FIG 10.

The biotinylated antiretroviral PAV-206 analog colocalizes in situ with ABCE1, a host component of assembly intermediates. (A) Schematic of the PLA approach for detecting colocalization of compound with ABCE1. 293T cells chronically infected with HIV-1 (293T-HIV) or uninfected 293T cells were treated with indicated amounts of PAV-818 (the biotinylated active compound), PAV-543 (the biotinylated inactive compound), or DMSO for 16 h. PLA was performed by incubation with primary antibodies (mouse anti-biotin and rabbit anti-ABCE1), followed by PLA secondary antibodies and other reagents as described in Fig. 6. Red spots indicate sites where biotinylated compound and ABCE1 are colocalized in situ. After PLA, IF was performed (green star) to mark intracellular ABCE1 with low-level green fluorescence. (B) The graph shows the average number of biotin-ABCE1 PLA spots per cell for each condition, with “+” indicating HIV-1-infected cells and “–” indicating uninfected cells. Ten fields were analyzed for each group (containing a total of 104 to 155 cells per group), with error bars showing the SEM. *, Significant difference in the number of biotin-ABCE1 PLA spots per cell when comparing treatment with PAV-818 versus PAV-543, both at 10 μM (P < 0.001). (C) A representative field for each group quantified in panel B is shown, except for DMSO treatment. Fields on the left show biotin-ABCE1 PLA spots alone in grayscale. To the right are the same fields shown as a merge of three color channels: biotin-ABCE1 PLA (red), ABCE1 IF (green), and DAPI-stained nuclei (blue). Scale bars, 10 μm.