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. 2025 Nov 26;5(1):100294. doi: 10.1016/j.cellin.2025.100294

HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

Jia-Qing Zeng 1, Zi-Lun Ruan 1, Qi Zhang 1, Xue-Mei Yi 1, Yun-Da Chen 1, Ming-Ming Hu 1, Shu Li 1,⁎
PMCID: PMC12744288  PMID: 41466838

Abstract

Upon infection, viral DNA/RNA is detected by cGAS/RIG-I-like receptors, triggering the adaptor MITA/STING- or VISA/MAVS-dependent innate antiviral immune response respectively. Both adaptors recruit the conserved TRAF3 and TRAF6 to activate TBK1-IRF3 and TAK1-NF-κB pathways respectively, leading to collaborative induction of antiviral effector genes. How the functions of TRAF3 and TRAF6 bifurcate in innate antiviral signaling remains enigmatic. We identified HTATSF1 as a positive regulator of virus-triggered innate antiviral response. Upon viral infection, HTATSF1 promotes HECTD3-catalyzed K63-linked polyubiquitination of TRAF3, leading to its recruitment of TBK1 and activation of IRF3. In contrast, HTATSF1 promotes recruitment of TAK1 to TRAF6 and activation of the TAK1-IKK-NF-κB axis independently of HECTD3. HTATSF1-deficiency impairs induction of downstream antiviral genes, and HTATSF1-deficient mice exhibit decreased cytokine production and increased mortality upon viral infection. Our findings demonstrate that HTATSF1 is an essential regulator of innate antiviral immune response by orchestrating the TRAF3-IRF3 and TRAF6-NF-κB pathways.

Keywords: TRAF3, TRAF6, HTATSF1, HECTD3, Polyubiquitination, Innate antiviral response

Graphical abstract

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Highlights

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    HTATSF1 is identified as a positive regulator of innate antiviral response.

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    HTATSF1 promotes TRAF3 polyubiquitination by HECTD3 and activation of TBK1-IRF3 axis.

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    HTATSF1 promotes IKK-NF-κB activation by linking TAK1 to TRAF6 independent of HECTD3.

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    HTATSF1-deficiency impairs innate antiviral response in mice.

1. Introduction

Innate immunity serves as the primary defense barrier against microbial pathogens. Upon pathogen invasion, pattern recognition receptors (PRRs) detect structurally conserved components of pathogens called pathogen-associated molecular patterns (PAMPs) to initiate innate immune response. For example, the cyclic GMP-AMP synthase cGAS senses viral and other cytosolic DNA, and the RIG-I-like receptors (RLRs) RIG-I and MDA5 recognize cytosolic viral RNA upon RNA virus infection (Crowl et al., 2017; Hu & Shu, 2018; Liu & Xu, 2025; Takeuchi & Akira, 2010). Upon sensing of viral and other cytosolic DNA, cGAS triggers the synthesis of the second messenger cGAMP, which binds to the ER membrane-associated adaptor protein MITA (also known as STING) (Gao et al., 2025; Ishikawa et al., 2009; Zhang & Zhong, 2022; Zhong et al., 2008). Similarly, sensing of viral RNA by RIG-I/MDA5 leads to their recruitment to the mitochondrial-associated adaptor protein VISA (also named MAVS, IPS-1, Cardif) (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). Subsequently, both MITA and VISA recruit tumor necrosis factor receptor-associated factor (TRAF) family proteins, including TRAF3 and TRAF6, which are differentially involved in regulating downstream signaling events. It has been demonstrated that TRAF3 plays an essential role in viral nucleic acid-triggered induction of type I interferons and interferon-stimulated genes (ISGs) by mediating activation of the TBK1-IRF3 axis (Häcker et al., 2006; Oganesyan et al., 2006; Zheng et al., 2023; Zhu et al., 2020), whereas TRAF6 is critical for activating the TAK1-IKK-NF-κB axis and induction of NF-κB-dependent inflammatory genes (Sun et al., 2004; Tang et al., 2018; Thomas, 2005). Coordinated induction of type I interferons, proflammatory cytokines and other effector genes ultimately results in innate immune and inflammatory response as well as promoting the later adaptive immune response against the invaded viruses (Chen et al., 2014; Wang et al., 2022, 2024; Zheng et al., 2023).

TRAF3 and TRAF6 play pivotal yet distinct functions in innate immune signaling and their regulations are critically important for fine-tuning innate antiviral response. It has been demonstrated that distinct linkage types of polyubiquitination modifications of TRAF3 selectively activate the transcription of type I interferons and proinflammatory cytokines (He et al., 2007; Oganesyan et al., 2006; Saha et al., 2006; Tseng et al., 2010). Specifically, K48-linked polyubiquitination promotes TRAF3 degradation via the proteasome pathway, thereby activating the non-canonical NF-κB signaling pathway via NIK-IKKα axis to enhance downstream inflammatory cytokine production (Matsuzawa et al., 2008; Vallabhapurapu et al., 2008). In contrast, K63-linked polyubiquitination facilitates the recruitment of TBK1, amplifying induction of type I interferons and other ISGs (Sharma et al., 2003). E3 ubiquitin ligases, including cIAP1/2, Peli1, and TRIAD3A, catalyze K48-linked polyubiquitination of TRAF3, triggering its proteasomal degradation (Nakhaei et al., 2009; Tseng et al., 2010; Xiao et al., 2013). In contrast, HECTD3 and TRIM24 mediate K63-linked polyubiquitination of TRAF3, thereby enhancing induction of type I interferons (Li et al., 2018; Zhu et al., 2020). On the other hand, K63-linked polyubiquitination of TRAF6 initiates the canonical NF-κB signaling pathway by recruiting and activating the TAK1 kinase complex (Häcker et al., 2006; Karin & Ben-Neriah, 2000). This activation subsequently induces phosphorylation of the IKK complex, leading to phosphorylation and proteasomal degradation of IκBα. Consequently, NF-κB is released and translocated to the nucleus, driving transcription of proinflammatory cytokine genes. Thus, TRAF3 and TRAF6 act as crucial adaptors that integrate signals from upstream PRRs and transduce these signals to distinct downstream kinases and transcription factors. However, the mechanisms of fine-tuned regulation of TRAF3 and TRAF6, particularly how the conserved TRAF3 and TRAF6 are differentially regulated for activating distinct downstream pathways in innate antiviral response remains enigmatic.

