NLRC4 promotes the cGAS‐STING signaling pathway by facilitating CBL‐mediated K63‐linked polyubiquitination of TBK1

TANK‐binding kinase 1 (TBK1) is crucial in producing type Ⅰ interferons (IFN‐Ⅰ) that play critical functions in antiviral innate immunity. The tight regulation of TBK1, especially its activation, is very important. Here we identify NLRC4 as a positive regulator of TBK1. Ectopic expression of NLRC4 facilitates the activation of the IFN‐β promoter, the mRNA levels of IFN‐β, ISG54, and ISG56, and the nuclear translocation of interferon regulatory factor 3 induced by cGAS and STING. Consistently, under herpes simplex virus‐1 (HSV‐1) infection, knockdown or knockout of NLRC4 in BJ cells and primary peritoneal macrophages from Nlrc4‐deficient (Nlrc4−/−) mice show attenuated Ifn‐β, Isg54, and Isg56 mRNA transcription, TBK1 phosphorylation, and augmented viral replications. Moreover, Nlrc4−/− mice show higher mortality upon HSV‐1 infection. Mechanistically, NLRC4 facilitates the interaction between TBK1 and the E3 ubiquitin ligase CBL to enhance the K63‐linked polyubiquitination of TBK1. Our study elucidates a previously uncharacterized function for NLRC4 in upregulating the cGAS‐STING signaling pathway and antiviral innate immunity.


| INTRODUCTION
Innate immune responses to viral infection start with the recognition of pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) by host pattern recognition receptors, including Toll-like receptors, Nucleotid-binding and oligomerization domain (NOD)-like receptors (NLRs), RIG-Ⅰ-like receptors, and several DNA sensors, among which cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a universal cytoplasmic DNA sensor in various cell types. 1 Once cGAS recognizes the pathogenic or aberrant DNA through its enzymatic activity in the cytoplasm, it catalyzes the synthesis of cGAMP. This second messenger binds to the endoplasmic reticulumanchored adaptor protein STING and induces its oligomerization and translocation to the Golgi apparatus. STING triggers the recruitment and phosphorylation of TANK-binding kinase 1 (TBK1), which further results in interferon regulatory factor 3 (IRF3) phosphorylation, dimerization, and nuclear translocation to induce type Ⅰ interferons (IFN-Ⅰ) production and subsequent a large number of IFN stimulated genes (ISGs) that have well-known antiviral and immune-regulatory functions. 2 To ensure appropriate immunity and prevent aberrant immune responses, the cGAS-STING signaling pathway is precisely and tightly regulated by various molecules, [3][4][5] and our previous study demonstrated that UNC93B1 attenuated the cGAS-STING signaling pathway by targeting STING for autophagy-lysosome degradation. 6 NLRs are well-known key components of the inflammasomes, including another two proteins, apoptosis-associated speck-like protein containing a CARD and procaspase-1, mediate interleukin (IL)-1β and IL-18 production and involve in proinflammatory responses. 7 NLRC4 is one of the NLR family proteins. Previous understanding of NLRC4 was confined to inflammasome, 8 while its function in the antiviral innate immunity and signaling pathways has yet to be found. Recent studies have shown the emerging roles of NLRs in the cGAS-STING-mediated DNA-sensing signaling pathway. 9 NLRC3 interacts with STING directly to prevent its trafficking to the perinuclear and punctate region, thus inhibiting STING-TBK1 association and IFN-I production induced by cytosolic DNA, cyclic di-GMP, and DNA viruses. 10 NLRX1 associates with STING to disrupt the interaction of STING and TBK1 and downregulate innate immunity during DNA virus infections. 11 NLRP2 suppresses IFN-I signaling via interacting with TBK1 to hinder TBK1-IRF3 association and IRF3 phosphorylation. 12 NLRP11 attenuates TBK1-induced IFNβ promoter activity in a dose-dependent manner at very low levels, implying that NLRP11 might participate in the cGAS-STING signaling pathway. 13 NLRP14 associates with nucleic acid-sensing components and dampens TBK1-dependent signaling by inducing its ubiquitination and degradation in a K48-ubiquitin-independent manner. 14 While substantial advances have been made recently to unveil the roles of NLRs in the cGAS-STING signaling pathway, the human NLR protein family consists of 22 members, suggesting that more NLRs might involve in the signaling pathway. We find that NLRC4 positively regulates the cGAS-STING signaling pathway by facilitating the interaction between TBK1 and the E3 ubiquitin ligase Cbl protooncogene (CBL) to enhance the K63-linked polyubiquitination and subsequent activation of TBK1. Furthermore, the replication of herpes simplex virus-1 (HSV-1), a DNA virus, is effectively inhibited by ectopic expression of NLRC4, and Nlrc4-deficient (Nlrc4 −/− ) mice exhibit higher mortality upon HSV-1 infection. These findings uncover a previously uncharacterized function for NLRC4 in activating TBK1, critical for IFN-I production in antiviral innate immunity.

