TRIM56 Promotes IRF3-dependent Antiviral Responses Downstream of TLR4

TRIM56 Promotes IRF3-dependent Antiviral Responses Downstream of TLR4 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Brief Communication TRIM56 Promotes IRF3-dependent Antiviral Responses Downstream of TLR4 Kui Li, Xiaohan Tong, Nan Li, Darong Yang, Z. Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8665144/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The ubiquitin ligase protein tripartite-motif containing 56 (TRIM56) positively regulates Toll-like receptor-3 (TLR3) signaling by forming a complex with Toll-Interleukin-1 receptor domain-containing adapter protein inducing interferon (IFN)-beta (TRIF), independent of its E3 ligase activity. Whether TRIM56 modulates other TLR pathways in innate immunity, however, is unclear. Herein, we show ectopic expression of TRIM56 augments activation of IFN regulatory factor-3 (IRF3)-dependent promoters following stimulation by lipopolysaccharide (LPS) in HEK293-TLR4-MD2-CD14 cells while leaving activation of NF-kB-dependent promoter unaffected, suggesting TRIM56 specifically promotes immune signaling through the TLR4-TRIF axis but not the MYD88 arm downstream of this TLR. Confirming its impact on endogenous antiviral responses in immune sentinel cells naturally harboring the TLR4 pathway, we demonstrated TRIM56 also enhanced LPS-induced expression of IFN-beta and IFN-stimulated genes (ISGs) and establishment of antiviral state in bone marrow-derived macrophages. Altogether, these data add to understanding of the role of TRIM56 in TLR-mediated innate immune responses. Given that TRIM56 is an ISG and that many immune adjuvants activate TLR4, the findings of this study could have implications for designing immunotherapies, especially those against viral infections. Biological sciences/Immunology/Innate immunity/Pattern recognition receptors/Toll-like receptors Biological sciences/Genetics/Gene expression TRIM56 TLR4 IRF3 interferon-stimulated genes antiviral response TRIF Figures Figure 1 Figure 2 Introduction Pattern recognition receptors (PRRs) such as the membrane-bound Toll-like receptors (TLRs) play important roles in initiating mammalian innate immune responses against microbial infections, including those by viruses. Upon engagement by their cognate ligands, PRRs recruit specific adaptor proteins, eliciting intracellular signaling pathways that converge on the activation of latent transcription factors, interferon (IFN)-regulatory factor-3 (IRF3) and NF-kB, which coordinately induce the expression of IFNs, IFN-stimulated genes (ISGs) and inflammatory mediators. Antiviral ISGs suppress viral replication in infected cells and establish an antiviral state in neighboring uninfected cells, buying time for the development of antigen-specific adaptive immunity. Additionally, the latter is shaped by secreted IFNs and inflammatory cytokines, which regulate the homing and activation of various immune cells 1 , 2 . Of the TLRs involved in orchestrating innate antiviral immunity, two enlist and depend on the Toll-Interleukin-1 receptor domain-containing adapter protein inducing IFN-beta (TRIF) – TLR3 and TLR4 – to activate IRF3-dependent antiviral response 1 . A member of the tripartite-motif containing (TRIM) family of E3 ubiquitin ligases, TRIM56 has garnered increasing attention for its direct antiviral effects against various RNA viruses as well as its roles in regulating immune signaling 2 – 9 . We have shown that TRIM56 promotes TLR3-mediated innate immune responses following stimulation by extracellular double-stranded RNA (dsRNA) or during hepatitis C virus infection 6 – 8 . Interestingly, TRIM56 executes this role in a non-canonical, E3 ligase-independent manner that hinges on its C-terminal portion forming a complex with TRIF, the TLR3 adaptor 6 , 7 . However, whether TRIM56 modulates innate immune signaling downstream of other TLRs, is unclear. In this study, we sought to determine the impact of TRIM56 on host responses downstream of TLR4, which requires TRIF to elicit IRF3-dependent expression of antiviral genes whereas critically depends on myeloid differentiation primary response 88 (MYD88) to activate NF-kB 10 . Materials and Methods Cells and TLR ligands HEK293 cells stably expressing human TLR4, MD2 and CD14 genes (HEK293-hTLR4-MD2-CD14, InvivoGen, San Diego, CA) and C57BL/6J bone marrow-derived macrophage (BMDM) cell line (BEI resources, Manassas, VA; referred to as B6Mφ) were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 