In this study, we performed genomic-wide screens for cellular factors that regulate viral replication and innate antiviral response. These screens identified the HIV Tat-specific factor 1 (HTATSF1) as a positive regulator of virus-triggered induction of downstream antiviral genes. HTATSF1-deficiency markedly attenuates transcription of type I interferon and proinflammatory cytokine genes triggered by the DNA virus herpes simplex virus 1 (HSV-1) and the RNA virus Sendai virus (SeV). HTATSF1-deficient mice exhibits decreased cytokine production and heightened susceptibility to lethal viral infection. Biochemical analysis reveals that HTATSF1 acts as a scaffold to link the E3 ligase HECTD3 to TRAF3 upon viral infection, resulting in K63-linked polyubiquitination of TRAF3 by HECTD3 and its recruitment of TBK1, leading to activation of the transcription factor IRF3. Additionally, HTATSF1 functions as a scaffold to link TAK1 to TRAF6 independent of HECTD3, which promotes activation of the TAK1-IKK-NF-κB axis. Our findings suggest that HTATSF1 plays a crucial role in innate antiviral signaling through orchestrating TRAF3-TBK1-IRF3 and TRAF6-TAK1-IKK-NF-κB pathways.

2. Results

2.1. HTATSF1 positively regulates virus-induced transcription of ISGs

Viral replication triggers induction of downstream antiviral genes and innate antiviral response, whereas innate antiviral response inhibits viral replication. We hypothesize that knockout of regulators of innate antiviral response would subsequently affect viral replication. To identify cellular factors involved in innate antiviral response, we designed a strategy in which genome-wide CRISPR-knockout is coupled with single-cell transcriptomics, therefore enabling us to analyze both viral nucleic acid abundance and levels of ISGs at single-cell resolution upon viral infection. We established genomic-wide CRISPR knockout library in human monocytic THP-1 cells, infected these cells with the DNA virus HSV-1 and performed single-cell transcriptomics analysis (Fig. S1). We performed a joint ranking of candidates based on enrichment ratio (descending) and adjusted p-value (ascending) in relation to HSV-1 viral loads, which enabled the identification of the top 50 candidate genes potentially regulating HSV-1 infectivity and/or innate antiviral response (Fig. 1(A), S2). To determine whether these candidate genes are involved in innate antiviral response, we individually edited these genes by the CRISPR-cas9 method in THP-1 cells and infected them with HSV-1 for 6 h. RT-qPCR analysis indicated that knockout of the pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) PRPF4B (ranked 7), vacuolar protein sorting 25 (VPS25) (ranked 11), and HTATSF1 (ranked 27) suppressed HSV-1-induced transcription of downstream IFNB1 and ISG56 genes to below 50% compared to the control group (Fig. 1(B)). VPS22, a homolog of VPS25, has been reported to promote the binding of IRF3 to the type I interferon promoter in the nucleus (Kumthip et al., 2017). In this study, we have focused on the characterization of HTATSF1 in innate antiviral response.

Fig. 1.

Fig. 1

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Identification of HTATSF1 as a positive regulator of innate antiviral signaling.(A) Enrichment of candidate genes in high-viral-load cells. Candidate genes were ranked by enrichment score. Each dot represents one gene, with black indicating significantly enriched genes and gray indicating non-enriched genes. HTATSF1 (ranked 27) is indicated. (B) Effects of knockout of the candidate genes on HSV-1-induced transcription of IFNB1 and ISG56 genes. The candidate genes in THP-1 cells were edited by the CRISPR-cas9 method with a combination of two independent sgRNAs for each gene. The cells (1 × 106) were infected with HSV-1 (MOI = 1) for 6 h, followed by RT-qPCR analysis to quantify the mRNA levels of IFNB1 and ISG56 genes. The x-axis represents edited candidate genes, while the y-axis indicates fold changes in IFNB1 or ISG56 mRNA level relative to that in control-edited cells. PRPF4B, VPS25 and HTATSF1 are indicated. (C) Effects of HTATSF1-deficiency on the transcription of downstream genes induced by HSV-1- or SeV in THP-1 cells. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for 3 or 6 h before RT-qPCR analysis to measure mRNA levels of the indicated genes. Deficiency of HTATSF1 in the THP-1 cells was confirmed by immunoblotting analysis with anti-HTATSF1. (D) Effects of HTATSF1-deficiency on the transcription of downstream genes induced by HSV-1- or SeV in BMDMs. Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl BMDM cells (1 × 106) were left uninfected or infected HSV-1 (MOI = 1) or SeV (MOI = 1) for 3 or 6 h before RT-qPCR analysis to measure mRNA levels of the indicated genes. Deficiency of HTATSF1 in the BMDMs was confirmed by immunoblotting analysis with anti-HTATSF1. For ((C) & (D)), data shown are mean ​± ​SEM (n ​= ​3 technical replicates) from one representative experiment, which were repeated for at least two times with similar results. ∗∗, P < 0.01 (unpaired t-test).

To determine whether HTATSF1 is general regulator of innate immune response to both DNA and RNA virus, we investigated the effects of knockout of HTATSF1 on the RNA virus SeV-triggered induction of downstream antiviral genes. RT-qPCR experiments indicated that HTATSF1-deficiency markedly inhibited both HSV-1 and SeV-triggered induction of downstream antiviral genes including IFNB1, ISG56, and IL6 (Fig. 1(C)), suggesting that HTATSF1 regulates both DNA and RNA virus-triggered innate immune response.

2.2. HTATSF1-deficiency impairs virus-triggered innate immune response in mice

We next determined whether HTATSF1 regulates innate antiviral response in mice. It has been previously shown that conventional knockout of Htatsf1 gene disrupts early embryonic development in mice (Corsini et al., 2018). Therefore, we employed flox mice carrying loxP sites flanking exons 3–6 for conditional gene targeting (Fig. S3A and B). The lysozyme 2 (Lyz2) promoter drives high and specific expression in myeloid cells. Mice carrying the Lyz2-Cre transgene express Cre recombinase that specifically recognizes and excises genomic sequences flanked by loxP sites in myeloid cells. By crossing Htatsf1fl/fl mice with Lyz2-Cre mice, we generated myeloid-specific conditional knockout mice (Lyz2-Cre;Htatsf1fl/fl). RT-qPCR analysis indicated that bone marrow-derived macrophages (BMDMs) isolated from Lyz2-Cre;Htatsf1fl/fl mice exhibited markedly decreased transcription of downstream antiviral genes including Ifnb1, Isg56, and Il6 upon HSV-1 or SeV stimulation compared to control cells (Fig. 1(D)). These results suggest that HTATSF1 is involved in DNA and RNA virus-triggered induction of downstream antiviral genes in mouse BMDMs.