| Mice
The mouse strain used in this study was C57BL/6 and Nlrc4 −/− C57BL/6 mice, about 8 weeks old and weighing approximately 20-25 g. All mice were housed under specific pathogen-free-grade conditions in the animal facilities of Fujian Medical University. The Nlrc4 −/− mice were generated by microinjection of Cas9 mRNA and sgRNAs into fertilized oocytes of C57BL/6 mice. 15 In brief, female mice aged 3-4 weeks were superovulated, and eggs were fertilized in vitro before the microinjection of the CRISPR/Cas9 system. The sgRNAs were synthesized by GenScript. The sgRNA sequences targetingNlrc4 were listed below: Primers for PCR genotyping of mice were synthesized (Sangon Biotech) with the following sequences: F (5′-CCCGAATGTAAAGCC TCTGA-3′), R (5′-GATGGCCAGTCAAAGATGGT′).

| Primary peritoneal macrophage (PM) isolation
The cells in the abdominal cavity of C57BL/6 mice were collected by intraperitoneal injection with 8 mL PBS and centrifugation. Cells were seeded and maintained in 1640 with 10% fetal bovine serum (FBS).
The adhered PMs were used for further experiments.

| Viral infection
Cells were cultured and infected with HSV-1 for different time points, then harvested for western blot (WB), quantitative real-time polymerase chain reaction (qRT-PCR), and viral plaque assay. About 1 × 10 7 PFU of HSV-1 was injected intravenously into each mouse, and survival rates were determined.

| Cells and viruses
HEK293T cells were purchased from the Stem Cell Bank, Chinese Academy of Sciences, BJ cells were kindly provided by Prof. Qi Chen (Fujian Normal University, China), and Vero cells were purchased from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle medium (HyClone), which was supplemented with 10% heat-inactivated FBS (PAN), 100 U/mL penicillin, and streptomycin in a 5% (vol/vol) CO 2 incubator at 37°C. HSV-1 strain F was propagated and titrated in Vero cells.

| Reagents and antibodies
Puromycin and radioimmunoprecipitation assay lysis buffers were purchased from Beyotime (Shanghai, China). Mouse anti-HA, anti-MYC, anti-FLAG, and anti-β-Actin monoclonal antibodies were purchased from Abmart. Rabbit anti-UL42 polyclonal antibody (pAb) was purchased by GL Biochem Ltd. Rabbit anti-TBK1 and anti-p-TBK1 antibodies were purchased from Cell Signaling Technology (Danvers). Mouse anti-NLRC4 mAb was purchased from Santa Cruz Biotechnology.

| Transfection and dual-luciferase reporter (DLR) assays
HEK293T cells were transfected with IFN-β-Luc and pRL-TK with or without expression plasmids, as indicated through standard calcium phosphate coprecipitation. After 24 h, DLR assays were performed with a luciferase assay kit (Promega).

