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. 2019 Nov 13;93(23):e01438-19. doi: 10.1128/JVI.01438-19

The E3 Ubiquitin Ligase TBK1 Mediates the Degradation of Multiple Picornavirus VP3 Proteins by Phosphorylation and Ubiquitination

Dan Li a,#, Wenping Yang a,#, Jingjing Ren a, Yi Ru a, Keshan Zhang a, Shaozu Fu a, Xiangtao Liu a, Haixue Zheng a,✉
Editor: Tom Gallagherb
PMCID: PMC6854495  PMID: 31534043

TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

KEYWORDS: FMDV, TBK1, phosphorylation, picornavirus VP3 proteins, ubiquitination

ABSTRACT

TANK-binding kinase 1 (TBK1) is essential for interferon beta (IFN-β) production and innate antiviral immunity. However, other, additional functions of TBK1 have remained elusive. Here, we showed that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. Further evidence showed that TBK1 could also be self-ubiquitylated in vivo. Importantly, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Mechanistically, TBK1 phosphorylated multiple picornavirus VP3 proteins at serine residues and ubiquitinated them via K63-linked ubiquitination at lysine residues. In addition, the C426 and C605 residues of TBK1 were not essential for TBK1 innate immunity activity; however, these residues were required for degradation of multiple picornavirus VP3 proteins and for its E3 ubiquitin ligase activity. Hence, our findings identified a novel role of TBK1 in regulating the virus life cycle and provided new insights into the molecular mechanisms of TBK1-mediated antiviral response.

IMPORTANCE TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

INTRODUCTION

Picornaviruses are ubiquitous and globally distributed, and they pose a threat to the health of humans and livestock. For example, enterovirus 71 (EV71) infection usually causes childhood exanthema (1); encephalomyocarditis virus (EMCV) is able to infect animal and humans, causing myocarditis and encephalitis (2); Seneca Valley virus (SVV) infection is associated with outbreaks of vesicular disease in sows as well as neonatal pig mortality (3); and foot-and-mouth disease virus (FMDV) is the causative agent of the economically most important animal viral disease worldwide (4). Picornaviruses are small, nonenveloped viruses that contain a single strand of positive-sense RNA (ssRNA) (5). All picornaviruses share a genome organization that is divided into three sections: the 5′ untranslated region (5′ UTR), the open reading frame (ORF) of the polyprotein, and the 3′ UTR. Most picornaviruses encode four capsid proteins (VP4, VP2, VP3, and VP1) (6).

The three major capsid proteins that constitute the external virion shell of picornaviruses, namely, VP1, VP2, and VP3, are considered to play a pivotal role in virus infection and host recognition (7). A previous study demonstrated that aspartic acid residue 70 of hepatitis A virus capsid protein VP3 contributes to an immunodominant antigenic site (8), and the interaction between nonstructural protein 2CATPase and capsid protein VP3 is required for enterovirus morphogenesis (9). Aspartic acid residue R56 in FMDV VP3 is associated with virus attenuation during infections in natural host animals (10). FMDV VP3 inhibits the expression of type I and type II interferon by reducing the mRNA level of virus-induced signaling adapter (VISA) and degrading the JAK1 protein (11, 12). It is unknown whether there are other host proteins that mediate the function of picornaviruses via its VP3 protein.

TANK-binding kinase 1 (TBK1) plays a key role in antiviral immunity (13, 14). Studies have revealed that herpes simplex virus (HSV) ICP27, UL46, γ134.5, and Us11 inhibit TBK1 to facilitate HSV replication (15–19). The NS5 protein of Zika virus (ZIKV) inhibits the activation of TBK1 to enhance its replication (20). Hepatitis C virus NS2 protease inhibits IκB kinase ε (IKKε) and TBK1 functions to inhibit the host cell antiviral response (21). HIV blocks interferon induction in human dendritic cells and macrophages by dysregulating TBK1 (22). Nevertheless, it remains unclear whether TBK1 regulates viral replication independently of its immune activity.

In this study, we found that TBK1 is an E3 ubiquitin ligase. TBK1 degrades multiple picornavirus VP3 proteins in a manner dependent on its kinase and E3 ubiquitin ligase activity. Our findings identified TBK1 as a new E3 ligase for K63-linked ubiquitination of multiple picornavirus VP3 proteins.

RESULTS

Overexpression of TBK1 results in the degradation of multiple picornavirus VP3 proteins.

The VP3 protein in FMDV is critical for recognition of the cellular heparin sulfate (HS) coreceptor of FMDV (23). Type I interferons (IFNs) play key roles in antiviral innate immune response. However, it is unclear if innate immune signaling molecules affect the expression of FMDV VP3. To identify candidate innate immune signaling molecules involved in regulating the FMDV VP3 protein, we carried out transient-transfection and Western blot assays. The results indicated that FMDV VP3 protein is degraded by TBK1 but not RIG-I (CARD), MITA (mediator of IRF3 activation), MDA5 (melanoma differentiation-associated gene 5), VISA, interferon regulatory factor 3 (IRF3), or IRF7 (Fig. 1A). Further experiments indicated that overexpression of TBK1 resulted in degradation of FMDV VP3 protein in a dose-dependent manner. In contrast, FMDV VP3 protein did not affect TBK1 expression (Fig. 1B). To determine whether TBK1 degrades other FMDV proteins, we cotransfected TBK1 with VP3, VP2, VP0, 3D, or 3A in 293T cells. The results suggested that TBK1 completely degraded FMDV VP3 protein and partly degraded FMDV VP2, VP0, and 3A proteins but did not affect the levels of FMDV 3D protein (Fig. 1C). We next investigated whether TBK1 might be a potential target for prophylactic and therapeutic intervention for improved containment of FMD. Whether FMDV VP3 protein was expressed before transfection with TBK1 (Fig. 1D) or after transfection with TBK1 (Fig. 1E), we observed that TBK1 degraded FMDV VP3 protein. Taken together, these results suggest that TBK1 mediates the degradation of FMDV VP3 protein.

