Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: The upregulation of TOR3A during RSV infection is facilitated by STAT1. ( A ) Flowchart overview of the infection of RAW264.7 cells with RSV followed by mass spectrometry analysis. RAW264.7 cells were infected with RSV (MOI = 1) for 12 h, with 2% DMEM (RSV solvent) as control. ( B ) Whole protein profiles of RAW264.7 cells after 12-hour RSV infection were analyzed. Cluster analysis identified differentially expressed interferon-stimulated genes (ISGs) proteins between RSV-infected and control groups. ( C ) KEGG analysis demonstrated activation of multiple infection-related signalling pathways following RSV infection. ( D ) GO enrichment analysis showing significant activation of cellular functions in response to RSV infection. ( E - F ) RT-qPCR analysis of Ifnb and Tor3a mRNA levels in RAW264.7 cells at 0, 6, 12, and 24 h post-RSV infection. ( G ) Western blot analysis of TOR3A protein expression in RAW264.7 cells at indicated time points (0, 12, 24 h) post-RSV infection. ( H ) RT-qPCR analysis of TOR3A mRNA levels in PBMCs from hospitalized RSV-bronchiolitis patients (n = 70) and controls (n = 40). ( I ) RT-qPCR analysis of TOR3A expression in PBMCs from RSV-infected children with mild (n = 48) or moderate to severe (n = 22) disease. ( J ) Analysis of the correlation between TOR3A mRNA levels and RSV viral copy number. ( K ) ROC analysis was performed to evaluate the predictive value of TOR3A expression for bronchiolitis in RSV-infected children. ( L ) Analysis of TOR3A mRNA levels from peripheral blood transcriptomic data ( GSE188424 ) in hospitalized RSV-infected children (n = 24) versus age-matched healthy controls (n = 24). ( M ) ROC curve analysis of the predictive value of TOR3A for RSV infection. ( N-O ) RAW264.7 cells were stimulated with IFN-β at a working concentration of 1000 U/ml. The mRNA and protein levels of TOR3A were measured by RT-qPCR and Western blot, respectively. ( P-Q ) RAW264.7 cells were pre-incubated with an IFNAR1-specific blocking antibody (100 ng/mL) for 2 h prior to RSV infection (12 h). The mRNA and protein levels of TOR3A were then measured by RT-qPCR and Western blot, respectively. ( R ) ChIP analysis revealed that STAT1 binds to the Tor3a promoter. ( S ) HEK293 T cells were co-transfected for 24 h with a luciferase reporter plasmid driven by the TOR3A promoter, a STAT1 expression plasmid, and empty vector, followed by a dual-luciferase reporter assay. Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (H, I, L, and S), one-way ANOVA followed by Dunett's multiple comparisons test (E, F, N, and P). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Infection, Mass Spectrometry, Solvent, Control, Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Concentration Assay, Incubation, Blocking Assay, Transfection, Luciferase, Plasmid Preparation, Reporter Assay