| WB, coimmunoprecipitation (Co-IP), and mass spectrometry (MS) experiments
Cells were transfected with plasmids as indicated and lysed on ice with lysis buffer. Then the lysates were incubated with corresponding antibodies and protein A/G-Agarose (Abmart) overnight at 4°C. The beads were washed three times with lysis buffer and then subjected to WB analysis. The MS experiment and data processing were performed by LifeInt Technology Co., Ltd.

| Immunofluorescence (IF) assays
Cells were transfected with plasmids as indicated and then washed with phosphate-buffered saline (PBS) three times. The cells were fixed in 4% paraformaldehyde and washed with PBS. After incubation for blocking nonspecific binding for 1 h at 37°C, primary antibodies were added and incubated for 8 h at 4°C. Samples were stained with suitable Alexa Fluor 488-or Alexa Fluor 594-conjugated secondary antibodies (Abmart). After then, the sample was stained with 4',6diamidino-2-phenylindole for 10 min at room temperature in the dark.
Images were acquired using an inverted fluorescent microscope (Zeiss).

| RNA isolation and qRT-PCR
Total RNA was extracted withTRIzol (Invitrogen) and subjected to reverse transcription to get the cDNA used as a qRT-PCR template to detect the mRNA levels. The primers used in qRT-PCR were listed below:   Figure 3H). These results suggested that NLRC4 promotes the cGAS-STING signaling pathway and antiviral innate immunity.

| NLRC4 targets TBK1
To identify the molecular target of NLRC4 in the cGAS-STING To further confirm the association between NLRC4 and TBK1, we performed IF assay in HEK293T cells transfected with NLRC4-MYC and TBK1-FLAG and found that NLRC4-MYC colocalized with TBK1-FLAG in the cytoplasm ( Figure 4I).
Moreover, the Co-IP experiments revealed that NLRC4-MYC interacted with TBK1-FLAG ( Figure 4J and K). We also examined the interaction between NLRC4 and TBK1 under physiological conditions. Human skin fibroblasts, BJ cells, were infected with or without HSV-1 and subjected to an endogenous Co-IP experiment. As shown in Figure 4L, NLRC4 interacted with TBK1 in both unstimulated and infected cells, and the interaction was comparable during viral infection. These results suggested that NLRC4 promotes the cGAS-STING signaling pathway by targeting TBK1.

| NLRC4 enhances the K63-linked polyubiquitination of TBK1
We next investigated the underlying molecular mechanisms of how NLRC4 promotes the cGAS-STING signaling pathway by targeting TBK1. HEK293T cells were transfected with TBK1-FLAG and increasing amounts of NLRC4-MYC and found that TBK1 protein and mRNA are comparable with increasing NLRC4 expression ( Figure 5A), indicating that NLRC4 did not affect TBK1 expression.
Since TBK1 activation requires dimerization, 16 K63-linked polyubiquitination, 17 and subsequent phosphorylation, 18 we next explored whether NLRC4 affected the dimerization of TBK1. TBK1-FLAG and TBK1-GFP were transfected into HEK293T cells with NLRC4-MYC, then subjected to a Co-IP experiment, and found that NLRC4 was

