FIG 6.

IN undermultimerization is stabilized by HIV-1 RNA and restored in viral particles. (A) Alpha 1 for direct binding between biotinylated TAR RNA (Bio-TAR) and IN_His. The schematic illustration at the top left indicates that streptavidin-coated donor (D) and anti-His acceptor (A) beads bind to the Bio-TAR–IN_His complex. The Alpha signal intensity between IN_His (IN^WT, IN^P26, and IN^RAKA at 10 nM) and Bio-TAR (from 0 to 1,000 nM) is shown at the bottom left. The intensities of IN^WT, IN^P26, and IN^RAKA from 0 to 10 nM Bio-TAR are fitted to saturated curves at the bottom right. The curves and mean K_D values ± SD were determined from representative fitting data from three independent experiments. (B) Alpha 2 for indirect IN multimerization between F-IN and IN_His. The schematic illustration indicates that anti-Flag-coated donor and anti-His coated acceptor beads bind to a dimer of F-IN and IN_His. Alpha signal ratios between F-IN and IN_His proteins (IN^WT, IN^P26, and IN^RAKA, respectively) by the addition of 0.1 to 1,000 nM concentrations of TAR RNA were fitted to sigmoid curves. Mean Hill slopes and log EC₅₀s ± SD were calculated by representative sigmoid curves from three independent experiments. (C) IN multimerization of HIV-1_NL4-3 IN^WT and IN^P26 viral particles generated in the presence or absence of 20 μM NCINI-3. The HIV-1 IN^WT and IN^P26 clones were purified by ultracentrifugation. Highly concentrated HIV-1 IN^WT and IN^P26 were cross-linked with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) BS3 and visualized by SDS-PAGE with anti-HIV-1 IN antibody. Monomers, dimers, tetramers, and high-order multimers of IN^WT and IN^P26 are shown as multimer ratios (percent) determined by measuring each band intensity using ImageJ. The ratios represent mean values ± SD from two independent experiments.