We next determined the importance of HTATSF1 in host defense against viral infection in mice. Following inoculation with HSV-1, SeV, or the RNA virus vesicular simplex virus (VSV), the production of serum cytokines including IFN-β, CXCL10, and IL-6 was significantly decreased in Lyz2-Cre;Htatsf1fl/fl mice compared with Htatsf1fl/fl mice (Fig. 2(A)). Given the distinct tissue tropism of these viruses, we analyzed viral loads in HSV-1-infected brains, SeV-infected spleens and VSV-infected lungs in the challenged mice. The genomic copy numbers of HSV-1 in the brains, SeV genomic copy numbers in the spleens and VSV genomic copy numbers in the lungs were markedly increased in Lyz2-Cre;Htatsf1fl/fl mice in comparison to Htatsf1fl/fl mice (Fig. 2(B)). In addition, Lyz2-Cre;Htatsf1fl/fl mice exhibited significantly higher mortality than Htatsf1fl/fl mice following infection with either HSV-1 or VSV (Fig. 2(C)). These results demonstrate that HTATSF1 plays an essential role in innate immune response to both DNA or RNA viruses in mice.

Fig. 2.

Fig. 2

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HTATSF1 is essential for host defense against DNA and RNA viruses in mice.(A) Measurement of virus-induced serum levels of IFN-β, CXCL10, and IL-6 in Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl mice. The mice (n = 6 for each group) were infected with HSV-1 (1 × 107 PFU), SeV (5 × 107 PFU) or VSV (1 × 108 PFU) for 6 h before ELISA measurement of the indicated cytokines. (B) Effects of HTATSF1-deficiency on viral genomic copy numbers. Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl mice (n = 6 for each group) were infected with HSV-1 (1 × 107 PFU), SeV (5 × 107 PFU) or VSV (1 × 108 PFU) for 2 days. Viral genomic copy numbers in the brains of HSV-1-infected mice were quantified by qPCR. Viral genomic copy numbers in the spleens of SeV-infected mice or lungs of VSV-infected mice were quantified by RT-qPCR respectively. (C) Effects of HTATSF1-deficiency virus-induced death. Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl mice were intraperitoneally injected with HSV-1 at 5 × 107 PFU per mouse (n = 7 for each group) or VSV at 5 × 108 PFU per mouse (n = 7 for each group), and the survival of mice was recorded daily for 12 days.

2.3. HTATSF1 mediates viral nucleic acid- but not IFN-β-triggered signaling

We next investigated how HTATSF1 regulates innate immune response to both DNA and RNA viruses. RT-qPCR analysis indicated that HTATSF1-deficiency dramatically inhibited transcription of downstream IFNB1, ISG56 and IL6 genes induced by transfected HT-DNA (dsDNA analog) and poly(I:C) (dsRNA analog) (Fig. 3(A)), suggesting that HTATSF1 regulates both cGAS- and RIG-I/MDA5-triggered signaling. Further experiments indicated that HTATSF1-deficiency also impaired cGAMP-triggered transcription of IFNB1, ISG56 and IL6 genes (Fig. 3(B)), suggesting that HTATSF1 mediates signaling downstream of cGAMP in the cGAS-MITA/STING axis. In contrast, HTATSF1-deficiency in THP-1 cells did not affect IFN-β-induced phosphorylation of the downstream transcription factor STAT1 (Fig. 3(C)) and transcription of ISGs including ISG56, CXCL10 and RIG-I (Fig. 3(D)). These results suggest that HTATSF1 regulates viral nucleic acid- but not type I interferon-triggered signaling events.

Fig. 3.

Fig. 3

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HTATSF1 mediates viral nucleic acid- but not IFN-β-triggered signaling.(A) Effects of HTATSF1-deficiency on transcription of downstream genes induced by viral nucleic acid mimics. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were untransfected or transfected with HT-DNA (2 μg/mL) or poly(I:C) (2 μg/mL) for the indicated times before RT-qPCR analysis to measure mRNA levels of the indicated genes. (B) Effects of HTATSF1-deficiency on transcription of downstream genes induced by cGAMP. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left untreated or treated with 2′3′-cGAMP (40 nM) for the indicated times before RT-qPCR analysis to measure mRNA levels of the indicated genes. (C) Effects of HTATSF1-deficiency on IFN-β-induced phosphorylation of STAT1. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left untreated or treated with IFN-β (100 ng/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. (D) Effects of HTATSF1-deficiency on transcription of downstream genes induced by IFN-β. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left untreated or treated with IFN-β (100 ng/mL) for 2 h before RT-qPCR analysis to measure mRNA levels of the indicated genes. Data shown are mean ± SEM (n = 3 technical replicates) from one representative experiment ((A), (B), (D)), which were repeated for at least two times with similar results. n.s., not significant; ∗∗, P < 0.01 (unpaired t-test).

2.4. HTATSF1 is associated with TRAF3 and TRAF6

Since HTATSF1 regulates both viral DNA- and RNA-triggered signaling, we investigated whether HTATSF1 interacts with components in these pathways. Transient transfection and co-immunoprecipitation (co-IP) assays indicated that HTATSF1 was associated with TRAF3 and TRAF6, but not cGAS, MITA, RIG-I, VISA, TBK1, IRF3, TRAF2, TAK1, IKKβ or p65 (Fig. 4(A)). Endogenous co-IP assays revealed that HTATSF1 constitutively interacted with TRAF3 and TRAF6 before and after HSV-1 or SeV infection (Fig. 4(B)). These results suggest that HTATSF1 mediates virus-triggered signaling by associating with TRAF3/6.

Fig. 4.

