Figure 2

TCERG1 depletion impairs elongation of HIV-1 transcripts. A. Schematic representation of the HIV-1 genome. The black lines indicate the position of each pair of primers used for qPCR experiments. B. Analysis by qPCR of the amount of nascent transcripts originated at the proximal (LTR-gag) and distal (nef) regions of the pNL4-3-wt strain in Jurkat cells with stable interference of TCERG1 (left panel). The results of three different experiments are represented in the histogram (means ± SEM; **, p < 0.01). TCERG1 mRNA interference was assessed by semi-qPCR and immunoblotting using β-actin as the housekeeping gene and loading control, respectively (right panels). C. Quantitative analysis of the amount of nascent transcripts originated at distal regions of the pNL4-3∆RT provirus. HEK293T cells were transfected with siRNAs against EGFP (siCTRL) or TCERG1 (siTCERG1) together with the pNL4-3∆RT plasmid and pXGH5 as a transfection control. Total RNA was extracted and qRT-PCR was performed using primers that amplify the env gene of the provirus (at positions 8010-8116). The graph shows the data from triplicates of three independent experiments as values relative to those obtained from cells without siRNAs transfectiona (means ± SEM; **, p < 0.01). D. Kinetics of RNAPII-dependent transcription elongation in the presence or absence of TCERG1. Control and TCERG1-knockdown T-Rex-HEK293 cells were transfected with pNL4-3∆RT and pXGH5 as a transfection control and treated 48 h later with 100 μM DRB for 3 h. qRT-PCR was performed using the primers that amplify the env gene of the provirus. The graph shows pre-mRNA levels at different times from the control and TCERG1-depleted cells. The data shown are the averages from triplicates of three independent experiments (mean ± SEM). The analysis of TCERG1 knockdown in T-Rex-HEK293 cells by Western blotting is shown on the right. CDK9 is used as loading control.