SIAH1 modulates antiviral immune responses by targeting deubiquitinase USP19

Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin‐specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus‐mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27‐linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63‐linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1‐mediated degradation of USP19 reversed USP19‐mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.

inflammatory responses.To achieve these functions, the immune system has evolved complex multilevel mechanisms, which include posttranslational modifications (PTMs) to control immune responses at the molecular level. 7,8It is well-documented that ubiquitination is one of the essential PTMs involved in the regulation of host type I IFN signaling. 9,10ditionally, as a dynamic and reversible process, ubiquitination is reversed by various deubiquitinating enzymes (DUBs) that cleave polyubiquitin chains.DUBs are a protease superfamily that can be further categorized into five families in humans based on their enzymatic activity domains. 11Ubiquitin-specific protein 19 (USP19) is a member of the ubiquitin-specific protease (USP) family and is known to be involved in a variety of cellular processes. 12The functions of USP19 include regulation of the unfolded protein response, [13][14][15] regulation of hypoxia by rescuing pathway molecules from degradation, 16 regulation of carcinogenesis and cancer progression, 17,18 and most importantly, suppression of host innate immune responses by targeting Beclin1, 19 TRAF3, 20 TRIF, 21 and TBK1. 22wever, despite much research and information on the role of USP19, studies on the factors that regulate USP19 are rare.
The seven in absentia homolog (SIAH) family of RING-domain proteins are RING type E3 ubiquitin ligases that consist of a catalytically active RING domain, a substrate binding domain, and two zinc finger motifs. 23Among the SIAH family, SIAH1 is known to be involved in a variety of cellular pathways, such as tumor suppression, hypoxia response, transcription regulation, oxidative stress, cell cycle regulation, and DNA damage response.In addition, SIAH1 targets numerous substrate proteins for degradation 23 : tumor suppressor protein kinase 2 (HIPK2), 24 P27 involved in the regulation of cell mortality 25 and tribbles homolog 3 (TRB3), 26 which is an important regulator of insulin signaling, lipid metabolism, and NF-κB signaling.These substrate proteins undergo ubiquitination and degradation by SIAH1.8][29][30] Although the functions of SIAH1 have been extensively studied, its role in regulating innate immune responses against viral infections has not been studied.
In this study, we report that SIAH1 is a novel positive regulator of host innate immune responses to viral infections.SIAH1 specifically interacts with the deubiquitinase USP19 and mediates the K27-linked ubiquitination and proteasomal degradation of USP19 in response to viral infections.These findings suggest that SIAH1-USP19 interaction and the early degradation of USP19 following a virus infection contribute to enhancing the host innate immune response to virus infections by positively regulating the type I IFN signaling pathway.

| Identification of SIAH1 as a positive regulator of type I IFN signaling
To determine the broader significance of ubiquitination in type I IFN pathway activation, we performed a cell-based gain-of-function screening by overexpressing 227 E3 ligases to evaluate their effect on IFN-β promoter activity.Hence, IFN-β, and TK-Renilla were cotransfected into human embryonic kidney (HEK293T) cells, along with each E3 ligase construct and checked the IFN-β luciferase activity.As shown in Supporting Information: Figure 1A, SIAH1 (E3 ligase no.143) enhanced the IFN-β luciferase activity compared to the control.Next, since SIAH1 is known to undergo autoubiquitination under native physiological conditions to limit their availability, 31 we checked the SIAH1 expression level in mouse macrophage cells (RAW264.7) and HEK293T cells after Influenza A virus (PR8-GFP) or Sendai virus (SeV) infection.As shown in Supporting Information: Figures 1B−1D, SIAH1 expression peaked rapidly in the early period after viral infections in both immune and epithelial cells.These findings suggest that SIAH1 is involve in type I IFN signaling and plays a unique role during viral infections.
Interestingly, we found that virus replication in SIAH1-knockdown CD14 + MDM, mBMDM, RAW264.7 and HEK293T cells was significantly higher than that of the control cells (Figures 1A,C,E IFN-β and IL-6 levels than the control (Supporting Information: Figure 2L).These findings indicate that SIAH1 positively regulates antiviral innate immune responses to DNA and RNA virus infections.

| SIAH1 positively regulates antiviral gene expression and type I IFN signaling
Next, we examined the transcription of IFN-related genes in SIAH1knockdown hCD14+ MDM and RAW264.7 cells after infection with PR8-GFP or VSV-GFP.Low levels of IFN-related gene transcription were found in the SIAH1 knockdown cells compared to the control cells (Figures 1M,N), which was consistent with our earlier findings.In contrast, when SIAH1 or a control vector stably overexpressed RAW264.7 cells infected with VSV-GFP, SIAH1 overexpression significantly increased IFN-related gene transcription (Supporting Information: Figure 4A).
Upon virus infection, viral PAMPs are recognized by host PRRs, which activate key signaling molecules in the type I IFN signaling pathway.Hence, we next investigated the effect of SIAH1 deficiency on virus-mediated activation of the downstream signaling pathway in RAW264.7 cells.The cells were stimulated with PR8-GFP or VSV-GFP, and samples were collected at given time intervals.Immunoblotting was used to evaluate whole-cell lysates (WCLs).After infecting SIAH1 siRNA knockdown or control cells with PR8-GFP, the phosphorylation levels of TBK1, IRF3, and STAT1 proteins were measured.As shown in Figure 1O, the levels of protein phosphorylation were lower in SIAH1 knockdown cells than in control cells.In contrast, VSV-GFP infection promoted the phosphorylation of TBK1, IRF3, and STAT1 in stable SIAH1-overexpressed RAW264.7 cells (Supporting Information: Figure 4B).These findings led us to conclude that SIAH1 positively regulates the type I IFN signaling pathway following viral infections.

