SENP5 enhances antiviral innate immunity by promoting AURKA-dependent STAT2 phosphorylation

Abstract

Background

Post-translational modifications (PTMs) play a critical role in regulating innate immune responses, including type I interferon (IFN) production and JAK-STAT pathway activation. However, the role of deSUMOylation in antiviral defense remains unclear.

Results

We identify Sentrin/SUMO-specific protease 5 (SENP5) as a positive regulator of the phosphorylation of STAT2 in JAK-STAT pathway via its deSUMOylation activity, thus inhibiting VSV or HSV-1 replication, and strengthening the antiviral activity of IFN-α. Further investigation reveals that SENP5-mediated deSUMOylation of Aurora kinase A (AURKA) alleviates its inhibition of STAT2 phosphorylation and antiviral innate immunity. Finally, AURKA suppresses STAT2 phosphorylation, and negatively regulates antiviral innate immunity by enhancing the activity of protein phosphatase 2 A (PP2A).

Conclusions

Our study demonstrates that SENP5 upregulates STAT2 phosphorylation via the AURKA–PP2A–STAT2 axis, and uncovers a previously uncharacterized role of SENP5 in regulating antiviral innate immunity.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02808-0.

Introduction

Upon viral invasion, the host innate immune system detects various pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and DNA sensors [1, 2]. This recognition of viral RNA or DNA triggers downstream adaptor proteins, such as MYD88, MAVS, or the cGAS/STING pathway, to initiate type I interferons (IFNs) production [3, 4]. Type I IFNs, which comprise multiple IFN-α subtypes, single IFN-β, and IFN-ε [5], bind to the interferon-α/β receptor (IFNAR) on neighboring cells, and induce the phosphorylation of Janus kinase 1 (JAK1) and tyrosine kinase 2 (TYK2) to initiate the activation of signal transducers and activators of transcription (STAT1 and STAT2). The phosphorylated STAT1 and STAT2 associate with interferon regulatory factor 9 (IRF9) to form the interferon-stimulated gene factor 3 (ISGF3) complex. ISGF3 translocates into the nucleus, and binds to interferon-stimulated response elements (ISREs), thereby inducing expression of interferon-stimulated genes (ISGs), such as ISG15, ISG56, and SAMHD1. Finally, these ISGs exhibit multiple antiviral functions to defend against viral replication [6, 7]. Type I IFN, particularly IFN-α, has been employed clinically to treat viral infections, such as chronic hepatitis B (CHB) infection [8, 9]. However, its efficacy is limited. Therefore, it is important to identify the key host factors that can strengthen the antiviral efficacy of IFN-α.

Post-translational modifications (PTMs), such as phosphorylation, acetylation, ubiquitination, and SUMOylation, play critical roles in regulating innate immune responses [10–13]. Deubiquitinating enzymes (DUBs) and ubiquitin-like proteases (ULPs), which comprise a diverse group of proteases, mediate the deconjugation of ubiquitin (Ub) and ubiquitin-like (UbL) modifiers, such as SUMO, from target proteins [14]. Emerging evidence highlights that DUBs and ULPs dynamically calibrate innate antiviral immunity through diverse mechanisms in both enzyme-activity- dependent and -independent manners [15]. However, their precise roles in modulating JAK-STAT signaling remain incompletely characterized.

In this study, we performed a systematic functional screening using small interfering RNA (siRNA) to investigate the roles of DUB and ULP family genes in regulating the JAK-STAT pathway. By this strategy, we identified Sentrin/SUMO-specific protease 5 (SENP5) as a positive regulator of STAT2 phosphorylation (p-STAT2) in active JAK-STAT signaling triggered by viral infection or IFN-α stimulation. Consequently, SENP5 inhibited the replication of various viruses and enhanced the IFN-α-mediated antiviral activity. We further demonstrated that SENP5-mediated deSUMOylation of Aurora kinase A (AURKA) alleviates the suppressive effect of AURKA on STAT2 phosphorylation. Our findings revealed SENP5 to be a novel regulator of antiviral innate immunity and provided new insights into SUMOylation-mediated regulation of JAK-STAT signaling.

Materials and methods

Cell culture and reagents

Human embryonic kidney 293T cells (HEK293T), human lung epithelial carcinoma A549 cells and human hepatoma cells Huh7 were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, New York, USA). Human monocytic THP-1 cells were cultured in RPMI 1640 medium (Gibco, New York, USA). All media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS).

Recombinant human interferon α-2a, AURKA inhibitor (Alisertib) and protein phosphatase 2 A (PP2A) inhibitor (Endothall) were purchased from MedChemExpress (New Jersey, USA). The proteasome inhibitor MG132 was purchased from Selleck Chemicals (Texas, USA).