Fig. 4

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HTATSF1 interacts with TRAF3 and TRAF6.(A) HTATSF1 is associated with TRAF3 and TRAF6. HEK293 cells (2 × 106) were transfected with the indicated plasmids before co-IP and immunoblotting analysis with the indicated antibodies. (B) Endogenous HTATSF1 is constitutively associated with TRAF3 and TRAF6. THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) for 3 or 6 h before co-IP and immunoblotting analysis with the indicated antibodies.

HTATSF1 contains an N-terminal RNA recognition motif (RRM), a middle U2AF homology motif (UHM) that recognizing short U2AF ligand motif (ULM) of target proteins, and a C-terminal acidic domain (Loerch et al., 2019). TRAF3 and TRAF6 are members of the TRAF family, which contains an N-terminal RING/ZnF (RZ) domain, a middle coiled-coil (CC) domain and a C-terminal TRAF domain that characterizes the TRAF proteins (Sun et al., 2020; Xu et al., 2019). We next performed domain mapping experiments to identify the critical structures mediating HTATSF1-TRAF3/6 interactions. Transient transfection and co-IP assays indicated that HTATSF1 interacted with the ZnF domain (aa117-273) of TRAF3 and the RZ domain (aa1-288) of TRAF6 (Fig. 5(A)), whereas the UHM domain of HTATSF1 was important for its interaction with both TRAF3 and TRAF6 (Fig. 5(B)). Consistently, reconstitution of HTATSF1 UHM domain markedly rescued HSV-1- and SeV-induced transcription of IFNB1 and ISG56 genes in HTATSF1-knockout THP-1 cells, though to a lesser degree compared to wild-type HTATSF1 (Fig. 5(C)). These results suggest that UHM of HTATSF1 and ZnF of TRAF3/6 are important for their interactions.

Fig. 5.

Fig. 5

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Domain mapping of HTATSF1 and TRAF3/6 interactions.(A) & (B) Domain mapping experiments. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before co-IP and immunoblotting analysis with the indicated antibodies. (C) HTATSF1 UHM is essential for its role in promoting innate antiviral signaling. HTATSF1-deficient THP-1 cells were reconstituted with full length HTATSF1 or HTATSF1 UHM. Control THP-1 cells (NC) cells (1 × 106) were reconstituted with an empty vector. The cells were then stimulated with HSV-1 (MOI = 1) or SeV (MOI = 1) for 6 h before RT-qPCR measurements of mRNA levels of IFNB1 and ISG56 genes. Data shown are mean ​± ​SEM (n ​= ​3 technical replicates) from one representative experiment, which were repeated for at least two times with similar results. ∗∗, P < 0.01 (unpaired t-test).

2.5. HTATSF1 promotes K63-linked polyubiquitination of TRAF3 by HECTD3

Previous studies have demonstrated that the functions of TRAF3 in innate antiviral signaling are regulated by polyubiquitination (Gao et al., 2021; Tseng et al., 2010). Overexpression of HTATSF1 markedly increased K63-linked but not the other lysine-linked polyubiquitination of TRAF3 (Fig. 6(A)), whereas overexpression of HTATSF1 had no marked effects on either K63- or K48-linked polyubiquitination of TBK1, the downstream kinase of TRAF3 (Fig. 6(B)). Conversely, HTATSF1-deficiency markedly decreased HSV-1- or SeV-induced K63-linked but not K48-lined polyubiquitination of TRAF3 in THP-1 cells (Fig. 6(C)). Mutagenesis indicated that mutation of K138 but not K154, K156, K160 or K168 to arginine impaired HTATSF1-triggered K63-linked polyubiquitination (Fig. 6(D)). We reconstituted TRAF3 or its K138R mutant in HTATSF1-deficient THP-1 cells. RT-qPCR analysis indicated that reconstitution of wild-type TRAF3, but not the TRAF3 K138R mutant, reversed the impaired transcription of IFNB1 and ISG56 resulting from HTATSF1-deficiency (Fig. S4A). These results suggest that HTATSF1 promotes K63-linked polyubiquitination of TRAF3 at K138.

Fig. 6.

Fig. 6

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HTATSF1 promotesK63-linkedpolyubiquitination of TRAF3 by HECTD3.(A) HTATSF1 promotes K63-linked polyubiquitination of TRAF3. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. WT, wild type; KR, K is mutated to R; KO, K only. (B) HTATSF1 does not regulate polyubiquitination of TBK1. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (C) HTATSF1-deficiency decreases K63- but not K48-linked polyubiquitination of TRAF3. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) or SeV (MOI = 5) for the indicated time before ubiquitination assays with the indicated antibodies. (D) HTATSF1 promotes K63-linked polyubiquitination of TRAF3 at K138. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (E) HECTD3 interacts with TRAF3 but not TRAF2 or TRAF6. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before co-IP and immunoblotting analysis with the indicated antibodies. (F) HTATSF1-deficiency decreases the interactions between TRAF3 and HECTD3 or TBK1. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) for 6 h before co-IP and immunoblotting analysis with the indicated antibodies. (G) HTATSF1-deficiency attenuates HECTD3-mediated K63-linked polyubiquitination of TRAF3. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (2 × 107) were transfected with the indicated plasmids for 18 h after PMA (100 ng/mL)-induced adhesion. The cells were then left uninfected or infected with HSV-1 (MOI = 5) for 6 h before co-IP and immunoblotting analysis with the indicated antibodies. (H) HECTD3-deficiency decreases K63-linked polyubiquitination of TRAF3. The control (gNC) and HECTD3-deficient (gHECTD3) THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) or SeV (MOI = 5) for the indicated time before ubiquitination assays with the indicated antibodies.

It has been previously shown that K63-linked polyubiquitination of TRAF3 at K138 by the E3 ligase HECTD3 is essential for its activation of the downstream TBK1-IRF3 axis (Li et al., 2018). We next investigated whether HTATSF1 links HECTD3 to TRAF3 for its K63-linked polyubiquitination. Transient transfection and co-IP assays indicated that HECTD3 interacted with TRAF3 but not TRAF2 or TRAF6 (Fig. 6(E)). Endogenous co-IP assays indicated that TRAF3 was constitutively associated with both HECTD3 and TBK1 in THP-1 cells before and after HSV-1 infection (Fig. 6(F)). However, HTATSF1-deficiency markedly decreased the basal as well as HSV-1-induced associations between TRAF3 and HECTD3 or TBK1 (Fig. 6(F)), suggesting that HTATSF1 links HECTD3 to TRAF3 and this link is important for recruitment of TBK1 to TRAF3. Consistently, knockout of HECTD3 dramatically reduced K63-linked polyubiquitination of TRAF3 (Fig. 6(H)), and overexpression of HECTD3 dramatically increased K63-linked polyubiquitination of TRAF3 in control cells, but had only minimal effects on K63-linked polyubiquitination of TRAF3 in HTATSF1-deficient cells (Fig. 6(G)). These results suggest that HTATSF1 links HECTD3 to TRAF3 for its K63-linked polyubiquitination and subsequent recruitment of TBK1 for activation.