| SIAH1 interacts with USP19 following virus infections
Following a virus infection, host sensors, adaptor molecules, kinases, and other components in the IFN signaling pathway are involved in activating downstream signaling. 1Therefore, to better understand the mechanism of SIAH1-mediated positive regulation in the antiviral innate immune responses, we attempted to identify a specific targeting molecule of SIAH1.We constructed a Strep-tagged full-length SIAH1 plasmid and used it as a bait to identify potential binding partners by Strep affinity purification coupled with mass spectrometry.Mass spectrometry results showed USP19 as a molecule interacting with SIAH1 (Supporting Information: Figure 5A).Furthermore, these results were solidified by a previous study that demonstrated the interaction between SIAH1 and USP19. 32USP19 is a well-known deubiquitinase enzyme.0][21] Therefore, we hypothesize that SIAH1, which is well known for the degradation of its substrate molecules, might degrade USP19 to control host innate immune signaling and to maintain immune homeostasis.First, we expressed USP19 in HEK293T cells alongside various doses of SIAH1 and discovered that USP19 expression was dose-dependently reduced in the presence of SIAH1 (Figure 2A).To determine whether SIAH1 degrades USP19 via the ubiquitinproteasome pathway, we performed a degradation experiment in the presence of different inhibitors.USP19 expression was regained in the presence of the proteasomal inhibitor MG132 but not in the presence of lysosomal inhibitors (chloroquine and NH 4 Cl) or caspase inhibitor Z-VAD (Figure 2B).These results suggest that SIAH1-mediated USP19 degradation depends on the proteasome pathway, which is also consistent with previous findings. 32xt, to evaluate the interaction between SIAH1 and USP19 under physiological conditions, HEK293T cells were infected with the SeV in a time-dependent manner.Immunoprecipitation (IP) tests revealed an endogenous interaction of SIAH1 with USP19 in HEK293T cells (Figure 2C).Consistent with the data in Supporting Information: Figure 1C, stabilization of SIAH1 and strong interactions between SIAH1 and USP19 occurred early after virus infection.Similar results were observed in Supporting Information: Figure 5B, where overexpressed SIAH1 interacted with endogenous USP19 after a virus infection.Additionally, confocal microscopic analysis using SIAH1 and USP19 transiently transfected HeLa cells showed that SIAH1 colocalized with USP19 in the cytosol (Supporting Information: Figure 5C).Confocal microscopy was also employed to confirm the time-dependent co-localization of SIAH1 and USP19.

This revealed substantial co-localization of SIAH1 and USP19 after
SeV infection of HeLa cells at the early stage of virus infection (Figure 2D).Furthermore, we created GST-tagged RING and substrate binding domain (SBD) of SIAH1-expressing plasmids to determine which SIAH1 domain was responsible for its interaction with USP19 (Figure 2E).Co-IP assay and western blot analysis revealed that the substrate binding domain of SIAH1 strongly interacted with USP19, but its RING domain showed no interaction with USP19 (Figure 2F).Next, we produced various domain constructs of USP19 (Figure 2G) and employed them in a Streppull-down assay with full-length SIAH1.Our results and previous studies confirm that SIAH1 interacts with the 462-473 amino acid (aa) region of USP19 (Figure 2H).Collectively, these results suggest that SIAH1 directly interacts with USP19 during the early stages of virus infection and leads USP19 for proteasomal degradation.

| SIAH1 mediates the degradation of USP19 via K27-linked polyubiquitination
The ubiquitin-proteasome system is a nonlysosomal proteolysis mechanism that is involved in intracellular protein degradation. 33 shown in Figure 2B, MG132 inhibits the degradation of USP19 by SIAH1, which suggests involvement of the ubiquitin-proteasome pathway in the process.As shown in Figure 3A, we found that USP19 was connected to both K27 and K29 ubiquitination when we expressed Strep-tagged USP19 and Flag-tagged SIAH1, together with distinct ubiquitin mutant plasmids that express only the indicated type.These results were further supported by IP assays and immunoblotting with K27 and K29 linkage-specific antibodies (Figure 3B).However, as previously mentioned, SIAH1 also undergoes auto-ubiquitination and degradation to maintain its availability. 23,34,35Since SIAH1 also pulled down with USP19 during the Strep-tagged USP19 pull-down in Figures 3A,B, we sought to assess whether SIAH1 undergoes auto-ubiquitination by K27-or K29-linked ubiquitination to eliminate the possibility of overlapping ubiquitination bands.Therefore, we transfected HEK293T cells with Flag-tagged SIAH1, as shown in Figure 3C.Flag-IP and immunoblot analysis revealed that SIAH1 undergoes K29-linked autoubiquitination.These results suggest that USP19 undergoes K27linked ubiquitination by SIAH1, and the auto-ubiquitination of SIAH1 is responsible for the K29 ubiquitination band in Figures 3A,B ubiquitination assay in the presence of SIAH1 using a K27 linkagespecific antibody (Figure 3D).In addition, an in vitro ubiquitination assay confirms that SIAH1, in collaboration with an E1 ubiquitin activating enzyme UBA1 and the E2 ubiquitin conjugating enzyme UbcH5c, efficiently transfer the ubiquitin to USP19, and consistent with the results presented in Figure 3A−D, K27-linked polyubiquitination was observed on USP19 in the presence of SIAH1 (Figure 3E).Next, to evaluate the USP19 ubiquitination under physiological conditions, we infected HEK293T cells with SeV in a time-dependent manner.As expected, USP19 IP and immunoblot analysis showed that USP19 rapidly undergoes K27-linked ubiquitination during the early periods of viral infections and reduced during the late periods of viral infections (Figure 3F).These results directly correlate with SIAH1 stability during the early periods of virus infection.Collectively, these results show that SIAH1 becomes stable during the early periods of virus infections and leads to K27-linked ubiquitination and proteasomal degradation of USP19.
To conclusively validate the crucial role of ubiquitination and subsequent degradation of USP19 in the antiviral response mediated by SIAH1, HEK293T cells were transfected with control-siRNA, SIAH1-siRNA or a combination of SIAH1-siRNA and USP19-siRNA.
Figure 3G shows the knockdown efficacy of endogenous SIAH1 and USP19 in each treatment cohort, as determined by western blot analysis.Following the knockdown, these cells were subsequently infected with PR8-GFP virus to assess the impact of these treatments on viral replication.Interestingly, we found that virus replication in SIAH1-knockdown was significantly higher than that of the control cells, and especially knockdown of SIAH1 in the USP19 knockdown condition, does not enhance the virus replication as in SIAH1 single knockdown condition (Figure 3H−I).Consequently, we confirm that siRNA-mediated SIAH1 knockdown dramatically reduced IFN-β and IL-6 production, but double knockdown of SIAH1 and USP19 does not reduce the IFN-β and IL-6 production (Figure 3J).These results solidify the necessity of ubiquitination and degradation of USP19 for the antiviral function of SIAH1.

