Fig 2: RELA coordinately activates expression of the HBP. Scrambled (Scr) or RELA-directed siRNA was transfected into hSAECs. Cells were mock-infected or RSV-infected (MOI = 1, 24 h) and qRT-PCR performed. Data are presented as fold change untreated controls normalized to peptidylprolyl isomerase A (PPIA) (A) GFPT1 mRNA, (B) GFPT2 mRNA, (C) PGM3 mRNA, (D) UAP1 mRNA, (E) GNPNAT1 mRNA. **, p < 0.01; *, p < 0.05, by post-hoc Tukey comparison. (F) Expression of RSV N transcript. n.s., not significant.

Fig 3: RSV and RELA regulate intracellular N-glycosylation of the integrin signaling pathway. N-glycoproteins were identified in cytoplasmic proteins from control and RSV-infected cells in the presence or absence of RELA silencing. (A) Agglomerative hierarchical clustering of 171 peptides significantly changed across the treatment conditions. Data was Z-scored across the row prior to clustering using Euclidian distance. The legend is at right. Each row represents an individual peptide; columns refer to different cell treatments/conditions. Note that the replicates for each treatment group cluster together and that the bulk of the N-glycopeptides are induced by RSV infection and are not observed in the RELA-depleted cells. (B) Volcano plot of peptides in RSV- versus mock-treated samples. X axis, fold change RSV versus mock; Y axis, log2 (ANOVA). Selected peptides in the integrin signaling pathway are labeled. (C) Volcano plot of peptides in RSV-infected cells WT versus RELA knockdown samples. X axis, fold change RSV versus mock; Y axis, log2 (ANOVA). Selected peptides in the integrin signaling pathway are labeled. (D) LFQ intensity for selected integrin (ITG), galectin-3 binding protein (LGALS3BP), ICAM1 and laminin subunit gamma 1 (LAMC1) peptides. **, p < 0.01 post-hoc.

Fig 4: OGG1 inducibly binds to HBP regulatory elements and recruits phospho-Pol II. XChIP was performed on control or RSV-infected hSAECs; data are expressed as a fold change over IgG control. (A) OGG1 binding to the GFPT2 intragenic enhancer. (B) OGG1 binding to the UAP1 promoter. (C–F), Cells were RSV infected in the presence of solvent (DMSO) or TH5487 as indicated. (C,D), XChIP for RELA binding. (E,F), XChIP for phospho-Ser2 CTD Pol II binding. *, p < 0.05; **, p < 0.01; n.s., not significant. Each symbol is an independent immunoprecipitation.

Fig 5: RELA binds to open chromatin domains in HBP core genes. For each gene (at left) is shown the chromatin accessibility determined by ATAC-Seq in uninfected (mock) or RSV-infected cells. The region assayed by Q-gPCR is indicated. The direction of gene transcription is indicated by arrow. At right is the Q-gPCR of mock- (CON) or RSV-infected cells analyzed by XChIP with RELA Ab. Each symbol is an independent immunoprecipitation. (A), GFPT2; (B), GNPNAT1; (C), PGM3; (D), UAP1.