HsIFNλ4 E154 induces more robust antiviral gene expression than the wt K154 variant.

A549 cells were treated with CM (1:3 dilution) for 24 hours from cells transfected with plasmids expressing the different HsIFNλs and EGFP. After isolation of RNA, transcriptome analysis was carried out by RNA-Seq. (A) Total number of significantly differentially-expressed genes in each experimental condition (x-axis) relative to each other condition (y-axis). Colour shaded by differences in numbers of transcripts between sample 1 (x-axis) and sample 2 (y-axis) are shown. (B) Violin plot of all significant, differentially-expressed genes (over two-fold) (log2 fold change for each condition compared to RNA from cells treated with EGFP). CM was obtained from cells transfected with HsIFNλ3op (red); HsIFNλ4 wt (green); HsIFNλ4 P70S (cyan), and HsIFNλ4 K154E (purple). (C) Heat map of all significantly differentially-expressed genes (over two-fold) (log10 Fragments Per Kilobase of transcript per Million mapped reads (FKPM) in each experimental condition including EGFP CM-stimulated cells. Genes shown as columns and values are not normalised to negative control. (D) Pathway analysis using IPA on all significantly differentially-expressed genes (>2 fold) for each variant compared to EGFP. The top five most significantly induced pathways are shown [-log(p value)]. (E) Comparison of differentially-expressed genes (significant and at least 2-fold difference) stimulated by the HsIFNλ4 variants (HsIFNλ4 wt in green, HsIFNλ4 P70S in cyan and HsIFNλ4 K154E in purple) illustrated by a Venn diagram showing shared and unique genes. Three examples in overlapping and unique areas of the Venn diagram are highlighted. (F) Raw gene expression values (FKPM+1) for representative genes from core, shared and K154E-‘specific’ groups for the different treatments. Data are shown as mean +/- SD of biological replicates (n = 3). Exemplary genes selected were: ISG15 and MX1 (core), UBA7 (not significantly induced by P70S), and ISG20 and IDO1 (apparently specific for K154E). All transcriptomic analysis and gene lists are available in S2 Data.