Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Distribution of transposable element (TE) classes among loci up-regulated by BT in bulk RNA-seq (counts of LTR, DNA, LINE, SINE, Satellite and Unknown elements). (B) Reactome terms enriched among BT-reactivated ERV loci. (C) GSEA enrichment plots in BT-versus DMSO-treated HT1080 cells. (D) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for dsRNA (green) and nuclei (DAPI, blue). BT treatment induces punctate cytosolic dsRNA accumulations. Scale bars, 5 µm. (E) RT-qPCR of IFNB1 mRNA in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (F) Secreted type I IFN measured by ELISA in the same HeLa cell lines and treatments as in (E). (G) Western blot of p-TBK1, total TBK1, MDA5, MAVS and RIG-I, the samples were same to (E). (H) Flow cytometric quantification of VSV-GFP infection in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (I-J) Left are survival curves of MAVS knockout C57BL/6J mice challenged with lethal VSV and HSV-1 after a single i.p. dose of BT (50 mg/kg). Right are viruses RNA levels in livers measured by RT-qPCR. RIP-qPCR detection of HERVK14-int RNA associated with RIG-I or MDA5 in RAW 264.7 cells treated with DMSO or BT for 12 h. (L) ELISA quantification of secreted type I IFN and RT-qPCR of IFNB1 mRNA. (M) Flow cytometry of VSV-GFP infection. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Rabbit antibodies against TBK1 (catalog no. 28397-1-AP), Beta Tubulin (catalog no. 10094-1-AP), RIG-1/DDX58 (catalog no. 25068-1-AP), MAVS (catalog no. 14341-1-AP) and IFIT3 (catalog no. 15201-1-AP), along with mouse antibodies targeting Beta Actin (catalog no. 66009-1-Ig) and the GFP tag (catalog no. 50430-2-AP), were procured from Proteintech.

Techniques: RNA Sequencing, Staining, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Heatmap of bulk RNA-seq in HT1080 cells treated with 100 µM BT versus DMSO for 12 hours. (B) Gene set enrichment analysis (left) and over-representation analysis (right) of BT-regulated genes, highlighting significant enrichment of antiviral and innate immunity pathways (GO and KEGG). (C) Volcano plot of differential expression in BT-versus DMSO-treated HT1080 cells (|log₂FC|>1, FDR<0.05). (D) RT-qPCR quantification of Ifnb1 and Oas2 mRNA in mouse bone marrow–derived macrophages (BMDMs) treated with indicated concentrations of BT for 12 hours. (E) Dose-dependent induction of IFNB1 mRNA (RT-qPR, top panels) and corresponding protein responses (western blots, bottom panels) in RAW 264.7, HT1080, HT29 and HeLa cells treated with the indicated BT concentrations for 12 hours. (F) RT-qPCR of IFNB1 and OAS2 in human PBMCs. (G) In vivo induction of Ifnb1 and Oas2 mRNA in heart, liver, lung, spleen and kidney of C57BL/6J mice 6 hours after a single intraperitoneal injection of BT (50 mg/kg). (H) RT-qPCR of Ifnb1 in RAW 264.7 cells following BT treatment (50, 75, 100 µM) for 12 hours in wild-tpye and knockout cells. Western blots showed absence of Viperin, IFIT3 and OAS2 induction in TBK1 knockout cells. (I) Flow cytometry of VSV-GFP infection in WT and TBK1 knockout RAW 264.7 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. Numbers indicated percentage of GFP positive cells. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Rabbit antibodies against TBK1 (catalog no. 28397-1-AP), Beta Tubulin (catalog no. 10094-1-AP), RIG-1/DDX58 (catalog no. 25068-1-AP), MAVS (catalog no. 14341-1-AP) and IFIT3 (catalog no. 15201-1-AP), along with mouse antibodies targeting Beta Actin (catalog no. 66009-1-Ig) and the GFP tag (catalog no. 50430-2-AP), were procured from Proteintech.

Techniques: RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Derivative Assay, Western Blot, In Vivo, Injection, Knock-Out, Flow Cytometry, Infection

