TBK1-stabilized ZNF268a recruits SETD4 to methylate TBK1 for efficient interferon signaling

Sufficient activation of interferon signaling is critical for the host to fight against invading viruses, in which post-translational modifications have been demonstrated to play a pivotal role. Here, we demonstrate that the human KRAB-zinc finger protein ZNF268a is essential for virus-induced interferon signaling. We find that cytoplasmic ZNF268a is constantly degraded by lysosome and thus remains low expressed in resting cell cytoplasm. Upon viral infection, TBK1 interacts with cytosolic ZNF268a to catalyze the phosphorylation of Serine 178 of ZNF268a, which prevents the degradation of ZNF268a, resulting in the stabilization and accumulation of ZNF268a in the cytoplasm. Furthermore, we provide evidence that stabilized ZNF268a recruits the lysine methyltransferase SETD4 to TBK1 to induce the mono-methylation of TBK1 on lysine 607, which is critical for the assembly of the TBK1 signaling complex. Notably, ZNF268 S178 is conserved among higher primates but absent in rodents. Meanwhile, rodent TBK1 607th aa happens to be replaced by arginine, possibly indicating a species-specific role of ZNF268a in regulating TBK1 during evolution. These findings reveal novel functions of ZNF268a and SETD4 in regulating antiviral interferon signaling. Sufficient activation of interferon signaling is critical for the host to fight against invading viruses, in which post-translational modifications have been demonstrated to play a pivotal role. Here, we demonstrate that the human KRAB-zinc finger protein ZNF268a is essential for virus-induced interferon signaling. We find that cytoplasmic ZNF268a is constantly degraded by lysosome and thus remains low expressed in resting cell cytoplasm. Upon viral infection, TBK1 interacts with cytosolic ZNF268a to catalyze the phosphorylation of Serine 178 of ZNF268a, which prevents the degradation of ZNF268a, resulting in the stabilization and accumulation of ZNF268a in the cytoplasm. Furthermore, we provide evidence that stabilized ZNF268a recruits the lysine methyltransferase SETD4 to TBK1 to induce the mono-methylation of TBK1 on lysine 607, which is critical for the assembly of the TBK1 signaling complex. Notably, ZNF268 S178 is conserved among higher primates but absent in rodents. Meanwhile, rodent TBK1 607th aa happens to be replaced by arginine, possibly indicating a species-specific role of ZNF268a in regulating TBK1 during evolution. These findings reveal novel functions of ZNF268a and SETD4 in regulating antiviral interferon signaling. The antiviral innate immune response is elicited swiftly upon the detection of invading viral nucleic acid through pathogen-associated molecular patterns (PAMPs) (1Brubaker S.W. Bonham K.S. Zanoni I. Kagan J.C. Innate immune pattern recognition: a cell biological perspective.Annu. Rev. Immunol. 2015; 33: 257-290Crossref PubMed Scopus (991) Google Scholar). Several RNA and DNA sensors, including RIG-I, MDA5, cGAS, and IFI16, have been identified for detecting cytosolic viral genomes (2Roers A. Hiller B. Hornung V. Recognition of endogenous nucleic acids by the innate immune system.Immunity. 2016; 44: 739-754Abstract Full Text Full Text PDF PubMed Google Scholar). Following the detection of viral nucleic acid, signaling is propagated via either mitochondria-located MAVS (3Seth R.B. Sun L. Ea C.-K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2617) Google Scholar) (also named IPS-1 (4Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. et al.IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2079) Google Scholar), VISA (5Xu L.-G. Wang Y.-Y. Han K.-J. Li L.-Y. Zhai Z. Shu H.-B. VISA is an adapter protein required for virus-triggered IFN-beta signaling.Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1556) Google Scholar), or Cardif (6Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. et al.Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (2013) Google Scholar)) or endoplasmic reticulum-located STING (7Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (2219) Google Scholar) (also named ERIS (8Sun W. Li Y. Chen L. Chen H. You F. Zhou X. et al.ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 8653-8658Crossref PubMed Scopus (618) Google Scholar), MITA (9Zhong B. Yang Y. Li S. Wang Y.-Y. Li Y. Diao F. et al.The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (1098) Google Scholar), or MPYS (10Jin L. Waterman P.M. Jonscher K.R. Short C.M. Reisdorph N.A. Cambier J.C. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals.Mol. Cell Biol. 2008; 28: 5014-5026Crossref PubMed Scopus (327) Google Scholar)). The activation of MAVS or STING recruits Tank-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), thus triggering phosphorylation-dependent activation and nuclear translocation of IRF3 (11Ikushima H. Negishi H. Taniguchi T. The IRF family transcription factors at the interface of innate and adaptive immune responses.Cold Spring Harb. Symp. Quant. Biol. 2013; 78: 105-116Crossref PubMed Scopus (189) Google Scholar), resulting in the transcription of interferon β (IFNβ) and other IFN-stimulated genes (ISGs) (12Chan Y.K. Gack M.U. RIG-I-like receptor regulation in virus infection and immunity.Curr. Opin. Virol. 2015; 12: 7-14Crossref PubMed Scopus (133) Google Scholar), which establishes an antiviral state of host cells and further promotes the induction of adaptive immunity (13Yoneyama M. Onomoto K. Jogi M. Akaboshi T. Fujita T. Viral RNA detection by RIG-I-like receptors.Curr. Opin. Immunol. 2015; 32: 48-53Crossref PubMed Scopus (327) Google Scholar). During the activation of interferon, TBK1 is an essential component and lies in the center of the IRF3 signaling cascade (14Häcker H. Karin M. Regulation and function of IKK and IKK-related kinases.Sci. STKE. 2006; 2006: re13Crossref PubMed Scopus (1077) Google Scholar). The activation of TBK1 is tightly regulated via various post-translational modifications (PTMs) (15Chiang C. Gack M.U. Post-translational control of intracellular pathogen sensing pathways.Trends Immunol. 2017; 38: 39-52Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). It has been reported that multiple PTMs, including phosphorylation (16Larabi A. Devos J.M. Ng S.-L. Nanao M.H. Round A. Maniatis T. et al.Crystal structure and mechanism of activation of TANK-binding kinase 1.Cell Rep. 2013; 3: 734-746Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), ubiquitination (17Song G. Liu B. Li Z. Wu H. Wang P. Zhao K. et al.E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.Nat. Immunol. 2016; 17: 1342-1351Crossref PubMed Scopus (126) Google Scholar, 18Lin M. Zhao Z. Yang Z. Meng Q. Tan P. Xie W. et al.USP38 inhibits type I interferon signaling by Editing TBK1 ubiquitination through NLRP4 signalosome.Mol. Cell. 2016; 64: 267-281Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 19Deng M. Tam J.W. Wang L. Liang K. Li S. Zhang L. et al.TRAF3IP3 negatively regulates cytosolic RNA induced anti-viral signaling by promoting TBK1 K48 ubiquitination.Nat. Commun. 2020; 11: 2193Crossref PubMed Scopus (30) Google Scholar), and acetylation (20Li X. Zhang Q. Ding Y. Liu Y. Zhao D. Zhao K. et al.Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.Nat. Immunol. 2016; 17: 806-815Crossref PubMed Google Scholar, 21Tang J. Yang Q. Xu C. Zhao H. Liu Y. Liu C. et al.Histone deacetylase 3 promotes innate antiviral immunity through deacetylation of TBK1.Protein Cell. 2021; 12: 261-278Crossref PubMed Scopus (17) Google Scholar), positively or negatively regulate TBK1 activity. Most recently, arginine methylation of TBK1 was also reported to promote TBK1 activation, further expanding our knowledge of TBK1 regulation (22Yan Z. Wu H. Liu H. Zhao G. Zhang H. Zhuang W. et al.The protein arginine methyltransferase PRMT1 promotes TBK1 activation through asymmetric arginine methylation.Cell Rep. 2021; 36109731Abstract Full Text Full Text PDF Scopus (16) Google Scholar). However, it remains unknown whether other non-canonical PTMs, such as lysine methylation, are involved in regulating TBK1 activity. Therefore, it would be interesting to explore the role of lysine methylation in regulating interferon signaling. KRAB-zinc finger proteins constitute the largest family of tetrapod-specific transcription factors (23Ecco G. Imbeault M. Trono D. KRAB zinc finger proteins.Development. 