Plasmids, small interfering RNAs (siRNAs) and transfection

The expression plasmids pCMV-SENP5-3×Flag, pCMV-AURKA-HA, pCMV-SUMO1/2/3/4 − 3×HA were constructed by Miaoling Biotechnology Company (Hubei, China). The expression plasmids pCMV-STAT2-HA and pCMV-STAT2-Flag were generously provided by Professor Haizhen Zhu (Hainan Medical University, Hainan, China). HBV expression plasmid was a vector carrying 1.1 copies of the HBV genome (genotype D). The pLKO.1-shSENP5 plasmid was generated using the primers listed in Table S1. The different mutants of SENP5 (C713L or C713S) and AURKA (K258R or T288A) were constructed by site-directed mutagenesis methods as previously described [16]. All primers are listed in Table S1.

The siRNA library targeting DUB and ULP family gene was designed and synthesized by RiboBio (Guangdong, China), and the siRNA targeting AURKA was synthesized by GenePharma (Shanghai, China). The sequences of targeting siRNAs are listed in Table S2. The indicated plasmids or siRNAs were transfected using Lipofectamine™ 3000 or Lipofectamine™ RNAiMAX (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions.

To produce lentivirus, pMD 2.G, psPAX2, and pLKO.1-shSENP5 were transfected into HEK293T cells. The virus released into the supernatant was harvested by centrifugation, and concentrated with Lentivirus Concentration Solution (Servicebio, Wuhan, China).

Virus and viral infections

The recombinant vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1) were kindly provided by Professor Yushun Wan (Chongqing Medical University, Chongqing, China). Viral titers were determined by plaque assay in HEK293T cells.

For viral infection experiments, the HEK293T, A549, or THP-1 cells were seeded in 12-well plates, and the indicated plasmid or targeting siRNA was transfected into the cells. At 48 h post-transfection, cells were infected with virus (MOI = 0.1) for 2 h. The inoculum was then replaced with fresh medium, and cells were collected at the indicated hours post-infection (h.p.i.) for subsequent Western blot or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis to assess the viral mRNA levels.

Interferon-stimulated response element (ISRE) luciferase reporter assay

HEK293T or Huh7 cells were co-transfected with specific siRNA targeting DUB/ULP family gene, along with ISRE luciferase reporter and Renilla luciferase-expressing plasmids previously described [17]. The cells were treated with IFN-α for 16 h and harvested for luciferase activity quantification using the Dual-Luciferase^® Reporter Assay System (Promega, Wisconsin, USA) according to the manufacturer’s protocol.

RNA extraction and RT-qPCR

Total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) and reverse-transcribed using a Takara reverse transcription kit (Takara, Dalian, China), according to the manufacturer’s instructions. The mRNA levels of indicated genes were detected by RT-qPCR using the FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland) on a CFX Connect Real-Time PCR Detection System. The relative level of target genes was normalized to GAPDH and calculated using the 2^–ΔΔCt method. All primers are listed in Table S1.

Western blot and Co-immunoprecipitation (Co-IP)

For Western blotting assay, cells were lysed in RIPA buffer containing Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The cellular proteins were separated by SDS-PAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 2 h and probed with the indicated primary antibodies. The following antibodies were used: SENP5 (Abcam, Cambridge, UK) (1:1000), AURKA (Bioss, Beijing, China) (1:1000), PP2A (CST, MA, USA) (1:1000), Flag (Beyotime, Shanghai, China) (1:1000), HA (Beyotime, Shanghai, China) (1:1000), VSV-G (Bioss, Beijing, China), HSV-1-gD (Bioss, Beijing, China), and the IFN (Type I/III) Signaling Pathway Antibody Sampler Kit (CST, MA, USA). After washing with PBST, the membranes were then probed with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (ZSGB-BIO, Beijing, China). The protein bands were detected by using an Enhanced Chemiluminescence (ECL) Substrate (Thermo Fisher Scientific, MA, USA), and the signal intensities of bands were quantified by ImageJ software.

For Co-IP assays, cell lysates were incubated with specific antibodies against Flag (Invitrogen, California, USA) or HA (Invitrogen, Carlsbad, California, USA) on a rotor at 4 °C for 2 h, then Protein A/G Magnetic Beads (MCE, New Jersey, USA) were added and incubated at 4 °C overnight. After being washed five times with PBST, the proteins bound to the beads were eluted by boiling in 1 × loading buffer for 10 min, and subjected to immunoblotting analysis.