µg/ml streptomycin at 37ºC with 5% CO2 in air atmosphere. We created B6Mφ pool that stably express FLAG-tagged human TRIM56 by retroviral gene transfer following described procedure 9 . Poly(I:C12U) (AA Blocks, San Diego, CA) and LPS (Invivogen, San Diego, CA) were added to culture medium at a final concentration of 30 µg/ml and 1 µg/ml, respectively, for stimulation of cells for different times as specified. Plasmids pcDNA3.1-TRIM56-V5 and its empty vector control pcDNA3.1-V5His 9 , reporter constructs containing repeated PRDI (p55C1B-Luc) 11 and repeated PRDII (PRDII-Luc) 12 from the IFN-beta promoter have been described. hZAP/IRF-E-Luc was constructed such that the ~ 0.3-kb long, distal region of human zinc-finger antiviral protein (hZAP) promoter encompassing the five IRF elements (IRF-Es) controls the expression of firefly luciferase reporter gene. It was engineered from the hZAP(-2486)-GL3 reporter construct 13 by Quikchange PCR mutagenesis deleting a ~ 2.1-kb long, internal region of hZAP promoter and joining the distal IRF-Es and the TATA box. The mutagenesis primers used were: CCTCTGTTGCCTTT GCTAGC GTCACACGCCTCAG (forward) and CTGAGGCGTGTGAC GCTAGC AAAGGCAACAGAGG (reverse). pRL-TK (Promega, Madison, WI) was used for normalization of transfection efficiency. Reporter Gene Assay The activities of IRF3-dependent PRDI and hZAP-IRF-E promoters and that of NF-kB-dependent PRDII promoter in transfected cells with and without LPS stimulation were determined using dual-luciferase assay as described 14 , 15 . RNA and Protein Analyses Total RNA extraction, cDNA synthesis, and quantitative reverse transcription-PCR (qRT-PCR) using SYBR green technology were conducted as described 8 , 15 , 16 . For immunoblotting, cell lysates were prepared and subjected to SDS-PAGE and subsequent immunoblotting as described 8 , 14 , 15 . Primer sequences and antibodies are described in Supplementary Information. VSV-Luc-based antiviral activity assay Cells with and without pre-stimulation by LPS for 8 h were challenged by a recombinant vesicular stomatitis virus expressing firefly luciferase (VSV-Luc) at MOI = 0.1 for 8 h, followed by cell lysis and luciferase assay as described 8 . Results and Discussion TRIM56 augments LPS-induced activation of IRF3-dependent promoters in HEK293-hTLR4-MD2-CD14 cells. Apart from being the sole adaptor protein for TLR3, TRIF is also employed by TLR4 and critical for induction of IRF3-dependent genes downstream of this pathway 10 . We hypothesized that the physical interaction between TRIM56 and TRIF 6 , 7 would influence TRIF-dependent signaling downstream of TLR4. To test this, we first determined the impact of transient expression of TRIM56 on LPS-induced activation of IRF3- and NF-κB-dependent promoters in HEK293-hTLR4-MD2-CD14 cells. In cells transfected with the empty vector, LPS stimulation weakly activated the 55C1B promoter (by 2.9-fold), which is driven by the IRF3-dependent PRDI motif from the IFN-β promoter. In comparison, LPS had a significantly greater effect in cells ectopically expressing TRIM56, stimulating the promoter by 9.8-fold (Fig. 1 A). The same can be said when we examined LPS-induced hZAP/IRF-E promoter (Fig. 1 B), which comprises a DNA fragment from human ZAP promoter encompassing five IRF elements that specifically bind activated IRF3 during viral infection 13 . Specifically, LPS stimulated hZAP/IRF-E promoter by 1.7-fold and 4.3-fold, in empty vector- and TRIM56 expression vector-transfected cells, respectively, compared with their own mock-stimulated controls. Taken together, these data show that TRIM56 augments transcription from IRF3-dependent promoters upon engagement of the TLR4 pathway, which is known to be dependent on TRIF. Notably, TRIM56 expression alone without LPS stimulation also significantly upregulated the hZAP/IRF-E promoter (by 4.7-fold) (Fig. 1 B). It will be interesting to determine in future studies how TRIM56 overexpression may increase ZAP levels and whether ZAP, an RNA-binding protein that promotes viral RNA degradation 17 , has any role in TRIM56-mediated direct antiviral effects against specific viruses 4 , 5 , 9 , 18 . By contrast, we observed robust activation of the NF-kB-dependent PRDII promoter by LPS stimulation in cells with and without TRIM56 ectopic expression, with no significant difference between the two (Fig. 1 C). Since NF-kB activation via the TLR4 pathway are predominantly MYD88-dependent 10 , the lack of an effect of TRIM56 on LPS-induced NF-kB activation