FIG 1.

FIG 1

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TBK1 induced the degradation of multiple picornavirus VP3 proteins. (A) Screening of signaling components for the degradation of FMDV VP3. 293T cells (2 × 105) were transfected with HA-VISA, HA-IRF3, HA-IRF7, HA-TBK1, HA-MITA, HA–RIG-I (CARD), HA-MDA5, and Flag-VP3 plasmids for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-HA antibodies. WB, Western blotting. (B) Overexpression of TBK1 induced FMDV VP3 degradation in a dose-dependent manner. 293T cells (2 × 105) were transfected with Flag-VP3 and HA-TBK1 plasmids (0 μg, 0.1 μg, 0.2 μg, or 0.4 μg) or HA-TBK1 and Flag-VP3 plasmids (0 μg, 0.1 μg, 0.2 μg, or 0.4 μg) for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-HA antibodies. (C) Effects of TBK1 on FMDV VP3, VP2, VP0, 3D, and 3A in 293T cells. 293T cells (2 × 105) were transfected with plasmids expressing HA-TBK1 and FMDV VP3, VP2, VP0, 3D, or 3A for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-HA antibodies. (D and E) Overexpression of TBK1 induced FMDV VP3 degradation. 293T cells (2 × 105) were transfected with HA-TBK1- or FMDV VP3-expressing plasmids for 12 h (D), and then the cells were transfected with FMDV VP3- or HA-TBK1-expressing plasmids, respectively, for 12 h (E). The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-HA antibodies. (F) MEFs are permissive for FMDV infection. PK-15 cells or MEFs (2 × 105) were infected with FMDV for the indicated times. FMDV genome replication was evaluated by RT-PCR. The data shown are representative of results from one of three independent experiments and are presented as means ± SD of results from three technical replicates. (G) Immunoblot analysis of FMDV VP3 expression levels in MEFs after FMDV infection for the indicated times. (H) Immunoblot analysis of TBK1 expression levels in TBK1−/− MEFs. (I) Knockout of TBK1 increased FMDV replication in MEFs. MEFs (2 × 105) were infected with FMDV for the indicated times. FMDV genome replication was evaluated by RT-PCR. The data shown are representative of results from one of three independent experiments and are presented as means ± SD of results from three technical replicates. **, P < 0.01. (J) Overexpression of TBK1 induced multiple picornavirus VP3 degradation in a dose-dependent manner. Experiments were performed as described for panel B. β-Actin was used as a loading control. For TBK1 and VP3 proteins, band intensities were determined by Image J software and normalized to that of β-actin. RT-PCR, real-time reverse transcription-PCR; SD, standard deviation. All Western blot results are representative of at least two independent experiments.

To determine whether mouse embryonic fibroblasts (MEFs) are susceptible to FMDV infection, MEFs and PK-15 cells were infected with FMDV. Quantitative real-time reverse transcription-PCR (RT-PCR) experiments indicated that MEFs supported FMDV replication at the indicated time points (Fig. 1F). In addition, we found that FMDV VP3 protein was expressed upon FMDV infection in MEFs (Fig. 1G). To investigate the functions of TBK1 in the anti-FMDV response, we obtained TBK1 knockout MEFs from Hong-Bing Shu (Wuhan University) and detected the expression of TBK1 in MEFs. The result showed that TBK1 was undetectable in TBK1−/− MEFs (Fig. 1H). To further explore the effect of TBK1 knockout on FMDV, we determined the number of FMDV genomic copies in TBK1 knockout MEFs. The data indicated that TBK1 knockout increased the severity of FMDV infection in MEFs (Fig. 1I). Collectively, these results suggest that knocking out TBK1 protein in MEFs enhances their susceptibility to FMDV infection.

Because VP3 proteins are orthologous and thus conserved among all picornaviruses (6), we determined whether TBK1 could degrade other picornavirus VP3 proteins. In transient-transfection and Western blot experiments, we observed that overexpression of TBK1 degraded EV71 VP3 protein, EMCV VP3 protein, and SVV VP3 protein (Fig. 1J). The data suggest that TBK1 can degrade multiple picornavirus VP3 proteins.

TBK1 interacts with FMDV VP3.

To investigate the molecular mechanisms for TBK1-mediated degradation of FMDV VP3 protein, we first determined whether overexpression of TBK1 is associated with FMDV VP3 protein. Transient-transfection and coimmunoprecipitation experiments indicated that TBK1 interacted with FMDV VP3 in double-transfected 293T cells (Fig. 2A). It was previously demonstrated that TBK1 is constitutively expressed in most cell types (24), while FMDV VP3 protein is undetectable under physiological conditions but is expressed in FMDV-infected cells. Therefore, we examined whether TBK1 is associated with VP3 protein in FMDV-infected cells. In coimmunoprecipitation experiments, endogenous TBK1 was observed to interact with endogenous FMDV VP3 protein under physiological conditions (Fig. 2B). These data suggest that TBK1 is constitutively associated with FMDV VP3 protein.

FIG 2.