Measuring phosphatase activity

Phosphatase activity was determined by measuring the release of inorganic phosphate (Pi) from phosphopeptide according to previous report [18]. Briefly, PP2A was immunoprecipitated from cell lysates using magnetic beads conjugated with an anti-PP2A antibody. The immunoprecipitated beads were incubated with the phosphopeptide substrate KRpTIRR (MedChemExpress, New Jersey, USA) in reaction buffer at 30 °C for 30 min. Released Pi was quantified using a malachite green-based phosphate assay kit (Elabscience, Wuhan, China). The phosphatase activity of PP2A was calculated using a standard curve.

Immunofluorescence microscopy

HEK293T cells were seeded on collagen-coated glass slides in 12-well plates. The cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.1% Triton X-100 and blocked with 5% BSA. Subsequently, the cells were incubated with primary antibodies against AURKA (Proteintech, IL, USA) and STAT2 (CST, MA, USA) at 4 °C overnight. After being washed three times with PBS, the cells were stained with Alexa Fluor 488 goat anti-rabbit (ZSGB-BIO, Beijing, China) and Alexa Fluor 594 goat anti-mouse (ZSGB-BIO, Beijing, China) secondary antibodies for 1 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) for 10 min, and then observed using a fluorescence microscope.

Statistical analysis

Data are presented as mean ± standard deviation (SD) from at least three independent experiments. Statistical significance was determined using Student’s t-test, one-way ANOVA, or two-way ANOVA, as appropriate, using GraphPad Prism (version 8.0). P < 0.05 was considered statistically significant: *P < 0.05, **P < 0.01, ***P < 0.001.

Results

Targeted screening identifies novel regulators of JAK-STAT pathway from DUBs and ULPs

Type I interferon (IFN-I) plays an important role in the antiviral innate immune response [19]. To systematically evaluate the potential functions of DUBs and ULPs in regulating IFN-I-induced JAK-STAT pathway, we conducted a screening of a siRNA library targeting 97 human DUB and ULP family genes by using the ISRE luciferase reporter assay. Then these selected candidates were further validated using the following assays: measurement of IFN-induced transcription of representative ISGs, and assessment of antiviral activity (Fig. 1A). A candidate screen based on ISRE promoter activity revealed that knockdown of previously unreported genes, including SENP5, USP35, and USP31, significantly attenuated ISRE activation in both HEK293T and Huh7 cells (Fig. 1B and S1). Furthermore, silencing SENP5, USP35, or USP31 suppressed the expression of classical ISGs such as ISG15, ISG54, and ISG56 in HEK293T, and SAMHD1 and APOBEC3G with antiviral function [20, 21] in Huh7 cells (Fig. 1C). To validate the potential antiviral function of SENP5, USP35, and USP31, we assessed their antiviral effects on the replication of VSV and HSV-1. As expected, knockdown of SENP5, USP35, or USP31 significantly increased the viral mRNA levels of VSV and HSV-1 (Fig. 1D). Therefore, employing candidate-selection methods, we identified SENP5, USP35, and USP31 as positive regulators of the JAK-STAT signaling and thus suppressing viral replication.

Fig. 1.

Candidate-selection assays identify SENP5 as a novel positive regulator of JAK-STAT signaling. A The schematic diagram of candidate-selection assays including ISRE activity, IFN-induced transcripts of represented ISGs and antiviral function assay. B HEK293T cells were co-transfected with the siRNAs targeting DUBs/ULPs, ISRE, and pTK-Renilla reporter plasmids for 48 h, and treated with IFN-α (1000 IU/mL, 16 h). The ISRE-luciferase activity was assessed using a dual-luciferase reporter assay system. C HEK293T or Huh7 cells were transfected with siRNA targeting USP31, USP35, or SENP5, and stimulated with IFN-α (1000 IU/mL). The mRNA levels of ISG15, ISG54, ISG56, APOBEC3G, and SAMHD1 were detected by RT-qPCR. D Effects of knockdown candidate gene (USP31, USP35, or SENP5) on the viral mRNA levels of VSV in HEK293T or HSV-1 in A549 cells. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01

