Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. The interaction of LSDV122 with IFNAR1 and IFNAR2 in mammalian overexpression system. HEK293T cells (5 × 10 6 ) 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 × 10 7 ) 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 × 10 4 ) 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 × 10 4 ) 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 × 10 7 ) 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 × 10 6 ) 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.

Article Snippet: Mouse monoclonal antibodies against HA (66006, Proteintech), Rabbit monoclonal antibodies against HA (H6908, Sigma), Mouse monoclonal antibodies against Flag (AP0530, Sigma), HRP-Flag (ZB15939, Servicebio), IgG (I5381 and I5006, Sigma), p-JAK1 (3331S, Cell Signaling Technology), JAK1 (A25841, ABclonal), p-TYK2 and TYK2 (9321S and 14193S, Cell Signaling Technology), p-STAT1 (9167S, Cell Signaling Technology), STAT1 (sc-417, Santa Cruz Biotechnology), p-STAT2 and STAT2 (88410S and 72604S, Cell Signaling Technology), IFNAR1 (A0575, ABclonal), IFNAR2 (53883s, Cell Signaling Technology), PE anti-DYKDDDDK Tag (Biolegend, 637310), Anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (8890, Cell Signaling Technology) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were purchased from the specified manufacturers.

Techniques: Over Expression, Transfection, Control, Western Blot, Plasmid Preparation, Expressing, Immunostaining, Confocal Microscopy, Infection, Immunoprecipitation

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. A schematic presentation of IFNAR1 and IFNAR2 truncations and their abilities to interact with LSDV122. − , no interaction; + , positive interaction. B. The interaction of IFNAR1 or IFNAR2 truncations with LSDV122. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. C. A schematic presentation of LSDV122 truncations and their abilities to interact with IFNAR1 and IFNAR2. D. The interaction of LSDV122 truncations with IFNAR1 or IFNAR2. HEK293T cells (5 × 10 6 ) were transfected with the indicated plasmids for 24 hours, followed by coimmunoprecipitation and immunoblotting analysis with the indicated antibodies. E. Effects of LSDV122 and its truncations on IFN-β-induced transcription of downstream genes. HEK293T cells (2 × 10 5 ) were transfected with either an empty vector, LSDV122 or its truncations for 20 hours. The cells were then left untreated or treated with IFN-β (100 ng/ml) for 10 hours before RT-qPCR experiments. Data shown are mean ± SD (n = 3) from one representative experiment. These experiments were repeated at least twice with similar results. ns nonsignificant, *P < 0.05, **P < 0.01 (unpaired t-test).

Article Snippet: Mouse monoclonal antibodies against HA (66006, Proteintech), Rabbit monoclonal antibodies against HA (H6908, Sigma), Mouse monoclonal antibodies against Flag (AP0530, Sigma), HRP-Flag (ZB15939, Servicebio), IgG (I5381 and I5006, Sigma), p-JAK1 (3331S, Cell Signaling Technology), JAK1 (A25841, ABclonal), p-TYK2 and TYK2 (9321S and 14193S, Cell Signaling Technology), p-STAT1 (9167S, Cell Signaling Technology), STAT1 (sc-417, Santa Cruz Biotechnology), p-STAT2 and STAT2 (88410S and 72604S, Cell Signaling Technology), IFNAR1 (A0575, ABclonal), IFNAR2 (53883s, Cell Signaling Technology), PE anti-DYKDDDDK Tag (Biolegend, 637310), Anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (8890, Cell Signaling Technology) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were purchased from the specified manufacturers.

Techniques: Transfection, Western Blot, Plasmid Preparation, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. Effects of LSDV122 on binding of IFN-I to IFNAR. HEK293T cells (2 × 10 5 ) 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 × 10 6 ) 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 × 10 7 ) 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.

Article Snippet: Mouse monoclonal antibodies against HA (66006, Proteintech), Rabbit monoclonal antibodies against HA (H6908, Sigma), Mouse monoclonal antibodies against Flag (AP0530, Sigma), HRP-Flag (ZB15939, Servicebio), IgG (I5381 and I5006, Sigma), p-JAK1 (3331S, Cell Signaling Technology), JAK1 (A25841, ABclonal), p-TYK2 and TYK2 (9321S and 14193S, Cell Signaling Technology), p-STAT1 (9167S, Cell Signaling Technology), STAT1 (sc-417, Santa Cruz Biotechnology), p-STAT2 and STAT2 (88410S and 72604S, Cell Signaling Technology), IFNAR1 (A0575, ABclonal), IFNAR2 (53883s, Cell Signaling Technology), PE anti-DYKDDDDK Tag (Biolegend, 637310), Anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (8890, Cell Signaling Technology) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were purchased from the specified manufacturers.

Techniques: Binding Assay, Transfection, Incubation, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Expressing, Immunoprecipitation, Control

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: 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.

Article Snippet: Mouse monoclonal antibodies against HA (66006, Proteintech), Rabbit monoclonal antibodies against HA (H6908, Sigma), Mouse monoclonal antibodies against Flag (AP0530, Sigma), HRP-Flag (ZB15939, Servicebio), IgG (I5381 and I5006, Sigma), p-JAK1 (3331S, Cell Signaling Technology), JAK1 (A25841, ABclonal), p-TYK2 and TYK2 (9321S and 14193S, Cell Signaling Technology), p-STAT1 (9167S, Cell Signaling Technology), STAT1 (sc-417, Santa Cruz Biotechnology), p-STAT2 and STAT2 (88410S and 72604S, Cell Signaling Technology), IFNAR1 (A0575, ABclonal), IFNAR2 (53883s, Cell Signaling Technology), PE anti-DYKDDDDK Tag (Biolegend, 637310), Anti-mouse IgG (H + L), F(ab’)2 Fragment (Alexa Fluor 594 Conjugate) (8890, Cell Signaling Technology) and Alexa Fluor 488 goat anti-rabbit IgG (H + L) (A11008, Invitrogen) were purchased from the specified manufacturers.

Techniques: Infection