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TBK1 interacts with FMDV VP3. (A) TBK1 interacts with FMDV VP3. 293T cells were transfected with FMDV VP3 by standard calcium phosphate precipitation for 12 h and were then transfected again with TBK1 by the use of Lipofectamine 2000 transfection reagent for 12 h prior to coimmunoprecipitation analysis (IP) and immunoblot analysis (IB). Abs, antibodies. (B) Endogenous associations between TBK1 and FMDV VP3. PK-15 cells (5 × 107) were left uninfected or infected with FMDV for the indicated times. Coimmunoprecipitation experiments were performed with anti-VP3, and the immunoprecipitates were analyzed by immunoblotting with anti-TBK1 and anti-VP3. The lysates were analyzed by immunoblotting with the indicated antibodies. (C) The K38 and S172 sites of TBK1 played a role in FMDV VP3 degradation. 293T cells (2 × 105) were transfected with HA-TBK1, HA-TBK1 (K38A), HA-TBK1 (S172A), HA-TBK1 (S716A), or Flag-VP3 for 24 h. The cell lysates were analyzed by immunoblotting with anti-Flag, anti-β-actin, or anti-HA antibodies. All Western blot results are representative of at least two independent experiments.

Having established that TBK1 mediates FMDV VP3 protein degradation, we next determined if kinase site mutants of TBK1 can still degrade FMDV VP3 protein. As shown in Fig. 2C, FMDV VP3 protein was degraded by TBK1 or TBK1 (S716A) but not a kinase-inactive TBK1 mutant (K38A) in which ATP-binding residue Lys38 was mutated to alanine or an autophosphorylation-inactive TBK1 mutant (S172A) (25). Collectively, these data suggest that degradation of FMDV VP3 by TBK1 depends on its kinase activation.

TBK1 degrades multiple picornavirus VP3 proteins via the proteasome pathway.

To investigate the mechanisms underlying the role of TBK1 in the stability of FMDV VP3 protein, we treated cells with various inhibitors to identify which protein degradation pathways were being utilized. Treatment with MG132, which inhibits the proteasome, but not with the lysosome inhibitor ammonium chloride (NH4Cl) or with autophagosome inhibitor 3-methyladenine (3-MA), markedly inhibited the degradation of FMDV VP3 protein (Fig. 3A), suggesting that overexpression of TBK1 leads to degradation of FMDV VP3 protein through a proteasome-dependent pathway. In addition, 293T cells were transfected by the use of TBK1 with FMDV VP3 and then treated with MG132. The result showed that TBK1 interacted with VP3 (Fig. 3B). Moreover, TBK1+/+ and TBK1−/− MEFs were treated with MG132 or vehicle before FMDV infection. We found that FMDV VP3 protein levels were higher in TBK1−/−, MG132-treated TBK1+/+, and MG132-treated TBK1−/− MEFs than in TBK1+/+ MEFs following FMDV infection (Fig. 3C). RT-PCR data showed that the mRNA levels of FMDV VP3 were not altered with TBK1 expression (Fig. 3D), indicating that the decrease in FMDV VP3 occurred at the protein level. Since TBK1 degraded FMDV VP3 through a proteasome-dependent pathway, we determined whether TBK1 could catalyze the ubiquitination of FMDV VP3. We found that TBK1 increased the ubiquitination of FMDV VP3 (Fig. 3E). Because proteins with the K48- or K63-linked polyubiquitin chains can be targeted for degradation in proteasome-dependent pathways, we next determined whether TBK1 catalyzed K48- or K63-linked ubiquitination of FMDV VP3. We observed that TBK1-catalyzed K63—rather than K48—ubiquitinated FMDV VP3 (Fig. 3F). In addition, we also found that knockout of TBK1 decreased the K63-linked ubiquitination of FMDV VP3 in MEFs followed by FMDV infection at the indicated times (Fig. 3G). Furthermore, we determined whether TBK1 degraded other picornavirus VP3 proteins by proteasome-dependent pathways. In transient-transfection and Western blot experiments, MG132 treatment markedly inhibited the degradation of multiple picornavirus VP3 proteins in 293T cells transfected with TBK1 and VP3 (Fig. 3H). In addition, we found that TBK1 increased the ubiquitination of EV71 VP3, EMCV VP3, and SVV VP3 proteins (Fig. 3I). These results suggest that TBK1 degraded multiple picornavirus VP3 proteins via ubiquitination.

FIG 3.