suggests that heightened TRIM56 expression does not promote signaling via MYD88. Consistent with this conclusion, our recent study has shown TRIM56 overexpression has no demonstrable effect on activation of the PRDII promoter by IL-1β 6 , which is also MYD88-dependent. TRIM56 enhances LPS-induced expression of Ifnb1 and antiviral ISGs in BMDMs. To confirm the effect of TRIM56 in immune sentinel cells that naturally harbor the TLR4 pathway, we conducted further experiments in B6Mφ cells with and without TRIM56 expression delivered by retroviral gene transfer. qRT-PCR demonstrated that when stimulated by Poly(I:C12U), a dsRNAs surrogate specifically engaging the TLR3 pathway without activating the cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs, RIG-I and MDA5) 19 , 20 , B6Mφ expressing TRIM56 (B6Mφ-hT56) had significantly greater induction of Ifnb1 and several representative antiviral ISGs – Isg15, Mda5, and Ifit1 – than B6Mφ without (Fig. 2 A). These data are consistent with our previous reports that TRIM56 promotes TLR3 signaling in non-immune cells HeLa, HT1080, HEK293-TLR3, and Huh7.5-TLR3 6–8 and suggest this conclusion also holds in an immune cell type. Time-course analyses of the expression of antiviral genes following LPS stimulation revealed a similar phenotype, with B6Mφ-hT56 cells exhibiting ~ 1-log or greater induction of Ifnb1, Isg15, Mda5, and Ifit1 than in control B6Mφ (Fig. 2 B). We additionally confirmed the heighted induction of MDA5 and ISG15 proteins, for which we had access to a sensitive antibody for detection, in B6Mφ-hT56 than in control B6Mφ following stimulation by either Poly(I:C12U) (Fig. 2 C, compare lanes 2 vs 1) or LPS (compare lanes 6 vs 5). In aggregate, these data ascertain our earlier reporter gene assay data from HEK293-hTLR4-MD2-CD14 cells (Fig. 1 ), illustrating that TRIM56 promotes induction of IRF3-dependent innate immune genes via TLR4 signaling in BMDMs. TRIM56 potentiates LPS-induced establishment of an antiviral state in BMDMs. We next sought to gauge the impact of TRIM56 on antiviral protection conferred by TLR4-TRIF signaling. B6Mφ and B6Mφ-hT56 cells were pre-stimulated by LPS or mock-stimulated, followed by challenge with VSV-Luc that expresses luciferase reporter as a readout for viral replication. As shown in Fig. S1 , enforced expression of TRIM56 per se marginally reduced VSV-Luc replication (compare B6Mφ and B6Mφ-hT56, by 1.6 fold), in keeping with our previous reports that TRIM56 alone has no significant antiviral activity against VSV in MDBK and HeLa cells 4 , 7 – 9 . Pre-stimulation by LPS induced a potent antiviral state in both control B6Mφ and B6Mφ-hT56 cells, curtailing viral replication by 29.6-fold and 68.0-fold, respectively, as compared with their unstimulated counterparts. Notably, LPS pre-stimulation was significantly more effective in B6Mφ-hT56 than in control B6Mφ. These data lend direct support to the notion that TRIM56 imparts heightened cellular antiviral state following engagement of the TLR4 pathway. In summary, this study demonstrates that TRIM56 promotes IRF3-dependent antiviral responses downstream of TLR4, adding to knowledge of the involvement of TRIM56 in TLR signaling and aiding a fuller understanding of the roles TRIM56 plays in innate immunity. Given that TRIM56 is an ISG upregulated during viral infection 9 and that many immune adjuvants activate TLR4, TRIM56 could present a node for developing immunotherapies, especially those against viral infections. Future studies in TRIM56-deficient animal models are warranted to probe this aspect and to investigate the physiological roles of TRIM56 in TLR signaling and other immune mechanisms. Declarations Competing Interests The authors declare no competing financial interests in relation to the work described. Author Contributions KL, XT and NLL conceived the study, XT and NLL collected data and were involved in data analysis, DY provided research material, ZAL contributed to data analysis and writing, KL, XT, and NLL wrote and edited the manuscript. Acknowledgements This study was supported by the UTHSC College of Medicine research incentive funds to K.L. Data Availability Statement All the data are contained within the article and supporting information. Raw data can be made available upon request. References Lester SN, Li K. Toll-like receptors in antiviral innate immunity. J Mol Biol 2014; 426(6): 1246–64. Li K, Lee JZ. The regulation of innate antiviral immunity by TRIM56. Infectious Diseases & Immunity 2025; 5(02): 120–126. Wang D, Li K. Emerging Roles of TRIM56 in Antiviral Innate Immunity. Viruses 2025; 17(1). Liu B, Li NL, Shen Y, Bao X, Fabrizio T, Elbahesh H et al. The C-Terminal Tail of TRIM56 Dictates Antiviral Restriction of Influenza A and B Viruses by Impeding Viral RNA Synthesis. J Virol 2016; 90(9): 4369–4382. Liu B, Li NL, Wang J, Shi PY, Wang T, Miller MA et al. Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against flaviviruses and coronavirus. J Virol 2014; 88(23): 13821–35. Liu BM, Li NL, Wang R, Li X, Li ZA, Marion TN et al. Key roles for phosphorylation and the Coiled-coil domain in TRIM56-mediated positive regulation of TLR3-TRIF-dependent innate immunity. J Biol Chem 2024; 300(5): 107249. Shen Y, Li NL, Wang J, Liu B, Lester S, Li K. TRIM56 is an essential component of the TLR3 antiviral signaling pathway. J Biol Chem 2012; 287(43): 36404–13. Wang D, Wang R, Li K. Impaired Antiviral Responses to Extracellular Double-Stranded RNA and Cytosolic DNA, but Not to Interferon-alpha Stimulation, in TRIM56-Deficient Cells. Viruses 2022; 14(1). Wang J, Liu B, Wang N, Lee YM, Liu C, Li K. 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Regulation of interferon regulatory factor 3-dependent innate immunity by the HCV NS3/4A protease. Methods Mol Biol 2009; 510: 211–26. Liu Y, Tong X, Wang R, Li ZG, Xie Z, Wang D et al. Cytokine-independent induction of LGP2/DHX58 in viral infection. J Gen Virol 2025; 106(10). Li ZA, Bajpai AK, Wang R, Liu Y, Webby RJ, Wilk E et al. Systems genetics of influenza A virus-infected mice identifies TRIM21 as a critical regulator of pulmonary innate immune response. Virus Res 2024; 342: 199335. Luo X, Wang X, Gao Y, Zhu J, Liu S, Gao G et al. Molecular Mechanism of RNA Recognition by Zinc-Finger Antiviral Protein. Cell Rep 2020; 30(1): 46–52 e4. Yang D, Li NL, Wei D, Liu B, Guo F, Elbahesh H et al. The E3 ligase TRIM56 is a host restriction factor of Zika virus and depends on its RNA-binding activity but not miRNA regulation, for antiviral function. PLoS Negl Trop Dis 2019; 13(6): e0007537. Theodoraki MN, Yerneni S, Sarkar SN, Orr B, Muthuswamy R, Voyten J et al. Helicase-Driven Activation of NFkappaB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment. Cancer Res 2018; 78(15): 4292–4302. Trumpfheller C, Caskey M, Nchinda G, Longhi MP, Mizenina O, Huang Y et al. The microbial mimic poly IC induces durable and protective CD4 + T cell immunity together with a dendritic cell targeted vaccine. Proc Natl Acad Sci U S A 2008; 105(7): 2574–9. Additional Declarations There is NO conflict of interest to disclose. There is NO conflict of interest to disclose. Supplementary Files TongetalTRIM56promotesTLR4IRF3supplinfo.pdf Tong et al supplementary information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:00:58","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64596,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8665144/v1/400d7e8ee873927f1b88417e.html"},{"id":101190624,"identity":"842a60c8-1343-4d41-a3ce-8572b31e09e8","added_by":"auto","created_at":"2026-01-27 07:00:58","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":567045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpact of ectopic expression of TRIM56 on LPS-induced activation of IRF3- and NF-kB-dependent promoters in HEK293-TLR4-MD2-CD14 cells. \u003c/strong\u003eActivation of IRF-3-dependent PRDI promoter (p55CIB-Luc, \u003cstrong\u003eA\u003c/strong\u003e) and hZAP-IRF-E promoter (\u003cstrong\u003eB\u003c/strong\u003e), or NF-κB-dependent PRDII promoter (\u003cstrong\u003eC\u003c/strong\u003e) in HEK293-TLR4-MD2-CD14 cells transiently expressing TRIM56 or an empty control vector, and mock-stimulated (empty bars) or stimulated by LPS (hatched bars) for 16 h.\u003cstrong\u003e \u003c/strong\u003eData are expressed as mean ± SD from two independent experiments. *, **, and *** denote p\u0026lt;0.05, p\u0026lt;0.01, and p\u0026lt;0.001, respectively (Student’s t-test).\u003c/p\u003e","description":"","filename":"TongetalT56TLR4Fig1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8665144/v1/a3430aaea19156ac99e51df3.jpg"},{"id":101190626,"identity":"2608fc4d-0533-4a60-8f1f-57c606cae7f8","added_by":"auto","created_at":"2026-01-27 07:00:58","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1404538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced antiviral gene expression in TRIM56-expressing bone marrow-derived macrophages following LPS stimulation. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e\u0026amp; \u003cstrong\u003eB\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eqRT-PCR analyses of the abundance of indicated gene transcripts in B6Mj with or without TRIM56-FLAG expression at indicated times post stimulation by Poly(I:C12U) (\u003cstrong\u003eA\u003c/strong\u003e) or LPS (\u003cstrong\u003eB\u003c/strong\u003e). * and *** denote p\u0026lt;0.05 and p\u0026lt;0.001, respectively (One-way ANOVA). (\u003cstrong\u003eC\u003c/strong\u003e) Immunoblotting of TRIM56 (anti-FLAG), GAPDH, MDA5, ISG15, and ACTIN protein levels in B6Mj with or without TRIM56-FLAG expression, mock-stimulated or stimulated by Poly(I:C12U) or LPS for 8 h. Quantification of the immunoblotting data by densitometry analysis (normalized to ACTIN) is presented as bar graphs on the right. Data are from two independent experiments. Error bars represent SD. *, **, and *** denote p\u0026lt;0.05, p\u0026lt;0.01, and p\u0026lt;0.001, respectively (Student’s t-test).\u003c/p\u003e","description":"","filename":"TongetalT56TLR4Fig2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8665144/v1/3d6a2c8cda9e71d2e764e7d0.jpg"},{"id":101398129,"identity":"9e56a1a4-147c-43ef-af78-42fbbe808592","added_by":"auto","created_at":"2026-01-29 09:39:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2554915,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8665144/v1/8303c66d-01d8-453f-ad4e-fb59afd277fe.pdf"},{"id":101190627,"identity":"f6352eaf-620b-49a6-a6c7-879480c8cadc","added_by":"auto","created_at":"2026-01-27 07:00:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":263051,"visible":true,"origin":"","legend":"Tong et al supplementary information","description":"","filename":"TongetalTRIM56promotesTLR4IRF3supplinfo.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8665144/v1/79e8b719c4c2fa63276f9f3c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.\nThere is NO conflict of interest to disclose.","formattedTitle":"TRIM56 Promotes IRF3-dependent Antiviral Responses Downstream of TLR4","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePattern recognition receptors (PRRs) such as the membrane-bound Toll-like receptors (TLRs) play important roles in initiating mammalian innate immune responses against microbial infections, including those by viruses. Upon engagement by their cognate ligands, PRRs recruit specific adaptor proteins, eliciting intracellular signaling pathways that converge on the activation of latent transcription factors, interferon (IFN)-regulatory factor-3 (IRF3) and NF-kB, which coordinately induce the expression of IFNs, IFN-stimulated genes (ISGs) and inflammatory mediators. Antiviral ISGs suppress viral replication in infected cells and establish an antiviral state in neighboring uninfected cells, buying time for the development of antigen-specific adaptive immunity. Additionally, the latter is shaped by secreted IFNs and inflammatory cytokines, which regulate the homing and activation of various immune cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Of the TLRs involved in orchestrating innate antiviral immunity, two enlist and depend on the Toll-Interleukin-1 receptor domain-containing adapter protein inducing IFN-beta (TRIF) \u0026ndash; TLR3 and TLR4 \u0026ndash; to activate IRF3-dependent antiviral response\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA member of the tripartite-motif containing (TRIM) family of E3 ubiquitin ligases, TRIM56 has garnered increasing attention for its direct antiviral effects against various RNA viruses as well as its roles in regulating immune signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. We have shown that TRIM56 promotes TLR3-mediated innate immune responses following stimulation by extracellular double-stranded RNA (dsRNA) or during hepatitis C virus infection\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Interestingly, TRIM56 executes this role in a non-canonical, E3 ligase-independent manner that hinges on its C-terminal portion forming a complex with TRIF, the TLR3 adaptor\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, whether TRIM56 modulates innate immune signaling downstream of other TLRs, is unclear. In this study, we sought to determine the impact of TRIM56 on host responses downstream of TLR4, which requires TRIF to elicit IRF3-dependent expression of antiviral genes whereas critically depends on myeloid differentiation primary response 88 (MYD88) to activate NF-kB\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCells and TLR ligands\u003c/h2\u003e \u003cp\u003eHEK293 cells