Fig. 3. The SARS-CoV-2 Alpha variant upregulates innate immune antagonists at the subgenomic RNA and protein level.

a, Top, the log₂ ratio of Alpha to VIC sgRNA normalized to total genomic RNA per time point and virus (from RNA-seq). Bottom, the log₂ ratio of summed peptide intensities per viral protein comparing Alpha to VIC (from proteomics analysis) (n = 3). Orf3a–d refers to Orf3a, Orf3b, Orf3c and Orf3d. S, spike protein; E, envelope protein; M, membrane protein. ND, not detected. b–d, Quantification of Orf9b (b), Orf6 (c) and N (d) sgRNA from the RNA-seq dataset (top) and summed peptides per viral protein (bottom). e, Quantification of Orf9b and N (left) or Orf6 (right) sgRNA abundance by RT–qPCR (24 hpi). f, Representative western blot of Orf6, N and S expression in infected Calu-3 cells (2,000 E copies per cell) at 24 hpi (n = 3). g, Pie chart depicting the proportion (shown as percentages) of total sgRNA mapping to each viral sgRNA for Alpha at 24 hpi. VIC percentages in parentheses. h, sgRNA log₂-normalized counts (dot height) projected onto their identified start sites on the SARS-CoV-2 genome (24 hpi). Canonical and two non-canonical sgRNAs (Orf9b and N*) are depicted. i, Scatter plot of sgRNA abundance in Alpha or VIC at 24 hpi. Grey dots indicate other non-canonical sgRNAs containing a leader sequence but no clear start codon. Mean ± s.e.m. (a–e). Two-way ANOVA with Tukey’s multiple comparisons post-hoc test (c–e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant.