Fig. 2: OASL chaperones the assembly of the RIPK3–ZBP1 necrosome complex.
a, Oasl1+/+ and Oasl1–/– primary fibroblasts were infected with MCMV-M45mutRHIM (m.o.i. = 5) for 0 or 8 h. Whole-cell lysates were immunoprecipitated using an anti-RIPK3 antibody, followed by immunoblot analysis using the indicated anti-ZBP1, anti-OASL1, anti-RIPK3 or anti-PKR antibody. b,c, In situ PLA using anti-RIPK3 and anti-ZBP1 antibodies in Oasl1+/+ and Oasl1–/– primary fibroblasts infected with MCMV-M45mutRHIM (m.o.i. = 5) for 0 or 6 h. Fluorescence microscopy was used to detect the discrete fluorescent PLA signals (red dots). Representative images of dots indicate the interaction of endogenous RIPK3 and ZBP1. Scale bar, 10 μm. c, Quantification of the dot signals of b per cell (n = 32 cells). Each dot indicates a single cell. d,e, HEK 293T cells were transfected with the indicated Flag, HA, V5 or untagged constructs, and cell lysates were immunoprecipitated with anti-RIPK3 (d) or anti-HA antibodies (e). Immunoprecipitates and whole cell extracts (input) were analysed by immunoblotting with the indicated antibodies. f, AH109 yeasts were transformed with pGBKT7-OASL1 or pACT2-ZBP1 constructs. Interactions were determined by triple dropout (Trp-Leu-His-) synthetic medium or secreted α-galactosidase activity assay using X-Gal. g, OASL1-mediated phosphorylation of the necroptosis effector MLKL. Oasl1–/– primary fibroblasts were complemented with empty vector (Vector), HA-tagged full-length OASL1, N-OAS domain or C-UBL domain and infected with MCMV-M45mutRHIM or HSV-1 (m.o.i. = 5) for the indicated times. Total cell lysates were subjected to immunoblot analysis with the indicated antibodies. h, Measurement of necrotic cell death on the basis of released LDH in Oasl1–/– primary fibroblasts reconstituted with empty vector or the indicated OASL1 constructs infected with HSV-1 (m.o.i. = 5) at various time points. Data are representative of two (b–f) or three (a,g,h) independent experiments. For c,h, data are presented as the mean ± s.e.m. and statistical analysis was performed using two-way ANOVA.