It has been well demonstrated that recruitment of TBK1 to TRAF3 results its activation, which further phosphorylates the transcription factor IRF3, leading to induction of type I interferon and other ISGs (Chen et al., 2014). Consistent with an essential role of HTATSF1 in promoting recruitment of TBK1 to TRAF3, immunoblotting analysis indicated that HTATSF1-deficiency impaired HSV-1- and SeV-induced phosphorylation of TBK1, IRF3 and STAT1 in THP-1 cells (Fig. 7(A), S5A) and BMDMs derived from Lyz2-Cre;Htatsf1fl/fl mice (Fig. 7(B), S5B). These results suggest that HTATSF1 plays an essential role in both DNA and RNA virus-triggered activation of the TBK1-IRF3 axis.

Fig. 7.

Fig. 7

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HTATSF1 is important forHSV-1-orSeV-triggeredphosphorylation of downstream signaling components.(A) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in THP-1 cells. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (B) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in BMDMs. Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl BMDM cells (1 × 106) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies.

2.6. HTATSF1 mediates the recruitment of TRAF6 to TAK1

Previously, it has been demonstrated that TRAF6 is essential for viral nucleic acid-triggered activation of TAK1-IKK-NF-κB axis (Sun et al., 2004). Since HTATSF1-deficiency impaired virus- and viral nucleic acid-triggered induction of IFNB1, ISG56 and IL6 gene (Fig. 1, Fig. 2, Fig. 3), which requires activation of both IRF3 and NF-κB, and HTATSF1 was associated with TRAF6 in co-IP assays (Fig. 4(A) and (B)), we next determined whether and how HTATSF1 regulates the TRAF6-TAK1-IKK-NF-κB axis. Ubiquitination assays indicated that overexpression of HTATSF1 did not promote TRAF6 polyubiquitination of any linkage types (Fig. 8(A)). Overexpression of HTATSF1 also did not promote polyubiquitination of TAK1, TAB1 or TAB2, which are components of the TAK1 complex (Fig. 8(B)). Consistently, neither HTATSF1-deficiency nor HECTD3-deficiency affected polyubiquitination of TRAF6 (Fig. 8(C)). However, HTATSF1-deficiency inhibited the recruitment of TAK1 to TRAF6 (Fig. 8(D)). We reconstituted TAK1 in HTATSF1-deficient THP-1 cells and found that reconstitution of TAK1 reversed the suppressed transcription of both IL6 and TNFA resulting from HTATSF1-deficiency (Fig. S4B). Immunoblotting analysis indicated that HTATSF1-deficiency impaired HSV-1- and SeV-triggerd phosphorylation of TAK1, IKKα/β, IκBα and p65, components in the TRAF6-TAK1-IKK-NF-κB axis, in THP-1 cells (Fig. 8(E), S6A) and BMDMs derived from Lyz2-Cre;Htatsf1fl/fl mice (Fig. 8(F), S6B). These results suggest that HTATSF1 plays an essential role in activating the TAK1-IKK-NF-κB axis following viral infection by linking TAK1 to TRAF6.

Fig. 8.

Fig. 8

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HTATSF1 mediates the recruitment of TAK1 to TRAF6 and its activation.(A) Overexpression of HTATSF1 has no marked effects on polyubiquitination of TRAF6. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (B) Overexpression of HTATSF1 has no marked effects on polyubiquitination of TAK1, TAB1 or TAB2. HEK293 cells (2 × 106) were transfected with the indicated plasmids for 18 h before ubiquitination assays with the indicated antibodies. (C) Deficiency of either HTATSF1 or HECTD3 has no marked effects on polyubiquitination of TRAF6. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) or SeV (MOI = 5) for the indicated times before ubiquitination assays with the indicated antibodies. (D) HTATSF1-deficiency decreases the interaction between TRAF6 and TAK1. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (2 × 107) were left uninfected or infected with HSV-1 (MOI = 5) for 6 h before co-IP and immunoblotting analysis with the indicated antibodies. (E) Effects of HTATSF1-deficiency on virus-induced phosphorylation of TAK1, IKKα/β, IκBα and p65 in THP-1 cells. The control (gNC) and HTATSF1-deficient (gHTATSF1) THP-1 cells (1 × 106) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (F) Effects of HTATSF1-deficiency on virus-induced phosphorylation of TAK1, IKKα/β, IκBα and p65 in BMDMs. Lyz2-Cre;Htatsf1fl/fl and Htatsf1fl/fl BMDM cells (1 × 106) were left un-infected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies.

3. Discussion

In this study, we identified HTATSF1 as a positive regulator of innate antiviral response to both DNA and RNA viruses. HTATSF1-deficiency suppresses transcription of downstream antiviral genes and impairs phosphorylation of signaling components in innate immune response following HSV-1 or SeV infection in human THP-1 and mouse BMDMs. HTATSF1-deficient mice show decreased production of serum cytokines including IFN-β, CXCL10, and IL-6, increased viral loads and are more susceptible to death upon HSV-1 or VSV challenge compared to wild-type mice. These results demonstrate that HTATSF1 is an important mediator of innate immune response to both DNA and RNA viruses.

HTATSF1 deficiency significantly impairs HSV-1- and SeV-induced phosphorylation of TBK1, IRF3 and STAT1, suggesting that HTATSF1 plays an essential role in virus-triggered activation of the IRF3 axis. We also observed that HTATSF1-deficiency inhibited virus-triggered phosphorylation of components in the TRAF6-TAK1-IKK-NF-κB axis. These data suggest that the observed in vivo phenotype (increased viral load and mortality) is likely a combined result of deficiencies in both arms of the immune response.

It has been well established that the cGAS-MITA/STING pathway plays an essential role in innate immune response to DNA virus, whereas the RLR-VISA/MAVS pathway is required for innate immune response to RNA virus (Hu & Shu, 2018). These pathways converge at the conserved TRAF family proteins, TRAF3 and TRAF6, which differentially regulate downstream TBK1-IRF3 and TAK1-IKK-NF-κB pathways respectively (Shi & Sun, 2018; Sun et al., 2004; Zhu et al., 2020). Several studies have shown that USP25, ATP1B1, and MYSM1 simultaneously target both TRAF3 and TRAF6 to regulate antiviral innate responses (Cao et al., 2021; Lin et al., 2015; Panda et al., 2015). Our current study suggests that HTATSF1 regulates the orchestration of TRAF3- and TRAF6-mediated downstream signaling events through distinct mechanisms.

HTATSF1 interacts with TRAF3 and TRAF6 but not other examined components in cGAS-MITA/STING and RLR-VISA/MAVS pathways in mammalian overexpression system. Endogenous HTATSF1 is constitutively associated with TRAF3 and TRAF6 before and after viral infection. These results suggest that HTATSF1 acts at the levels of TRAF3 and TRAF6 in innate antiviral signaling. Our results show that HTATSF1 promotes K63-linked polyubiquitination of TRAF3 but not TRAF6. We further found that the E3 ligase HECTD3 is responsible for catalyzing K63-linked polyubiquitination of TRAF3 upon viral infection. Overexpression of HTATSF1 promotes the association of HECTD3 and TRAF3, whereas knockout of HTATSF1 impairs the basal and virus-induced association of HECTD3 and TRAF3 and reduces K63-linked polyubiquitination of TRAF3 upon viral infection. Deficiency of either HTATSF1 and HECTD3 impairs recruitment of TBK1 to TRAF3 upon viral infection. These results suggest that HTATSF1 acts as a scaffold protein facilitating recruitment of the E3 ligase HECTD3 to TRAF3 upon viral infection, and the K63-linked polyubiquitination of TRAF3 by HECTD3 enables its recruitment of downstream kinase TBK1, leading to activation of the TBK1-IRF3 axis.

In contrast, deficiency of HTATSF1 and HECTD3 has no marked effects on polyubiquitination of TRAF6, TAK1, TAB1 and TAB2. Deficiency of HTATSF1 but not HECTD3 impairs the recruitment of TAK1 to TRAF6 as well as activation of the TAK1-IKK-NF-κB axis upon viral infection. These results suggest that HTATSF1 plays an essential role in regulating TRAF6-TAK1-IKK-NF-κB axis independent of HECTD3. Since HTATSF1 is constitutively associated with TRAF6 before and after viral infection, whereas TAK1 is recruited to TRAF6 and activated upon viral infection, the simplest explanation for our results is that HTATSF1 acts as a link for recruitment of TAK1 to TRAF6 after viral infection. Collectively, our findings suggest that HTATSF1 promotes TRAF3- and TRAF6-mediated IRF3 and NF-κB activation pathways through distinct mechanisms, and acts as a versatile regulator of innate immune response to both DNA and RNA viruses.

4. Materials and methods

4.1. Reagents, antibodies, cells and viruses

2′3′-cGAMP (tlrl-nacga23-02, Invitrogen); lipofectamine 2000 (52887, Invitrogen); polybrene (TR-1003-G, Millipore); puromycin (Thermo); RNAiso plus (9109, Takara Bio); SYBR Green Mix (172–5274, Bio–Rad); geneticin (G-418) (11811-031, Gibco); ELISA kits for murine IFN-β (42400, PBL), CXCL10 (EMC121, NeoBioscience), IL-6 (431304, BioLegend); recombinant hIFN-β (300-02BC, PEPRO TECH); poly(I:C) (tlrl-pic-5, Invitrogen); herring testis (HT)-DNA (D6898, Sigma); human IL-1β (211-11B-100, PEPRO TECH) were purchased from the indicated companies.

Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1S172 (ab109272), TBK1 (ab40676), p-IRF3S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3S396 (4947), p-STAT1Y701(9167), TRAF3 (4729), p-TAK1T184/187 (4508), p-IκBαS32/36 (9246), IκBα (9242), p-IKKα/βS176/177 (2078), IKKβ (2370), and p-p65S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

HEK293 cells and THP-1 cells were obtained from ATCC. Herpes simplex virus-1 (HSV-1), Sendai virus (SeV) and vesicular stomatitis virus (VSV) were previously described (Li et al., 2010; Lian et al., 2018a, 2018b).

4.2. Constructs

Mammalian expression plasmids for Flag-tagged TRAF3 and its mutants, TRAF6 and its mutants, TAK1, TAB1, TAB2; HA-tagged cGAS, MITA, RIG-I, VISA, TRAF2, TRAF3, TRAF6, TBK1, IRF3, TAK1, IKKβ, p65, Ub and its mutants were previously described (Lei et al., 2019; Li et al., 2010; Lian, Wei, et al., 2018; Wu et al., 2019; Yi et al., 2022; Zhang et al., 2025; Zhong et al., 2008). Mammalian expression plasmids for Flag-, HA- or Myc-tagged HTATSF1, HECTD3 and their mutants were constructed by standard molecular biology techniques. Guide RNA (gRNA) plasmids targeting Htatsf1 or Hectd3 were constructed in a lenti-CRISPR-V2 vector, which was provided by Dr. Shu-Wen Wu (Wuhan University).

4.3. Mice

The Htatsf1fl/+ mice were generated by GemPharmatech Co. Ltd (Nanjing, China) by CRISPR/Cas9-mediated gene editing. Briefly, CRISPR/Cas9 system was microinjected into the fertilized eggs of C57BL/6JGpt mice together with the targeting vector with two loxp sites flanking the exon 3-exon 6 of the Htatsf1 gene (Fig. S2A). The fertilized eggs were transplanted to obtain positive F0 mice which were confirmed by PCR and sequencing. Stable F1 generation mice were obtained by mating positive F0 generation mice with C57BL/6JGpt mice. Lyz2-Cre mice have been previously described (Sun et al., 2025). The Lyz2-Cre mouse strain specifically expresses Cre recombinase in myeloid-lineage cells. When crossed with Htatsf1fl/fl mice, it generates myeloid-specific conditional knockout mice (Lyz2-Cre;Htatsf1fl/fl). The Lyz2-Cre;Htatsf1fl/fl or Htatsf1fl/fl littermates were used throughout the study. Genotyping by PCR was performed using the following primers.

