTCGCTGTCTGGCTGCT-3’ [36]; ChIP-qPCR-IL6-F: 5’- TCTGCAAGATGCCACAAGGT-3’, ChIP-qPCR-IL6-R: 5’- TGAAGCCCACTTGGTTCAGG-3’ [70]. Plaque assay MDCK cells were seeded in 6-well plates to be just 80–90% confluent the following day. A 10-fold serial dilution of samples from virus infection assay were prepared in infection media. Cells were washed, inoculated with 800 μL diluted samples and incubated at 37°C for 1 h. Cells were then washed and overlaid with 2 mL/well of media (2×F12-DMEM (Gibco) containing 2% Agar (Oxoid), 0.3% BSA, 0.1% NaHCO 3 (Sigma-Aldrich) and 1 μg/mL TPCK-treated trypsin). After a 48–72 h incubation, the cells were fixed with 4% paraformaldehyde for 1 h. The agar layer was then gently removed, and stained with 0.1% crystal violet solution (Sigma-Aldrich). Plaques were subsequently counted and viral titers were expressed as PFU/mL.

Statistics All data are shown as mean values and standard deviations of biological replicates (the details are given in the table and figure legends). Statistical significance of the differences between groups was estimated by using two-tailed Student’s t-test, two-tailed Student’s t-test with Welch’s correction, one-way ANOVA or a two-way ANOVA with GraphPad Prism 6. P values was depicted as follows: ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Supporting information S1 Fig. The translocation of SPHK2 in A549 cells after IAV, ZIKV or EV71 infection. A549 cells were uninfected or infected with influenza A/WSN/33 (H1N1) virus (WSN), Zika virus (ZIKV) or Enterovirus 71 (EV71) at an MOI of 0.5 for 24 h. (A) Cells were fixed and stained using DAPI for nuclei (Blue), as well as anti-viral protein (IAV NP, ZIKV NS1 or EV71 VP1) antibodies (Green) and anti-SPHK2 antibodies (Red). The cells were visualized by confocal laser scanning microscopy. (B) The protein levels of SPHK2 in uninfected, ZIKV or EV71 infected A549 cells were detected by Western blotting, β-actin and Lamin B were used as loading controls for cytoplasmic and nuclear proteins, respectively. Results are representatives of three independent experiments. https://doi.org/10.1371/journal.ppat.1010794.s001 (TIF) S2 Fig. The growth rate of SPHK2 overexpressed or knock out A549 cells. (A) A549 cells were transfected with pCMV-Myc or Myc-SPHK2, cells with mock transfection were as CTRL, and cell proliferation was assessed by trypan blue exclusion analyses. (B) The mRNA and protein levels of SPHK2 in knockout A549 cells were detected separately by qPCR and Western blotting. (C) The cell proliferation of A549 cells and the SPHK2 knock out A549 cells (sgSPHK2-1) were monitored by trypan blue exclusion analyses. Results are representatives of three independent experiments. https://doi.org/10.1371/journal.ppat.1010794.s002 (TIF) S3 Fig. The growth rate and SPHK2 activity in A549 cells transfected with Myc-SPHK2 (WT) or Myc-SPHK2 mutant (G212E). (A) A549 cells were transfected with Myc-SPHK2 (WT) or Myc-SPHK2 mutant (G212E), cells with mock transfection were as CTRL, and cell proliferation was assessed by trypan blue exclusion analyses. (B) SPHK2 activity in SPHK2-WT or SPHK2-G212E overexpressed A549 cell normalized to the CTRL is displayed, meanwhile, Myc-tagged SPHK2-WT, SPHK2-G212E protein and endogenous SPHK2 protein levels in SPHK2-WT or SPHK2-G212E overexpressed A549 cell lysates were detected by Western blotting, and β-Actin was as an internal control. Data are means ± SD of three independent experiments. ***, P<0.001. https://doi.org/10.1371/journal.ppat.1010794.s003 (TIF) S4 Fig. The mRNA levels of TNF-α and IL6 in SPHK2 overexpressed or knock out A549 cells after IAV infection. (A) A549 cells were transfected with pCMV-Myc (-) or SPHK2-expressing plasmids, and 24 h after transfection, cell were infected with WSN virus at an MOI of 0.5. The mRNA levels of TNF-α and IL6 were analyzed by qPCR at 24 hpi, which was normalized to GAPDH. (B) A549 cells (WT) or SPHK2 knockout A549 cells (KO) were infected with WSN virus at an MOI of 0.5 for 24 h. The mRNA levels of TNF-α and IL6 were analyzed by qPCR at 24 hpi, which was normalized to GAPDH. Data are means ± SD of three independent experiments. ns, not significant. https://doi.org/10.1371/journal.ppat.1010794.s004 (TIF) S5 Fig. SPHK2 activity in shCTRL or shHDAC1 A549 cells infected with IAV and IFN-β promoter activity in Myc-SPHK2 transfected shCTRL or shHDAC1 A549 cells with IAV