Fig. 8

Effects of MAP3K4, PTPN13, and SERPINB6 RNA interference on HIV-1 integration in memory CD4⁺ T cells. RNA interference experiments were performed on memory CD4⁺ T-cells as described in the Fig. 7 legend. a Shown is the experimental flow chart. Briefly, cells were stimulated via CD3/CD28 for 2 days, washed and nucleofected with siRNA pools (1 µM) specific for MAP3K4, PTPN13, and SERPINB6 or a non-targeting siRNA (NT). Nucleofected cells were cultured in the presence of IL-2 (5 ng/ml) for 24 h and then exposed to the HIV NL4.3BAL-GFP strain (50 ng HIV-p24/ml) for 3 h at 37 °C. Infected cells were cultured in the presence of IL-2 (5 ng/ml) for 3 days. b The yield of siRNA silencing was determined by real-time RT-PCR quantification of MAP3K4, PTPN13, and SERPINB6 mRNA expression in cells nucleofected with targeting (MAP3K4, PTPN13, SERPINB6) versus non-targeting (NT) siRNA. Shown are results obtained with cells from two to five different donors. c Levels of integrated HIV-DNA were quantified by nested real-time PCR in cells harvested at day three post-infection. HIV-DNA copy numbers were normalized relative to CD3 expression. Shown is relative HIV-DNA integration in targeting versus NT siRNA conditions (normalized to the maximal value considered to be 100 % in NT condition); the values above graphs are integrated HIV-DNA copies per 10⁶ cells in NT nucleofected cells (mean ± SD of triplicate wells). Paired t test values are indicated on the graphs