5'arm-F1: 5′-CATCTGAGTTCGGACCCTATAACC-3’;

5'arm-R1: 5′-TACAGTCAAGTGGGCATTGCC-3’;

3'arm-F2: 5′-GCATCGCATTGTCTGAGTAGGTG-3’;

3'arm-R2: 5′-CTCTCAGGTCTCCAATCCAACTG-3’.

Amplification of the WT 5'arm allele with primers F1 and R1 generates a 384-bp fragment, whereas amplification of the flox allele with primers F1 and R1 generates a 489-bp fragment. Amplification of the WT 3'arm allele using primers F2 and R2 does not generate a fragment, whereas amplification of the flox allele using primers F2 and R2 generates a 436-bp fragment. All mice were housed in the specific pathogen-free animal facility at the Medical Research Institute of Wuhan University and all animal experiments were approved by the Animal Care Committee at the Medical Research Institute of Wuhan University.

4.4. Preparation of primary mouse cells

Bone marrow-derived macrophages (BMDMs) were prepared as previously described (Gao et al., 2024; Yi et al., 2022). Briefly, mouse BMDMs (1 × 107) were cultured for 5 days in 100-mm dishes with 8 mL of M-CSF (10 ng/mL)-supplemented conditional medium.

4.5. Transfection

Transfection was performed as previously described (Zhang et al., 2025). HEK293 cells were transfected by the standard calcium phosphate precipitation method. THP-1 cells were transfected with Lipofectamine 2000. An empty control plasmid was added to ensure that each transfectant received the same amount of total DNA.

4.6. Genome-wide CRISPR screening of antiviral regulators using Perturb-seq

THP-1 monoclonal cells were selected as the model system for genome-wide CRISPR screening of host antiviral regulators. A genome-wide CRISPR knockout library (Brunello; Addgene #73179) was packaged in HEK-293 cells and transduced into THP-1 cells at a multiplicity of infection (MOI) of 0.3, maintaining 500 × coverage. The transduced cells underwent puromycin selection (2 μg/mL for 6–10 days) before HSV-1 infection (MOI = 5, 6 h). Single-cell transcriptomes and sgRNA identities were captured using the 10 × Genomics Chromium 5’ platform with CRISPR Guide Capture (Wuhan Biobank Service). Four biological replicates were generated, yielding a total of 46,649 high-quality single cells (approximately 12,000 cells per replicate). In parallel with Perturb-seq, bulk RNA-seq was performed in THP-1 cells under HSV-1-stimulated and mock conditions to (i) confirm target gene expression in this cell line, (ii) calibrate inclusion thresholds for single-cell analyses, and (iii) benchmark infection-induced transcriptome changes. Sequencing data were processed using a combined workflow of 10x Genomics Cell Ranger (v7.1) for alignment to a human GRCh38 and HSV-1 reference, followed by downstream analyses in R (v4.3.2). Bulk RNA-seq data were processed using STAR (v2.7.10a) for alignment to GRCh38, and transcript quantification was performed with RSEM (v1.3.3) to obtain TPM values. Cells were retained if they contained 2000–10,000 detected genes, fewer than 100,000 total UMI counts, and < 20% mitochondrial reads. Genes were included in downstream analyses only if they satisfied both: (i) > 2500 UMIs across all cells in the single-cell expression matrix, and (ii) expression level TPM > 5 in bulk THP-1 RNA-seq under either HSV-1-stimulated or mock conditions. For perturbation assignments, sgRNA–cell pairs were retained if the sgRNA count exceeded 50 for the corresponding target gene.

To identify candidate genes whose knockout increases viral replication, HSV-1 transcripts in each cell were quantified. Viral replication was quantified by HSV-1 transcript UMIs per cell. (i) High-viral-load cells were defined as the top 5% of the distribution (>650 UMIs). (ii) For each gene, the enrichment ratio was calculated as the proportion of its sgRNA counts in high-viral-load cells relative to its sgRNA counts in all cells. (iii) Next, to determine whether sgRNAs for a specific gene were over-represented among high-viral-load cells beyond random expectation, we assessed the significance of each gene's enrichment using Fisher's exact tests, comparing the representation of each gene's sgRNA in high-load vs. non-high cells against the distribution for non-targeting controls (NTCs) sgRNAs, and adjusted by Benjamini–Hochberg correction. (iv) A gene was considered “enriched” only if it satisfied both criteria: statistical significance (FDR < 0.05) and an effect size exceeding NTCs. (v) We restricted the analysis to gene perturbations represented in at least five cells to ensure adequate sampling depth and reduce statistical instability associated with sparse observations. Together, this strategy enabled a systematic identification of host genes whose knockout leads to markedly higher HSV-1 replication, implicating these genes as potential antiviral restriction factors. Finally, we prioritized the candidate antiviral regulators by jointly ranking them based on effect size (enrichment ratio, descending) and statistical confidence (adjusted p-value, ascending). The top 50 candidate genes were selected for downstream experimental validation.

4.7. Quantitative PCR

Total RNA was isolated for qPCR analysis to measure the mRNA abundance of the indicated genes. The data are shown as the relative abundance of the indicated mRNA derived from human or mouse cells and normalized to the level of human ACTB or mouse Actb respectively. Gene-specific primer sequences were as follows:

Human ACTB: 5′-CACCATTGGCAATGAGCGGTTC-3’ (forward) and 5′-AGGTCTTTGCGGATGTCCACGT-3’ (reverse);

Human IFNB1: 5′-CTTGGATTCCTACAAAGAAGCAGC-3’ (forward) and 5′-TCCTCCTTCTGGAACTGCTGCA-3’ (reverse);

Human ISG56: 5′-GCCTTGCTGAAGTGTGGAGGAA-3’ (forward) and 5′-ATCCAGGCGATAGGCAGAGATC-3’ (reverse);

Human IL6: 5′-TTCTCCACAAGCGCCTTCGGTC-3’ (forward) and 5′-TCTGTGTGGGGCGGCTACATCT-3’ (reverse);