FIG 3

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TBK1 degrades multiple picornavirus VP3 proteins via the proteasome pathway. (A) TBK1 mediates the proteasomal degradation of FMDV VP3. 293T cells (2 × 105) were transfected with the indicated plasmids. At 18 h after transfection, the cells were treated with the indicated inhibitors (MG132 [100 μM], 3-MA [0.5 μg/μl], NH4Cl [25 mM]) for 6 h before immunoblot analysis. (B) TBK1 interacts with FMDV VP3 in 293T cells. 293T cells were transfected with TBK1 and FMDV VP3 for 18 h, and then the cells were treated with MG132 (100 μM) for 6 h before coimmunoprecipitation and immunoblot analysis. (C) Immunoblot analysis of FMDV VP3 in TBK1+/+ and TBK1−/− MEFs treated with MG132 (100 μM) for 1 h and then infected with FMDV for the indicated times. DMSO, dimethyl sulfoxide. (D) The mRNA level of FMDV VP3 was unaffected by TBK1. 293T cells (2 × 105) were transfected with the VP3 and TBK1 plasmids (0 μg, 0.5 μg, 1.0 μg, or 2.0 μg) for 24 h. Relative mRNA levels of FMDV VP3 were determined by RT-PCR. GAPDH was used as an internal reference. The data shown are representative of results from one of three independent experiments and are presented as means ± SD of results from three technical replicates. NS, no significant differences. (E) Overexpression of TBK1 promoted the ubiquitination of FMDV VP3. 293T cells (2 × 106) were transfected with Myc-VP3 by standard calcium phosphate precipitation for 12 h, and then the cells were again transfected with Flag-TBK1 and HA-ubiquitin (HA-Ub) by the use of Lipofectamine 2000 transfection reagent for 12 h prior to coimmunoprecipitation and immunoblot analysis. The immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (upper panel). Protein expression was analyzed by immunoblotting with the indicated antibodies (lower panels). (F) TBK1 promoted K63-linked but not K48-linked ubiquitination of FMDV VP3. 293T cells (2 × 106) were transfected with Myc-VP3 by standard calcium phosphate precipitation for 12 h, and then the cells were transfected again with Flag-TBK1 and HA-tagged Lys-48-only or Lys-63-only ubiquitin plasmids by the use of Lipofectamine 2000 transfection reagent for 12 h. Cell lysates were immunoprecipitated with an anti-Myc antibody and then analyzed by immunoblotting with an anti-Myc antibody (upper panel). The expression levels of related proteins were examined by immunoblotting with the indicated antibodies (lower panels). (G) Effects of TBK1 knockout on the K63-linked ubiquitination of FMDV VP3. The wild-type and TBK1 knockout MEFs were infected with FMDV for the indicated times or left uninfected. The cell lysates were immunoprecipitated with anti-VP3. The immunoprecipitates were denatured and reimmunoprecipitated with anti-VP3 and then analyzed by immunoblotting with a K63-linkage-specific ubiquitin antibody (upper panel). The expression of the related proteins was examined by immunoblotting with the indicated antibodies (lower panels). (H) TBK1 mediates the proteasomal degradation of multiple picornavirus VP3 proteins. Experiments were performed as described for panel A. β-Actin was used as a loading control. For TBK1 and VP3 proteins, band intensities were determined by the use of Image J software and normalized to that of β-actin. (I) Overexpression of TBK1 promoted the ubiquitination of multiple picornavirus VP3 proteins. Experiments were performed as described for panel F. RT-PCR, real-time reverse transcription-PCR; SD, standard deviation. All Western blot results are representative of at least two independent experiments.

The key residue determinants of TBK1 and FMDV VP3 in TBK1-mediated degradation of FMDV VP3.

To determine which key residues of TBK1 are responsible for the degradation of FMDV VP3 protein, we performed sequence analysis and identified four conserved cysteine residues in TBK1: C423, C426, C471, and C605 (Fig. 4A). We then generated a series of TBK1 mutants in which the cysteine residues were mutated to alanine sequentially or simultaneously and examined their effect on FMDV VP3 degradation. As shown in Fig. 4B and C, mutation of C426 or C605 to alanine, double mutation of both C426 and C605 to alanine (C426/605A), mutation of K38 to alanine, and mutation of S172 to alanine but not C423A, C471A, and S716A remarkably reduced TBK1-mediated degradation of FMDV VP3 protein. In addition, to determine major residues of FMDV VP3 that are targeted for degradation by TBK1, we identified two conserved lysine residues of FMDV VP3: K20 and K118 (Fig. 4D). We therefore constructed FMDV VP3 mutants in which the lysine residues were mutated to arginine individually and assessed their impact on degradation by TBK1. Our data showed that TBK1 caused the degradation of FMDV VP3 and FMDV VP3 K20R mutant but not FMDV VP3 K118R (Fig. 4E). These results suggest that the C426/605 site of TBK1 and the K118 site of VP3 play important roles in TBK1-mediated VP3 degradation.

FIG 4.

FIG 4

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The key sites of TBK1 for inducing the degradation of multiple picornavirus VP3 proteins. (A) Alignment of conserved cysteine sequences of human, mouse, cattle, monkey, swine, and rat TBK1. (B and C) Effects of TBK1 and its mutants on the expression of FMDV VP3 protein. 293T cells (2 × 105) were transfected with the indicated plasmids for 24 h before immunoblot analysis. (D) Alignment of conserved lysine sequences of different serotypes of FMDV VP3. (E) TBK1 induced FMDV VP3 K20R degradation but not FMDV VP3 K118R degradation. 293T cells (2 × 105) were transfected with the indicated plasmids for 24 h before immunoblot analysis. (F and G) Overexpression of TBK1 C426/605A (F), but not TBK1 L352/I353A (G), abolished the TBK1-mediated degradation of multiple picornavirus VP3 proteins in a dose-dependent manner. Experiments were performed as described for panel B. β-Actin was used as a loading control. For TBK1 C426/605A, TBK1 L352/I353A, and VP3 proteins, band intensities were determined by Image J software and normalized to that of β-actin. All Western blot results are representative of at least two independent experiments.

To confirm the roles of TBK1 C426/605A on the stability of picornavirus VP3 proteins, we cotransfected different doses of TBK1 C426/605A plasmids with multiple picornavirus VP3 plasmids. We found that TBK1 C426/605A did not affect the stability of picornavirus VP3 proteins (Fig. 4F). It was previously demonstrated that ubiquitin-like domain (ULD)-mutated TBK1 (TBK1 L352/I353A) failed to activate IFN-β and NF-κB promoters and to induce the gene transcription of IFN-β and RANTENS (26, 27). To investigate the effects of TBK1 L352/353A on the expression of multiple picornavirus VP3 proteins, we constructed a TBK1 L352/353A plasmid. In transient-transfection and Western blot experiments in 293T cells, we found that overexpression of TBK1 L352/353A degraded multiple picornavirus VP3 proteins in a dose-dependent manner (Fig. 4G). Collectively, these results suggest that the amino acid residues C426/605, but not L352/353, play a key role in TBK1-mediated degradation of multiple picornavirus VP3 proteins.

Mutations in C426/605 residues of TBK1 did not affect its immune activation function.

