Additional file 1: Figure S1. of Development of 5‘ LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals

High levels of DNA methylation of proviral 5’ LTR remain stable after repeated cellular stimulation of the 2D12 cell line. Capacity to decrease 5’ LTR DNA methylation level associated with increased reactivation capacity of the highly methylated proviral promoter was assessed. Repeated stimulations of the 2D12 cell line with TNF-α and PMA were performed with or without the selection of EGFP-positive cells by FACS-sorting. In contrast to the increase of DNA methylation of the proviral 5’ LTR in the H12 cell line, the high levels of 5’ LTR DNA methylation in the 2D12 cell line did not decrease after repeated cellular stimulations neither did percentage of EGFP-positive 2D12 cells increase. At the time of each stimulation, HIV-1 provirus reactivation after 24-h of TNF-α and PMA treatment was assessed according to the percentage of EGFP-positive cells. Bisulfite sequence determined the level of 5’ LTR CpG methylation. Bisulfite sequencing of the 5’ LTR was performed at 24 days after each stimulation, when the cells were restored to the non-stimulated, steady state. (A) Latent HIV-1 provirus CpG methylation levels in the 2D12 5’ LTR sequences after repeated stimulations of cells. The percentage of methylated CpGs in the 5’ LTR was determined by bisulfite sequencing. The number of successive stimulations is depicted on the x-axis whereas the mean percentage of methylated CpGs is depicted on the y-axis. The solid line shows activations without selection of EGFP-positive cells, and the dashed line indicates activations followed by selection of EGFP-positive cells. (B) Latent HIV-1 provirus reactivation in the 2D12 cell line after repeated stimulations of cells. The 2D12 cell line was stimulated with TNF-α and PMA for 24 h, and the percentage of EGFP-positive cells was determined by FACS. The number of successive stimulations is depicted on the x-axis, and the percentage of EGFP-positive cells is depicted on the y-axis. The solid line shows activations without selection of EGFP-positive cells whereas the dashed line indicates activations followed by selection of EGFP-positive cells. (TIF 313 kb)