| SIAH1 enzyme activity is required for USP19 ubiquitination
Prior reports have identified that the SIAH1 C44S mutant abrogates the E3 ligase activity of SIAH1. 29 ) were treated with MG132 for 6 h before harvest.WCLs were subjected to Strep-PD assay followed by immunoblotting with indicated antibodies.(B) HEK293T cells were transfected with HA-tagged ubiquitin, Strep-tagged USP19 and Flag-tagged SIAH1.MG132 was added to the cells for 6 h before harvest.WCLs were subjected to Strep-PD assay and immunoblotted with indicated antibodies.(C) HEK293T cells were transfected with HA-ubiquitin together with Flag-tagged empty vector or SIAH1.MG132 was added to the cells for 6 h before harvest.WCLs were immunoprecipitated with anti-Flag antibodies and immunoblotted with indicated antibodies.(D) HEK293T cells were transfected with Flag-tagged empty vector or SIAH1.MG132 was added to the cells for 6 h before harvest.WCLs were immunoprecipitated with anti-USP19 antibodies and immunoblotted with indicated antibodies.(E) In vitro ubiquitination assay for USP19.HEK293T cells were transfected with Strep-tagged USP19, GST-tagged SIAH1, or SIAH1 C44S individually.immunoprecipitants were incubated with a reaction mixture containing ubiquitin, E1, and UbcH5c (E2), followed by immunoblotting with an anti-K27 linkage specific antibody.(F) HEK293T cells were infected with SeV (1MOI) in time dependent manner.MG132 was added to the cells for 6 h before harvest.WCLs were immunoprecipitated with IgG (control antibody) or anti-USP19 antibody and immunoblotted with indicated antibodies.(G) HEK293T cells were transfected with control-siRNA, SIAH1-siRNA or USP-siRNA as indicated and WCLs were immunoblotted with indicated antibodies.(H−J) HEK293T cells were treated with control-siRNA, SIAH1-siRNA or USP-siRNA as indicated and were infected with PR8-GFP.GFP expression, GFP absorbance, and virus titer were taken at 12 and 24 hpi (H, I), and secreted levels of IFN-β and IL-6 levels were measured by specific ELISA (J).Data information: *p < 0.05, and **p < 0.01 (two-tailed Student's t-test).Data are representative of three independent experiments, each with similar results, and expressed as the mean ± SD of two biological replicates.WCLs, whole-cell lysates.
USP19.These findings suggest that the E3 ligase activity of SIAH1 is required for the K27-linked ubiquitination of USP19.In vitro ubiquitination also showed consistent results, in which ubiquitination of USP19 did not occur in the presence of SIAH1 C44S, as it did in the presence of SIAH1 (Figure 3E).Ultimately, these findings demonstrate that the E3 ligase activity of SIAH1 induces K27-linked ubiquitination of USP19, leading to its proteasomal degradation.USP19, each containing the SIAH1-binding residue (Figure 5A).We then examined the degradation of the USP19 constructs in the presence and absence of MG132.We found that only the 1-489 aa fragment of USP19 does not undergo proteasomal degradation by SIAH1, which confirms that lysine residues in the 450−850 aa region of USP19 are important for SIAH1-mediated ubiquitination and proteasomal degradation (Figure 5B).To identify the specific lysine residue/s in this region, we used a ubiquitin site prediction algorithm (UbiPred). 37We created mutants in which arginine replaced lysine based on the predicted results (K489R, K490R, K571R, K610R, K651R, K681R, K724R, and K735R).Then, with each mutant, we examined SIAH1-mediated degradation and compared it to the wild type.As shown in Supporting Information: Figures 6A and 6B, none of the mutants were able to withhold the SIAH1-mediated degradation or K27-linked ubiquitination, which is similar to the wild type.A Western blot analysis was performed to assess the degradation dynamics of transiently overexpressed USP19 mutants.The results revealed that the SIAH1-mediated degradation of the K489R/ K490R/K610R mutant (designated USP19 3 K) was significantly hampered compared to the wild-type counterpart (Figure 5C).Next, to further confirm the effect of this mutant on SIAH1-mediated degradation, we checked the degradation of USP19 and USP19 3 K with increasing doses of SIAH1.As shown in Figure 5D, despite the observed dose-dependent degradation of USP19 in response to SIAH1, the mutant variant USP19 3 K was resistant to this degradation process.Furthermore, the ubiquitination assay performed on USP19 and its mutant variants yielded concordant results, demonstrating similar ubiquitination patterns in the USP19 WT and the K490R/K571R/K610R mutant.In contrast, USP19 3 K exhibited reduced levels of K27-linked ubiquitination when exposed to SIAH1, highlighting a distinctive and attenuated ubiquitination response compared to wild-type USP19 and the K490R/K571R/K610R mutant (Figure 5E).These findings demonstrate that Lys489, Lys490, and Lys610 of USP19 together act as specific sites for K27-linked ubiquitination by SIAH1.We also confirmed that USP19 3 K has the same effect as USP19 in terms of virus infection functionality, as virus replication was greater in the presence of both USP19 and USP19 3 K than in the absence of either (Figures 5F,G).The virus infection-enhancing properties of USP19 3 K remained unaltered in the presence of SIAH1 expression, while the virus infectionenhancing properties of USP19 were substantially attenuated when SIAH1 was coexpressed (Figures 5F,G).After viral infections, USP19 3 K inhibited IFN-β and IL-6 expression but was unaffected by SIAH1 expression (Figure 5H).These observations underscore the impaired proteolytic efficiency of SIAH1 toward the USP19 3 K mutant, suggesting the critical role that the lysine residues at positions 489, 490, and 610 play in mediating the K27-linked ubiquitination and degradation processes.

