Fig. 4

Expression of mPML in PML-KO MEFs restores restriction of HIV-1 and SIV_mac. a Western blotting (WB) analysis of mPML overexpression in MEFs. PML cDNA was isolated from WT cells and transduced into both WT and PML-KO MEFs. The empty vector (EV) was transduced as a control. mPML expression was analyzed by WB of extracts from EV-transduced MEF-WT cells (WT + EV), mPML-transduced MEF-WT cells (WT + PML), EV-transduced PML-KO cells (KO + EV), and mPML-transduced PML-KO MEF cells (KO + PML). The WB was performed using an anti-mPML monoclonal antibody (upper panel) followed by an anti-actin antibody (lower panel) as a loading control. The arrow points to mPML, as judged from its expected size, whereas the heavier bands are presumably SUMOylated forms. The positions of the molecular size markers are indicated on the left. b Analysis of retrovirus infectivity in the transduced MEFs. The cells were infected with multiple doses of either HIV-1_NL-GFP or SIV_mac-GFP, and the percentage of GFP-positive cells was measured at 2 dpi by FACS (P ≤ 0.001, one-tailed paired Student’s t test for KO + PML vs. KO). c FACS plots from transduced MEFs infected with HIV-1_NL-GFP. WT and PML-KO MEF cells transduced with either EV or mPML were infected with HIV-1_NL-GFP. The percentage of infected cells and mean fluorescence intensities determined at 2 dpi are indicated for each plot. d Down-regulation of LTR-driven GFP expression following overexpression of mPML in PML-KO MEFs. WT and PML-KO MEFs were stably transduced with either mPML or EV, as a control, then infected with multiple doses of HIV-1_NL-GFP (left panel), SIV_mac-GFP (middle panel) or B-MLV_GFP (right panel). The MFI was measured by FACS at 2 dpi (P < 0.01, one-tailed paired Student’s t test for KO + PML vs. KO after HIV-1_NL-GFP or SIV_mac-GFP infection)