Ribosome profiling (Ribo-seq) data were obtained from Finkel et al., 2020; mass spectrometry data were obtained from Davidson et al., 2020 and Bezstarosti et al., 2020. Reads were trimmed to their first (5′) nucleotide to minimize statistical dependence while preserving reading frame. Results are shown after pooling samples treated with harringtonine (SRR11713360, SRR11713361) or lactimidomycin (SRR11713358, SRR11713359) at 5 hr post-infection. (A) Ribosome profiling coverage (read depth) near translation initiation sites, measured as mean reads per million mapped reads. Only reads of length 29–31 nucleotides were used, chosen for their enrichment at the start sites of highly expressed annotated genes (Figure 2—figure supplement 4). Light blue backgrounds denote fully (internal) overlapping genes. Annotated genes show an accumulation of 5′ ends of protected reads upstream of the gene’s start site (vertical gray dotted lines), peaking near the ribosome P-site offset of −12 nt (red = maximum depth). Distributions are largely consistent across individual samples (Figure 2—figure supplement 1). The ranges of the y-axes vary according to expression level, with the most highly expressed gene (N; Figure 2—figure supplement 2) having the largest range. (B) Correlation between protein expression as estimated by mass spectrometry and ribosomal profiling. ‘iBAQ prop’ refers to the relative (proportion of maximum) protein intensity-based absolute quantification (iBAQ) value (Schwanhäusser et al., 2011). Genes are denoted by shape and ordinally colored by iBAQ prop from high (red) to low (blue). ‘Upstream peak’ refers to the maximum read depth observed at the approximate P-site offset (red bars in A), while mean read depths were measured across each gene using 30 nt reads separately for in-frame (codon position 1) and out-of-frame (codon positions 2 and 3) sites (non-overlapping gene regions only, except for ORF9b). (C) Reading frame of ribosome profiling reads in the ORF3c/ORF3d region of ORF3a. Solid lines show the fraction of reads in each frame, summed across samples in sliding windows of 30 nt (step size = 1 nt; read length = 30 nt). Color denotes frame: green = reference frame (ORF3a); burgundy = ss13, the forward-strand frame encoding ORF3c, whose codon position 3 overlaps codon position 1 of ORF3a; and gold = ss12, the forward-strand frame encoding ORF3d, whose codon position 2 overlaps codon position 1 of ORF3a. Values are shown for the central nucleotide of each window, with shaded regions corresponding to 95% binomial confidence intervals. Alternative frame translation is suggested where a given frame (solid line) exceeds its average across the remainder of the gene (horizontal dashed line; non-OLG regions of ORF3a). Vertical gray dotted lines indicate gene start and end sites. Gray bars show read depth for each window, with a maximum of 2889 reads at genome position 25442.