Extended Data Fig. 5: (Related to Fig. 3). Characterization of HSV-1 ICP27-triggered aberrant host cell transcription.

a, Aberrant transcription regions were defined for each mRNA as regions spanning individual introns (Intronic), regions upstream or downstream of annotated gene boundaries (Upstream, Downstream) or transcription in the divergent orientation from the same promoter (Divergent). Transcription within each region was normalized to reference constitutive exon(s), to calculate relative levels. b, Proportion of aberrant transcription regions with significant changes in normalized transcription levels in FLAG-ZBP1 MEFs and FLAG-ZBP1 HT-29 cells infected with HSV-1 (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. c, Temporal dynamics of aberrant transcription changes in HSV-1-infected HFF cells, estimated using 4sU-seq data. 4sU RNA labelling time windows are indicated on the left; 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 Sequence Read Archive (accession PRJNA256013). d, Proportion of aberrant transcription regions with significant changes in normalized transcription levels in HFF cells infected with either wild-type HSV-1 (WT, strain 17) or a matched ICP27-null mutant (ΔICP27). 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 Sequence Read Archive (accession PRJNA637636). e, Z-RNA accumulation in ICP27-transfected MEFs with or without leptomycin B (LepB, 5 nM) treatment was assessed at 6 h post-transfection. f, Fluorescence intensity of Z-RNA signal in e. n = 33 cells (no treatment), n = 39 cells (LepB treatment). g, A-RNA accumulation in MEFs transfected with FLAG-ICP27 construct was assessed 6 h post-transfection. Cells were either treated with RNase A, or were left untreated, before staining. Nuclei are outlined with dashed white lines. h, Fluorescence intensity of A-RNA signal in g. n = 61 cells (vector), n = 50 cells (ICP27-transfected), n = 53 cells (ICP27-transfected + RNase A). i, Schematic of ICP27 domain organization and location of the n504 mutation. LRR: leucine-rich putative nuclear export sequence (NES), D/E: acidic region, NLS: nuclear localization signal, RGG: arginine-/glycine-rich RNA binding motif, R2: second arginine-rich region, KH1-3: three predicted hnRNP K homology (KH) domains, CCHC: zinc-finger-like domain. The n504 mutation introduces a premature stop codon, generating a C-terminally truncated ICP27 mutant which is compromised in its ability to bind CPSF. j, Z-RNA formation in MEFs transfected with empty vector, FLAG-ICP27 or FLAG-ICP27 n504 mutant constructs assessed at 6 h post-transfection. k, Fluorescence intensity of Z-RNA signal in j. n = 34 cells (vector), n = 37 cells (ICP27-transfected), n = 35 cells (ICP27 n504-transfected). l, RNA eluted from Z22 or control IgG antibody pulldowns from ICP27 transfected cells were examined by qPCR for Z-RNAs formed within the 3′ extended transcripts downstream of Nabp1, Btbd3 or Ptbp1 loci, or in a de novo transcript generated from the H2ac18/19 locus or in the opposite orientation from the Hmga1 gene. Data were normalized to Input. m, RNA eluted from anti-FLAG antibody pulldowns of either FLAG-ZBP1 or FLAG-ZBP1 ΔZα mutant MEFs transfected with ICP27 were examined by qPCR for Z-RNAs formed in 3′ extended transcripts downstream of Nabp1, Btbd3 or Ptbp1 loci, or in a de novo transcript generated from the H2ac18/19 locus or in the opposite orientation from the Hmga1 gene. Data were normalized to Input. n, Kinetics of cell death in IFNβ pre-treated (100 ng/ml, 16 h) primary Zbp1^+/+ and Zbp1^–/– MEFs infected with HSV-1 RHIM mut (MOI = 5) and HSV-1 RHIM mut ΔICP27 (MOI = 15). o, Protein levels of ZBP1, RIPK3, and MLKL in HT-29 cells stably reconstituted with either empty vector (Vec) or FLAG-ZBP1 (FLAG-ZBP1), following IFNβ treatment (100 ng/ml, 48 h). p, Kinetics of cell death in Vec and FLAG-ZBP1 HT29 cells infected with either HSV-1 RHIM mut (MOI = 5) or HSV-1 RHIM mut ΔICP27 (MOI = 15). q, Protein levels of ZBP1, RIPK3 and MLKL in HS68 human fibroblasts in the presence or absence of IFNβ treatment (100 ng/ml, 48 h). r, Kinetics of cell death in hIFNβ pretreated (100 ng/mL, 16 h) HS68 cells infected with either HSV-1 RHIM mut (MOI = 5) or HSV-1 RHIM mut ΔICP27 (MOI = 15). s, Kinetics of cell death in WT and Zbp1–/– primary MEFs which were either left untreated, or were pre-treated with IFNβ (100 ng/ml, 16 h), followed by infection with HSV-1 RHIM mut virus (MOI = 5). t, Immunoblot showing induction of endogenous ZBP1 after infection of primary WT (Zbp1+/+) or Zbp1–/– MEFs with HSV-1 RHIM mut virus (MOI = 5, 8 h). Blotting for the HSV-1 protein ICP0 shows equal levels of virus replication in Zbp1+/+ and or Zbp1–/– MEFs. Data are mean ± s.d. (n = 3 biologically independent samples in l, m; n = 4 in n, p, r, s). Two-tailed unpaired t-test with Welch’s correction (f, l). One-way ANOVA with Dunnett’s multiple comparisons test (h, k, m). *P < 0.05, **P < 0.005, ***P < 0.0005. (p < 0.0001 in f, h, k). Data are representative of at least two independent experiments (e, g, j, o, q, t) or three independent experiments (n, p, r, s).