Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 negatively regulated IFN-β induced by H13N2 infection. (a, B) luciferase reporter assays were used to assess the impact of FGF8 overexpression on IFN-β and ISRE promoter activity in A549 cells infected with H13N2 at an MOI of 1. (C-F) FGF8-overexpressing A549 cells were infected with H13N2 at an MOI of 1. At 12 hours post-infection (hpi), IFN-β levels in the cell supernatant were measured using ELISA (C), and IFN-β mRNA levels were evaluated by RT-qPCR (d). At 24 hpi, the mRNA levels of interferon-stimulated genes MX1 (e) and IFIT1 (f) were assessed by RT-qPCR. (G-J) stable FGF8-knockdown A549 cells were infected with H13N2 at an MOI of 1. At 12 hpi, IFN-β levels in the cell supernatant were quantified by ELISA (G), and IFN-β mRNA levels were evaluated using RT-qPCR (H). At 24 hpi, the mRNA levels of MX1 (i) and IFIT1 (J) were assessed by RT-qPCR. (K and L) Western blot analysis evaluated RIG-I, p-TBK1, and p-IRF3 expression in A549 cells with FGF8 overexpression (L) or knockdown (K) at 12 hours after H13N2 infection (MOI = 1). Band intensities were quantified by densitometric analysis. Statistical analysis was performed using two-tailed unpaired Student’s t-tests, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Infection, Luciferase, Over Expression, Activity Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: FGF8 drives ubiquitin – proteasomal degradation of RIG-I. (a) FGF8 inhibits RIG-I-mediated signaling. A luciferase reporter assay was performed to evaluate the effect of FGF8 overexpression on IFN-β promoter activation induced by RIG-I. (B and C) FGF8 does not affect RIG-I transcription. RIG-I mRNA levels were quantified by RT-qPCR in FGF8-overexpressing A549 cells at 0, 6, and 12 hours post-infection with H13N2 (b) or H1N1 (C) at an MOI of 1. (d) dose-dependent reduction of RIG-I protein. A549 cells were transfected with increasing amounts of Flag-FGF8 plasmid for 24 hours, followed by infection with H13N2 (MOI = 1) for 12 hours. RIG-I protein levels were analyzed by Western blot, and band intensities were quantified by densitometry. (e) FGF8 reduces RIG-I stability. FGF8-overexpressing A549 cells were infected with H13N2 (MOI = 1) and treated with cycloheximide (CHX, 50 µg/mL) for the indicated time periods. Protein levels were analyzed by Western blot, and the relative abundance of HA-RIG-I was quantified to assess protein degradation rates. (F and G) proteasome inhibition restores RIG-I levels. A549 cells infected with H13N2 (f) or H1N1 (G) at an MOI of 1 were treated with DMSO, chloroquine (CQ, 50 µM), or MG132 (10 µM) for 6 hours. RIG-I expression was analyzed by Western blot, with relative protein levels quantified by densitometry. (H and I) FGF8 promotes K48-linked ubiquitination of RIG-I. HEK-293T cells were co-transfected with the indicated plasmids and treated with MG132 for 6 hours. (H) Total ubiquitination of RIG-I was assessed by immunoprecipitation with anti-HA antibody followed by immunoblotting (ib) with anti-Myc. (i) K48- or K63-linked ubiquitination was analyzed using specific ubiquitin mutants. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Luciferase, Reporter Assay, Over Expression, Activation Assay, Quantitative RT-PCR, Infection, Transfection, Plasmid Preparation, Western Blot, Inhibition, Expressing, Immunoprecipitation, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: Identification of the ubiquitination site on RIG-I targeted by FGF8. (a) diagram illustrating the truncated constructs of RIG-I. (b) HEK-293T cells were co-transfected with specified plasmids and exposed to MG132 for 6 hours. Western blot analysis was conducted to assess the ubiquitination of various RIG-I truncation constructs. (C) Western blot analysis identified the ubiquitination site on RIG-I targeted by FGF8, and band intensities were quantified by densitometry to assess the degradation of each mutant. (d) a dual-luciferase assay was conducted in HEK293T cells co-transfected with specified RIG-I mutants and FGF8 to evaluate the impact of FGF8 on IFN-β promoter activity. Error bars indicate the mean ± SEM from three independent experiments. Two-tailed unpaired Student’s t-tests were used. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Ubiquitin Proteomics, Construct, Transfection, Western Blot, Mutagenesis, Luciferase, Activity Assay, Two Tailed Test

