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. 2018 Dec 6;14(12):e1007432. doi: 10.1371/journal.ppat.1007432

Fig 3. Distinct HIV-1 gp120s are differentially sensitive to CCR5 dimerization.

Fig 3

A Membranes from HEK 293 cells transfected with FLAG/SNAP-tagged WT-CCR5 or L196K-CCR5 cDNA show similar receptor expression levels. Fluorescence intensity (at 620 nm) of the SNAP substrate BG-Lumi4-Tb bound to receptors increases linearly with membrane quantity, in contrast to non specific binding (NSB) to parental membranes. B FRET efficacy was measured between SNAP- and CLIP-tagged receptors labeled with lumi4-Tb and d2, respectively, and expressed as the ratio of the fluorescence intensities at 665 nm (d2) and 620 nm (Lumi4-Tb) (λex = 320 nm). HEK cells were transfected in such a way that WT-CCR5, L196K-CCR5 and L208K-CCR5 have similar cell surface expression levels and that the FRET acceptor(CLIP)/donor(SNAP) ratio is non-saturating and kept constant in all experiments. Mutation of Leu-196 reduces CCR5 dimerization, in contrast to Leu-208 that resides outside the dimerization interface [52] (n = 3). C, Saturation binding curves of the CCR5 chemokine 125I-CCL3 to FLAG/SNAP-tagged WT-CCR5- or L196K-CCR5-expressing membranes. Results indicate that L196K-CCR5 has a 36%-fold lower Bmax value than WT-CCR5 (2.5 vs 3.9 pmole/mg of protein, respectively). In the range of the 125I-CCL3 concentrations tested, however, similar affinity constant values were deduced from the curves for WT- and L196K-CCR5 (KD = 0.36±0.18 and 0.47±0.13 nM, respectively). D Saturation binding curves of 35S-gp120 #25 or #34 to FLAG/SNAP-tagged WT-CCR5- or L196K-CCR5-expressing membranes. E Specific binding of 10nM 35S-gp120/sCD4 to WT-CCR5-, L196K-CCR5- or L196C-CCR5-expressing membranes. * P<0.05; ** P <0.01; *** P <0.001, vs binding to WT-CCR5 in unpaired two-tailed Student t test. Results (means ± SEM, n = 3–6) are expressed as % specific binding relative to 35S-gp120 #34 binding to WT-CCR5. F Binding of mAbs 2D7, 45531 or anti-Flag M2 to FLAG/SNAP-tagged WT- or L196K-CCR5-expressing A3.01 or HEK cells was revealed by AlexaFluor 647-conjugated secondary Ab and flow cytometry analysis (n = 3).