2017; 144: 2719-2729Crossref PubMed Scopus (193) Google Scholar). At present, studies on KRAB-zinc finger protein family members are mainly focused on their functions as transcription factors in the fields of biological evolution, embryonic development, and cancer (24Yang P. Wang Y. Macfarlan T.S. The role of KRAB-ZFPs in transposable element repression and Mammalian evolution.Trends Genet. 2017; 33: 871-881Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 25Lupo A. Cesaro E. Montano G. Zurlo D. Izzo P. Costanzo P. KRAB-zinc finger proteins: a repressor family displaying multiple biological functions.Curr. Genomics. 2013; 14: 268-278Crossref PubMed Scopus (171) Google Scholar), and the total number of studies is minimal compared to the large gene number (23Ecco G. Imbeault M. Trono D. KRAB zinc finger proteins.Development. 2017; 144: 2719-2729Crossref PubMed Scopus (193) Google Scholar, 25Lupo A. Cesaro E. Montano G. Zurlo D. Izzo P. Costanzo P. KRAB-zinc finger proteins: a repressor family displaying multiple biological functions.Curr. Genomics. 2013; 14: 268-278Crossref PubMed Scopus (171) Google Scholar). In other areas of biology, little is known about whether KRAB-zinc finger proteins have functions other than acting as transcription factors. Species-specific regulation has brought increasing attention to understanding human innate immunity (14Häcker H. Karin M. Regulation and function of IKK and IKK-related kinases.Sci. STKE. 2006; 2006: re13Crossref PubMed Scopus (1077) Google Scholar, 26Crowl J.T. Gray E.E. Pestal K. Volkman H.E. Stetson D.B. Intracellular nucleic acid detection in autoimmunity.Annu. Rev. Immunol. 2017; 35: 313-336Crossref PubMed Scopus (144) Google Scholar). Traditionally, the mouse is widely used as a model animal in investigating innate immune systems. A large number of milestone discoveries are found using mouse models (27Motwani M. Pesiridis S. Fitzgerald K.A. DNA sensing by the cGAS-STING pathway in health and disease.Nat. Rev. Genet. 2019; 20: 657-674Crossref PubMed Scopus (650) Google Scholar). Despite the importance of mouse models, growing evidence has shown that the human innate immune system differs in many ways from the mouse system (28Burleigh K. Maltbaek J.H. Cambier S. Green R. Gale M. James R.C. et al.Human DNA-PK activates a STING-independent DNA sensing pathway.Sci. Immunol. 2020; 5eaba4219Crossref PubMed Scopus (101) Google Scholar, 29Jin S. Tian S. Luo M. Xie W. Liu T. Duan T. et al.Tetherin suppresses type I interferon signaling by targeting MAVS for NDP52-mediated selective autophagic degradation in human cells.Mol. Cell. 2017; 68: 308-322.e4Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). For example, Burleigh et al. (28Burleigh K. Maltbaek J.H. Cambier S. Green R. Gale M. James R.C. et al.Human DNA-PK activates a STING-independent DNA sensing pathway.Sci. Immunol. 2020; 5eaba4219Crossref PubMed Scopus (101) Google Scholar) reported that though both human and mouse primary fibroblasts and primary MEF cell lines could respond to DNA transfection characterized by STING degradation, HSPA8 phosphorylation could only be activated in the human fibroblasts. In addition, Jin and colleagues showed that type Ⅰ interferon production was elevated in human Tetherin KO cells but repressed in plasmacytoid dendritic cells in Tetherin KO mice (29Jin S. Tian S. Luo M. Xie W. Liu T. Duan T. et al.Tetherin suppresses type I interferon signaling by targeting MAVS for NDP52-mediated selective autophagic degradation in human cells.Mol. Cell. 2017; 68: 308-322.e4Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). ZNF268a is primarily a nucleus-resident protein and is long considered to be a transcriptional regulator (30Sun Y. Gou D. Liu H. Peng X. Li W. The KRAB domain of zinc finger gene ZNF268: a potential transcriptional repressor.IUBMB Life. 2003; 55: 127-131Crossref PubMed Scopus (14) Google Scholar). In this study, we report a novel role of ZNF268a in facilitating antiviral innate immune signaling in the cytoplasm. We find that ZNF268a