(a, bottom) Map of the major human adenovirus 5 early E2, E3, and E4 mRNAs. Deletions in the E3 regions of dl312 and the E1A-DM vector are shown by cross-hatched horizontal bars. Shared E3 sequence used as the E3 gene body is indicated by the gray bar above E3. Vertical stripes highlighted in yellow indicate promoter-proximal regions. Global Run-On sequencing (GRO-seq) counts from primary human bronchial-tracheal epithelial cells infected with wt+dl312 or DM vectors at 12 hr post-infection were plotted on the Ad5 genome with H3K18ac, H3K27ac, and H3K9ac ChIP-seq data (Hsu et al., 2018). GRO-seq tracks are shown for the two viral DNA strands (+, transcribed to the right; and –, transcribed to the left), with two different y-axis scales to allow visualization of high- and low-amplitude peaks. The double-headed arrows in the GRO-seq plots in the E4 region refer to gene body regions discussed in the text. (b) Average fold change in pause index for E2early, E3, and E4 in cells expressing DM-E1A compared to wt E1A. Pause index is the ratio of reads in the promoter region (transcription start site [TSS] to +200) to reads in the gene body (+200 to TTS). Error bars represent standard deviation of three biological replicates. Paired t-test comparing wt E1A and DM-E1A for E2, E3, and E4. * indicates a significant difference (p-value<0.05) between cells expressing wt E1A and DM-E1A. ‘ns’ indicates no significant difference.