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A inhibits IFN-I production and facilitates RSV replication in vitro . ( A ) TOR3A expression was detected by Western blot in RAW264.7 cells stably transfected with shTOR3A. ( B-C ) RT-qPCR analysis of Tor3a and RSV-F mRNA levels in control and shTOR3A cells at 0, 6, 12, and 24 h post-RSV infection. ( D ) After 24 h of RSV-mCherry infection, RSV replication was visualized by fluorescence microscopy in shTOR3A and control cells (scale bar, 100 μm; top: bright field image; bottom: fluorescence image). ( E-F ) RT-qPCR analysis of Ifnb and Isg15 mRNA levels in control and shTOR3A cells at 0, 6, 12, and 24 h post-RSV infection. ( G-I ) RAW264.7 cells with TOR3A knockdown (shTOR3A) were transfected with an empty vector (EV) or a TOR3A expression plasmid (shTOR3A + TOR3A), followed by RSV infection for 6 h. Tor3a , Ifnb , and Isg15 mRNA levels were measured by RT-qPCR. ( J-K ) RT-qPCR analysis of IFNB and ISG15 mRNA levels in HEK293 T cells at 12 and 24 h post-RSV infection, following 24 h of transfection with Myc-TOR3A. ( L ) RT-qPCR analysis of RSV-F mRNA levels in HEK293 T cells at 24 h post-RSV infection, following 24 h of transfection with Myc-TOR3A. ( M ) Fluorescence microscopy analysis of RSV infection in HEK293 T cells at 24 h post-infection, following 24 h of transfection with Myc-TOR3A. Scale bar, 100 μm. ( N-O ) RT-qPCR analysis of IFNB and ISG15 mRNA levels in HEK293 T cells at 12 and 24 h post-SeV infection, following transfection with Myc-TOR3A for 24 h. ( P ) After overexpressing Myc-TOR3A for 24 h in HeLa cells, the cells were infected with SeV for 12 h and stained with IRF3 (red) and Myc (green) antibodies (scale bar, 10 μm). ( Q) Western blot analysis of VSV-G protein expression at 6 and 12 h post-VSV-GFP infection in HEK293 T cells transfected with Myc-TOR3A for 24 h ( R ) After transfecting HEK293 T cells with Myc-TOR3A for 24 h, the cells were infected with VSV-GFP for 12 h, and the replication of VSV-GFP was visualized by fluorescence microscopy (scale bar, 100 μm). ( S ) After HEK293 T cells were transfected with Myc-TOR3A for 24 h, they were infected with VSV for 24 h. The virus-containing supernatant was then collected for plaque assay, and the results were subjected to statistical analysis. ( T ) TOR3A knockdown efficiency was confirmed by Western blot in HEK293 T cells transfected with si-NC or siTOR3A (si-1, si-2) for 48 h. ( U ) RT-qPCR analysis of IFNB mRNA levels in HEK293 T cells after 12 h of Poly(I:C) (1 μg/mL) transfection, following 36 h of TOR3A siRNA transfection. ( V ) Fluorescence microscopy of RSV replication in HEK293 T cells at 24 h post-infection, following 36 h of transfection with TOR3A siRNA. Scale bar, 100 μm. Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (B, C, E, F, J, K, L, N, O and S), two-way ANOVA followed by Sidak's multiple comparisons test (G, H, I and U). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: In Vitro, Expressing, Western Blot, Stable Transfection, Transfection, Quantitative RT-PCR, Control, Infection, Fluorescence, Microscopy, Knockdown, Plasmid Preparation, Staining, Virus, Plaque Assay

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A deficiency augments IFN-I production and restrains virus replication in vivo . ( A ) Establishment of an intranasal RSV infection model in mice. ( B ) ELISA of serum IFN-β in WT versus TOR3A KO mice post-RSV infection (1 × 10⁶ PFU, i.n.). ( C-D ) RT-qPCR analysis of RSV-F and RSV-M2-1 mRNA levels in the lungs of RSV-infected WT and TOR3A KO mice (n = 3 per group). ( E ) Plaque assay of RSV titres in lung homogenate supernatants from RSV-infected WT and TOR3A KO mice. ( F ) Western blot analysis of M2-1 protein expression in lung tissues from RSV-infected WT and TOR3A KO mice. ( G ) H&E staining and pathological scoring of lung sections from PBS- or RSV-treated WT and TOR3A KO mice. Scale bar, 100 μm. ( H ) Establishment of a VSV intraperitoneal infection model in mice. ( I ) RT-qPCR analysis of VSV load in the lungs of WT and TOR3A KO mice (n = 3 per group) after infection (4 × 10⁸ PFU). ( J ) Plaque assay of VSV titres in lung homogenate supernatants from VSV-infected WT and TOR3A KO mice. ( K ) Western blot analysis of VSV-G protein expression in lung tissues from VSV-infected WT and TOR3A KO mice. (L) H&E staining and histopathological scoring of lung sections from PBS- or VSV-treated WT and TOR3A KO mice. Scale bar, 100 μm. (M) Survival of WT and TOR3A KO mice (n = 20 per group) after intraperitoneal infection with VSV (4 × 10⁸ PFU). Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (B, C, D, E, G, I, J, and K) or survival curve (L) followed by Log-rank (Mantel-Cox) test. ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Virus, In Vivo, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Plaque Assay, Western Blot, Expressing, Staining