can specifically target the critical kinase TBK1 and help maintain the interaction between TBK1 and MAVS/STING to facilitate the activation of IRF3. Mechanistically, cytoplasmic ZNF268a can be degraded by the Tollip-mediated selective autophagy system in the absence of viral challenge. However, when viral infection occurs, TBK1 interacts with cytosolic ZNF268a and directly phosphorylates Serine 178 of ZNF268a, which could promote the protein stability of ZNF268a and significantly increase the expression level of ZNF268a in the cytoplasm. With the stabilization of ZNF268a, it can recruit lysine methyltransferase SETD4 to TBK1, which directly induces the mono-methylation of lysine 607 in TBK1, thus maximizing TBK1 activation. Unexpectedly, we demonstrate that ZNF268a Serine 178 and TBK1 lysine 607 are conserved residues across primates but are absent or highly variable in rodents, likely suggesting a species-specific regulation of innate immunity in human beings. Previously, we demonstrated a positive role of ZNF268a in regulating virus-induced proinflammatory cytokines (31Liu Y. Yin W. Wang J. Lei Y. Sun G. Li W. et al.KRAB-zinc finger protein ZNF268a deficiency attenuates the virus-induced pro-inflammatory response by preventing IKK complex assembly.Cells. 2019; 8: 1604https://doi.org/10.3390/cells8121604Crossref PubMed Scopus (7) Google Scholar). To further investigate whether ZNF268a is essential in facilitating effective host interferon response against viral infection, we first used our previously established ZNF268a KO HEK293T cell line (31Liu Y. Yin W. Wang J. Lei Y. Sun G. Li W. et al.KRAB-zinc finger protein ZNF268a deficiency attenuates the virus-induced pro-inflammatory response by preventing IKK complex assembly.Cells. 2019; 8: 1604https://doi.org/10.3390/cells8121604Crossref PubMed Scopus (7) Google Scholar) to test the effect of ZNF268a on interferon response induced by poly(I:C), which is a synthetic analog of double-stranded RNA (dsRNA) and commonly used as a molecular pattern associated with viral infections-induced interferon transcription. Lack of ZNF268a attenuated IFN-β transcription 6 h post poly(I:C) transfection (Fig. 1A). Next, we stimulated the cells with SeV for 12 h and measured the transcripts of IFN-β and ISGs of the cultured cells by quantitative real-time PCR. Knock-out of ZNF268a largely abrogated IFN-β, ISG54, ISG56, CXCL10, and CCL5 transcription induced by SeV in HEK293T cells during the time points indicated in Figure 1B. In addition, we tested other ISGs including IFITM1, ISG20, IFI27, RARRES3, OASL, MX1, IFI16, HERC5, DDX60, and UBE2L6, among which the expression of MX1, IFI16, HERC5, DDX60 and UBE2L6 is not affected by ZNF268a deficiency (Fig. S1). Furthermore, down-regulation of ZNF268a in PMA-induced THP-1 cells caused up to 75% lower expression of IFN-β, ISG54, ISG56, and CXCL10 in response to DNA virus HSV-1 infection (Fig. 1C). Consistent with the qPCR data, ELISA results showed that ZNF268a-deficient HEK293T cells produced significantly less IFN-β in response to SeV infection (Fig. 1D). Accordingly, SeV genome copies were much higher in ZNF268a KO HEK293T than in WT cells (Fig. 1E). In addition, the deletion of ZNF268a impaired HEK293T cells’ resistance to GFP-VSV infection, as shown by stronger GFP intensity in ZNF268a KO cells by microscopy (Fig. 1F). Collectively, these data indicated a positive role for ZNF268a in regulating the RNA/DNA viral infection-induced interferon response. To better understand how ZNF268a regulates the innate immune responses to viral infection, we compared the cell response to SeV infection in WT or ZNF268a KO HEK293T cells by performing IFN-β/ISRE promoter dual luciferase assay. As shown in Figure 2, A and B, ZNF268a deficiency significantly reduced the activation of both promoters, indicating that the deletion of ZNF268a impaired the virus-induced interferon signaling pathway. On the other hand, we observed decreased activation of IRF3 upon SeV infection in ZNF268a KO HEK293T cells, as revealed by native PAGE that IRF3 dimerization was decreased in ZNF268a KO HEK293T cells (Fig. 2C), which corresponded