stably expressing human TLR4, MD2 and CD14 genes (HEK293-hTLR4-MD2-CD14, InvivoGen, San Diego, CA) and C57BL/6J bone marrow-derived macrophage (BMDM) cell line (BEI resources, Manassas, VA; referred to as B6Mφ) were maintained in Dulbecco\u0026rsquo;s Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin and 100 \u0026micro;g/ml streptomycin at 37\u0026ordm;C with 5% CO2 in air atmosphere. We created B6Mφ pool that stably express FLAG-tagged human TRIM56 by retroviral gene transfer following described procedure\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePoly(I:C12U) (AA Blocks, San Diego, CA) and LPS (Invivogen, San Diego, CA) were added to culture medium at a final concentration of 30 \u0026micro;g/ml and 1 \u0026micro;g/ml, respectively, for stimulation of cells for different times as specified.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmids\u003c/h3\u003e\n\u003cp\u003epcDNA3.1-TRIM56-V5 and its empty vector control pcDNA3.1-V5His\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, reporter constructs containing repeated PRDI (p55C1B-Luc)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and repeated PRDII (PRDII-Luc)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e from the IFN-beta promoter have been described. hZAP/IRF-E-Luc was constructed such that the ~\u0026thinsp;0.3-kb long, distal region of human zinc-finger antiviral protein (hZAP) promoter encompassing the five IRF elements (IRF-Es) controls the expression of firefly luciferase reporter gene. It was engineered from the hZAP(-2486)-GL3 reporter construct\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e by Quikchange PCR mutagenesis deleting a\u0026thinsp;~\u0026thinsp;2.1-kb long, internal region of hZAP promoter and joining the distal IRF-Es and the TATA box. The mutagenesis primers used were: CCTCTGTTGCCTTT\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCTAGC\u003c/span\u003eGTCACACGCCTCAG (forward) and CTGAGGCGTGTGAC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGCTAGC\u003c/span\u003eAAAGGCAACAGAGG (reverse). pRL-TK (Promega, Madison, WI) was used for normalization of transfection efficiency.\u003c/p\u003e\n\u003ch3\u003eReporter Gene Assay\u003c/h3\u003e\n\u003cp\u003eThe activities of IRF3-dependent PRDI and hZAP-IRF-E promoters and that of NF-kB-dependent PRDII promoter in transfected cells with and without LPS stimulation were determined using dual-luciferase assay as described\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eRNA and Protein Analyses\u003c/h3\u003e\n\u003cp\u003eTotal RNA extraction, cDNA synthesis, and quantitative reverse transcription-PCR (qRT-PCR) using SYBR green technology were conducted as described\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. For immunoblotting, cell lysates were prepared and subjected to SDS-PAGE and subsequent immunoblotting as described\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Primer sequences and antibodies are described in Supplementary Information.\u003c/p\u003e\n\u003ch3\u003eVSV-Luc-based antiviral activity assay\u003c/h3\u003e\n\u003cp\u003eCells with and without pre-stimulation by LPS for 8 h were challenged by a recombinant vesicular stomatitis virus expressing firefly luciferase (VSV-Luc) at MOI\u0026thinsp;=\u0026thinsp;0.1 for 8 h, followed by cell lysis and luciferase assay as described\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e \u003cb\u003eTRIM56 augments LPS-induced activation of IRF3-dependent promoters in HEK293-hTLR4-MD2-CD14 cells.\u003c/b\u003e Apart from being the sole adaptor protein for TLR3, TRIF is also employed by TLR4 and critical for induction of IRF3-dependent genes downstream of this pathway\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. We hypothesized that the physical interaction between TRIM56 and TRIF\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e would influence TRIF-dependent signaling downstream of TLR4. To test this, we first determined the impact of transient expression of TRIM56 on LPS-induced activation of IRF3- and NF-κB-dependent promoters in HEK293-hTLR4-MD2-CD14 cells. In cells transfected with the empty vector, LPS stimulation weakly activated the 55C1B promoter (by 2.9-fold), which is driven by the IRF3-dependent PRDI motif from the IFN-β promoter. In comparison, LPS had a significantly greater effect in cells ectopically expressing TRIM56, stimulating the promoter by 9.8-fold (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The same can be said when we examined LPS-induced hZAP/IRF-E promoter (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which comprises a DNA fragment from human ZAP promoter encompassing five IRF elements that specifically bind activated IRF3 during viral infection\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Specifically, LPS stimulated hZAP/IRF-E promoter by 1.7-fold and 4.3-fold, in empty vector- and TRIM56 expression vector-transfected cells, respectively, compared with their own mock-stimulated controls. Taken together, these data show that TRIM56 augments transcription from IRF3-dependent promoters upon engagement of the TLR4 pathway, which is known to be dependent on TRIF.