| SIAH1 inhibits the USP19-mediated deubiquitination of Beclin1, TRAF3, and TRIF to positively regulate the host immune response
The deubiquitinase enzyme USP19 is a known negative regulator of host antiviral innate immunity.0][21] USP19 stabilizes Beclin1 by removing K11-linked ubiquitin chains, and stabilizing Beclin1 interrupts the interaction between RIG-I and MAVS to suppress cellular antiviral innate immunity. 19P19 suppresses cellular antiviral innate immune signaling by removing K63-linked ubiquitin chains from TRAF3. 20Furthermore, USP19 removes K27-linked ubiquitin chains from TRIF, which impairs the TLR3/4-mediated innate immune signaling. 21Since our findings show that USP19 undergoes degradation by SIAH1, we checked the effect of SIAH1 on USP19-mediated deubiquitination of Beclin1, TRAF3, and TRIF using a HEK293T cotransfection experiment.As expected, the results showed a distinct reduction of K11-linked ubiquitination of Beclin1, K63-linked ubiquitination of TRAF3, and K27-linked ubiquitination of TRIF in the presence of USP19.These ubiquitination events were subsequently restored with SIAH1 in a dose-dependent manner (Figures 6A,C,E).However, SIAH1 is incapable of rescuing the deubiquitination of Beclin1, TRAF3, and TRIF, which had been affected by USP19 3 K (Figures 6B,D,F).
Together, these results confirm that SIAH1-mediated degradation of USP19 enhances antiviral innate immune responses by promoting K11-linked ubiquitination of Beclin1, K63-linked ubiquitination of TRAF3, and K27-linked ubiquitination of TRIF.

| Deubiquitination of MAVS by USP19 is counteracted by SIAH1 to positively regulate the type I IFN pathway
Using systematic functional screening of DUB family members involved in regulating antiviral immunity, Liu et al. found the possibility of USP19 interfering with host innate immune signaling by targeting MAVS. 38Building on this understanding, our study aimed to explore the involvement of USP19 in the negative regulation of the host antiviral innate immune response through its interaction with MAVS, and to assess how SIAH1-mediated degradation of USP19 might impact its functional role in this context.
First, we checked the interaction between USP19 and MAVS by cotransfection and confocal microscopy in overexpressed and endogenous conditions following time-dependent SeV infection.
The findings elucidate a distinct interaction and co-localization between USP19 and MAVS (Figure 7A and Supporting Information: Figure 7).However, this co-localization diminishes notably during the early stages of a viral infection (Figure 7B).Next, we created GSTtagged MAVS domain constructs (Figure 7C) and used them in a GSTpull-down assay with full-length USP19 to determine the domain within MAVS that interacts with USP19.Full-length and N-terminal truncated MAVS domain constructs (1−540, 1−470, 180−540, 450−540), in which all constructs contain the transmembrane (TM) domain of MAVS, strongly interacted with USP19; however, the TM mutant fragment (1−470) lost its ability to interact with USP19 (Figure 7D).These findings demonstrate that the TM domain of MAVS is critical for interaction with USP19.Prior investigations have provided evidence suggesting the essential role of the TM domain within MAVS.This domain is crucial for mediating MAVS selfassociation, and subsequent aggregation, a process known to ultimately trigger the activation of the type I IFN pathway. 39,40erefore, to evaluate the impact of USP19 on MAVS selfassociation, we conducted experiments using HEK293T cells cotransfected with Flag-and V5-tagged MAVS, in combination with increasing doses of USP19.As shown in Figure 7E, USP19 was unable to impede MAVS self-association, thus failing to inhibit the activation of the host immune response by inhibition of selfassociation.
A recent study demonstrated that upon virus infection, TRIM31 interacts with MAVS at the mitochondria to catalyze K63-linked polyubiquitination of MAVS, which promotes MAVS aggregation and activation of type I IFN signaling. 41Hence, we conducted an investigation into the impact of USP19 on the K63-linked ubiquitination of MAVS, given its role as a DUB.Under conditions of plasmid overexpression, our findings demonstrate that USP19 effectively inhibits the K63-linked ubiquitination of MAVS in a dose-dependent manner (Figure 7F).A similar inhibition can be observed in SeV

