Skip to main content
Fig. 5 | Genome Biology

Fig. 5

From: Widespread activation of antisense transcription of the host genome during herpes simplex virus 1 infection

Fig. 5

Evidence for inhibition of sense transcription by BBC3as. a Single-molecule FISH. HSV-1-infected cells (6 h) and non-infected controls (0 h). As a reference, non-infected controls were stained for an example mRNA (CCNA2) and lncRNA (NEAT1). Note that because of lower (mRNA) or higher (lncRNA) expression, exposure times and image settings had to be adjusted and are not directly comparable between panels a and b. b Comparison of BBC3as in HeLa and NHDF cells. Nuclear foci in infected (6 h) HeLa (upper row) and NHDF (lower row) cells. c Profiling sense and antisense transcripts in pulse sequencing data. Shown are log2 fold changes compared to mock-treated cells at different timepoints. Black line, median of all genes; gray area, 25/75% quantiles; unlabeled light blue/light red represent the other four sense/antisense transcripts from a. d BBC3 locus. Transcription start of sense and antisense RNA and guide RNAs used for CRISPR cell lines are indicated. The putative BBC3as promoter region and subregions used for the promoter assay in d are shown as green bars. Note that only the full and outer constructs contain the TATAAA sequence. e Reporter assay of the putative BBC3as promoter. The sequences indicated in c were cloned in front of the Firefly luciferase, with the Renilla luciferase on the same plasmid for normalization. Shown are chemiluminescence Renilla/Firefly ratios from two biological replicates scaled to the 0-h timepoint. Error bars represent standard deviations. f RT-qPCR on total RNA. RNA was isolated from HeLa cells labeled for 20 minutes with 4sU. Shown is total RNA from uninfected cells and 6 h post-infection, subjected to random hexamer-directed RT followed by qPCR with primer pairs as indicated. Experiments were performed with two measurements each from two biological replicates and normalized to the wild-type HeLa 0-h value using the D. melanogaster spike-in RNA. Error bars represent standard deviations. g RT-qPCR on newly synthesized RNA. Labeled, i.e., newly synthesized RNA, was isolated and subjected to RT-qPCR as in f. Significance levels were calculated using a t-test with equal variance levels. **p ≤ 0.01; not significant (n.s.), p > 0.05. h Nanostring nCounter assays on newly synthesized RNA. Experiments were performed with one measurement each from two biological replicates and scaled to the wild-type HeLa 0-h value after normalization using housekeeping genes. Error bars represent standard deviations. Significance levels were calculated using a t-test with equal variance levels. **p ≤ 0.05; not significant (n.s.) p > 0.05. i RNA-sequencing coverage profiles of the BBC3 locus of wild-type HeLa cells and the two BBC3as promoter knockout cell lines

Back to article page