Journal: Virulence

Article Title: FGF8-mediated TRIM16 regulation promotes K48-linked ubiquitination and degradation of RIG-I to facilitate Influenza a virus immune evasion

doi: 10.1080/21505594.2026.2677346

Figure Lengend Snippet: TRIM16 mediated RIG-I degradation and promoted influenza virus replication. (a) Co-immunoprecipitation analysis was performed in cells transfected with Flag-TRIM16 and HA-RIG-I, with or without H13N2 infection (MOI = 1), to verify the interaction. (b) immunofluorescence microscopy showing the localization of TRIM16 (green) and RIG-I (red) in cells infected with H13N2 or mock-infected (NC). Nuclei were stained with DAPI (blue). Note that TRIM16 and RIG-I show diffuse distribution in the NC group but form co-localized puncta (yellow) upon H13N2 infection. Scale bar: 5 μm. (C) in vitro ubiquitination assay to verify the direct E3 ligase activity of TRIM16 using wt and ΔB-Box mutant proteins. (d) in vitro ubiquitination assay to determine the linkage specificity of TRIM16-mediated RIG-I ubiquitination using K48-only and K63-only ubiquitin mutants. (e) bioinformatic analysis using PONDR revealed the presence of intrinsically disordered regions (IDRs) in the FGF8 protein sequence. (f) fluorescence microscopy of A549 cells transfected with EGFP-FGF8 (green). Nuclei were stained with DAPI. Scale bar represents 10 μm. (G) TurboID-based proximity labeling assay was performed in cells expressing FGF8-TurboID. Biotinylated proteins were captured using streptavidin beads, and the pulled-down proteins were analyzed by Western blot to detect the presence of RIG-I and TRIM16. (H and I) validation of TRIM16 knockdown. RT-qPCR (H) and Western blot (i) confirmed the silencing efficiency in A549 cells. (J) control and TRIM16-silenced A549 cells were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein levels (NP, PB1, PB2) were analyzed by Western blot, and band intensities were quantified by densitometry. (K) RT-qPCR analysis of IFN-β mRNA levels in TRIM16-silenced A549 cells 12 hours post-infection with H13N2 (MOI = 1). (L) Western blot confirmation of TRIM16 overexpression (OE-TRIM16). (M) A549 cells overexpressing TRIM16 were infected with H1N1 or H13N2 (MOI = 0.5) for 24 hours. Viral protein expression was analyzed by Western blot and quantified by densitometry. Error bars indicate the mean ± SEM from three independent experiments. Statistical analysis was performed using two-tailed unpaired Student’s t-tests. ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: To characterize the direct E3 ligase activity of TRIM16 and its specific ubiquitin linkage in a cell-free system, recombinant human UbcH5b (HY- P79449 , MedChemExpress) and RIG-I protein (TP317615, OriGene) were employed as the E2 conjugating enzyme and substrate, respectively.

Techniques: Virus, Immunoprecipitation, Transfection, Infection, Immunofluorescence, Microscopy, Staining, In Vitro, Ubiquitin Proteomics, Activity Assay, Mutagenesis, Sequencing, Fluorescence, Labeling, Expressing, Western Blot, Biomarker Discovery, Knockdown, Quantitative RT-PCR, Control, Over Expression, Two Tailed Test