Next, we mutated C426 and C605 of TBK1 to alanine to evaluate the functions of these residues in TBK1-mediated activation of innate immunity. We observed that both TBK1 C426/605A and TBK1 increased IRF3 phosphorylation, autophosphorylation of TBK1, and the molecular weight of IRF3 (Fig. 5A). Similarly, both TBK1 C426/605A and TBK1 increased VISA phosphorylation (Fig. 5B). It was reported previously that K63-linked ubiquitination of TBK1 is critical for TBK1 activation (28, 29). This ubiquitination of TBK1 was greatly increased in cells expressing the C426/605 mutant (Fig. 5C). Taken together, these observations suggest that TBK1 C426/605A does not affect TBK1 innate immunity activation or block TBK1 K63 ubiquitination but instead leads to the accumulation of K63 ubiquitination, even though this highly ubiquitinated TBK1 is inactive.

FIG 5.

FIG 5

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The mutation of C426/605 residues did not affect TBK1 activity. (A) Effects of TBK1 and TBK1 C426/605A on TBK1 activation and IRF3 phosphorylation. 293T cells (2 × 105) were transfected with the indicated plasmids for 24 h before immunoblot analysis. (B) Effects of TBK1 and TBK1 C426/605A on VISA phosphorylation. Experiments were performed as described for panel A. (C) K63-linked ubiquitination of TBK1 was evaluated by cotransfection of K63-specific ubiquitin, and a marked accumulation of TBK1 K63-linked ubiquitination was detected in TBK1 C426/605A. (D to G) Interactions among TBK1, IRF3, IKKε, MITA, and TBK1 or TBK1 mutants. Transient transfection and coimmunoprecipitation were performed with the indicated plasmids and antibodies. (H and I) Effects of overexpressing TBK1 and its mutants on activation of the IFN-β promoter (H) and ISRE (I). 293T cells (1 × 105) were transfected with the indicated expression (0.1 μg each) and reporter (0.1 μg each) plasmids for 24 h before luciferase assays were performed. The data shown are from a single experiment representative of three independent experiments and are presented as means ± SD of results from three technical replicates. EV, empty vector; WT, wild type; SD, standard deviation. All Western blot results are representative of at least two independent experiments.

To elucidate the molecular basis of TBK1 C426/605A, we assessed TBK1 homodimerization and heterodimerization changes upon mutating C426/605 residues to alanine. Coimmunoprecipitation assays showed that the association with IKKε was unaffected in the TBK1 C426/605A mutants (Fig. 5D and E). Similarly, the C426/605 mutants of TBK1 did not exhibit altered associations with the IRF3 substrate (Fig. 5F) or interactions with the MITA adaptor/substrate (Fig. 5G). We next examined whether the effect of TBK1 mutants on FMDV VP3 depended on its immune response. We found that the mutants of TBK1 did not exhibit altered TBK1-triggered activation of the IFN-β promoter and the interferon-sensitive response element (ISRE) by reporter assays (Fig. 5H and I). All of these observations suggest that the TBK1 C426/605A mutant did not affect TBK1 dimerization and activation.

The C426/605 sites of TBK1 and lysine site of VP3 play an important role in TBK1-mediated degradation of VP3.

To confirm the role of TBK1 C426/605A in degradation of VP3, we evaluated VP3 protein ubiquitination by TBK1 C426/605A. As shown in Fig. 6A, TBK1 C426/605A did not alter the ubiquitination (and K63-linked ubiquitination) of VP3. Furthermore, we studied the involvement of the VP3 K118R mutant in TBK1 degradation of VP3. We found that TBK1-mediated ubiquitination of the VP3 K118R mutant was decreased; however, K63 ubiquitination of VP3 K118R was not affected (Fig. 6B). Sequence analysis identified conservative lysine residues in EV71 VP3, EMCV VP3, and SVV VP3 proteins, which are EV71 VP3 K130, EMCV VP3 K123, and SVV VP3 K130. We made those three picornavirus VP3 proteins mutants in which the lysine residues were mutated to arginines and examined their K63-linked ubiquitination by TBK1. As shown in Fig. 6C, TBK1 increased K63-linked ubiquitination of picornavirus VP3 proteins, whereas lysine-to-arginine mutants of VP3 proteins exhibited no TBK1-catalyzed K63-linked ubiquitination. These data collectively demonstrate that the C426/605 sites of TBK1 play an important role in TBK1-mediated ubiquitination of FMDV VP3 and that the K118, K130, K123, and K130 sites located in the VP3 domain of FMDV, EV71, EMCV, and SVV, respectively, are involved in the TBK1-mediated degradation of picornavirus VP3 proteins.

FIG 6.

FIG 6

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TBK1 C426/605A did not affect ubiquitination of FMDV VP3, and TBK1 did not affect ubiquitination of FMDV VP3 K118R. (A) TBK1 C426/605A did not affect ubiquitination of FMDV VP3. 293T cells (2 × 106) were transfected with the indicated plasmids. At 24 h after transfection, cell lysates were immunoprecipitated with an anti-Myc antibody. The immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (upper panel). Protein expression was analyzed by immunoblotting with the indicated antibodies (lower panel). (B) TBK1 did not affect K63 ubiquitination of FMDV VP3 K118R. The experiments were performed as described for panel A. (C) TBK1 increased K63 ubiquitination of multiple picornavirus VP3 proteins. 293T cells were transfected with multiple picornavirus VP3 proteins or their mutants by standard calcium phosphate precipitation for 12 h, and then the cells were transfected again with Flag-TBK1 and HA-tagged Lys-63-only ubiquitin plasmids by the use of Lipofectamine 2000 transfection reagent for 12 h. The immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (upper panel). Protein expression was analyzed by immunoblotting with the indicated antibodies (lower panel). (D) The interaction between TBK1 and Myc-VP3 or its mutants. 293T cells were transfected with TBK1 and Myc-VP3 or its mutants for 18 h. The cells were treated with MG132 (100 μM) for 6 h before coimmunoprecipitation was performed with the indicated plasmids and antibodies. (E) The interaction between TBK1 or its mutants and Flag-VP3. The experiments were performed as described for panel D. All Western blot results are representative of at least two independent experiments. WT, wild type.

