Extended Data Fig. 8: (Related to Fig. 4). IAV NS1 mediates IAV-induced host cell Z-RNA formation and ZBP1-driven cell death.
From: Host cell Z-RNAs activate ZBP1 during virus infections
a, Proportion of aberrant transcription regions with significant changes in normalized transcription levels in FLAG-ZBP1 MEFs and FLAG-ZBP1 HT-29 cells infected with IAV H1N1 strain A/Puerto Rico/8/1934 (MOI = 2, 8 h post-infection). The total number of tested regions associated with expressed mRNAs is indicated on the right. Changes exceeding 10% were classified as strong. b, Proportion of aberrant transcription regions with significant changes in normalized transcription levels in the nucleus of A549 cells infected with either wild-type IAV (WT, H1N1 strain A/WSN/1933) or a matched NS1-null mutant (ΔNS1) at 16 h post-infection. The total number of tested regions associated with expressed mRNAs is indicated on the right. Changes exceeding 10% were classified as strong. Sequencing data were obtained from the European Nucleotide Archive (accession PRJEB75711). c, RNA eluted from Z22- or control IgG antibody pulldowns from cells transfected with an NS1 expression construct were examined by qPCR for Z-RNAs formed within 3′ extended transcripts downstream of 2310057M21Rik and Gm14419. Data were normalized to Input. d, RNA eluted from anti-FLAG antibody pulldowns of either FLAG-ZBP1 or FLAG-ZBP1 ΔZα mutant MEFs transfected with an NS1 expression construct were examined by qPCR for Z-RNAs formed within 3′ extended transcripts downstream of 2310057M21Rik and Gm14419. Data were normalized to Input. e, Levels of IAV NS1 and NP in MEFs infected with IAV (MOI = 5) or IAV ΔNS1 (MOI = 5) at 8 h after infection. f, g, RNA eluted from Z22- (f), anti-FLAG antibody- (g) or IgG antibody pulldowns from FLAG-ZBP1 MEFs infected with IAV (MOI = 5) and IAV ΔNS1 (MOI = 5) were examined by qPCR for Z-RNAs formed within 3′ extended transcripts downstream of 2310057M21Rik, Gtpbp4, Vbp1 and Gm14419 loci. Data were normalized to Input. h, i, RNA eluted from Z22- (h), anti-ZBP1 antibody- (i) or IgG antibody pulldowns from primary MEFs infected with IAV (MOI = 5) and IAV ΔNS1 (MOI = 5) were examined by qPCR for Z-RNAs formed in 3′ extended transcripts downstream of 2310057M21Rik, Gtpbp4, Vbp1 and Gm14419 loci. Data were normalized to Input. j, Kinetics of cell death in primary Zbp1+/+ and Zbp1–/– MEFs infected with IAV (MOI = 5) and IAV ΔNS1 (MOI = 5). k, Kinetics of cell death in Vec or FLAG-ZBP1 HT29 cells infected with IAV (MOI = 5) and IAV ΔNS1 (MOI = 5). Data are mean ± s.d. (n = 3 biologically independent samples per group in c, d, f- i; n = 4 in j, k). Two-tailed unpaired t-test with Welch’s correction (c). One-way ANOVA with Dunnett’s multiple comparisons test (d). Two-way ANOVA with Tukey’s multiple comparisons test (f- i) *P < 0.05, **P < 0.005, ***P < 0.0005. Data are representative of three independent experiments (j, k).