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV proteins regulate RIG-I-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with GST-RIG-IN to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting and luciferase activity assays. ( B and C ) HEK293T cells were transfected with GST-RIG-IN and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA , IFNB1 ( B ), and ISG15 , OAS1 ( C ) were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the RIG-IN-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Activity Assay, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 promotes RIG-I degradation via autophagy. ( A ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, cells were analyzed by immunoblotting with RIG-I antibody. Gray-scale statistical analysis of RIG-I relative to β-Actin is displayed on the right. ( B ) HEK293T cells were transfected with Flag-VP2, along with or without GST-RIG-IN. At 48 hpt, cells were analyzed by immunoblotting with the IFIT1 and IFIT3 antibodies. Gray-scale statistical analysis of IFIT1, IFIT3, and RIG-I-N relative to β-actin is displayed on the right. ( C ) HEK293T cells were transfected with Flag-VP2 or vector, along with poly(I:C). At 24 hpt, the mRNA levels of host DDX58 were examined using qPCR, with GAPDH serving as the internal reference control. ( D ) HEK293T cells were co-transfected with VP2 and RIG-I plasmids. At 24 hpt, cells were treated with the inhibitors MG132 (10 μM), CQ (10 μM), and 3-MA (10 mM) for 12 h. The cell lysates were analyzed by immunoblotting. ( E ) HEK293T cells were co-transfected with GFP-LC3B (Green), Flag-tag (Orange), and HA-RIG-I (Red) plasmids for 48 h and subjected to immunofluorescence staining. Nuclei were stained with DAPI. Scale bars, 10 µm. ( F ) HEK293T cells were transfected with siCon and siATG5 and cultured for 24 h, then co-transfected with VP2 and RIG-I plasmids for 24 h. The cells were analyzed by immunoblotting. Gray-scale statistical analysis of RIG-I relative to β-Actin is displayed on the right. Data from independent experiments ( n ≥ 3) were statistically analyzed using one- or two-way ANOVA with multiple comparison correction (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 and ns, not significant).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, FLAG-tag, Immunofluorescence, Staining, Cell Culture, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV proteins regulate MDA5-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Activity Assay, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 suppresses IFN-I production by targeting the upstream of TBK1. ( A–D ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV VP2 protein, along with MAVS ( A ), TRAF3 ( B ), TBK1 ( C ), and IRF3 ( D ) expressing plasmids to induce IFN-I production. At 24 hpt, cells were subjected to the luciferase activity assay and immunoblotting analysis using the phosphorylation and total antibodies of IRF3 and TBK1, along with the tag antibodies. Gray-scale statistical analysis of phosphorylation relative to total protein is conducted. ( E and F ) HEK293T cells were transfected with plasmids expressing ALSV VP2 protein, along with the indicated signaling molecules. At 48 hpt, cells were subjected to immunoblotting analysis. The relative levels of the indicated proteins normalized to β-Actin are shown. Data from independent experiments ( n ≥ 3) were statistically analyzed using two-way ANOVA with multiple comparison correction (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 suppresses IFN-I production by targeting the upstream of TBK1. ( A–D ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV VP2 protein, along with MAVS ( A ), TRAF3 ( B ), TBK1 ( C ), and IRF3 ( D ) expressing plasmids to induce IFN-I production. At 24 hpt, cells were subjected to the luciferase activity assay and immunoblotting analysis using the phosphorylation and total antibodies of IRF3 and TBK1, along with the tag antibodies. Gray-scale statistical analysis of phosphorylation relative to total protein is conducted. ( E and F ) HEK293T cells were transfected with plasmids expressing ALSV VP2 protein, along with the indicated signaling molecules. At 48 hpt, cells were subjected to immunoblotting analysis. The relative levels of the indicated proteins normalized to β-Actin are shown. Data from independent experiments ( n ≥ 3) were statistically analyzed using two-way ANOVA with multiple comparison correction (*** P < 0.001 and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Luciferase, Activity Assay, Western Blot, Phospho-proteomics, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV proteins regulate MDA5-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Activity Assay, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 promotes RIG-I degradation via autophagy. ( A ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, cells were analyzed by immunoblotting with RIG-I antibody. Gray-scale statistical analysis of RIG-I relative to β-Actin is displayed on the right. ( B ) HEK293T cells were transfected with Flag-VP2, along with or without GST-RIG-IN. At 48 hpt, cells were analyzed by immunoblotting with the IFIT1 and IFIT3 antibodies. Gray-scale statistical analysis of IFIT1, IFIT3, and RIG-I-N relative to β-actin is displayed on the right. ( C ) HEK293T cells were transfected with Flag-VP2 or vector, along with poly(I:C). At 24 hpt, the mRNA levels of host DDX58 were examined using qPCR, with GAPDH serving as the internal reference control. ( D ) HEK293T cells were co-transfected with VP2 and RIG-I plasmids. At 24 hpt, cells were treated with the inhibitors MG132 (10 μM), CQ (10 μM), and 3-MA (10 mM) for 12 h. The cell lysates were analyzed by immunoblotting. ( E ) HEK293T cells were co-transfected with GFP-LC3B (Green), Flag-tag (Orange), and HA-RIG-I (Red) plasmids for 48 h and subjected to immunofluorescence staining. Nuclei were stained with DAPI. Scale bars, 10 µm. ( F ) HEK293T cells were transfected with siCon and siATG5 and cultured for 24 h, then co-transfected with VP2 and RIG-I plasmids for 24 h. The cells were analyzed by immunoblotting. Gray-scale statistical analysis of RIG-I relative to β-Actin is displayed on the right. Data from independent experiments ( n ≥ 3) were statistically analyzed using one- or two-way ANOVA with multiple comparison correction (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 and ns, not significant).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, FLAG-tag, Immunofluorescence, Staining, Cell Culture, Comparison

