Epigenetic Regulation of HIV-1 Latency by Cytosine Methylation
Figure 5
Cytosine methylation maintains HIV-1 latency in polyclonal Jurkat T cells.
(A) Flow cytometry of Jurkat T cells infected with HIV-1 R7/E−/GFP clone. Gates indicate GFP-positive (productively infected) and GFP-negative (latently infected and uninfected) cells. (B) Quantitative PCR to measure HIV-1 DNA in infected Jurkat cells. For polyclonal cell populations, days after infection are indicated. For J-Lat clones, cell line is indicated. Levels of HIV-1 DNA were normalized to cellular DNA. Y-axis indicates fold over uninfected Jurkat negative control. Experiment was performed in triplicate and error bars indicate standard deviation. (C) HIV-1 expression over time in a polyclonal population of latently infected and uninfected Jurkat T cells. GFP fluorescence was measured by flow cytometry. The time point at which cells were treated with aza-CdR plus TNF-α is indicated on the x-axis. (D) Bisulfite-mediated methylcytosine mapping of HIV-1 CpG islands in polyclonal Jurkat T cells that are latently infected (GFP-negative, upper panel) or productively infected (GFP-positive, lower panel). Asterisks indicate cytosines with a statistically significant greater level of methylation in the GFP-negative population. (E) Latent HIV-1 reactivation in a polyclonal population of latently infected and uninfected Jurkat T cells treated with aza-CdR, TNF-α, or aza-CdR plus TNF-α. HIV-1 expression was measured by flow cytometry for GFP, and the percentage of cells that express GFP is displayed. Error bars indicate standard deviation of three experiments.