SENP5 exhibits potent antiviral activity against VSV and HSV-1

SENP5 was selected for further investigation due to its robust enhancement of the IFN-induced JAK-STAT signaling and antiviral activity. As shown in Fig. 2A and Fig. S2A, SENP5 expression levels gradually increased following VSV or HSV-1 infection, indicating that SENP5 may be related to viral infection. To further address the exact antiviral role of SENP5 against viruses, we transfected HEK293T cells with siRNA targeting SENP5, followed by VSV or HSV-1 infection. Knockdown of endogenous SENP5 resulted in increased levels of VSV mRNA and VSV-G protein in HEK293T cells (Fig. 2B). Conversely, we found that overexpression of SENP5 resulted in decreased levels of VSV mRNA and VSV-G protein in a dose-dependent manner (Fig. 2C). Consistently, VSV replication, as indicated by increased VSV-GFP fluorescence, was enhanced in SENP5-knockout cells (209.5 ± 6.9% of control) (Fig. 2D), further confirming the inhibitory role of SENP5. Similar results were observed in A549 cells or THP-1 cells infected with HSV-1, suggesting that SENP5 inhibits VSV and HSV-1 replication independent of cell type (Fig. 2E and F and S2B). Next, as IFN-α is a clinically approved drug to treat viral infection, we determined the role of SENP5 in IFN-α-mediated antiviral effects. IFN-α treatment remarkably reduced VSV mRNA levels, and SENP5 overexpression enhanced this inhibitory effect on VSV replication (Fig. 2G). Furthermore, SENP5 overexpression, combined with IFN-α treatment, reduced HBV core-associated DNA levels in the cytoplasm and HBsAg in the supernatant (Fig. 2H), whereas SENP5 knockdown attenuated the anti-HBV effects of IFN-α (Fig. S2C). These data demonstrate that SENP5 not only inhibits the replication of VSV and HSV-1, but also enhances the antiviral activity of IFN-α.

Fig. 2.

SENP5 suppresses viral replication and potentiates antiviral activity of IFN-α. A HEK293T cells were infected with VSV (MOI = 0.5) for indicated time points, then the levels of SENP5 were detected by Western blotting. B-C HEK293T were transfected with siRNAs targeting SENP5 (B) or Flag-SENP5 plasmid (C), and infected with VSV (MOI = 0.1, 12 h). The levels of VSV mRNA and VSV-G protein were detected by RT-qPCR (upper) and Western blotting (bottom). D HEK293T cell lines with stable knockout SENP5 (SENP5-KO) were infected with VSV-GFP (MOI = 0.005) for 30 h. The fluorescence of VSV-GFP was detected by fluorescence microscopy (upper), and the fluorescence intensity was analyzed with the ImageJ software (bottom). E-F A549 cells were transfected with siSENP5 (E) or Flag-SENP5 plasmid (F), and infected with HSV-1 (MOI = 0.1, 12 h). The levels of HSV-1 mRNA were detected by RT-qPCR. G HEK293T cells were transfected with Flag-SENP5, followed by VSV infection and IFN-α treatment (100 IU/mL, 12 h). The levels of VSV mRNA were detected by RT-qPCR. H Huh7 cells were co-transfected with HBV replication plasmid and Flag-SENP5 plasmid, followed by IFN-α treatment (1000 IU/mL, 48 h). The relative levels of cytoplasmic HBV core-associated DNA and secreted HBsAg were detected by qPCR and ELISA assay. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

SENP5 increases the phosphorylation of STAT2 to enhance antiviral immunity

Next, we investigated whether SENP5 enhances antiviral immunity by regulating the production of IFN-Ⅰ. As shown in Fig. 3A, knockdown of SENP5 barely affected HSV-1 induced IFN-β mRNA levels and protein secretion in THP-1 cells, indicating that SENP5 does not affect the production of IFN-β upon viral infection. We therefore speculated that SENP5 prompts antiviral immunity by modulating the IFN-α-mediated JAK-STAT signaling pathway. In IFN-α treated HEK293T cells, silencing SENP5 with siSENP5 did not affect the transcriptional levels of IFNAR1, JAK1, STAT1, and STAT2 in the JAK-STAT pathway (Fig. S3A). However, silencing SENP5 significantly reduced IFN-α-induced phosphorylation of STAT2 at Tyr690 (p-STAT2, Y690) in HEK293T, Huh7 and A549 cells (Fig. 3B and C and S3B), without affecting total STAT2 protein levels. In contrast, SENP5 overexpression markedly increased the phosphorylation levels of STAT2 (Fig. 3D and E). Notably, neither knockdown nor overexpression of SENP5 significantly altered the protein levels or phosphorylation status of other components in the JAK–STAT signaling pathway, such as IFNAR1, TYK2, JAK1 and STAT1. Similarly, in VSV-infected A549 cells, the p-STAT2 levels were significantly decreased in SENP5-silenced cells at 3 and 6 hours post-infection (h.p.i.), while ectopic expression of SENP5 enhanced p-STAT2 levels at indicated time points (Fig. 3F and G). These data provide evidence that SENP5 positively regulates antiviral immunity by upregulating the phosphorylation of STAT2 in the JAK-STAT pathway upon viral infection or IFN-α stimulation.

Fig. 3.