infection-induced endogenous MAVS ubiquitination in a USP19
dose-dependent manner (Figure 7G).Next, to assess whether the deubiquitinase activity of USP19 is important for the deubiquitination of MAVS, we checked the ubiquitination of MAVS in the presence of an enzymatic-activity deficient USP19 mutant (USP19 C506S).As shown in Figure 7H, USP19 C506S is unable to inhibit K63-linked ubiquitination of MAVS as USP19 WT, suggesting the importance of deubiquitinase activity of USP19 in inhibiting MAVS ubiquitination.
Since our findings have established that SIAH1 degrades USP19, our subsequent objective was to investigate whether SIAH1 exerts an influence on the ubiquitination of MAVS.Consequently, we cotransfected MAVS and USP19 together with increasing doses of SIAH1.The results demonstrate that the USP19-mediated inhibition of MAVS ubiquitination is restored in a dose-dependent manner with the introduction of SIAH1 (Figure 8A).In contrast, SIAH1 was unable to restore the ubiquitination of MAVS that had been inhibited by the USP19 3 K mutant (Figure 8B).
After virus infection, the K63-linked ubiquitination of MAVS increases the development of prion-like aggregates of MAVS. 41This knowledge prompted us to investigate the effect of USP19 and SIAH1 on the aggregation of MAVS. Figure 8C   To date, USP19 is known to downregulate the host immune response by targeting Beclin1, TRAF3, and TRIF for deubiquitination.

