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Figure 5 | Retrovirology

Figure 5

From: A single-residue change in the HIV-1 V3 loop associated with maraviroc resistance impairs CCR5 binding affinity while increasing replicative capacity

Figure 5

Receptor binding properties of wild-type and modified gp120 monomers derived from the MVC-Sens and MVC-Res viral isolates. a Competition of mAb Q4120 binding to CD4-expressing HEK 293T cells by increasing concentrations of the indicated purified monomeric gp120. Results were normalized for nonspecific binding (0%) and specific binding in the absence of glycoprotein (100%, B0) and were fitted to a one-site competitive binding model. A representative experiment performed in duplicate is shown (n = 3). b Equilibrium saturation binding of the 35S-labeled gp120 of MVC-Sens, MVC-Sens(P/S) (i.e. MVC-Sens wherein Pro-308 is substituted by Ser), MVC-Res(V3S) (i.e. MVC-Res whose V3 loop is replaced by that of MVC-Sens) or MVC-Res(−A) (i.e. MVC-Res lacking the Ala insertion). Curves represent specific binding of glycoproteins to crude membranes from CCR5-expressing HEK 293T cells, determined in the presence of 400 nM sCD4, and obtained by subtracting from total binding the non specific binding measured in the presence of 10 μM TAK779 or using parental HEK cells. Data were fitted to a one-site binding model. Representative experiments performed in duplicate are shown (n = 3–4). c Specific binding of 10 nM of the indicated 35S-labeled gp120 monomers (+400 nM sCD4) to CCR5-expressing HEK cells, in the presence (+) or absence (−) of 10 μM MVC, was calculated as in panel (b), and then expressed as percent of MVC-Sens gp120 binding in the absence of MVC (100%). The binding of glycoproteins measured in the presence of 10 μM TAK779 was considered as nonspecific binding in these experiments. Of note, in some cases, glycoproteins showed levels of binding that came slightly lower than this nonspecific binding, explaining why “negative” specific binding are plotted in the panel. Results represent mean ± SD of 2–5 independent experiments performed in duplicate. d The panel represents specific binding on CD4-expressing HEK cell membranes of the indicated 35S-gp120 used at a concentration equal to their Ki value for CD4 deduced from the displacement experiments of mAb Q4120 binding shown in panel (a) (see text). One experiment out of two is shown. e Binding of the indicated concentrations of MVC-Sens (closed symbols and straight lines) or MVC-Res (open symbols and dashed lines) gp120 to 17b (squares and diamonds) or E51 (circles and triangles) mAbs immobilized on a CM4 sensorchip, alone (triangles and diamonds) or after preincubation with 200 nM sCD4 (circles and squares). Of note, triangles and diamonds are superimposed at the bottom of the panel due to marginal binding in the absence of sCD4. Open and closed squares are also superimposed due to similar binding of MVC-Sens and MVC-Res gp120s to mAb 17b. f and g Competition of 10 nM 35S-gp120Bx08 (f) or 35S-gp120Sens (g) binding to CCR5-expressing membranes by increasing concentrations of the indicated unlabeled gp120 was carried out in the presence of an excess concentration of sCD4 (1,000 nM). Results were normalized for nonspecific binding determined in the presence of 10 μM TAK779 (0%) and specific binding in the absence of competitor (100%, B0) and were fitted to a one-site competitive binding model. Representative experiments out of 2–3 independent experiments run in duplicate are shown.

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