SENP5 specifically enhances STAT2 phosphorylation. A THP-1 cells were transduced with lentiviruses containing shRNA to SENP5 (shSENP5), and infected with HSV-1. The relative levels of mRNA (upper) and secreted IFN-β protein (bottom) in the medium were measured by RT-qPCR and ELISA assay, respectively. B-C SENP5-silenced HEK293T (B) or Huh7 (C) cells were stimulated with IFN-α (1000 IU/mL) for 0.5 h. The protein levels of key molecules in the JAK-STAT pathway were detected by Western blotting. D-E Parallel analyses of key molecules in the JAK-STAT pathway were conducted in HEK293T (D) or Huh7 (E) cells overexpressing Flag-SENP5 and in the presence of IFN-α stimulation (1000 IU/mL, 0.5 h). F-G A549 cells were transfected with siSENP5 (F) or Flag-SENP5 plasmid (G), followed by infection with VSV (MOI = 3) for indicated times. The levels of p-STAT2 and STAT2 protein were detected by Western blotting. The data are presented as the mean ± SD of three independent experiments. ns: not significant

SENP5 promotes IFN-α-induced STAT2 phosphorylation and antiviral responses through its deSUMOylation activity

SENP5 is a SUMO-specific protease and cleaves SUMO2/3 modifications from substrate proteins [22]. Functional assays in HEK293T cells co-transfected with HA-SUMO3 revealed that overexpression of wild-type (WT) SENP5, but not the catalytically inactive mutants C713S or C713L [23], significantly reduced HA-SUMO3 protein levels (Fig. S4A), confirming that Cys713 is critical for the deSUMOylation activity of SENP5. To investigate whether the regulation of the IFN-α-induced JAK-STAT signaling by SENP5 depends on its deSUMOylation activity, we analyzed IFN-α-induced p-STAT2, the transcript levels of ISG15 and ISG54 following ectopic expression of SENP5 WT or the C713L mutant. As shown in Fig. 4A and B, WT SENP5 enhanced IFN-α-induced p-STAT2, mRNA levels of ISG15 and ISG54, while the C713L mutant abrogated these effects. Consistently, exogenous expression of SENP5-C713L mutant failed to enhance the anti-HBV activity of IFN-α (Fig. 4C).

Fig. 4.

DeSUMOylation activity of SENP5 is essential for regulating JAK-STAT signaling and antiviral immunity. A HEK293T cells were transfected with Flag-tagged SENP5-WT or SENP5-C713L mutant plasmid, and stimulated with IFN-α (1000 IU/mL, 0.5 h). The levels of p-STAT2 were detected by Western blotting. B HEK293T cells were stimulated with IFN-α (1000 IU/mL, 7 h), and the levels of ISG15 and ISG54 mRNA were detected by RT-qPCR. C Huh7 cells were co-transfected with HBV replication plasmid and Flag-tagged SENP5-WT or C713L mutant plasmid, followed by IFN-α treatment (1000 IU/mL, 48 h). The levels of HBV core-associated DNA were detected by qPCR. D A549 cells were transfected with Flag-tagged SENP5 WT or SENP5 C713L mutant plasmid, and infected with VSV. The levels of ISG15 and ISG54 mRNA were detected by RT-qPCR. E-F The levels of VSV mRNA and VSV-G protein were detected by RT-qPCR and Western blotting, respectively. G The SENP5-KO HEK293T cells were rescued with SENP5-WT or C713L plasmid, followed by infection with VSV-GFP (MOI = 0.005) for 30 h. The fluorescence of VSV-GFP was detected by fluorescence microscopy (left), and the fluorescence intensity was analyzed with the ImageJ software (right). The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Next, we evaluated whether SENP5-mediated inhibition on VSV replication also depends on its deSUMOylation activity. Similarly, overexpression of WT SENP5, but not the C713L mutant, markedly increased the mRNA levels of ISG15 and ISG54 induced by VSV infection (Fig. 4D). Furthermore, overexpression of C713L mutant failed to suppress intracellular VSV mRNA or VSV-G protein levels (Fig. 4E and F). Consistently, in SENP5-KO HEK293T cells, the inhibitory effect on VSV-GFP replication was restored upon reconstitution with wild-type SENP5 (39.8 ± 9.8% of control), but barely rescued by the SENP5-C713L catalytic mutant (90.6 ± 11% of control) (Fig. 4G). Collectively, these results indicate that the deSUMOylation activity of SENP5 is essential for regulating the JAK-STAT pathway and antiviral immunity.