infection. (A) SPHK2 activity in IAV infected and Myc-SPHK2 transfected shCTRL A549 cell lysates or shHDAC1 A549 cell lysates normalized to the CTRL is displayed. (B) shCTRL or shHDAC1 A549 cells were transfected with pCMV-Myc, Myc-SPHK2, and then infected with WSN virus at an MOI of 0.5. IFN-β activity was measured by using luciferase reporter assays. Data are means ± SD of three independent experiments. ***, P<0.001; ****, P<0.0001; ns, not significant. https://doi.org/10.1371/journal.ppat.1010794.s005 (TIF) S6 Fig. The colocalization analysis of SPHK2 with overexpressed HDAC1 or TET3 after IAV infection. (A) A549 cells were transfected with Myc-HDAC1 or Flag-TET3, after 24 h transfection, cells were infected with influenza A/WSN/33 (H1N1) virus (WSN) at an MOI of 0.5 for 24 h. Cells were fixed and stained using DAPI for nuclei (Blue), as well as FITC-anti-influenza A virus NP antibodies (Green), anti-Myc or anti-Flag antibodies (Yellow) and anti-SPHK2 antibodies (Red). The cells were visualized by confocal laser scanning microscopy. (B) shHDAC1 A549 cells were mock-infected or infected with WSN at an MOI of 0.5 for 24 h. Cells were fixed and stained using DAPI for nuclei (Blue), as well as FITC-anti-influenza A virus NP antibodies (Green) and anti-SPHK2 antibodies (Red). The cells were visualized by confocal laser scanning microscopy. Results are representatives of three independent experiments. https://doi.org/10.1371/journal.ppat.1010794.s006 (TIF) S7 Fig. The interaction of SPHK2 (FL), SPHK2 mutant (G212E) or its truncated fragments with endogenous HDAC1, TET3 and IFN-β promoter. (A) A549 cells transfected with pCMV-Myc, Myc-SPHK2-FL and SPHK2 truncated fragments (Myc-SPHK2-F1, Myc-SPHK2-F2, and Myc-SPHK2-F3) were immunoprecipitated using anti-Myc antibody, then SPHK2-FL, SPHK2-F1, Myc-SPHK2-F2 and SPHK2-F3 proteins in immunoprecipitated complex were analyzed by immunoblotting using Myc-antibody or SPHK2 antibody. (B) A549 cells transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-G212E or SPHK2 truncated fragments (Myc-SPHK2-F1, Myc-SPHK2-F2, and Myc-SPHK2-F3) were immunoprecipitated using anti-Myc antibody, and then Myc-tagged SPHK2, HDAC1, and TET3 proteins were analyzed by immunoblotting. (C) A549 cells were transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-G212E or SPHK2 truncated fragments, and IFN-β promoter enrichment for Myc-SPHK2-FL, Myc-G212E or SPHK2 truncated fragments was detected by ChIP-qPCR. Data are means ± SD of three independent experiments. *, P<0.05; **, P<0.01. https://doi.org/10.1371/journal.ppat.1010794.s007 (TIF) S8 Fig. The TGF-β1 and IL6 promoter enrichment in SPHK2 overexpressed or IAV infected A549 cells. (A) A549 cells were transfected with pCMV-Myc, Myc-SPHK2, 24 h post transfection, TGF-β1 and IL6 promoter enrichment for Myc-SPHK2 was detected by ChIP-qPCR, TGF-β1 and IL6 promoter enrichment for TET3 was as a positive control. (B) A549 cells were infected with IAV WSN at MOI of 0.5, after 24 h infection, TGF-β1 and IL6 promoter enrichment for SPHK2 was detected by ChIP-qPCR. Data are means ± SD of three independent experiments. ns, not significant. https://doi.org/10.1371/journal.ppat.1010794.s008 (TIF) S9 Fig. The interaction of SPHKs with TET3 in A549 cells co-transfected with Flag-tagged TET3, Myc-SPHK1 or Myc-SPHK2. A549 cells co-transfected with Flag-TET3, Myc-SPHK1 or Myc-SPHK2 were immunoprecipitated using anti-Myc antibody, then Myc-tagged SPHK1, SPHK2 and Flag-tagged TET3 proteins were analyzed by immunoblotting. (B) HEK293 cells treated with SPHK1 inhibitor SK1-I were transfected with pCMV-Myc, Myc-SPHK2 and then infected with WSN virus at an MOI of 0.5. IFN-β activity was measured by using luciferase reporter assays. Data are means ± SD of three independent experiments. ***, P<0.001. https://doi.org/10.1371/journal.ppat.1010794.s009 (TIF)

Acknowledgments We thank Dr. Wenbao Qi of the Key Laboratory of Veterinary Vaccine Innovation of the Ministry of Agriculture for providing the influenza virus A/PR/8/34 (H1N1), Dr. Bumsuk Hahm of University of Missouri-Columbia, Dr. Wenjun Song of Guangzhou Medical University, Dr. Fenyong Liu of University of California, Berkeley, and Dr. Hua Zhu of Rutgers New Jersey Medical School for critical comments and technical assistance.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010794

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