USP19-mediated removal of K11-linked ubiquitin chains from
Beclin1 results in the stabilization of Beclin1, which then interferes with the RIG-I and MAVS interaction, leading to the downregulation of host antiviral innate immune responses. 19Several E3 ligases, including cIAP1, cIAP2, and TRIM24, are known to facilitate cellular antiviral responses by triggering K63-linked ubiquitination of TRAF3, 42,43 and it was confirmed that USP19 inhibits this ubiquitination by removing K63 ubiquitin chains from TRAF3. 20In addition, the K27-linked polyubiquitination of TRIF is critical for its recruitment to TLR3/4 following pathway stimulation, and it is known that USP19 deubiquitinates TRIF. 21Interestingly, in this study, we found that USP19 deubiquitinates MAVS, thereby suppressing the host antiviral innate immune response.MAVS has previously been shown to produce functional prion-like aggregates in response to viral infection, which are necessary for RLR signaling activation, 40 and TRIM31 enhanced the formation of such prion-like MAVS aggregation by cartelizing the K63-linked polyubiquitination of MAVS during virus infection. 41Consequently, we confirmed a novel role played by USP19 in reversing TRIM31-mediated K63-linked ubiquitination and aggregation of MAVS (Figures 7 and 8) during a virus infection.
Previous investigations have shown that SIAH1 knockout mice exhibit growth retardation, early lethality, and spermatogenetic defects, indicating an essential function of this enzyme during development. 44SIAH family members have been reported to be involved in several signaling pathways, including oxidative stress, hypoxia response, cell cycle regulation, tumor suppression, oxidative stress, and spermatogenesis. 35As an E3 ligase, SIAH1 primarily controls these cellular pathways by ubiquitination and degradation of its substrate molecules, which regulate protein localization, stability, and other cellular functions.Studies have shown that SIAH1 plays a role in regulating innate immune signals against virus infection based on its ability to bind to numerous viral proteins.For example, SIAH1 interacts with and degrades Kaposi's sarcoma-associated herpesvirus ORF45, 27 and SIAH1 promotes polyubiquitination and proteasomal degradation of the HSV-immediate early protein ICP0. 28SIAH1 also degrades the Hepatitis B virus X protein (HBx) 29 and promotes ubiquitination and proteasomal degradation of the Avian reovirus (ARV) p10 protein. 30These findings show that SIAH1 directly targets viral proteins, but its role in targeting host proteins against viral infection is still unknown.In this study, we selected SIAH1 from the IFN-β luciferase assay using an E3 ligase library.Our large-scale pulldown and mass-spectrometry analysis revealed that USP19 is one of the binding molecules for SIAH1 (Supporting Information: Figure 5A).
Consequently, we identified SIAH1 as a novel positive regulator of host antiviral innate immune response by targeting USP19 for proteasomal degradation.Some RING-type E3 ligases are known to be ubiquitinated by the autocatalytic process, which leads to proteasome-dependent degradation and reduced E3 activity. 45Similarly, under native physiological conditions, SIAH1 is known to maintain its availability through autoubiquitination and degradation. 23,34,35Additionally, previous studies have shown that SIAH1 expression levels and activity are stabilized under stressed and virus-infected conditions. 28,46In this study, we also confirmed that SIAH1 is stabilized in immune and epithelial cells early following viral infections (Supporting Information: Figures 1B-1D), and stabilization of SIAH1 correlated with the interaction of SIAH1 with USP19 early after virus infection (Figures 2C,D).SIAH1 stability decreased late after virus infection, leading to the stabilization of USP19.This phenomenon of SIAH1 may be a feature of maintaining host immune homeostasis by preventing excessive production of IFNs and proinflammatory cytokines, which can lead to the development of chronic inflammatory disorders, autoimmune illnesses, and certain malignancies. 47evious studies have found that the PxAxVxP motif of the substrate protein is critical for interaction with SIAH1 through sequence alignment and mutagenesis study. 48USP19 contains this motif between 462 and 473 aa, and we constructed a deletion mutant of the USP19 462−473 aa region.With a domain binding assay, we confirmed that the substrate binding domain of SIAH1 interacts with the 462−473 aa motif of USP19 and the induction of ubiquitination and degradation of USP19.To investigate the precise lysine residue/s in USP19 required for SIAH1mediated ubiquitin conjugation, we used the UbiPred tool to predict which lysine residues within the selected 450−850 aa region of USP19 are associated with ubiquitination (Figures 5A,B), and we found that SIAH1-mediated ubiquitin conjugation occurs simultaneously at Lys489, Lys490, and Lys610 of USP19.Each lysine is equally important for this ubiquitin conjugation, as single and double mutations did not affect the ubiquitination and degradation of USP19 (Figure 5C−E).This case is very rare and can be said to be a sophisticated control mechanism for innate immune regulation.
Figure 2G).Next, based on the above findings, we investigated the antiviral role of SIAH1 overexpression.Ectopic overexpression of SIAH1 was confirmed by western blot (Supporting Information: Figure2H) and as expected, SIAH1 stably overexpressing RAW264.7 cells reduced the replication of VSV-GFP and HSV-GFP viruses (Supporting Information: Figures3A and 3C), and elevated IFN-β and IL-6 secretion (Supporting Information: Figures3B and 3D).Furthermore, transient transfection and ectopic overexpression of SIAH1 in HEK293T cells resulted in a significant reduction of VSV-GFP replication (Supporting Information: Figures2I and 2J) and enhanced host antiviral innate immune response by increasing the production of IFN-β and IL-6 compared to control cells (Supporting Information: Figure2K).In addition, in HEK293T cells transiently transfected with SIAH1, treatment with poly(I:C) and poly(dA:dT) produced higher . These results were further confirmed by an endogenous USP19 F I G U R E 2 SIAH1 interacts with deubiquitinase enzyme USP19 and lead to its proteasomal degradation.(A) HEK293T cells were transfected with Strep-tagged USP19 plasmid in the presence of increasing doses of Flag-tagged SIAH1.WCLs were subjected to immunoblotting with indicated antibodies.(B) HEK293T cells were transfected with Strep-tagged USP19 together with Flag-tagged SIAH1 and were treated with MG123, NH 4 Cl, Z-VAD or chloroquine for 6 h before harvest.WCLs were subjected to immunoblotting with indicated antibodies.(C) HEK293T cells were infected with SeV (1MOI) in time dependent manner.MG132 was added to the cells for 6 h before harvest.WCLs were immunoprecipitated with IgG (control antibody) or anti-SIAH1 antibody and immunoblotted with indicated antibodies.(D) Confocal microscopy was conducted to examine the time-dependent co-localization of SIAH1 (red) and USP19 (green) in HeLa cells upon SeV infection (1MOI) in the presence of MG132.Arrows indicate the colocalized SIAH1 and USP19 protein.(E, F) Schematic representation of the domain constructs of SIAH1 (E).HEK293T cells were transfected with GST-tagged SIAH1 domain constructs together with Strep-tagged USP19.MG132 was added to the cells for 6 h before harvest.WCLs were subjected to GST pull-down (PD) assay, followed by immunoblotting with indicated antibodies (F).(G, H) Schematic representation of the domain constructs of USP19 (G).HEK293T cells were transfected with Strep-tagged USP19 domain constructs together with Flag-tagged SIAH1.MG132 was added to the cells for 6 h before harvest.WCLs were subjected to Strep-PD assay, followed by immunoblotting with indicated antibodies (H).Data are representative of three independent experiments, each with similar results.WCLs, whole-cell lysates.F I G U R E 3 (See caption on next page).
To assess the necessity of SIAH1-E3 ligase activity in the ubiquitination and subsequent degradation of USP19 during viral infections, we first constructed a SIAH1 mutant with cysteine to serine substitution at the 44th codon in the RING domain (SIAH1 C44S).Subsequently, HEK293T cells overexpressing either wild-type SIAH1 or SIAH1 C44S were subjected to PR8-GFP viral infection, and the impact on virus replication and cytokine production was examined.The findings revealed that HEK293T cells expressing SIAH1 C44S exhibited heightened viral replication and diminished cytokine production compared to HEK293T cells overexpressing wild-type SIAH1 (Figure 4A−C), indicating that SIAH1 modulates host antiviral innate immune responses through its E3 ligase activity.Next, we assessed the effect of SIAH1 C44S on USP19.As shown in Figure 4D, our investigation revealed that the dose-dependent expression of the E3 ligase activitydeficient mutant, SIAH1 C44S, did not result in a substantial reduction in the protein level of USP19.To further demonstrate that SIAH1 regulates USP19 at the post-translational level, we determined the effects of SIAH1 and the SIAH1 C44S mutant on USP19 protein in the presence of cycloheximide (CHX), which is a protein synthesis inhibitor.The relative stability of the USP19 protein was examined at the indicated interval after treatment with CHX to block protein synthesis.As shown in Figure 4E, the co-expression of USP19 with SIAH1 induces USP19 degradation.In contrast, SIAH1 C44S does not induce the degradation of USP19.Next, we examined the K27-linked ubiquitination of USP19 in the presence of SIAH1 or SIAH1 C44S.As shown in Figure 4F, in contrast to the K27-linked ubiquitination of USP19 mediated by SIAH1, the expression of SIAH1 C44S did not elicit the K27-linked ubiquitination of F I G U R E 3 USP19 undergo K27-linked ubiquitination and proteasomal degradation by SIAH1.(A) HEK293T cells were transfected with Flagtagged empty vector or SIAH1 with Strep-tagged USP19 and each different HA-tagged ubiquitin mutants (indicated lysine [K] only, other K mutated to arginines [R]

