Fig 2: TERRA triggers a MAVS-dependent IFN response.a, Experimental timeline. Growing (PD35) IMR90E6E7 expressing WT ZBP1(S)-Flag with either TRF1(?N) or VP16-TRF1(?N) were treated with doxycycline (1 µg ml-1) prior to two sequential siRNA transfections. Samples were collected at day 8 post-doxycycline. b, Immunoblotting of cells as in a at day 8 post-doxycycline. GAPDH loading control. Three independent experiments were performed. c, Scatter plots showing RT-qPCR analysis of ISGs in cells as in b. Expression levels were normalized to control cells TRF1(?N) transfected with non-targeting control (siCTR) siRNA. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Three independent experiments were performed. d, Line chart showing growth curves analysis of IMR90E6E7 that have bypassed senescence expressing control sgRNAs (sgGFP) or sgRNAs targeting MAVS or STING. Two independent experiments were performed. e, Immunoblotting of IMR90E6E7 as in a. Protein extracts were collected at day 30 when control cells entered crisis. GAPDH loading control. Two independent experiments were performed. f, Heatmap representing RNA-sequencing data for ISGs in IMR90E6E7 as in a. RNA extracts were collected at day 30 when control cells entered crisis. ISGs that are significantly downregulated (sgMAVS versus sgGFP) with a fold change > 1.5 and false-discovery rate <0.05 are shown. The complete list of ISGs includes 390 genes and is based on the RNAseq analysis of 5 cell types transfected with 20 viruses from 9 virus classes51. One experiment was performed. Abbreviations: ZBP1(S): short isoform; sgRNA: single guide RNA; CTR: control; WT: wild-type; PDs: population doublings. Source data

Fig 3: CNTN1 specifically interacts with USP25.(A) Overexpressed CNTN1 interacts with USP25. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis. (B) Overexpressed USP25 interacts with HA-MAVS. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis. (C) Endogenous USP25 interacts with CNTN1. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis. (D) USP25 interacts with MAVS directly. Purified GST-USP25 was used to pull-down purified His-MAVS. (E) CNTN1 interacts with USP25 directly. Purified GST-CNTN1 was used to pull-down purified His-USP25. (F) Endogenous MAVS interacts with USP25. HEK293 cells were left uninfected or infected with H5N6 virus for 24 h before co-immunoprecipitation and immunoblotting analysis. (G) CNTN1 colocalizes with USP25 in the cell cytoplasm. U2OS cells were transfected with a Flag-CNTN1 plasmid for 24 h, and subsequently left uninfected or infected with H5N6 virus for 12 h before staining with mito-tracker. In the zoomed images, the yellow color indicated the colocalization of Flag-CNTN1 and USP25. (H) USP25 colocalizes with MAVS in the cell cytoplasm. U2OS cells were transfected with a Flag-USP25 plasmid for 24 h, and subsequently left uninfected or infected with H5N6 virus for 12 h before staining with mito-tracker. In the zoomed images, the yellow color indicated the colocalization of Flag-USP25 and MAVS, the purple color indicated the colocalization of mitochondria and MAVS. (I) CNTN1-deficiency inhibits the interaction between USP25 and MAVS. CNTN1+/+ or CNTN1-/- HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis. (J) MAVS-deficiency inhibits the interaction between USP25 and CNTN1. MAVS+/+ or MAVS-/- HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis.

Fig 4: CNTN1 facilitates USP25 to degrade MAVS through the proteasomal pathway.(A) USP25 inhibits K63-linked ubiquitination of MAVS. HEK293 cells transfected with Myc-MAVS, HA-ubiquitin, or its mutants (K48O, K63O), together with a control or USP25 plasmids, were pre-treated with MG132 (10 μM) for 6 h, and the cells were then subjected to denature-IP and immunoblotting analysis with the indicated antibodies. (B) USP25+/+ and USP25-/- cells transfected with Myc-MAVS, HA-ubiquitin (K63O), were left uninfected or infected with SeV for 12 h. The cells were then subjected to denature-IP and immunoblotting analysis with the indicated antibodies. (C) USP25+/+ and USP25-/- cells were left uninfected or infected with SeV for 12 h. Twenty-four hours later, cells were harvested for SDD-AGE or SDS-PAGE followed by immunoblotting analysis with the indicated antibodies. (D) USP25-knockdown inhibits CNTN1-triggered K63-linked ubiquitination of MAVS. HEK293 cells were transfected with the indicated plasmids and USP25 siRNA or NC for 24 h. The cells were then treated with MG132 for 6 h before being used in ubiquitination assays with the indicated antibodies. (E) CNTN1+/+ or CNTN1-/- HEK293 cells were transfected with Myc-MAVS and HA-ubiquitin (K63O), together with a control or USP25 plasmids. The cells were then subjected to denature-IP and immunoblotting analysis with the indicated antibodies. (F) USP25-knockdown inhibits CNTN1-mediated degradation of MAVS. HEK293 cells were transfected with the indicated plasmids and USP25 siRNA or NC for 24 h before immunoblotting analysis.

Fig 5: ZBP1(S) levels increase during crisis dependent on cGAS, STING and MAVS.a, Graphical representation of human ZBP1 protein showing the positions of Za1, Za2 and RHIM domains with reference to the two main ZBP1 isoforms, ZBP1(L) and ZBP1(S). b, Scatter plots with bars showing RT-qPCR analysis of ZBP1 in IMR90E6E7 and WI38SV40LT at the indicated PDs. Expression levels were normalized to growing cells. Bars represent mean ± s.d. of technical replicates. n: number of technical replicates. Two-tailed Student’s t-test, *** p < 0.001. Three independent experiments were performed. c, Immunoblotting of IMR90E6E7 and WI38SV40LT at the indicated PDs. GAPDH loading control. Three independent experiments were performed. d, Immunoblotting of crisis (PD108) IMR90E6E7 transduced with two individual lentiviral shRNA vectors targeting either ZBP1(L) by binding to a sequence within the Za1 or a sequence present in both ZBP1 isoforms. Protein extracts were collected at day 6 post-shRNA transfection. GAPDH loading control. Two independent experiments were performed. e, Scatter plots showing RT-qPCR analysis of ZBP1 in crisis (PD106) IMR90E6E7 transfected with siRNA targeting IFN receptors and main nucleic-acid sensors and adapters. RNA extracts were collected at day 4 post-siRNA transfection. Expression levels were normalized to siCTR cells. Bars represent mean ± s.d. from technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Three independent experiments were performed. Abbreviations: ZBP1(L): long isoform; ZBP1(S): short isoform; CTR: control; PDs: population doublings. Source data