| NLRC4 facilitates CBL-mediated K63-linked polyubiquitination of TBK1
As NLRC4 is not an E3 ubiquitin ligase, we hypothesized that NLRC4 might function as a mediator to recruit an E3 ubiquitin ligase. Therefore, we sought to identify the E3 ubiquitin ligase responsible for TBK1 K63-linked polyubiquitination. HEK293T cells were transfected with NLRC4-MYC or empty vector plasmid, then subjected to Co-IP and protein MS. We identified several E3 ubiquitin ligases as candidates, including CBL, HUWE1, RNF40, and TRIM28, by analyzing the protein composition of IP products. Next, we assessed the effect of these E3 ubiquitin ligases on TBK1-NLRC4induced IFN-β transcription and found that both CBL and HUWE1, rather than TRIM28 or RNF40, promoted IFN-β transcription ( Figure 6A). Thus, we focused on CBL and HUWE1. To confirm the interaction between NLRC4 and CBL or HUWE1, Co-IP assays were performed in HEK293T cells transfected with NLRC4-MYC and CBL-FLAG or HUWE1-FLAG, and the results stated that both CBL-FLAG and HUWE1-FLAG indeed interacted with NLRC4-MYC ( Figure 6B and C). Then, we investigated whether NLRC4 acts with CBL or HUWE1 to induce the K63-linked polyubiquitination of TBK1. The co-expression of CBL-MYC, but not HUWE1-EGFP, and NLRC4-MYC further promoted TBK1 polyubiquitination ( Figure 6D and E), indicating that NLRC4 and CBL functioned synergistically to induce the K63-linked polyubiquitination of TBK1. We also assessed whether CBL is involved in the TBK1-NLRC4 interaction. As shown in Figure 6F, CBL-FLAG is associated with TBK1-GFP, further enhanced by the ectopic expression of NLRC4-MYC. In addition, we demonstrated that NLRC4 and CBL augmented the transcription of IFN-β induced by TBK1 but not IRF3/5D ( Figure 6G and H). These results suggested that NLRC4 promotes the interaction between TBK1 and CBL, which may account for the K63-linked polyubiquitination and subsequent activation of TBK1. USP2b, RNF144B, and UBE2S-USP15 inhibit the K63-linked polyubiquitination of TBK1. [39][40][41][42] Although much attention has been focused on regulating TBK1, multiple potential molecules that target TBK1 and the underlying molecular mechanisms have yet to be fully characterized.

| DISCUSSION
This study identified a previously unrecognized role for NLRC4 in the positive regulation of K63-linked polyubiquitination of TBK1. However, NLRC4 is not an E3 ubiquitin ligase. It is reported that NLRX1 promotes the K48-linked polyubiquitination and proteasomal degradation of MAVS through recruiting PCBP2-AIP4. 43  h, then subjected to IP, and precipitates were analyzed by WB. (F) HEK293T cells were transfected with CBL-FLAG, TBK1-GFP, and NLRC4-MYC for 24 h, then subjected to IP, and precipitates were analyzed by WB. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. ubiquitination and subsequent proteasomal degradation of RIG-I, 45 and CBL negatively regulates the NF-κB signaling pathway by promoting the K48-linked polyubiquitination of TRAF6. 46 Meanwhile, CBL enhances the K63-linked polyubiquitination of tyrosine kinase EphrinA2 to facilitate clathrin-mediated internalization of associated virus, 47 and CBL mediates the K63-linked polyubiquitination of RIPK1 to promote RIPK1 intermediate formation and TNF-αinduced apoptosis. 48 In this study, we identified that CBL interacted with NLRC4, and CBL facilitated TBK1-induced IFN-β mRNA level and TBK1 K63-linked polyubiquitination. Moreover, NLRC4 facilitates the interaction between TBK1 and CBL. These results indicated that NLRC4 positively regulates the cGAS-STING signaling pathway by promoting CBL-mediated TBK1 K63-linked polyubiquitination.
Since TBK1 activation undergoes dimerization, K63-linked polyubiquitination, and phosphorylation, [16][17][18] the phosphorylation of TBK1 would be affected by its ubiquitination. As expected, our results demonstrated that NLRC4 KD or knockout inhibited the phosphorylation of TBK1 during HSV-1 infection. On the other hand, NLRC4-CBL-mediated K63-linked polyubiquitination of TBK1 might prevent the dephosphorylation of TBK1 mediated by TRIM18-PPM1a or other molecules, 49 implying that host-appropriate immunity is tightly and precisely regulated.
In conclusion, our study identifies a previously unrecognized role for NLRC4 in the positive regulation of the cGAS-STING signaling pathway and antiviral innate immunity, elucidates the underlying molecular mechanisms by which NLRC4 enhances the interaction between TBK1 and CBL to facilitate the K63-linked polyubiquitination and activation of TBK1 (Figure 7), and provides scientific and theoretical bases for the regulation of host antiviral innate immunity.

AUTHOR CONTRIBUTIONS
Mengzhou Xue and Chunfu Zheng conceived and designed the study.