In transient-transfection and coimmunoprecipitation experiments, mutation of the K118 site of VP3 did not block the association between TBK1 and VP3 (Fig. 6D). Furthermore, mutation of the C426/605 sites of TBK1 did not block the association between TBK1 and VP3 (Fig. 6E). These results suggest that the K118 site of VP3 and the C426/605 sites of TBK1 do not participate in the association between TBK1 and VP3.

TBK1 catalyzes the phosphorylation of multiple picornavirus VP3 proteins at serine residues.

Since the TBK1 K38A mutant could not degrade the FMDV VP3 protein, we hypothesized that TBK1 could phosphorylate the FMDV VP3 protein. We next determined the residues of FMDV VP3 protein that are phosphorylated by TBK1. The GPS3.0 program predicted that FMDV VP3 contains one consensus TBK1 phosphorylation residue: S153. We next determined whether TBK1 would affect serine phosphorylation of the FMDV VP3 protein. In transient-transfection and coimmunoprecipitation experiments, TBK1 dose-dependently enhanced the phosphorylation of FMDV VP3 protein at serine residues as determined by incubation with an antibody against phosphorylated serine (p-Ser). This effect was blocked by treatment with calf intestinal alkaline phosphatase (CIP) (Fig. 7A). To determine whether TBK1 indeed targets Ser153 of FMDV VP3 protein for phosphorylation, we constructed a FMDV VP3 S153A mutant. In transient-transfection and coimmunoprecipitation experiments, FMDV VP3 S153A exhibited no TBK1-catalyzed serine phosphorylation (Fig. 7B). Taken together, these results suggest that TBK1 phosphorylates FMDV VP3 protein at Ser153.

FIG 7.

FIG 7

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TBK1 phosphorylates multiple picornavirus VP3 proteins at serine residues. (A) FMDV VP3 protein is serine phosphorylated by TBK1. 293T cells were transfected with FMDV VP3 (3 μg) and TBK1 expression plasmids (0 μg, 2.5 μg, or 5 μg) for 18 h. The cells were treated with MG132 (100 μM) for 6 h. Cell lysates were immunoprecipitated with anti-Myc, and the immunoprecipitates were treated with buffer or calf intestine phosphatase (CIP) and then analyzed by immunoblotting with the indicated antibodies. (B) TBK1 phosphorylates FMDV VP3 at S153. The experiments were performed as described for panel A. (C) Alignment of conserved serine sequences of multiple picornavirus VP3 proteins. (D) Effects of TBK1 on the phosphorylation of multiple picornavirus VP3 proteins and their mutants. The experiments were performed as described for panel A. (E) Effects of TBK1 on FMDV VP3 and its mutant degradation. 293T cells were transfected with the indicated plasmids for 24 h and then analyzed by immunoblotting with the indicated antibodies. (F) Effects of TBK1 on multiple picornavirus VP3 proteins and their mutant degradation. The experiments were performed as described for panel E. All Western blot results are representative of at least two independent experiments.

The Ser153 site of VP3 proteins is highly conserved in multiple picornaviruses (Fig. 7C). Mutagenesis experiments indicated that TBK1 phosphorylates wild-type picornavirus VP3 proteins but not picornavirus VP3 mutants EV71 VP3 S163A, EMCV VP3 S156A, and SVV VP3 S163A (Fig. 7D). In addition, we found that TBK1 degraded FMDV VP3 but not FMDV VP3 S153A (Fig. 7E). Similarly, TBK1 degraded wild-type picornavirus VP3 proteins but not the picornavirus VP3 mutants VP3 SA (S156R and S163R) and VP3 KR (K130R and K156R) (Fig. 7F). These results suggest that TBK1 targets serine residues phosphorylation of picornavirus VP3 proteins.

TBK1 is an E3 ubiquitin ligase.

Since the results reported above showed that TBK1 degraded multiple picornavirus VP3 proteins by proteasome-dependent pathways, we reasoned that TBK1 is an E3 ubiquitin ligase. To test the ubiquitylation of TBK1, we used rabbit reticulocyte lysate, which contains ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) (30). Results from in vitro ubiquitination assays demonstrated that TBK1 catalyzed the polyubiquitination of itself in vitro (Fig. 8A). Similarly, we also found that TBK1 increased ubiquitination of VP3 protein by in vitro ubiquitination assays (Fig. 8B). In overexpression experiments, TBK1 dose-dependently promoted the polyubiquitination of itself (Fig. 8C). Next, we performed in vitro ubiquitination assays to determine the E2-mediated polyubiquitination of TBK1. The results indicated that incubating UbcH5c, but not UbcH1-3, UbcH5a, UbcH5b, UbcH6-8, UbcH10, or UbcH13, with TBK1 yielded substantial polyubiquitylation of TBK1 (Fig. 8D). In addition, mutant TBK1 C426/605A, but not TBK1 K38A and TBK1 L352/I353A, failed to mediate polyubiquitination of itself (Fig. 8E). These results indicate that TBK1 is an E3 ubiquitin ligase in cooperation with the E2 enzyme UbcH5c.

FIG 8.

