Fig 2: IFNAR2 knockdown inhibits glioma pathobiology. (A) Western blot of IFNAR2 after shRNA knockdown (representative blot with densitometry normalized to GAPDH; n = 3; unpaired two−tailed t test). (B, C) CCK-8 assays showing proliferation capacity in U87 and U251 cells with IFNAR2 knockdown, n=3. (D, E) Clonogenic potential of IFNAR2-deficient U87 and U251 cells, n=3. (F–I) Transwell migration and invasion assays demonstrating impaired motility in IFNAR2-knockdown cells, cells/field; n=3. (J) Histological analyses of orthotopic glioma shNC, shIFNAR2#1, and shIFNAR2#2 groups. (K) Quantification of orthotopic xenograft volumes. (L) KM curves of glioma-bearing mice. **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ### P< 0.001.

Fig 3: A model of antagonization of IFN-I signaling by LSDV122.Following LSDV infection, the viral protein LSDV122 interacts with both subunits of the IFN-I receptor, IFNAR1 and IFNAR2, via their respective transmembrane and intracellular domains. This impairs the assembly of the IFNAR complex and recruitment of downstream kinases JAK1 and TYK2 to the complex, leading to suppression of IFN-I-triggered induction of ISGs and antiviral response.

Fig 4: LSDV122 disrupts IFNAR complex formation and kinase recruitments.A. Effects of LSDV122 on binding of IFN-I to IFNAR. HEK293T cells (2 × 105) were transfected with the indicated plasmids for 24 hours. The cells were then incubated with Flag-tagged IFN-α2 or IFN-β (+, 200 ng; ++, 800 ng) for 30 minutes at 4°C, followed by surface staining with an anti-Flag antibody. The binding of IFN-α2 or IFN-β to the cell surface was analyzed by flow cytometry. B. Effects of LSDV122 on IFNAR complex formation and its recruitment of downstream kinases. HEK293T cells (5 × 106) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. C. Effects of LSDV122 on assembly of endogenous IFNAR complex and its downstream kinases. HEK293T cells (2 × 107) were transfected with an empty vector or LSDV122-expressing plasmid for 24 hours. The cells were either left untreated or treated with IFN-β (200 ng/ml) for 30 minutes, then harvested for immunoprecipitation using control IgG, anti-IFNAR1 or anti-IFNAR2 antibodies. The lysates and immunoprecipitates were subjected to immunoblotting analysis with the indicated antibodies. These experiments were repeated at least twice with similar results.

Fig 5: LSDV122 interacts with IFNAR1 and IFNAR2.A. The interaction of LSDV122 with IFNAR1 and IFNAR2 in mammalian overexpression system. HEK293T cells (5 × 106) were transfected with the indicated plasmids for 24 hours and then lysed for coimmunoprecipitation with control IgG or anti-HA antibody, followed by immunoblotting analysis with the indicated antibodies. B. The interaction of LSDV122 with endogenous IFNAR1 and IFNAR2. HEK293T cells (2 × 107) were transfected with an empty vector or LSDV122-expressing plasmid for 24 hours and then lysed for coimmunoprecipitation with anti-Flag antibody, followed by immunoblotting analysis with the indicated antibodies. C. Co-localization of LSDV122 with IFNAR1 and IFNAR2. HeLa cells (2 × 104) were transfected with the indicated plasmids for 20 hours and then fixed for immunostaining before subjected to confocal microscopy. D. Subcellular distribution of LSDV122 during LSDV infection. MDBK cells (6 × 104) were infected with LSDV-GFP (MOI = 2) for 18 hours and then fixed for immunostaining before subjected to confocal microscopy. E. The interaction of endogenous LSDV122 with IFNAR1 and IFNAR2 during LSDV infection. MDBK cells (5 × 107) were infected with LSDV (MOI = 2) for 18 hours and the cells were harvested for immunoprecipitation with pre-immune serum or anti-LSDV122 serum. The lysates and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. F. The interactions of LSDV122 and its homologous proteins with IFNAR1 and IFNAR2 in mammalian overexpression system. HEK293T cells (5 × 106) were transfected with the indicated plasmids for 24 hours and then lysed for coimmunoprecipitation with control IgG or anti-Flag antibody, followed by immunoblotting analysis with the indicated antibodies. These experiments were repeated at least twice with similar results.