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNotably, TRIM56 expression alone without LPS stimulation also significantly upregulated the hZAP/IRF-E promoter (by 4.7-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). It will be interesting to determine in future studies how TRIM56 overexpression may increase ZAP levels and whether ZAP, an RNA-binding protein that promotes viral RNA degradation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, has any role in TRIM56-mediated direct antiviral effects against specific viruses\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBy contrast, we observed robust activation of the NF-kB-dependent PRDII promoter by LPS stimulation in cells with and without TRIM56 ectopic expression, with no significant difference between the two (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Since NF-kB activation via the TLR4 pathway are predominantly MYD88-dependent\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, the lack of an effect of TRIM56 on LPS-induced NF-kB activation suggests that heightened TRIM56 expression does not promote signaling via MYD88. Consistent with this conclusion, our recent study has shown TRIM56 overexpression has no demonstrable effect on activation of the PRDII promoter by IL-1β\u003csup\u003e6\u003c/sup\u003e, which is also MYD88-dependent.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTRIM56 enhances LPS-induced expression of Ifnb1 and antiviral ISGs in BMDMs.\u003c/b\u003e To confirm the effect of TRIM56 in immune sentinel cells that naturally harbor the TLR4 pathway, we conducted further experiments in B6Mφ cells with and without TRIM56 expression delivered by retroviral gene transfer. qRT-PCR demonstrated that when stimulated by Poly(I:C12U), a dsRNAs surrogate specifically engaging the TLR3 pathway without activating the cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs, RIG-I and MDA5)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, B6Mφ expressing TRIM56 (B6Mφ-hT56) had significantly greater induction of Ifnb1 and several representative antiviral ISGs \u0026ndash; Isg15, Mda5, and Ifit1 \u0026ndash; than B6Mφ without (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These data are consistent with our previous reports that TRIM56 promotes TLR3 signaling in non-immune cells HeLa, HT1080, HEK293-TLR3, and Huh7.5-TLR3\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e and suggest this conclusion also holds in an immune cell type. Time-course analyses of the expression of antiviral genes following LPS stimulation revealed a similar phenotype, with B6Mφ-hT56 cells exhibiting\u0026thinsp;~\u0026thinsp;1-log or greater induction of Ifnb1, Isg15, Mda5, and Ifit1 than in control B6Mφ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We additionally confirmed the heighted induction of MDA5 and ISG15 proteins, for which we had access to a sensitive antibody for detection, in B6Mφ-hT56 than in control B6Mφ following stimulation by either Poly(I:C12U) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, compare lanes 2 vs 1) or LPS (compare lanes 6 vs 5). In aggregate, these data ascertain our earlier reporter gene assay data from HEK293-hTLR4-MD2-CD14 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), illustrating that TRIM56 promotes induction of IRF3-dependent innate immune genes via TLR4 signaling in BMDMs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTRIM56 potentiates LPS-induced establishment of an antiviral state in BMDMs.