to the attenuated level of phospho-S396 IRF3 when ZNF268a was depleted (Fig. 2D). Following viral infection, IRF3 nuclear translocation was also repressed in ZNF268a deficient HEK293T cells (Fig. 2E). In line with the imaging data, the biochemical fractionation assay also indicated that the level of nuclear IRF3 accumulated stronger and faster in ZNF268a wild-type cells than in ZNF268a knockout cells (Fig. 2F). However, notably, we did not observe a concurrent decrease in TBK1 phosphorylation of S172 (Fig. 2D), suggesting that the phosphorylation-dependent activation of TBK1 was largely unaffected by ZNF268a depletion. Furthermore, re-introduction of the full-length construct of ZNF268a partially restored IFN-β promoter activation upon SeV infection (Fig. 2G). Poor expression level could be the cause for the partial restore (Fig. S2), as we noticed similar examples in other KRAB-ZNF proteins such as ZFP809, which was reported to express poorly in differentiated HEK293A but regularly in undifferentiated embryonic stem cells (32Wolf D. Goff S.P. Embryonic stem cells use ZFP809 to silence retroviral DNAs.Nature. 2009; 458: 1201-1204Crossref PubMed Scopus (282) Google Scholar, 33Wang C. Goff S.P. Differential control of retrovirus silencing in embryonic cells by proteasomal regulation of the ZFP809 retroviral repressor.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E922-E930PubMed Google Scholar). In general, the result demonstrated that the impaired IRF3 signaling activation was due to the specific loss of ZNF268a. To further identify which component of the interferon signaling pathway is targeted by ZNF268a, we overexpressed RIG-I, cGAS + STING, MAVS, TBK1, IRF3 WT or its constitutively active form IRF3 5D constructs individually, in HEK293T cells. We found that ZNF268a deficiency inhibited RIG-I, cGAS + STING, MAVS, and TBK1 overexpression-induced activation of IFN-β promoters (Fig. 3A) but had little effect on IRF3 WT or IRF3 5D-induced activation of ISRE promoter (Fig. 3B), suggesting that ZNF268a could potentially target TBK1 to affect interferon signaling. To confirm the role of ZNF268a at the level of TBK1, we examined the interaction of Flag-tagged ZNF268a with HA-tagged TBK1. Immunoprecipitation assay using either Flag (Fig. 3C) or HA (Fig. 3D) antibody demonstrated that Flag-ZNF268a was associated with HA-TBK1. Due to the lack of suitable ZNF268a antibody for immunoprecipitation, we exogenously expressed Flag-tagged ZNF268a in cells and used Flag antibody to perform immunoprecipitation and immunoblotted for endogenous TBK1. The result showed that Flag-ZNF268a was indeed able to bind endogenous TBK1 (Fig. 3E), further supporting the association of ZNF268a and TBK1. Since ZNF268a can associate with TBK1 without affecting TBK1 phosphorylation, we speculated that the attenuated interferon responses observed in ZNF268a KO HEK293T cells could be due to impaired assembly of the TBK1 signaling complex or decreased Lys63-linked ubiquitination since it is reported to be critical for TBK1 activation (17Song G. Liu B. Li Z. Wu H. Wang P. Zhao K. et al.E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1.Nat. Immunol. 2016; 17: 1342-1351Crossref PubMed Scopus (126) Google Scholar, 34Zhang Q. Meng F. Chen S. Plouffe S.W. Wu S. Liu S. et al.Hippo signalling governs cytosolic nucleic acid sensing through YAP/TAZ-mediated TBK1 blockade.Nat. Cell Biol. 2017; 19: 362-374Crossref PubMed Scopus (141) Google Scholar). Detection of TBK1 Lys63-linked ubiquitination in ZNF268a WT and KO HEK293T cells with or without viral infection showed no significant difference, excluding the possibility of ZNF268a in regulating TBK1 activation via K63 ubiquitination (Fig. 3F). In contrast, co-immunoprecipitation assays showed a robustly diminished association between TBK1 and MAVS, STING or IRF3 in ZNF268a KO HEK293T cells (Fig. 3, G and H). Thus, we concluded that TBK1 is the target of ZNF268a in regulating virus-induced activation of IRF3 signaling. As shown in Figure 1B, the transcriptional level of ZNF268a remains unaltered during viral infection. We further evaluated if its protein level changed