Figure 2.

Variable efficiency of HIV-1 transmission mediated by different DC-SIGN-expressing cell lines (A) DC-SIGN expression in Raji and K562 cell lines. Parental cells and DC-SIGN transfectants were stained with MAb against DC-SIGN as described [5]. On all histograms, the gray curve represents staining with an isotype control antibody, whereas the filled black curve represents DC-SIGN MAb staining. The mean fluorescence intensity of DC-SIGN staining is shown in the inset of the histograms. One representative experiment out of three is shown. Cells maintained consistent DC-SIGN expression levels throughout the analyses. (B) Adhesion of HIV-1 to DC-SIGN-expressing cells. Cells were incubated with pseudotyped HIV-Luc/ADA containing 20 ng of CA-p24 for 2 h at 37°C, washed extensively, lysed with 0.5% Triton X-100, and quantified with p24 ELISA kits. HIV-1 absorbed by Raji/DC-SIGN cells was normalized as 100% (170 pg of recovered CA-p24 in this experiment). The relative percentage of adsorbed p24 was the average of three separate samples. One representative experiment of six is shown. (C) Adhesion of ICAM-3 to DC-SIGN-expressing cells. The percentage of the cells bound to ICAM-3 was measured by flow cytometry using a fluorescent bead adhesion assay as described [5]. Adhesion of ICAM-3 to DC-SIGN-negative parental cells was less than 2%. Mouse IgG represents an isotype control antibody. One representative experiment of three is shown. (D) Capture and transmission of HIV-Luc/ADA by different donor cells. Donor cells pulsed with HIV-1 (1 × 10⁵ IU) were washed before coculturing with Hut/CCR5 target cells as described for Figure 1. DC-SIGN-negative parental cells were used as controls. Donor cells were preincubated with either mannan (20 μg/ml) or MAb against DC-SIGN (10 μg/ml), respectively, before virus addition. Mouse IgG (10 μg/ml) was used a control antibody. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second. (E) DC-SIGN enhancement of trans-infection by HIV-Luc/ADA. Donor cells pulsed with HIV-1 (1 × 10⁴ IU) were cocultured with Hut/CCR5 target cells without removing unbound virus present in the culture medium. DC-SIGN-negative parental cells were used as controls. Donor cells were preincubated with either mannan (20 μg/ml) or MAb against DC-SIGN (10 μg/ml), respectively, before virus addition. Mouse IgG (10 μg/ml) was used as a control antibody. Each data set represents the mean of three separate wells of infected cells. One representative experiment out of three is shown. cps, counts per second.