FIG 8

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TBK1 is an E3 ubiquitin ligase. (A) In vitro self-ubiquitylation of TBK1. TBK1 protein was obtained by in vitro transcription and translation and was then incubated with biotin-Ub (Bio-Ub), E1, and the E2s (rabbit reticulocyte lysate). Polyubiquitination of TBK1 was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of TBK1 were analyzed by the use of immunoblots with anti-TBK1 (bottom panels). (B) Effects of TBK1 on ubiquitination of VP3 protein in vitro. The experiments were performed as described for panel A. (C) Overexpression of TBK1 promoted polyubiquitination of itself in a dose-dependent manner. 293T cells (2 × 106) were transfected with Flag-TBK1 (0 μg, 2.5 μg, or 5 μg) and HA-Ub (1 μg) together with a control plasmid (5 μg, 2.5 μg, or 0 μg). At 24 h after transfection, cells were subjected to immunoprecipitation (IP) with anti-Flag under denatured conditions and the immunoprecipitates were analyzed by immunoblotting with anti-HA (upper panel). The whole-cell lysates were analyzed by immunoblotting with anti-Flag (lower panels). Ub, ubiquitin. (D) TBK1 is directly ubiquitinated by itself in the presence of the E2 (UbcH5c). TBK1 was translated in vitro, and the indicated E2s were added for ubiquitination assays. Ubiquitin-conjugated TBK1 was detected by immunoblotting with HRP-streptavidin (upper panel). Before ubiquitination analysis, the levels of the translated proteins were detected with the indicated antibodies (lower panels). (E) Effects of TBK1 and its mutants on polyubiquitination in vitro. TBK1 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, and UbcH5c were incubated with TBK1 or its mutants, followed by ubiquitination and immunoblot analysis as described for panel D. All Western blot results are representative of at least two independent experiments.

DISCUSSION

It has become clear that virus-triggered induction of type I IFNs is crucial for the early innate antiviral response as well as for late-stage adaptive immunity. Here, we investigated whether innate immune molecules affect picornaviruses. By performing transient-transfection and Western blot experiments, we revealed a key role for TBK1 in regulating the expression of multiple picornavirus VP3 proteins in a manner dependent on its kinase and E3 ubiquitin ligase activity.

Previous studies have identified three major classes of E3, termed the HECT (homologous to E6-associated protein C terminus), RING finger, and U-box (a modified RING motif without the full complement of Zn2+-binding ligands) E3s. In addition, two subclasses of RING E3s have been defined: RIR (RING in between RING-RING) domain E3s and multiprotein complex (CRL [Cullin-RING]) E3s (31, 32). Ubiquitination is catalyzed by a three-enzyme cascade consisting of the E1 Ub-activating enzyme, the E2 Ub-conjugating enzyme, and the E3 Ub protein ligase (33). In the study, we found that TBK1 is a novel E3 ubiquitin ligase. First, TBK1 has no conserved HECT, RING finger, or U-box domains. Second, we performed in vitro ubiquitylation assays and found that TBK1 underwent self-ubiquitylation, an indication of E3 ligase activity. Third, we also performed in vivo ubiquitylation assays and found that TBK1 could be self-ubiquitylated in 293T cells. Fourth, TBK1 underwent self-ubiquitylation in vitro when combined with E2 enzyme UbcH5C.

Usually, proteasomes recognize and degrade proteins that have been modified with K48-linked polyubiquitin chains (34). Interestingly, we found that TBK1 degraded the FMDV VP3 protein by K63 ubiquitination. In contrast to the well-studied K48 linkage type, little is known about the regulation and roles of K63 ubiquitination; only a few targets have been characterized in yeast (35). In the current study, we confirmed that TBK1 is a novel E3 ubiquitin ligase. Further studies are needed to determine whether TBK1 alone degrades target proteins by K63 ubiquitination and to further characterize the function of TBK1 as an E3 ubiquitin ligase.

IKK-related kinases contain IKKα, IKKβ, IKKε, and TBK1 (36). They have high sequence similarity and structural similarity (37). IKK-related kinases contain three domains: a kinase domain (the ULD) and two coiled-coil domains (26). The K38 is essential for kinase activation of IKK-related kinases (38). In this study, we found that the C425/605 amino acid residues in TBK1 play important roles in the E3 ubiquitin ligase activity and that TBK1 degrades multiple picornavirus VP3 proteins in a manner dependent on its kinase and E3 ubiquitin ligase activity. The C605 amino acid residues of IKK-related kinases are conserved, and we speculate that IKK-related kinases represent a novel class of E3 ubiquitin ligases that degrade other proteins in a manner dependent on their kinase and E3 ubiquitin ligase activities.

Protein phosphorylation and ubiquitination regulate the activity and stability of target proteins and are important for signal transduction and multiple physiological processes. Viral infection activates the protein kinase A catalytic subunits (PKACs), which in turn phosphorylate VISA at T54, thereby impairing VISA aggregation and leading to its K48-linked polyubiquitination and degradation by the E3 ligase MARCH5 (39). The serine-threonine kinase CK1ε interacts with and phosphorylates TRAF3 at Ser349, which thereby promotes the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of kinase TBK1 to TRAF3 (40). Protein kinase DYRK2 phosphorylates Ser527 of TBK1, which is essential for the recruitment of NLRP4 and for degradation of TBK1 by E3 ubiquitin ligase DTX4 (41). The EL1-like casein kinase suppresses abscisic acid signaling and responses by phosphorylating and destabilizing abscisic acid receptors PYR/PYLs in Arabidopsis (42). In this study, we found that TBK1 degraded multiple picornavirus VP3 proteins in a manner dependent on its kinase and E3 ubiquitin ligase activity and that the kinase activity of TBK1 was connected with its E3 ubiquitin ligase activity in degradation of multiple picornavirus VP3 proteins. First, either TBK1 K38A or TBK1 C426/605A inhibited TBK1-mediated degradation of multiple picornavirus VP3 proteins. Second, MG132 treatment abolished the ability to perform TBK1-mediated degradation of multiple picornavirus VP3 proteins. Third, TBK1 was shown to function as a kinase and an E3 ubiquitin ligase.

