Fig. 3: Orf6 expression is a major determinant of enhanced innate immune antagonism by emerging VOCs.

a,b, Western blot of Alpha (a) or BA.5 (b) reverse genetic (RG) virus infections in Calu-3 cells at 24 h.p.i. ± 5 μM ruxolitinib (Rux). c, Replication of RG viruses parental Alpha WT and ΔOrf6 in Calu-3 cells infected with 2,000 E copies per cell over time. d, Gene expression in cells from c over time. e, Quantification of IRF3 nuclear translocation detected by single-cell fluorescence microscopy over time. f–i, HAEs were infected with 1,500 E copies per cell of the indicated variants ± 5 μM ruxolitinib. f, Viral release into apical washes over time. g,h, Apical release in HAEs infected with Alpha WT (g) or ΔOrf6 ± 5 μM ruxolitinib (h). i, Gene expression in cells from f. Three biological replicates shown. j, Replication of RG viruses BA.5 WT, ΔOrf6 and Orf6 D61L isolates in Calu-3 cells infected with 2,000 E copies per cell over time. k, IFNB expression in cells from j. l, Gene expression of Calu-3 cells at 24 h.p.i. m, Western blot of STAT1-pY701, STAT1-pS727, total STAT1, IRF3-pS396, total IRF3 and β-actin at 24 h.p.i. n–r, Quantification of five independent western blots showing IRF3-pS396 (n), total IRF3 (o), STAT1-pS727 (p), STAT1-pY701 (q) and total STAT1 (r) over β-actin at 24 h.p.i. s,t, Quantification of IRF3 (s) and STAT1 (t) nuclear translocation detected by single-cell fluorescence microscopy at 24 h.p.i. u,v, Replication of BA.5 WT and ΔOrf6 in HAEs infected with 1,500 E copies per cell in the absence (u) or presence (v) of 5 μM ruxolitinib. For c and d, two-way analysis of variance (ANOVA) and Bonferroni post-test were used. For e, s and t, data from 1,500 cells per condition are shown as box-and-whisker blots indicating 10th–90th percentile. In infected cultures, translocation was determined in N+ cells. Groups were compared by Kruskal–Wallis test. For k, l and n–r, one-way ANOVA with Dunnett’s post-test was used. For f–i, u and v, unpaired two-tailed Student’s t-test was used. Replicate measurements from one of three independent experiments. Fold change over mock is shown. Mean ± s.e.m. or individual datapoints are shown.

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