DeSUMOylation of AURKA by SENP5 promotes STAT2 phosphorylation

Next, we continued to explore the mechanism underlying the SENP5-mediated regulation of STAT2 phosphorylation and antiviral immunity via its deSUMOylation activity. A previous immunoprecipitation-mass spectrometry (IP-MS) analysis using SENP5 as bait protein identified 310 significantly enriched interacting proteins [24], and cross-referencing with the BioGRID database revealed three overlapping candidate proteins: DPP9, CARM1, and AURKA (Fig. 5A). STAT2 phosphorylation was remarkably changed by silencing the endogenous expression of AURKA, suggesting AURKA may participate in regulating STAT2 phosphorylation (Fig. S4B). Co-IP confirmed the interaction between SENP5 and AURKA (Fig. S4C), consistent with previous studies [25]. Furthermore, overexpression of WT SENP5, but not the C713L mutant, reduced the SUMOylation of AURKA WT but not the K258R mutant, confirming that SENP5 specifically deSUMOylates AURKA at lysine 258 (K258) (Fig. S4D) [25]. Based on these findings, we hypothesized that AURKA might be involved in SENP5-mediated regulation of STAT2 phosphorylation.

Fig. 5.

SENP5-AURKA-STAT2 axis regulates STAT2 phosphorylation and viral replication. A Venn diagram overlaying SENP5-interacting proteins (IP-MS, blue; n = 310) with STAT2 interactors (BioGRID, pink; n = 82). B HEK293T cells were co-transfected with HA-AURKA and Flag-STAT2 plasmids, the interaction between HA-AURKA and Flag-STAT2 was analyzed by co-immunoprecipitation. C-D HEK293T cells were transfected with siRNA targeting AURKA (C) or HA-AURKA plasmid (D), and stimulated with IFN-α (1000 IU/mL, 0.5 h). The levels of p-STAT2 and p-STAT1 were detected by Western blotting. E-F The effect of knockdown of AURKA on the level of VSV-G protein (E) and mRNA (F). G-H The effect of overexpression of AURKA on the level of VSV-G protein (G) and mRNA (H). The data are presented as the mean ± SD of three independent experiments. **P < 0.01

The Co-IP assay confirmed that AURKA interacts with STAT2 (Fig. 5B). Functionally, knockdown of endogenous AURKA enhanced IFN-α-induced p-STAT2, whereas overexpression of AURKA markedly suppressed p-STAT2 (Fig. 5 C and D), indicating that AURKA negatively regulates IFN-α-induced STAT2 phosphorylation. Consistently, knockdown of endogenous AURKA significantly reduced VSV mRNA and VSV-G protein levels in HEK293T cells (Fig. 5E and F), as well as HSV-1 mRNA levels in A549 cells (Fig. S5A). Conversely, overexpression of HA-AURKA promoted the replication of VSV and HSV-1 (Fig. 5G and H, S5B and S5C). These results collectively demonstrate that AURKA negatively regulates p-STAT2 in the JAK-STAT pathway, thereby facilitating the replication of VSV and HSV-1.

SENP5 regulates antiviral responses through AURKA-dependent mechanisms

Next, we investigated whether the suppression of antiviral immunity by AURKA depends on its kinase activity. In IFN-α-stimulated HEK293T cells, overexpression of AURKA WT significantly suppressed the levels of p-STAT2 and ISG15 transcripts. However, this inhibition was completely abolished by pretreatment with the AURKA kinase inhibitor (Alisertib), indicating that the regulatory effect of AURKA on p-STAT2 and ISG15 mRNA levels depends on its kinase activity (Fig. S5D and S5E). Consistently, the ability of WT AURKA to increase VSV-G protein levels was abolished by Alisertib pretreatment (Fig. S5F). Similarly, analysis of AURKA mutants revealed that both the kinase-deficient mutant (T288A) and SUMOylation-deficient mutant (K258R) lost their inhibitory effect on IFN-α-induced p-STAT2 and ISG15 transcripts (Fig. 6A and B). Accordingly, the promoting effect of AURKA on the VSV-G protein levels was diminished upon expression of K258R or T288A mutants (Fig. 6C). Collectively, these results suggest that AURKA suppresses the IFN-induced JAK-STAT pathway and host antiviral response through its kinase activity which is regulated by SENP5-mediated deSUMOylation.

Fig. 6.

SENP5 regulates JAK-STAT signaling and antiviral responses in an AURKA-dependent manner. A-C HEK293T cells were transfected with HA-tagged AURKA-WT, K258R or T288A mutant plasmid, followed by stimulation with IFN-α (1000 IU/mL) for 0.5 h (A), or 16 h (B), or VSV (MOI = 0.1, 12 h) (C). D A549 cells were transfected with AURKA-WT, K258R or T288A mutant plasmids or treated with a PP2A inhibitor Endothall (20 µM) as a control. The PP2A phosphatase activity was analyzed by a malachite green-based phosphate quantitation assay. E-F A549 cells were transfected with siRNA targeting AURKA in the presence or absence of Endothall treatment (20 µM), followed by stimulation with IFN-α (500 IU/mL) for 4 h (E), or 16 h (F). G-I HEK293T cells were transfected with Flag-SENP5 plasmid in the presence or absence of endogenous AURKA knockdown. The levels of p-STAT2 (A, E, G), VSV-G protein (C, I), and ISG15 mRNA (B, F, H) were measured by Western blotting and RT-qPCR, respectively. The data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant

As protein phosphatase 2 A (PP2A) is identified to regulate the dephosphorylation of STAT2 [26], we continued to investigate whether AURKA mediates the phosphorylation of STAT2 via PP2A. Endothall (a PP2A selective inhibitor) treatment increased the levels of IFN-α-induced STAT2 phosphorylation and ISG15 mRNA (Figs. S5G and S5H). Furthermore, overexpression of WT AURKA increased the activity of PP2A, while the kinase-deficient mutant (T288A) and SUMOylation-deficient mutant (K258R) of AURKA abrogated these effects (Fig. 6D). Finally, Endothall treatment partially attenuated the enhancement trend of IFN-α-induced STAT2 phosphorylation and ISG15 mRNA expression in AURKA knockdown cells (compare Endothall treatment group and PBS group) (Fig. 6E and F). These findings demonstrate that AURKA increases the activity of PP2A, and reduces the IFN-induced STAT2 phosphorylation.

Finally, we explored whether AURKA mediates the regulatory effect of SENP5 on antiviral immunity. Consistent with the above findings, overexpression of SENP5 enhanced IFN-α-induced p-STAT2 and ISG15 transcripts, while knockdown of AURKA completely abrogated this effect (Fig. 6G and H). Similarly, the ability of SENP5 to suppress VSV-G protein (Fig. 6I) and HSV-1 mRNA levels (Fig. S5I) during viral infection was blocked in AURKA-depleted cells. These findings demonstrate that SENP5 potentiates the IFN-induced JAK-STAT signaling pathway and antiviral immunity in an AURKA-dependent manner.

Discussion

IFN-I functions as an approved drug to control viral infection [5, 27]. In particular, IFN-α has been proven to have the capacity to achieve a functional cure for HBV in clinical practice [28–30]. However, its clearance effect and durability are limited [9]. It is critical to identify the key host factor that strengthens IFN-I-induced JAK-STAT signaling pathway for developing new strategies to enhance the antiviral effect of IFN-α [8]. Numerous studies have demonstrated that DUBs and ULPs are critical for mediating innate antiviral immunity through diverse mechanisms [13, 15]. However, a comprehensive understanding of their roles in regulating JAK-STAT signaling pathway is still unclear. In this research, we performed an siRNA-based screen by individually knocking down 97 DUB and ULP genes, and employed functional readouts including IFN-α- driven ISRE activity, ISG transcriptions, and antiviral efficacy for validation. Based on this strategy, we identified SENP5 as a specific positive regulator of JAK-STAT pathway that restricts viral replication, highlighting the central role of SUMOylation in antiviral innate immunity.

Similar to ubiquitination, SUMOylation is a dynamic and reversible PTM [11], and deSUMOylation is catalyzed by SENPs [31]. SENPs, which are categorized into subfamilies (SENP1/2, SENP3/5, SENP6/7), have been reported to modulate innate immunity in different manners. As negative regulators of IFN-I signaling, SENP1 and SENP2 suppress IFN-I signaling by directly deSUMOylating MAVS or IRF3 [32, 33]. SENP6 modulates deSUMOylation of USP8, and enhances IFNAR2 ubiquitination and degradation, thus attenuating IFN-I antiviral activity [34]. Conversely, SENP7 mediates the deSUMOylation of cGAS, thus boosting the expression of antiviral genes to restrict HSV-1 infection [35]. Although SENP5 has established roles in DNA damage repair [36], cell cycle control [37] and tumorigenesis [38], its exact function in regulating innate immunity remains unclear. In this research, we evaluated the potential function of SENP5 in antiviral innate immunity at the cellular level. First, SENP5 increased the ISG15 and ISG54 transcriptions induced by viral infection or IFN-α (Figs. 1 and 4). This suggests that SENP5 can promote JAK-STAT signaling. Second, SENP5 restricted VSV and HSV-1 replication and strengthened the antiviral activity of IFN-α via its catalytic activity (Fig. 4). Mechanistically, SENP5 specifically potentiated phosphorylation of STAT2 (Figs. 3 and 4). These results suggest a positive regulatory role of SENP5 in innate immunity by strengthening IFN-I signaling responses.