Together, our findings have established TBK1 as a regulator of precise control of picornaviruses by regulation of the phosphorylation and ubiquitination of picornavirus VP3 proteins. This should open fresh perspectives on the function of these long-studied kinase families. Moreover, our study has shed light on the mechanism responsible for the specificity and diversity achieved by the complicated regulation of the picornavirus life cycle, which makes TBK1 a potential target for treating picornavirus infection.

MATERIALS AND METHODS

Reagents.

Mouse monoclonal antibodies against Flag, Flag-horseradish peroxidase (Flag-HRP), Myc (Sigma), hemagglutinin (HA) (Covance), and β-actin (Sigma); rabbit polyclonal antibody against TBK1 (Abcam); and rabbit monoclonal antibody against K63-linkage-specific polyubiquitin chain (Cell Signaling Technology) were purchased from the corresponding manufacturers. A mouse anti-VP3 polyclonal antibody was prepared in our laboratory using conventional methods. iQ SYBR green real-time supermix (Bio-Rad), GammaBind G Plus-Sepharose (Amersham Biosciences), and Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) were purchased from the indicated manufacturers.

Viruses and cells.

Human embryonic kidney (HEK) 293T cells (ATCC) and porcine kidney cells (PK-15) (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). TBK1−/− and wild-type mouse embryonic fibroblasts (MEFs) were provided by the indicated investigator (Hong-Bing Shu, Wuhan University). Type O FMDV was prepared in our laboratory and propagated in PK-15 cells, and the supernatants of infected cells were harvested and stored at –80°C for further studies.

Constructs.

Mammalian expression plasmids encoding VP0, VP2, 3D, 3A, VP3, and VP3 mutants were constructed by PCR amplification of their cDNA from PK-15 cells infected with FMDV and subsequent cloning into cytomegalovirus (CMV) promoter-based vectors containing a Flag tag. Mammalian expression plasmids encoding HA-TBK1, Flag-TBK1, HA-MDA5, HA-VISA, HA-IRF7, HA-IRF3, HA–RIG-1 (CARD), and HA-MITA were described previously (43–45).

Transfection and reporter gene assays.

293T cells (∼1 × 105) were seeded on 48-well plates and transfected the following day by standard calcium phosphate precipitation. In the same experiment, empty control plasmid was added to ensure that all transfections received the same amount of total DNA. To normalize for transfection efficiency, 10 ng of pRL-TK Renilla luciferase reporter plasmid was added to each transfection. Luciferase assays were performed with a dual-specific luciferase assay kit (Promega). Firefly luciferase activities were measured and normalized to Renilla luciferase activities.

Coimmunoprecipitation and Western blot analysis.

For transient-transfection and coimmunoprecipitation experiments, 293T cells (1 × 106) were transfected with the respective plasmids for 24 h and were then lysed in 1 ml of lysis buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X, 1 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation, a 0.4-ml aliquot of lysate was incubated with 0.2 μg of the indicated antibodies or control IgG and 25 μl of a 1:1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for 2 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were analyzed by Western blotting as previously described (46).

For endogenous coimmunoprecipitation experiments, cells (5 × 107) were infected with FMDV (multiplicity of infection [MOI] = 1.0) for the indicated time. Cells were then lysed in 5 ml lysis buffer, and the lysate was incubated with 1 μl of the indicated antiserum or preimmune control serum. The subsequent procedures were carried out as described above.

Ubiquitination assays.

For in vivo ubiquitination experiments, we adopted a two-step immunoprecipitation strategy (47). The first round of immunoprecipitation was performed as described above. The immunoprecipitates were reextracted in lysis buffer containing 1% SDS and denatured by heating for 5 min. The supernatants were diluted with regular lysis buffer until the concentration of SDS decreased to 0.1%. The diluted supernatants were reimmunoprecipitated with the indicated antibodies, and the immunoprecipitates were analyzed by immunoblotting with the indicated antibodies.

For in vitro ubiquitination experiments, proteins were expressed with a TNT quick-coupled transcription/translation system kit (Promega, Madison, WI) according to the manufacturer’s instructions. Ubiquitination was analyzed with a ubiquitination kit (Enzo Life Sciences, Farmingdale, NY) by following the manufacturer’s protocols.

RT-PCR.

Total RNA was isolated from cells using TRIzol reagent (Takara) and subjected to RT-PCR analysis to measure mRNA expression. The mRNA levels of specific genes were normalized to the level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA. The gene-specific primer sequences were as follows: for VP3, 5′-GGCAAAGTGTTCAACCCCC-3′ and 5′-CACCCTGTCCGAATCCGT-3′; for GAPDH, 5′-GAGTCAACGGATTTGGTCGT-3′ and 5′-GACAAGCTTCCCGTTCTCAG-3′.

Viral RNA was extracted from FMDV-infected cells with TRIzol. Genomic copy numbers of FMDV were determined by the use of a previously described quantitative RT-PCR assay (48, 49).

Statistical analysis.

The significance of differences between samples was assessed using an unpaired two-tailed Student's t test. The variance was estimated by calculating standard deviations (SD), and the calculated SD values are represented by error bars. All experiments were performed independently at least three times, with results from a representative experiment being shown (**, P < 0.01).

ACKNOWLEDGMENTS

We thank Hong-Bing Shu (Wuhan University) for providing plasmids.

This work was supported by Gansu Provincial Science and Technology Department of China grant 17JR5RA323; by National Natural Science Foundation of China grants 31672585, 31572542, and U1501213; and by Chinese Academy of Agricultural Sciences grant Y2017JC55.

We declare that we have no competing interests.

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