Effects of the H171T IN substitution on HIV-1 replication and recombinant IN activities. (A) p24 production of HIV-1NL4-3 and HIV-1NL4-3(H171T IN) plotted as percent WT with standard deviations shown for n = 3 independent experiments. (B) Single round infection of HIV-1NL4-3 and HIV-1NL4-3(H171T IN) determined by luciferase expression and plotted as percent WT infectivity with standard deviations shown for three independent experiments. (C) SEC of WT and H171T INs. The peaks corresponding to tetrameric (T) and monomeric (M) forms of IN are indicated. (D) HTRF-based LEDGF/p75 independent integration assay and (E) HTRF-based LEDGF/p75 dependent integration assays showing stimulation of WT and H171T IN activities at indicated LEDGF/p75 concentrations. Total HTRF signal is plotted with standard deviation for three independent experiments shown. (F) HTRF-based assays to determine LEDGF/p75 binding affinities for WT (opened boxes) or H171T (closed circles) INs. Error bars indicate standard deviation for three independent experiments.