Human HTATSF1: 5′-CGTCAAATCACTGCCCAGGCAT-3’ (forward) and 5′-GACAGAATCTGAACGCCTAAGGC-3’ (reverse);

Human HECTD3: 5′-ATCGAGATCCGCATCGTGGAGT-3’ (forward) and 5′-TAGTTGGCTGGAACAGGTCTGC-3’ (reverse);

Mouse Actb: 5′-CATTGCTGACAGGATGCAGAAGG-3’ (forward) and 5′-TGCTGGAAGGTGGACAGTGAGG-3’ (reverse);

Mouse Ifnb1: 5′-GCCTTTGCCATCCAAGAGATGC-3’ (forward) and 5′-ACACTGTCTGCTGGTGGAGTTC-3’ (reverse);

Mouse Isg56: 5′-TACAGGCTGGAGTGTGCTGAGA-3′(forward) and 5′-CTCCACTTTCAGAGCCTTCGCA-3’ (reverse);

Mouse Il6: 5′-TCTGCAAGAGACTTCCATCCAGTTGC 3’ (forward) and 5′-AGCCTCCGACTTGTGAAGTGGT-3’ (reverse);

HSV-1 genome: 5′-CCCTCTCCAAGGTTCCGTTC-3’ (forward) and 5′-TGCCTTTCAAACCGACCAGT-3’ (reverse).

SeV genome: 5′-GTGATTTGGGCGGCATCT-3’ (forward) and 5′-GATGGCCGGTTGGAACAC-3’ (reverse); VSV genome: 5′-AGGGAACTGTGGGATGACTG-3’ (forward) and 5′-GAACACCTGAGCCTTTGAGC-3’ (reverse).

4.8. CRISPR‒Cas9 knockout

The procedures for genome engineering using the CRISPR‒Cas9 system have been previously described (Lin et al., 2018; Luo et al., 2016). Briefly, double-stranded oligonucleotides corresponding to target sequences were cloned into the lenti-CRISPR-V2 vector and cotransfected with packaging plasmids (psPAX2 and pCMV-VSV-G) into HEK293 cells. The culture medium was replaced with new medium without antibiotics at 12 h after transfection. Two days after transfection, the viruses were harvested and used to infect THP-1 cells. The infected cells were selected with puromycin (2 μg/mL) for at least 6 days. The following sequences in the indicated genes were targeted:

gHTATSF1#1: 5′-ACCTCCACAAGAAAAAGCCC-3’;

gHTATSF1#2: 5′-AATGCTCCTGAGGCCAACAG-3’;

gHECTD3#1: 5′-AGCTTGTAGAGCACCTCTCG-3’;

gHECTD3#2: 5′-CGGCGGTTGATGTATAGGCG-3’.

4.9. Stable cell lines

HEK293 cells plated on 100-mm dishes were transfected with the indicated retroviral plasmids together with the pGag-pol and the pVSV-G plasmids. The culture medium was replaced with new medium without antibiotics at 12 h after transfection. After additional 48 h, the viruses were harvested to infect THP-1 cells in the presence of polybrene (2 μg/mL). The infected THP-1 cells were selected with Geneticin (G-418) (400 μg/mL) for at least 6 days before experiments.

4.10. Co-immunoprecipitation and immunoblot analysis

Cells were lysed in NP-40 lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride). For each immunoprecipitation experiment, a 0.4-mL aliquot of lysate was incubated with the indicated antibody or control IgG (0.5 μg) and 30 μL of a 1:1 slurry of Protein G Sepharose beads (GE Healthcare) at 4 °C for 3 h. The sepharose beads were washed three times with 1 mL of lysis buffer containing 0.5 M NaCl. The precipitates were fractionated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE), and immunoblot analysis was performed with the indicated antibodies. For endogenous co-immunoprecipitation experiments, the indicated cells were stimulated with virus for the indicated time or left untreated before co-immunoprecipitation and immunoblotting analysis.

4.11. Ubiquitination assays

The immunoprecipitates were re-extracted in lysis buffer containing 1% SDS and denatured by heating for 10 min. The samples were centrifuged at 12,000×g ​for 1 min, and then the supernatants were diluted with lysis buffer until SDS concentration was decreased to 0.1%. The supernatants were then subjected to reimmunoprecipitation with the indicated antibodies. The immunoprecipitates were analyzed by immunoblots with the indicated ubiquitin antibodies.

4.12. Digitonin permeabilization

cGAMP (1 μg/mL) was used to treat THP-1 cells (1 × 106) pretreated with a digitonin permeabilization solution (50 mM HEPES pH 7.0, 100 mM KCl, 3 mM MgCl2, 0.1 mM DTT, 85 mM sucrose, 0.2% BSA, 1 mM ATP, 0.1 mM GTP and 2 μg/mL digitonin) at 37 °C for 20 min. Two or 4 h later, the THP-1 cells were collected and subjected to RT-qPCR analysis.

4.13. Statistical analysis

GraphPad Prism software was used for statistical analyses. Quantitative data in histograms are shown as the means ± SEM. Data were analyzed by the log-rank (Mantel‒Cox) test or Student's unpaired t-test. The number of asterisks represent the degree of significance with respect to the P value. Statistical significance was set to P ​< ​0.01, ∗∗.

CRediT authorship contribution statement

Jia-Qing Zeng: Writing – original draft, Investigation, Formal analysis, Conceptualization. Zi-Lun Ruan: Investigation. Qi Zhang: Investigation. Xue-Mei Yi: Investigation. Yun-Da Chen: Investigation. Ming-Ming Hu: Conceptualization. Shu Li: Writing – original draft, Supervision, Project administration, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We thank Shi-Han Wang, Ya-Li Lei, Shu-Zhen Yang, Yu Zhang and other members of our laboratory for technical help and discussions. This work was supported by grants from the State Key R&D Program of China (2024YFA1306500, 2022YFA1304900), the National Natural Science Foundation of China (32188101), the Major Project of Guangzhou National Laboratory (GZNL2024A01014, GZNL2024A01016), the Fundamental Research Funds for the Central Universities (2042022dx0003), and Natural Science Foundation of Wuhan (2024040701010031).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cellin.2025.100294.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Multimedia component 1
mmc1.pdf (328.5KB, pdf)

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