Figure 5.

PHF13 expression prior to infection increases the number of integrated proviral genomes and infected cells. (a) U2OS-C5 cells were treated with 1 µg ml⁻¹ doxycycline or left untreated for 24 h before cells were infected with 100 ng p24 VSVG pseudotyped HIV-1 NL4-3 IRES-eGFP. Twenty-four hours postinfection cells were analysed by flow cytometry. The mean percentage of GFP+ cells from three independent experiments was calculated and the resulting data were normalized to untreated cells (100%). Further shown are representative FACS plots from one experiment and the corresponding immunoblot to control for efficient PHF13 overexpression. (b) The same set-up as in (a), however Jurkat-TAg cells were microporated with pCG-PHF13-IRES-BFP or BFP-only expression plasmids. Twenty-four hours post-infection cells were analysed by flow cytometry and the mean percentage of PHF13 (BFP+)/HIV-1 infected (GFP+) double-positive cells of three independent experiments was calculated and normalized to the BFP-only control (100%). (c,d) Genomic DNA of U2OS-C5 and Jurkat-TAg cells treated as described in (a) and (b) was isolated and analysed for the relative number of integrated proviruses as described in the methods section. Mean fold of relative proviral copies of (c) three replicates with two independent virus stocks in U2OS-C5 cells (n = 6) and (d) two replicates with two independent stocks (n = 4) in Jurkat-TAg cells are presented. To control for complete inhibition of integration, doxycycline induced and infected U2OS-C5 cells were also treated with 250 nM Raltegravir (c). Error bars indicate the standard deviation. Statistics were calculated with an unpaired two-tailed Student's t-test. *p < 0.05; **p < 0.01.