2. 7 |
SIAH1 conjugates K27-linked ubiquitin at lysine 489, 490, and 610 of USP19 Thus far, the findings show that SIAH1 interacts with USP19 and causes K27-linked ubiquitination.Ubiquitination usually results in the binding of the ubiquitin molecule to a specific lysine residue of the substrate.36Hence, to determine the specific region within USP19 that is ubiquitinated by SIAH1, we generated five deletion mutants of F I G U R E 4 Enzymatic activity of SIAH1 is essential for the ubiquitination of USP19.(A−C) HEK293T cells were transfected with Flag-tagged empty vector, SIAH1 or SIAH1 C44S and infected with PR8-GFP (1MOI).GFP expression (A), GFP absorbance and virus titer was taken at 12 and 24 hpi (B), and secreted levels of IL-6 and IFN-β levels were measured by specific ELISA (C).(D) HEK293T cells were transfected with Streptagged USP19 with increasing doses of Flag-tagged SIAH1 C44S.WCLs were immunoblotted with indicated antibodies.(E) HEK293T cells were transfected with Strep-tagged USP19 with Flag-tagged empty vector, SIAH1, or SIAH1 C44S.Cells were treated with Cyclohexylamine (CHX) at the indicated time before harvesting the cells.WCLs were subjected to immunoblotting with indicated antibodies.(F) HEK293T cells were transfected with HA-tagged ubiquitin, Strep-tagged USP19, with Flag-tagged SIAH1 or SIAH1 C44S.MG132 was added to the cells for 6 h before harvest.WCLs were subjected to Strep-PD assay and immunoblotted with indicated antibodies.Data information: *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).Data are representative of three independent experiments, each with similar results, and expressed as the mean ± SD of two biological replicates.WCLs, whole-cell lysates.
The above experiment suggests redundancy in the USP19 ubiquitinconjugation sites.Therefore, we constructed double lysine mutants of USP19, in which several combinations of two lysine residues in the F I G U R E 5 SIAH1 targets Lys489, Lys490, and Lys610 of USP19 for K27-linked ubiquitination.(A, B) Schematic representation of the domain constructs of USP19 (A).HEK293T cells were transfected with Flag-tagged SIAH1 with Strep-tagged USP19 domain constructs in the presence and absence of MG132.WCLs were subjected to immunoblotting with indicated antibodies (B).(C) HEK293T cells were transfected with Strep-tagged USP19 WT, USP19 K489R K490R K610R (3 K), USP19 K490R K571R K610R together with Flag-tagged empty vector or SIAH1.WCLs were immunoblotted with indicated antibodies.(D) HEK293T cells were transfected with Strep-tagged USP19 WT or USP19 3 K together with increasing doses of Flag-tagged SIAH1.WCLs were subjected to immunoblotting with indicated antibodies.(E) HEK293T cells were transfected with Strep-tagged USP19 WT, USP19 3 K, USP19 K490R K571R K610R together with HA-tagged ubiquitin and Flag-tagged SIAH1.MG132 was added to the cells for 6 h before harvest.WCLs were subjected to Strep-PD assay and immunoblotted with indicated antibodies.(F−H) HEK293T cells were transfected with Strep-tagged USP19 WT, USP19 3 K together with Flag-tagged empty vector or SIAH1 and infected with PR8-GFP (1MOI).GFP expression (F), GFP absorbance and virus titer was taken at 12 and 24 hpi (G), and secreted levels of IL-6 and IFN-β levels were measured by specific ELISA (H).Data information: **p < 0.01, ***p < 0.001, ns, not significant (two-tailed Student's t-test).Data are representative of three independent experiments, each with similar results, and expressed as the mean ± SD of two biological replicates.WCLs, whole-cell lysates.450−850 aa region of USP19 were mutated (K489R/K490R and K571R/K610R).As shown in Supporting Information: Figures 6C and 6D, SIAH1-dependent degradation or ubiquitination was unchanged due to the double lysine mutation.These results led us to generate triple-lysine mutants of USP19.Two triple-lysine mutants of USP19 K489R/K490R/K610R and K490R/K571R/K610R were constructed.

F
I G U R E 6 USP19 mediated deubiquitination of Beclin1, TRAF3 and TRIF were inhibited by SIAH1.(A, B) HEK293T cells were transfected with HA-tagged K11 specific ubiquitin with Flag-tagged Beclin1 and Strep-tagged (A) USP19 WT (B) USP19 3 K, together with increasing doses of Myc-tagged SIAH1.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(C, D) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin with Flag-tagged TRAF3 and Strep-tagged (C) USP19 WT (D) USP19 3 K, together with increasing doses of Myc-tagged SIAH1.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(E, F) HEK293T cells were transfected with HA-tagged K27 specific ubiquitin with Flag-tagged TRIF and Strep-tagged (E) USP19 WT (F) USP19 3 K, together with increasing doses of Myc-tagged SIAH1.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.Data are representative of three independent experiments, each with similar results.WCLs, whole-cell lysates.
shows the USP19-mediated inhibition of SeV infection-induced MAVS aggregation (Figure8C, lane 3), and SIAH1 expression recovers the aggregation in a dose-dependent manner.