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A targets RIG-I and inhibits its function in the IFN-I pathway. ( A ) Western blot analysis of IFN-I pathway proteins in HeLa cells after 12 h RSV infection, following 24 h transfection with Myc-TOR3A or empty vector (EV). ( B ) Western blot analysis of IFN-I pathway proteins in HeLa cells after 12 h SeV infection, following 24 h transfection with Myc-TOR3A or empty vector (EV). ( C ) Western blot analysis of RIG-I, p-TBK1, TBK1, and TOR3A in shTOR3A and control RAW264.7 cells at 12 h post-RSV infection. ( D ) Western blot analysis of Flag-RIG-I and gradient Myc-TOR3A expression in HEK293 T cells after 48 h of co-transfection. ( E ) Western blot analysis of RIG-I protein levels in HeLa cells transfected with increasing amounts of Myc-TOR3A for 48 h. ( F ) Schematic diagram of TOR3A domains and the ATPase double mutant TOR3A-DM (K173A/E236Q). ( G ) Western blot analysis of RIG-I and MDA5 in HeLa cells after 48 h transfection with wild-type (WT) or double mutant (DM) Myc-TOR3A. ( H ) Co-IP analysis of the interaction between Myc-TOR3A and Flag-RIG-I in HEK293 T cells after 48 h of co-transfection. ( I ) Endogenous Co-IP of TOR3A and RIG-I interaction in RAW264.7 cells after 12 h of RSV infection. ( J ) Endogenous Co-IP of TOR3A and RIG-I interaction in THP-1 cells after 12 h of RSV infection. ( K ) Co-localization of Myc-TOR3A and Flag-RIG-I in HEK293 T cells after 48 h co-transfection. Cells were immunostained for Myc (green) and Flag (red) and imaged by confocal microscopy. Co-localization was quantified by Pearson's correlation coefficient (r). Scale bar, 10 μm. ( L ) Co-localization of endogenous RIG-I (green) and TOR3A (red) in mouse primary PMs at 6 h post-SeV infection. Images were acquired by confocal microscopy, and co-localization was quantified using Pearson's correlation coefficient (r). Scale bar, 25 μm. Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (A, B, and C). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Western Blot, Infection, Transfection, Plasmid Preparation, Control, Expressing, Cotransfection, Mutagenesis, Co-Immunoprecipitation Assay, Confocal Microscopy

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A downregulates RIG-I in a ubiquitin-proteasome pathway-dependent manner. ( A ) RT-qPCR analysis of endogenous RIG-I mRNA levels in HEK293 T cells after 48 h of transfection with increasing amounts of Myc-TOR3A plasmid. ( B ) Western blot analysis of Flag-RIG-I protein stability in HEK293 T cells co-expressing Myc-TOR3A after treatment with cycloheximide (CHX, 50 μg/mL) for indicated times. ( C ) Western blot analysis of RIG-I protein stability in RSV-infected (12 h) WT and TOR3A KO PMs after treatment with cycloheximide (CHX) for the indicated times. ( D ) Western blot analysis of Flag-RIG-I protein levels in HEK293 T cells co-expressing Myc-TOR3A after 6 h treatment with the proteasome inhibitor MG132 (10 μM). ( E ) Western blot analysis of Flag-RIG-I protein levels in HEK293 T cells co-expressing Myc-TOR3A after 6 h treatment with the lysosomal inhibitor chloroquine (CQ). ( F ) Co-IP and Western blot analysis of exogenous RIG-I ubiquitination in HEK293 T cells co-expressing Myc-TOR3A, Flag-RIG-I, and HA-ubiquitin for 48 h. ( G ) HEK293 T cells were co-transfected with Myc-TOR3A, Flag-RIG-I, and various HA-tagged ubiquitin mutants for 48 h, followed by MG132 treatment for 6 h. RIG-I ubiquitination was analyzed by Co-IP and Western blot. ( H ) Schematic diagram of the structural domain of RIG-I. ( I ) Following co-transfection with Myc-TOR3A and Flag-RIG-I domain plasmids for 48 h and MG132 treatment for 6 h, Co-IP and Western blot were performed to assess both the domain-specific interaction with TOR3A and the K48-linked ubiquitination of each RIG-I domain. Statistical significance was determined by student's t-test (B and C), one-way ANOVA followed by Dunett's multiple comparisons test (A). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Expressing, Infection, Co-Immunoprecipitation Assay, Cotransfection

