Fig 2: Efficient viral RNA splicing prevents dsRNA formation. (A) HeLa cells were transfected with control siRNA (solid bars) or siRNA targeting both RALY and hnRNPC (dashed bars) for 24 h before being infected with WT Ad5 (Ad5, black), or Ad5 mutants lacking E1B55K (ΔE1B, purple) or the E4 region (ΔE4, red). At 32 hpi total RNA was harvested and qRT-PCR was performed. Spliced and unspliced viral transcripts were detected using the method shown in 6D and the ratio of these RNA species depicted as splicing efficiency. Data are shown for three independent biological replicates, bars depict mean, and error bars depict standard deviation. Significance was analyzed by unpaired two-tailed t-test. (B) Immunoblot with exactly matching conditions to those shown in (A). (C) HeLa cells were transfected with control siRNA (siCTRL) or siRNA targeting both RALY and hnRNPC (siRBP) for 24 h before being infected with WT Ad5, ΔE1B or ΔE4. Cells were fixed at 32 hpi and stained with 9D5 anti-dsRNA antibody (green) and cellular protein that marks VRCs (USP7, magenta). Nuclei were stained with DAPI, and nuclear periphery are outlined by a dashed white line. Scale bar (white line) denotes 10 μm. (D) Nuclear dsRNA from (C) was quantified as MNFI and every cell displayed as a dot. The dsRNA positive threshold was defined as 4 standard deviations over the MNFI of dsRNA in uninfected cells and displayed as a dashed line. Cells below the threshold were colored grey, while cells positive for nuclear dsRNA were colored black, and the mean and standard deviation of these positive cells is denoted by red error bars. The percentage of total cells expressing dsRNA during infection with each virus is shown below their name on the x-axis. Significance was analyzed by unpaired two-tailed Mann–Whitney t-test. For all experiments, significance was shown as P-value >0.5 (not-significant, n.s.), * P< 0.05, ** P< 0.01 and *** P< 0.001.