F
I G U R E 7 USP19 interacts with MAVS and deubiquitinates MAVS by removing K63-linked ubiquitin chains from MAVS.(A) HEK293T cells were transfected with GST-tagged MAVS together with Strep-tagged USP19 or control vector.WCLs were subjected to Strep-PD assay and immunoblotted with indicated antibodies.(B) HeLa cells were infected with SeV (1MOI) in a time dependent manner, followed by confocal microscopy assay with anti-USP19 (green) and anti-MAVS (red) antibodies.Nuclei were stained with DAPI (blue).(C, D) Schematic representation of the domain constructs of MAVS (C).HEK293T cells were transfected with GST-tagged MAVS domain constructs together with Strep-tagged USP19.WCLs were subjected to GST-PD assay, followed by immunoblotting with indicated antibodies (D).(E) HEK293T cells were transfected with V5-tagged and Flag-tagged MAVS together with increasing doses of Strep-tagged USP19.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(F) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin with Flag-tagged MAVS and increasing doses of Strep-tagged USP19 WT.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(G) HEK293T cells were transfected with increasing doses of Strep-tagged USP19 WT and infected with SeV (1MOI).WCLs were immunoprecipitated with anti-MAVS antibody and immunoblotted with indicated antibodies.(H) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin with Flag-tagged MAVS and increasing doses of Strep-tagged USP19 C506S.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.Data are representative of three independent experiments, each with similar results.WCLs, whole-cell lysates.Because TRIM31 is responsible for MAVS K63-linked ubiquitination, we next tested whether USP19 could reverse TRIM31-mediated MAVS K63-linked ubiquitination.Our findings show that USP19 reverses the TRIM31-mediated K63-ubiquitination of MAVS in a dose-dependent manner (Figure 8D).These observations were further substantiated through an in vitro ubiquitination assay involving MAVS, TRIM31, and USP19.In this assay, USP19 was observed to impede the TRIM31-mediated ubiquitination of MAVS, a hindrance that was subsequently alleviated in the presence of SIAH1 (Figure 8E).Overall, these findings suggest that SIAH1 mediates the degradation of USP19 and promotes the K63-linked ubiquitination and aggregation of MAVS, thereby promoting activation of the type I IFN signaling.3 | DISCUSSION Rapid PTMs, such as ubiquitination, are essential for the transduction of certain intracellular signals.Consisting of more than 700 members, E3 ligases play a pivotal role in regulating immune responses to F I G U R E 8 SIAH1 promote the TRIM31 mediated K63-linked ubiquitination that was inhibited by USP19.(A) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin, Flag-tagged MAVS, Strep-tagged USP19 WT together with increasing doses of Myc-tagged SIAH1.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(B) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin, Flag-tagged MAVS, Strep-tagged USP19 3 K together with increasing doses of Myc-tagged SIAH1.WCLs were immunoprecipitated with anti-Flag antibody and immunoblotted with indicated antibodies.(C) HeLa cells were transfected with Strep-tagged USP19 WT with increasing doses of GST-tagged SIAH1 and infected with SeV (1MOI).Following that, crude mitochondria were isolated from the cells and subjected to the semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) and immunoblotted with anti-MAVS antibody for MAVS aggregation detection.The samples were used for the SDS-PAGE and immunoblotted with indicated antibodies.(D) HEK293T cells were transfected with HA-tagged K63 specific ubiquitin, V5-tagged MAVS and Flag-tagged empty vector or TRIM31 together with increasing doses of Strep-tagged USP19.WCLs were immunoprecipitated with anti-V5 antibody and immunoblotted with indicated antibodies.(E) In vitro ubiquitination assay for MAVS.HEK293T cells were transfected with V5-tagged MAVS, Flag-tagged TRIM31, Strep-tagged USP19, and GST-tagged SIAH1 individually.immunoprecipitants were incubated with a reaction mixture containing ubiquitin, E1, and UbcH5a (E2), followed by immunoblotting with an anti-K63 linkage specific antibody.(F) Schematic representation of the summery of the study (created with BioRender).Data are representative of three independent experiments, each with similar results.WCLs, whole-cell lysates.maintain host immune homeostasis.By screening the E3 ligase library, we selected a novel E3 ligase, SIAH1 and found that it is a potent positive regulator of host immune responses to viral infections.First, we showed that knocking down SIAH1 in immune and epithelial cells increases virus replication while suppressing type I IFNs and proinflammatory cytokine secretion in response to viral infections.Consistent with these findings, overexpression of SIAH1 showed the opposite phenotype and signaling.Second, we found that the substrate binding domain of SIAH1 directly interacts with the 462-473 aa region of the deubiquitinase USP19 during the early stage of a virus infection.Third, USP19 undergoes K27-linked ubiquitination and proteasomal degradation by SIAH1 and is directly associated with the E3 ligase activity of SIAH1.Fourth, USP19 undergoes SIAH1-mediated K27-linked ubiquitination, specifically at lysine positions 489, 490, and 610.Fifth, USP19 interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for the negative regulation of type I IFN signaling.Finally, we found that SIAH1mediated degradation of USP19 reverses the USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of type I IFN signaling.Our findings imply that SIAH1 is critical in regulating USP19 and increasing antiviral innate immune responses during the early periods of viral infections.