CCR5 structural plasticity shapes HIV-1 phenotypic properties
Fig 5
Role of CCR5 dimerization in HIV-1 entry into T-cells.
A Fusion kinetics of BlaM-vpr-containing virus #25 or 34 with CD4+ A3.01 T-cells expressing FLAG/SNAP-tagged WT-CCR5 or L196K-CCR5. Data points represent means ± SEM of 2 independent determinations (out of 5). B Gating strategy of A3.01 cells expressing WT-CCR5 (red) or L196K-CCR5 (blue) at a low (GMFI = 111 and 123 for WT-CCR5 and L196K-CCR5, respectively), intermediate (599 and 533) or high level (2903 and 2382). Shown are representative flow cytometry plots of cell side scatter vs receptor expression level revealed with the AlexaFluor 647-conjugated anti-FLAG mAb M2. On the whole cell populations, both receptors were expressed at similar expression levels (GMFI = 362 and 312 for WT-CCR5 and L196K-CCR5, respectively). Comparable results were obtained using the unconjugated M2 revealed by an AlexaFluor 647-conjugated goat anti-mouse IgG (GAM) (GMFI = 5871 and 3342) C, D and E The virus-cell fusion experiments shown in A were analyzed with A3.01 T-cells expressing low (C), intermediate (D) or high (E) cell surface level of either WT- or L196K-CCR5. F Levels of fusion at 3 h for the indicated viruses were measured with A3.01 T-cells expressing low (L) or high (H) level of either WT- (red bars) or L196K-CCR5 (blue bars). Results are means ± SEM of 3 independent determinations (except for the non-M and M-tropic viruses JR-CSF and JR-FL where n = 1). In panels A, C, D, E and F, results are expressed as percents of fusion relative to the maximum extent of fusion (Fmax) of virus #34 with L196K-CCR5-expressing cells. This Fmax value, expressed as the percentage of cells containing BlaM-vpr, ranged between 40–60% and did not vary with the receptor expression level.