during viral infection. We transfected Flag-tagged ZNF268a into HEK293T cells and challenged the cells with SeV. We found a drastically elevated protein expression of cytoplasmic ZNF268a in the infected cells (Fig. 4A). Similar results were repeatedly obtained in VSV-infected HEK293T cells (Fig. 4B). We also measured the degradation rate of cytoplasmic ZNF268a using the CHX chase assay. The result showed that viral infection substantially extended the half-life of cytoplasmic ZNF268a (Fig. 4C). The fact that TBK1 physically interacts with ZNF268a leads us to hypothesize that TBK1-ZNF268a interaction could be critical for the virus-induced stabilization of ZNF268a. To examine this hypothesis, we analyzed the ZNF268a protein level in HEK293T cells with a deficiency in TBK1. Deletion of TBK1 blocked virus-induced cytoplasmic ZNF268a protein elevation (Fig. 4D). The cytoplasmic upregulation of ZNF268a raised the possibility that exposure to the viral nucleic acid can release ZNF268a from its constant degradation by cell quality control systems, which mainly consist of the ubiquitin-proteasome system and the autolysosome system. To further determine the mechanism by which the ZNF268a protein level is increased after infection, we treated cells with proteasome or lysosome inhibitors. As the result in Figure 4E showed, lysosome degradation inhibition by chloroquine, Baflomycin A1, or NH4Cl restored the ZNF268a protein expression, whereas proteasome degradation inhibition by MG132 did not. Colocalization of ZNF268a with lysosome marker LAMP1 further confirmed cytoplasmic ZNF268a is constantly targeted for degradation in lysosome (Fig. 4F). Moreover, we found that ZNF268a could specifically associated with selective autophagic receptor Tollip (Fig. 4G), suggesting a tight control of the protein level of ZNF268a in resting cells and potentially explaining why transient transfected ZNF268a construct expresses poorly in cells. Together, these data show that viral infection can prevent lysosomal degradation of ZNF268a in a TBK1-dependent manner. In order to further understand how TBK1 mediates ZNF268a protein stabilization, we restored TBK1 WT expression in TBK1 KO HEK293T cells and found an accumulation of cytoplasmic ZNF268a, confirming the critical role of TBK1 in stabilizing cytoplasmic ZNF268a (Fig. 5A). Since TBK1 is a well-known serine/threonine kinase, we speculated that the interaction of TBK1 and ZNF268a could lead to the phosphorylation of ZNF268a, further resulting in its stabilization. Bioinformatic analysis predicted three potential phospho-sites located in the N-terminus of ZNF268a, including S119, S237, and S178 (Fig. 5B upper, sites in green). We next performed mass spectrometry analysis of Flag-tagged ZNF268a in the presence or absence of viral infection (Fig. 5B lower). The analysis identified Ser9, Ser33, and Ser178 as specific phosphorylation sites (Fig. 5B upper, sites in red). In vitro kinase assay using recombinant TBK1 and the N-terminus of ZNF268a (in which the whole KRAB domain and surrounded sequences were included while all ZnF motifs were excluded, so we termed this truncated protein as GST-KRAB for convenience) showed that increasing amount of TBK1 triggered stronger smear bands of ZNF268a N-terminus (Fig. 5C), which was not detected upon incubation of the reaction products with λ-phosphatase (Fig. 5D), indicating TBK1 was able to phosphorylate ZNF268a at its N-terminus. As both the bioinformatic prediction and the MS result identified Ser178 phosphorylation of ZNF268a (Fig. 5B upper), we focused on this amino acid in our following experiments. To confirm that TBK1 phosphorylates ZNF268a at Ser178, we generated three independent antibodies recognizing Ser178-phosphorylated ZNF268a. Immunoblot analysis with these antibodies showed that TBK1 efficiently phosphorylated ZNF268a N-terminus WT at Ser178 in vitro, whereas phosphorylation of ZNF268a N-terminus S178A was not detected (Fig. 5E). Notably, in HEK293T cells, VSV infection induced Ser178 phosphorylation of immunoprecipitated Flag-ZNF268a (Fig. 5F), whereas loss of TBK1 abrogated this effect (Fig. 5G). To