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A targets RIG-I for degradation via the E3 ubiquitin ligase STUB1. ( A ) Mass spectrometry analysis of Myc-TOR3A-interacting proteins co-immunoprecipitated from HEK293 T cells identifies the E3 ubiquitin ligase STUB1 (n = 2). ( B ) Prediction of potential E3 ubiquitin ligases for RIG-I (DDX58) using the UbiBrowser database. ( C ) Co-IP and Western blot analysis of the interaction between Flag-RIG-I and HA-STUB1 in HEK293 T cells 8 h after co-transfection. ( D ) Co-immunoprecipitation (Co-IP) and Western blot analysis of the interaction between Myc-TOR3A and HA-STUB1 in HEK293 T cells after 48 h of co-transfection. ( E ) Co-IP of STUB1 in HeLa cells, followed by Western blot analysis to detect co-precipitated RIG-I and TOR3A. ( F ) Confocal microscopy analysis of the co-localization among HA-STUB1, Myc-TOR3A, and Flag-RIG-I in HeLa cells after 48 h of transfection. Scale bar, 10 μm. ( G ) Western blot analysis of RIG-I protein levels in RSV-infected (12 h) RAW264.7 cells with STUB1 knockout, with or without TOR3A overexpression. ( H ) Western blot analysis of exogenous Flag-RIG-I levels in HEK293 T cells after sequential transfection: first with STUB1-targeting siRNAs (si1, si2) for 24 h, followed by co-transfection with Myc-TOR3A and Flag-RIG-I plasmids for 48 h. ( I ) HEK293 T cells were co-transfected with HA-STUB1 and various Flag-tagged RIG-I domain plasmids for 48 h. Cell lysates were then subjected to co-immunoprecipitation (Co-IP) to assess both the interaction between STUB1 and each RIG-I domain, and the K48-linked ubiquitination levels on these domains. ( J ) Western blot analysis of Flag-RIG-I protein levels in HEK293 T cells co-expressing HA-STUB1 with either wild-type (WT) or site-mutated Flag-RIG-I plasmids for 48 h. ( K ) Co-IP analysis of K48-linked ubiquitination on wild-type (WT) or K146R mutant Flag-RIG-I in HEK293 T cells co-expressing HA-STUB1, following MG132 treatment. ( L ) RT-qPCR analysis of IFNB mRNA in RIG-I KO HEK293 T cells, reconstituted with wild-type or K146R mutant RIG-I and Myc-TOR3A, after RSV infection. Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (L). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Ubiquitin Proteomics, Mass Spectrometry, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Cotransfection, Confocal Microscopy, Transfection, Infection, Knock-Out, Over Expression, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Emerging Microbes & Infections

Article Title: TOR3A represses type I interferon production and limits viral clearance during respiratory syncytial virus infection

doi: 10.1080/22221751.2026.2637961

Figure Lengend Snippet: TOR3A facilitates the E3 ligase STUB1 in ubiquitinating RIG-I. ( A ) Western blot analysis of Flag-RIG-I protein levels in HEK293 T cells co-expressing HA-STUB1 and Myc-TOR3A for 48 h. ( B ) Co-IP and Western blot analysis of the interaction between HA-STUB1 and Flag-RIG-I in HEK293 T cells co-expressing Myc-TOR3A for 48 h. ( C ) Co-IP and Western blot analysis of K48-linked ubiquitination on Flag-RIG-I in HEK293 T cells co-expressing HA-STUB1 and Myc-TOR3A. ( D ) Western blot analysis of RIG-I, TOR3A, and STUB1 protein levels in wild-type versus TOR3A-knockout HEK293 T cells. Co-IP and Western blot analysis of the STUB1-RIG-I interaction in the same cell lines. ( E ) Co-IP and Western blot analysis of K48-linked polyubiquitination of RIG-I in wild-type versus TOR3A-knockout HEK293 T cells. ( F ) Western blot analysis of RIG-I protein levels in wild-type and TOR3A-knockout HEK293 T cells after 48 h transfection with the HA-STUB1 plasmid. ( G ) Structural model of TOR3A generated using PyMOL software. ( H ) Co-IP and Western blot analysis of the interaction between Flag-RIG-I and Myc-TOR3A domains in HEK293 T cells. ( I ) Co-IP and Western blot analysis of the interaction between HA-STUB1 and Myc-TOR3A domains in HEK293 T cells after MG132 treatment. ( J ) Western blot analysis of TOR3A, RIG-I, and STUB1 expression, and Co-IP/Western blot analysis of their ternary complex formation, in primary macrophages (PMs) at 12 h post-RSV infection. ( M ) Analysis of RIG-I ubiquitination by immunoprecipitation in RSV-infected (12 h) wild-type versus TOR3A-knockout PMs. ( L ) A model illustrating how TOR3A regulates innate immune signalling against RSV infection. Data are representative of three independent experiments and presented as mean ± SD. Statistical significance was determined by Student's t-test (C). ns: no significance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: The C57BL/6 TOR3A KO line was generated by Biomodel Organism Science & Technology Development Co., Ltd. (Shanghai, China) using CRISPR/Cas9.

Techniques: Western Blot, Expressing, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Knock-Out, Transfection, Plasmid Preparation, Generated, Software, Infection, Immunoprecipitation