Fig. 1

dCA promotes a repressive chromatin environment at the HIV promoter in HeLa-CD4 chronically infected cells. a dCA reduces viral transcription overtime in NL4-3 chronically infected HeLa-CD4 cells. Cells were split on average every 3 days in the presence of ART with or without 10 nM dCA. Capsid production in the supernatant was quantified via p24 ELISA. Data are representative of three independent experiments. b SAHA treatment activated viral production in chronically infected HeLa-CD4 cells. After day 280 of culture, cells (highlighted with “⦿/☒” in panel A) treated with ART and ART + dCA were stimulated with 2.5 µM SAHA for 24 h. Capsid production in the supernatant was quantified by a p24 ELISA. Data are average of 3 independent experiments, error bars represent standard deviation (SD) of 3 independent experiments (ND, not detected). c SAHA treatment increased viral mRNA levels in chronically infected HeLa-CD4 cells. Cell samples from panel B were also used for RNA extraction, and cDNAs from extracted total RNA were quantified by RT-PCR using primers to the Nef region. Results were normalized as the number of viral mRNA copies per GAPDH mRNA. Viral mRNA generated in the ART control was set to 100%, error bars represent SD of 3 independent experiments. d Viral production in HeLa-CD4 cell line upon treatment interruption. Treatment of dCA was stopped on day 360 and the cells were further maintained in ART for another 55 days. Capsid production was quantified via p24 ELISA. Data are representative of three independent experiments. e Distribution of RNAP II on HIV genome DNA. ChIP assay to RNAP II was performed using cells samples from panels B-C. Results are represented as the percentage of input and subtracted the background of the isotype IgG control. Data are average of 3 independent experiments, and error bars represent SD of 3 experiments for each primer set. f Chromatin structure of the HIV LTR upon dCA treatment was determined by MNase protective assays. The amount of the MNase digested PCR product was normalized to that of the undigested product using the ∆C(t) method (y-axis), which is plotted against the midpoint of the corresponding PCR amplicon (x-axis). The X-axis represents base pairs units with 0 as the start of HIV LTR. Error bars represent the SD of 3 independent experiments. g H3 occupancy on HIV promoter DNA determined by ChIP to H3. Results are presented as percent immunoprecipitated DNA over input, after background subtraction of the isotype IgG control. The ORF of GAPDH was used as control. Data are average of 3 independent experiments and error bars represent the SD of 3 experiments for each primer set. h The level of H3 lysine 27 acetylation (H3K27Ac) on HIV promoter DNA determined by H3K27Ac ChIP. The results are presented as acetylation of H3K27 over total Histone H3 level, after subtraction of the isotype IgG control background. The promoter of RPL-10 was used as the control. Data are average of 3 independent experiments and error bars represent the SD of 3 experiments for each primer set. i The recruitment of PBAF complex on HIV promoter DNA determined by ChIP to BAF180. Results are presented as percent immunoprecipitated DNA over input, after isotype IgG control background subtraction. The promoter of GAPDH was used as the control. Data are average of 3 independent experiments and error bars represent SD of 3 experiments for each primer set. Statistical significance was determined using unpaired t-test (ns, no significance, *P < 0.05, **P < 0.01)