Fig. 1: The IFN-stimulated protein OASL is required for efficient virus-induced necroptosis.
a, Mouse primary fibroblasts were mock-infected or infected with MCMV-M45mutRHIM virus (multiplicity of infection (m.o.i.) = 5) for 6 h. RIPK3 protein complexes were enriched and immunoprecipitated (IP) using RIPK3 antibody-conjugated agarose beads and analysed by mass spectrometry. Functional-related or biological-related proteins are grouped in boxes. b, HEK 293T cells were transfected with the indicated constructs, and cell lysates were immunoprecipitated with V5-specific antibody. Immunoprecipitates and whole cell extracts (input) were analysed by immunoblotting with the indicated antibodies. c, Cell death kinetics of Oasl1+/+ and Oasl1–/– primary fibroblasts infected with MCMV-WT or MCMV-M45mutRHIM (m.o.i. = 5). Necrotic cell death was measured on the basis of the uptake of Sytox Green and quantified in real-time from 4 to 12 h.p.i. (n = 4 biological replicates). d, Left: microscopy analysis of cell death in Oasl1+/+ and Oasl1–/– primary fibroblasts infected with MCMV-M45mutRHIM at 16 h.p.i. Arrows indicate cells with necrotic features after infection. Scale bar, 20 μm. Right: quantification of necrotic cell death by measuring the release of LDH from Oasl1+/+ and Oasl1–/– primary fibroblasts infected with MCMV-M45mutRHIM (m.o.i. = 5) for 8 h. e, Quantification of necrotic cell death by measuring intracellular ATP levels (n = 4) and LDH release (n = 3) in supernatants of Oasl1+/+ and Oasl1–/– primary fibroblasts infected with HSV-1 (m.o.i. = 5). f, Immunoblot analysis of RIPK3 and MLKL phosphorylation (P-RIPK3 and P-MLKL, respectively) in Oasl1+/+ and Oasl1–/– primary fibroblasts infected with MCMV-M45mutRHIM (left) or HSV-1 (right) at m.o.i. = 5 for the indicated hours. Infected cells were collected and subjected to immunoblotting with the indicated antibodies. g, Viral replication of MCMV-M45mutRHIM and HSV-1 in Oasl1+/+ (n = 4 for MCMV-M45mutRHIM, n = 3 for HSV-1) and Oasl1–/– (n = 2) primary fibroblasts were determined by plaque assays by titrating culture supernatants at the indicating time points. Data are representative of two (b,f,g) or three (c–e) independent experiments. For c–e,g, data are presented as the mean ± s.e.m. Statistical analyses were performed using two-tailed unpaired t-test (d) or two-way analysis of variance (ANOVA) (c–e,g). NS, not significant.
