FIG 5.

Chimeric transmembrane proteins containing the PSGL-1 cytoplasmic tail cocluster with HIV-1 Gag more efficiently than their native counterparts. (A) T cells with a polarized morphology (P2 cells) were dually immunostained for PSGL-1 and CD46. As indicated by arrows, PSGL-1 localized to uropods, whereas CD46 localized to the entire cell surface. (B) Schematic representation of the chimeric constructs. (C) The mean fluorescence intensities of HeLa cells expressing transfected ICAM-3 constructs were determined as described in the legend to Fig. 3C. Note that the mean fluorescence intensities of HeLa cells transfected with plasmids carrying ICAM-1 or CD46 were not determined due to endogenous expression of these proteins. (D) HeLa cells were transfected with plasmids carrying WT Gag-mEos3.2 and the indicated constructs and imaged as described in the legend to Fig. 1. Representative reconstructed images and cross-correlation curves are shown. Bars = 500 nm. Gag-mEos3.2 is shown in green, and the indicated proteins are shown in red. (E) The analysis of total coclustering was performed as described in the legend to Fig. 1B using cross-correlation curves for images of a total of 10 cells per condition from 2 independent experiments. Values shown indicate means ± SEMs. P values were calculated for each PCT construct relative to its wild-type counterpart. ***, P < 0.0005. (F) HeLa cells were transfected with a WT HIV-1 molecular clone (pNL4-3) and expression plasmids carrying wild-type ICAM-1 or ICAM-1/PCT and cultured for 2 days. Cell and virus lysates were collected and subjected to immunoblotting analysis using HIV-Ig and anti-ICAM-1. Note that wild-type ICAM-1 was detected in virus lysates but at a much smaller amount than ICAM-1/PCT. The results shown are representative of the results from three independent experiments. The normalized ICAM-1/p24 ratio shown at the bottom was calculated and averaged from the results of the three experiments. *, P < 0.05.