STAT2 is an essential factor in the IFN-I-stimulated JAK-STAT signaling pathway and plays a pivotal role in antiviral responses [39]. Previous reports have demonstrated the activation of STAT2 is modulated through PTMs, including phosphorylation and acetylation [40, 41]. The typical activation of STAT2 primarily involves the upstream JAK1/TYK2-mediated phosphorylation at Y690, enabling its dimerization and inducing subsequent canonical ISGs expression to exert antiviral functions [42]. Additionally, other kinases such as MAPK12 regulate the early phosphorylation of STAT2 at Y690 following Influenza A virus infection, which is independent of the JAK1/TYK2 pathway [43]. Besides phosphorylation at Y690, serine and threonine phosphorylation of STAT2 have also been reported, which may positively or negatively affect the function of STAT2 [44, 45]. However, the full spectrum of host factors regulating the activation of STAT2 remains incompletely elucidated. In this study, we demonstrated that SENP5 specifically potentiates STAT2 phosphorylation and bolsters antiviral responses via its deSUMOylating activity. A previous study showed that SENP5 mediates the deSUMOylation of AURKA at K258, thereby suppressing its kinase activity [25]. As a serine/threonine kinase, AURKA plays a critical role in regulating cell division [46]. Recently, extensive research has indicated that dysregulation of AURKA is frequently observed in various malignancies and contributes to oncogenesis, making it a potential target for cancer therapy [47, 48]. However, the role of AURKA in controlling antiviral innate immunity is less reported. Here, we demonstrated AURKA increased the activity of PP2A, and thereby reduced IFN-induced STAT2 phosphorylation, leading to impaired antiviral immunity (Fig. 6). Inhibition of AURKA kinase activity by Alisertib, an established AURKA inhibitor used in anti-cancer regimens, potentiated IFN-α signaling and suppressed VSV and HSV-1 replication, indicating that the AURKA-PP2A-STAT2 signaling axis may be a new antiviral target. Furthermore, the antiviral effect of SENP5 against VSV and HSV-1 was abrogated in AURKA-depleted cells, indicating AURKA acts as a key molecular brake linking SENP5-mediated deSUMOylation to regulating innate immunity (Fig. 7).

Fig. 7.

SENP5 promotes the JAK-STAT signaling and antiviral responses via deSUMOylation of AURKA. SENP5 is upregulated by virus infection, and regulates the deSUMOylation of AURKA. The deSUMOylation of AURKA reduced the activity of PP2A, relieving the AURKA-mediated suppression on STAT2 phosphorylation

This study has several limitations. First, this research was conducted in cell cultures. Further research employing mouse models and clinical samples of virus-infected patients would be helpful for confirming our findings. Second, while we tested the antiviral function of SENP5 on VSV, HSV-1 or HBV, the effects on other RNA or DNA viruses, including influenza virus and adenovirus, remain to be investigated. Third, although we elucidated the role of AURKA in controlling the antiviral innate immunity, the precise molecular mechanism by which AURKA regulates PP2A activity and STAT2 phosphorylation requires further investigation.

In conclusion, our findings elucidate a previously unknown function of SENP5 for inhibiting viral replication and strengthening the antiviral function of IFN-α, which will contribute to a deeper understanding of deSUMOylation-mediated regulation in antiviral innate immunity. Furthermore, the SENP5-AURKA-PP2A-STAT2 axis may represent a novel target for clinical antiviral drug development.

Abbreviations

AURKA

Aurora kinase A

Co-IP

Co-immunoprecipitation

DUBs

Deubiquitinating enzymes

HSV-1

Herpes simplex virus type 1

IFN-I

Type I interferon

ISG

Interferon-stimulated gene

ISRE

Interferon-stimulated response element

PP2A

Protein Phosphatase 2A

PTMs

Post-translational modifications

SENP5

Sentrin/SUMO-specific protease 5

siRNA

small interfering RNA

STAT2

Signal transducer and activator of transcription 2

ULPs

Ubiquitin-like proteases

VSV

Vesicular stomatitis virus

Authors’ contributions

Shuyi Song: methodology, investigation, formal analysis, writing and original draft preparation. Yuwei Kan: methodology, investigation, formal analysis, writing and original draft preparation. Minghui Zhou: methodology, investigation, formal analysis. Yanjun Jiang: investigation, formal analysis. Yitian Tang: investigation. Hongchun Luo: funding acquisition, formal analysis, conceptualization. Yuan Hu: funding acquisition, conceptualization, supervision, formal analysis, writing-reviewing and editing. All authors read and approved the manuscript.

Funding

This work was supported by Chongqing Venture and Innovation Support Program for Overseas Returnees (cx2023026 to YH), and Natural Science Foundation of Chongqing (CSTB2024NSCQ-MSX0406 to YH, cstc2021jcyjmsxmX0202 to HCL), and CQMU Program for Youth Innovation in Future Medicine (W0160 to YH and HCL).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.