confirm the importance of ZNF268a Ser178 phosphorylation in stabilizing ZNF268a, we performed a CHX chase assay to determine the degradation rate of ZNF268 WT and S178D phospho-mimetic mutant. As expected, ZNF268a S178D degraded slower than its WT counterpart (Fig. 5H). Of note, when ZNF268a Serine 178 was mutated to aspartic acid, the interaction of ZNF268a and Tollip was almost abolished (Fig. 5I), implying that ZNF268a S178 phosphorylation-mediated stabilization was likely via diminishing the interaction with Tollip, thus preventing the targeted degradation. To further investigate the effect of ZNF268a Ser178 phosphorylation on antiviral innate immunity, we compared IRF3 dimerization in ZNF268a WT, S178D, and S178A overexpressed ZNF268a KO HEK293T cells. SeV infection could induce IRF3 dimerization in WT and S178D rescued cells, but this dimerization was compromised in S178A-expressed cells (Fig. S5A). In line with the effects of IRF3 dimerization, IFN-β transcription was restored in WT ZNF268a transfected cells, but this restoration was utterly lost in S178A expressing cells (Fig. S5B). These data suggested that ZNF268a S178 phosphorylation plays an essential role in the antiviral interferon signaling transduction. To systemically elucidate how ZNF268a regulates the TBK1 signaling complex, we performed mass spectrometry to identify potential ZNF268a interacting partners during viral infection. As depicted in Fig. S4A, we subjected Flag-tagged ZNF268a overexpressed cytoplasmic fraction but not whole cell lysate to immunoprecipitation to avoid potential interference of nuclear binding partners of ZNF268a, followed by MS identification of ZNF268a interacting proteins. Via this approach, we identified 422 ZNF268a interacting proteins when cells were infected by SeV (Fig. S4B). Among these protein candidates, we chose 90 to further verify their potential roles in antiviral innate immunity based on our interest in post-translational modifications. We tested these proteins by knocking down their expression via a pool of three individual siRNAs per target in a luciferase reporter assay (Fig. S4C). Notably, downregulation of SETD4, the only identified methyltransferase, attenuated the activation of IFN-β reporter following SeV infection (Fig. S4C, the lowest panel). Then, we constructed HA-tagged SETD4 and performed a Co-IP experiment to further verify its interaction with ZNF268a. Our result suggested that overexpressed SETD4 was sufficient to associate with ZNF268a even at resting state (Fig. 6A). Immunofluorescence analysis also confirmed the co-localization of exogenously expressed ZNF268a and SETD4 in the cytoplasm (Fig. 6B). Importantly, we detected clear interaction between SETD4 and TBK1 via Co-IP both in the uninfected and the SeV-infected HEK293T cells (Fig. 6C). IF assay also supported the conclusion that SETD4 bound TBK1 by demonstrating that they co-localized in cells (Fig. 6D). Furthermore, we also found SETD4 interacted with TBK1 in a ZNF268a-dependent manner, as ZNF268a depletion strongly impaired this interaction (Fig. 6E). Interestingly, analysis of SETD4 protein complex by sucrose gradient ultracentrifugation showed that the Flag-tagged SETD4 in the lysate of non-infected cells, mainly sediments as either a higher-order complex or a lower-order complex (Fig. 6F). Virus infection strongly shifted SETD4 from the lightest or heaviest fractions to the middle-molecular weight fractions, also in a ZNF268a-dependent manner (Fig. 6F). These results suggested that the SETD4’s behaviors, including TBK1 association and incorporation into higher order molecular complex, were critically dependent on ZNF268a. Together, we concluded that ZNF268a mediates the recruitment of SETD4 to TBK1 and associated complexes upon viral infection. To further examine the function of SETD4 in antiviral innate immunity, we knocked down SETD4 via siRNAs in HEK293T cells (Fig. 7A) and measured ISRE reporter activation by viral infection. siRNA-mediated knockdown of SETD4 resulted in drastically decreased activation of the ISRE promoter (Fig. 7B). In ZNF268a WT HEK293T cells, overexpression of SETD