\u003c/b\u003e We next sought to gauge the impact of TRIM56 on antiviral protection conferred by TLR4-TRIF signaling. B6Mφ and B6Mφ-hT56 cells were pre-stimulated by LPS or mock-stimulated, followed by challenge with VSV-Luc that expresses luciferase reporter as a readout for viral replication. As shown in Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, enforced expression of TRIM56 \u003cem\u003eper se\u003c/em\u003e marginally reduced VSV-Luc replication (compare B6Mφ and B6Mφ-hT56, by 1.6 fold), in keeping with our previous reports that TRIM56 alone has no significant antiviral activity against VSV in MDBK and HeLa cells\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Pre-stimulation by LPS induced a potent antiviral state in both control B6Mφ and B6Mφ-hT56 cells, curtailing viral replication by 29.6-fold and 68.0-fold, respectively, as compared with their unstimulated counterparts. Notably, LPS pre-stimulation was significantly more effective in B6Mφ-hT56 than in control B6Mφ. These data lend direct support to the notion that TRIM56 imparts heightened cellular antiviral state following engagement of the TLR4 pathway.\u003c/p\u003e \u003cp\u003eIn summary, this study demonstrates that TRIM56 promotes IRF3-dependent antiviral responses downstream of TLR4, adding to knowledge of the involvement of TRIM56 in TLR signaling and aiding a fuller understanding of the roles TRIM56 plays in innate immunity. Given that TRIM56 is an ISG upregulated during viral infection\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and that many immune adjuvants activate TLR4, TRIM56 could present a node for developing immunotherapies, especially those against viral infections. Future studies in TRIM56-deficient animal models are warranted to probe this aspect and to investigate the physiological roles of TRIM56 in TLR signaling and other immune mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests in relation to the work described.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eKL, XT and NLL conceived the study, XT and NLL collected data and were involved in data analysis, DY provided research material, ZAL contributed to data analysis and writing, KL, XT, and NLL wrote and edited the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by the UTHSC College of Medicine research incentive funds to K.L.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eAll the data are contained within the article and supporting information. Raw data can be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLester SN, Li K. Toll-like receptors in antiviral innate immunity. \u003cem\u003eJ Mol Biol\u003c/em\u003e 2014; 426(6): 1246\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi K, Lee JZ. The regulation of innate antiviral immunity by TRIM56. \u003cem\u003eInfectious Diseases \u0026amp; Immunity\u003c/em\u003e 2025; 5(02): 120\u0026ndash;126.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Li K. 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Whether TRIM56 modulates other TLR pathways in innate immunity, however, is unclear. Herein, we show ectopic expression of TRIM56 augments activation of IFN regulatory factor-3 (IRF3)-dependent promoters following stimulation by lipopolysaccharide (LPS) in HEK293-TLR4-MD2-CD14 cells while leaving activation of NF-kB-dependent promoter unaffected, suggesting TRIM56 specifically promotes immune signaling through the TLR4-TRIF axis but not the MYD88 arm downstream of this TLR. Confirming its impact on endogenous antiviral responses in immune sentinel cells naturally harboring the TLR4 pathway, we demonstrated TRIM56 also enhanced LPS-induced expression of IFN-beta and IFN-stimulated genes (ISGs) and establishment of antiviral state in bone marrow-derived macrophages. Altogether, these data add to understanding of the role of TRIM56 in TLR-mediated innate immune responses. Given that TRIM56 is an ISG and that many immune adjuvants activate TLR4, the findings of this study could have implications for designing immunotherapies, especially those against viral infections.\u003c/p\u003e","manuscriptTitle":"TRIM56 Promotes IRF3-dependent Antiviral Responses Downstream of TLR4","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 07:00:53","doi":"10.21203/rs.3.rs-8665144/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9145e2d-08d9-40f7-99b4-5b8ad4a41cda","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61589582,"name":"Biological sciences/Immunology/Innate immunity/Pattern recognition receptors/Toll-like receptors"},{"id":61589583,"name":"Biological sciences/Genetics/Gene expression"}],"tags":[],"updatedAt":"2026-01-28T17:45:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 07:00:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8665144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8665144","identity":"rs-8665144","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}