Extended Data Fig. 4: BA.5 efficiently expresses SARS-CoV-2 innate antagonists during airway epithelial cell infection.

(a) Western blot of Calu-3 cells treated with poly(I:C), vehicle control lipofectamin2000 (L2K) or 5 μM ruxolitinib (Rux) where indicated. STAT1-pY701, STAT1-pS727, total STAT1, IRF3-pS396, total IRF3, and β-actin are shown at indicated time points. (b, c) Quantification of (b) IRF3 and (c) STAT1 nuclear translocation detected by single-cell fluorescence microscopy in Calu-3 cells stimulated with poly(I:C) or L2K. Data from 1500 cells/condition are shown. (d) Western blot of Orf9b, Orf6, spike and β-actin at 24hpi in Calu-3 cells infected with the indicated variants at 2000 E copies/cell. (e) Representative western blot of infected Calu-3 cells ±5 μM ruxolitinib. Non-specific bands detected by polyclonal anti-spike primary antibody are indicated. (f-k) Quantification of viral protein expression from five independent western blots of infected Calu-3 cells at 48hpi ±5 μM ruxolitinib. (f, g) Orf6, (h, i) N and (j, k) spike were normalized to β-Actin over BA.2. (l) Western blot of Calu-3 cells infected with BA.1, BA.2 and two independent BA.5 isolates at 48hpi. (m, n) Calu-3 cells were infected with BA.1, BA.2 and two independent BA.5 isolates and (m) replication measured. (n) Expression of IFNB, CXCL10 and IFIT1 is shown at 24hpi in cells from (m). (o) Representative western blot of Orf6 and N expression by HAEs infected with 1500 E copies/cell of BA.2 or BA.5 over time. (p) Viral replication in Calu-3 cells by RT-qPCR at 24hpi. (q) Orf6 and N sgRNA expression in cells from (n). For b, box and whisker blots show 10–90 percentile and groups were compared at each time point as indicated using a Kruskal-Wallis test. For f-k, n, p-q, one-way ANOVA with Dunnett’s post-test was used to compare BA.2 with other variants. For m, two-way ANOVA with a Bonferroni post-test was used to compare variants with BA.2 at each time point. Colors indicate comparator (BA.1, blue BA.5, purple; BA.5 (B), pink). For f-k, m-n, p-q, replicate measurements from one of two independent experiments are shown. Fold change over mock is shown. Mean + /-SEM or individual datapoints are shown. hpi, hours post infection. sgRNA, subgenomic RNA.

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