Fig. 1
Cellular stimulation contributes to de novo DNA methylation of proviral 5’ LTR in the H12 cell line. a Flowchart of repeated stimulations of the H12 cell line with TNF-α and PMA performed with or without the selection of EGFP-negative cells by FACS-sorting. 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. We assume the level of 5’ LTR DNA methylation controls the reactivation efficiency, however, the changes in 5’ LTR DNA methylation levels are not instant. Therefore, the levels of 5’ LTR methylation are determined immediately before the next stimulation cycle, e.g., the % of the 5’ LTR methylation depicted at the stimulation number 2 (Fig. 1c) is determined immediately before the 2nd stimulation cycle, when the cells after the first stimulation re-established the non-stimulated, basal phenotype and displayed only basal levels of EGFP and CD69. Three cycles of cellular stimulations followed by the selection of EGFP-negative cells by FACS-sorting were performed, including three measurements of % EGFP-positive cells and four determinations of % CpG methylation. The last % CpG methylation was measured after the third stimulation, when the cells restored the non-stimulated steady state. Five cycles of cellular stimulations without selection of EGFP-negative cells were performed, including five measurements of % EGFP-positive cells and five determinations of % CpG methylation. The last % CpG methylation was measured 24 days after the fourth stimulation when the cells restored non-stimulated steady state. (The % CpG methylation 24 days after the fifth stimulation was not determined). b Latent HIV-1 provirus reactivation in the H12 cell line after repeated stimulations of cells. The H12 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-negative cells whereas the dashed line indicates activations followed by selection of EGFP-negative cells. c Latent HIV-1 provirus CpG methylation levels in the H12 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-negative cells, and the dashed line indicates activations followed by selection of EGFP-negative cells. The last point of the solid line applies to Fig. 1de. d The CpG methylation profile of the 5’ LTR in non-stimulated H12 cells before the fifth cycle of 24-h stimulation with TNF-α and PMA. The cycles of cellular stimulations were performed without selection of EGFP-negative cells. A schematic representation of the CpG dinucleotide distribution in 5’ LTR of the pEV731 vector together with the corresponding transcription factor binding sites is shown. Analysis of the promoter molecules is shown as a linear array of open circles representing non-methylated CpG residues and closed circles representing methylated CpG residues. Each line represents one sequenced molecule of the 5’ LTR. The methylation level is presented as a mean percentage of methylated CpGs (mCpGs) in HIV-1 promoters. e Distribution of the 5’ LTR molecules according to the proportion of methylated CpGs contained within. Non-stimulated H12 cells before the fifth cycle of 24-h stimulation with TNF-α and PMA are depicted. The cycles of cellular stimulations were performed without selection of EGFP-negative cells. The percentage of the respective molecules is shown on the x-axis. The total counts of methylated CpGs per molecule are shown on the y-axis