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. 2015 Nov 20;11(11):e1005274. doi: 10.1371/journal.ppat.1005274

Fig 5. VER-155008 caused a significant reduction in viral transcripts, viral DNA and progeny in TREx BCBL1-RTA cells.

Fig 5

(A) Cells were reactivated for 24 h in the presence of control DMSO (0.1%) or a range of increasing inhibitor concentrations, total RNA was isolated and qRT-PCR carried out. Early (orange colour), late (red colour) and ori-Lyt viral transcripts were all significantly reduced at 1 μM VER-155008 compared to the levels found in DMSO-treated samples. All samples were normalised to GAPDH. Results show the mean of three biological replicates with error bar as standard deviation. (B) The stability of GAPDH transcript was assessed in unreactivated TREx BCBL1-RTA cells in the presence of the transcriptional inhibitor actinomycin D (AcD) (2.5 μg/ml) or control DMSO (0.25%). Cells were collected over the time points indicated and total RNA was extracted followed by qRT-PCR. After 6 h AcD treatment, the amount of GAPDH transcripts was reduced by more than half (CT = 18.09) compared with DMSO-treated cells (CT = 16.24). A further reduction was observed at 8 h treatment (CT = 19.68). The average of two biological replicates with error bar as standard deviation is shown. (C) The amount of GAPDH transcripts did not significantly decrease when using VER-155008 for 24 h at concentrations lower than 3 μM compared to DMSO-treated samples. In contrast, concentrations higher than 3 μM significantly showed a CT lower than DMSO samples, indicating compromised cellular transcription at these inhibitor concentrations. The same samples in which viral transcripts had been quantified were used to plot all CT values. Results show the mean of three biological replicates with error bar as standard deviation. (D) Cells were reactivated for 72 h, total DNA was isolated and real-time qPCR was performed. Viral DNA load was significantly decreased at 2 μM VER-155008. Results show the mean of three biological replicates with error bar as standard deviation. (E) TREx BCBL1-RTA cells were reactivated for 72 h in the presence of VER-155008 at 2.5 μM or vehicle drug DMSO (0.1%). The culture medium was then incubated for 24 h with HEK-293T cells followed by total RNA extraction and qRT-PCR. A significant decrease in viral progeny was seen in inhibitor-treated cells. Results show the mean of three biological replicates with error bar as standard deviation.