Fig. 4

Characterization of HIV-1 RNA populations in HeLa cells expressing either wild-type or U1 D2upEx snRNA by ONT sequencing. HeLa cells were transfected with either the wild-type U1 snRNA or the modified U1 D2upEx snRNA enhancing SS D2 usage. 48 h later, cells were harvested, RNA was extracted and cDNA libraries were prepared. ONT sequencing and analyses were performed as described in Fig. 1. a Usage of SD and SA sites are expressed as a percentage of the occurrence of all SS within each condition. Data are presented as mean (n = 3). P values were calculated using an unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). b Isoform expression levels were calculated for both wild-type and U1 D2upEx snRNA conditions. Heatmap represents the fold enrichment of isoforms expressed in U1 D2upEx snRNA expressing cells over wild-type U1 snRNA condition according to the coloured scale. For strongly downregulated RNAs that were not detectable anymore by ONT sequencing in U1D2upEx snRNA conditions, a correction of 0.0075% corresponding to the lowest  % calculated for the detection of a single read, was added. c Relative expression of 12 viral isoforms were measured by qPCR in WT and U1D2upEx snRNA expressing samples and fold changes were calculated as in b. US RNA levels were estimated at D1. Correlation curve was plotted using a linear regression model supplied by Prism 7. Pearson correlation coefficient r is indicated. p < 0.0001