Figure 5

vEnv regulates the miR181A2/P300/CBP-associated factor (PCAF) expression, and increases HIV LTR histone H3 Acetylation. (A) The miR181A2 mRNA level was reduced in J-Lat 6.3 T cells treated with Env(X4)-VLP for 24 hrs, as compared to the untreated cells (n = 3) (left panel). The reduced miR181A2 mRNA levels in PBMCs from 3 donors treated with Env-VLP, as compared to the untreated cells (n = 2) (right panel). (B) The PCAF mRNA (left panel) and protein levels (right panel) in J-Lat 6.3 T cells treated with Env-VLP or untreated (n = 3). (C) The HIV gag transcription levels in J-Lat 6.3 T cells overexpressed with miR181A2 or miR181A2-3p (inhibitor), as compared to the cells transduced with empty lentiviral vectors (n = 3). J-Lat 6.3 T cells were transduced with lentiviral vectors encoding miR181A2, miR181A2-3p or transduced with empty vector for 24 hrs and kept on culture for another 72 hrs. Then, cells were collected and the HIV comparative transcription (gag/GAPDH) was measured by RT-PCR. (D) The PCAF mRNA (left panel) and protein levels (right panel) in miR181A2 or miR181A2-3p overexpressing J-Lat 6.3 T cells (n = 3). (E) The schematic diagram of the positions of the nucleosomes bound to the HIV-1 LTR and the location of the primers used for the real-time PCR in the ChIP assay. (F) The detection of histone H3 acetylation ratio in HIV LTR -109- + 82 by CHIP (n = 3). J-Lat 6.3 T cells were treated with Env(X4)-VLPs for 24 hrs. In parallel, cells treated with histone deacetylase inhibitor VOR or VPA were included as positive controls and the untreated cells act as a negative control. After treatment, cells were lysed and analyzed by CHIP assay (left panel). Also, the global histone H3 acetylation in J-Lat 6.3 T cells treated with Env(X4)-VLP, VOR, VPA or untreated were detected by western blot with anti-histone H3 acetylated antibody. Data are mean and sd. Ns, not significant p > 0.05. (Two-tailed unpaired t-test).