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Heatmap of bulk RNA-seq in HT1080 cells treated with 100 µM BT versus DMSO for 12 hours. (B) Gene set enrichment analysis (left) and over-representation analysis (right) of BT-regulated genes, highlighting significant enrichment of antiviral and innate immunity pathways (GO and KEGG). (C) Volcano plot of differential expression in BT-versus DMSO-treated HT1080 cells (|log₂FC|>1, FDR<0.05). (D) RT-qPCR quantification of Ifnb1 and Oas2 mRNA in mouse bone marrow–derived macrophages (BMDMs) treated with indicated concentrations of BT for 12 hours. (E) Dose-dependent induction of IFNB1 mRNA (RT-qPR, top panels) and corresponding protein responses (western blots, bottom panels) in RAW 264.7, HT1080, HT29 and HeLa cells treated with the indicated BT concentrations for 12 hours. (F) RT-qPCR of IFNB1 and OAS2 in human PBMCs. (G) In vivo induction of Ifnb1 and Oas2 mRNA in heart, liver, lung, spleen and kidney of C57BL/6J mice 6 hours after a single intraperitoneal injection of BT (50 mg/kg). (H) RT-qPCR of Ifnb1 in RAW 264.7 cells following BT treatment (50, 75, 100 µM) for 12 hours in wild-tpye and knockout cells. Western blots showed absence of Viperin, IFIT3 and OAS2 induction in TBK1 knockout cells. (I) Flow cytometry of VSV-GFP infection in WT and TBK1 knockout RAW 264.7 cells treated with BT (50, 75, 100 µM) and infected (MOI = 0.1) for 12 h. Numbers indicated percentage of GFP positive cells. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Rabbit antibodies against TBK1 (catalog no. 28397-1-AP), Beta Tubulin (catalog no. 10094-1-AP), RIG-1/DDX58 (catalog no. 25068-1-AP), MAVS (catalog no. 14341-1-AP) and IFIT3 (catalog no. 15201-1-AP), along with mouse antibodies targeting Beta Actin (catalog no. 66009-1-Ig) and the GFP tag (catalog no. 50430-2-AP), were procured from Proteintech.

Techniques: RNA Sequencing, Quantitative Proteomics, Quantitative RT-PCR, Derivative Assay, Western Blot, In Vivo, Injection, Knock-Out, Flow Cytometry, Infection

Journal: bioRxiv

Article Title: Betrixaban Activates cGAS and ERVs to Promote Dual Nucleic-Sensing Antiviral Immunity

doi: 10.1101/2025.08.08.669242

Figure Lengend Snippet: (A) Distribution of transposable element (TE) classes among loci up-regulated by BT in bulk RNA-seq (counts of LTR, DNA, LINE, SINE, Satellite and Unknown elements). (B) Reactome terms enriched among BT-reactivated ERV loci. (C) GSEA enrichment plots in BT-versus DMSO-treated HT1080 cells. (D) Confocal micrographs of RAW 264.7 cells treated with DMSO or 50 µM BT for 12 h, stained for dsRNA (green) and nuclei (DAPI, blue). BT treatment induces punctate cytosolic dsRNA accumulations. Scale bars, 5 µm. (E) RT-qPCR of IFNB1 mRNA in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (F) Secreted type I IFN measured by ELISA in the same HeLa cell lines and treatments as in (E). (G) Western blot of p-TBK1, total TBK1, MDA5, MAVS and RIG-I, the samples were same to (E). (H) Flow cytometric quantification of VSV-GFP infection in HeLa wild-type (WT), MDA5 knockout, RIG-I knockout and MAVS knockout cells treated with BT. (I-J) Left are survival curves of MAVS knockout C57BL/6J mice challenged with lethal VSV and HSV-1 after a single i.p. dose of BT (50 mg/kg). Right are viruses RNA levels in livers measured by RT-qPCR. RIP-qPCR detection of HERVK14-int RNA associated with RIG-I or MDA5 in RAW 264.7 cells treated with DMSO or BT for 12 h. (L) ELISA quantification of secreted type I IFN and RT-qPCR of IFNB1 mRNA. (M) Flow cytometry of VSV-GFP infection. Data are shown as mean ± SEM. N.S., not significant, p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: Rabbit antibodies against TBK1 (catalog no. 28397-1-AP), Beta Tubulin (catalog no. 10094-1-AP), RIG-1/DDX58 (catalog no. 25068-1-AP), MAVS (catalog no. 14341-1-AP) and IFIT3 (catalog no. 15201-1-AP), along with mouse antibodies targeting Beta Actin (catalog no. 66009-1-Ig) and the GFP tag (catalog no. 50430-2-AP), were procured from Proteintech.

Techniques: RNA Sequencing, Staining, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay, Western Blot, Infection, Flow Cytometry