V1V2 loop shielding is intact in heterotrimers consisting of WT and V1V2-deleted subunits. (A–C) The capacity of the V1V2 loop to shield the V3 loop was assessed by studying binding of the V3 loop–specific mAbs 19b and 1-79 to chimeric Env trimers expressed on transfected 293-T cells. Env subunits used to generate the heterotrimers differed only in the presence/absence of the V1V2 loop. (A) Schematics indicating the theoretical number of 19b- and 1-79–accessible epitopes for all potential heterotrimer conformations assuming either self (green shaded area) or neighboring protection (blue shaded area) for the two indicated Env pairs. Blue squares denote V3 loop sensitive to mAbs 19b and 1-79. Gray shaded areas indicate V1V2 loops. (B) Predicted binding curves for V3 loop mAbs under the binding scenario depicted in A using our mathematical model. Green denotes self and blue denotes neighboring protection when heterotrimers are intact and confer V1V2 shielding. The gray line indicates the predicted binding pattern if heterotrimers have lost the capacity for V1V2 shielding. By varying expression ratios of the two respective Env proteins (indicated on the x axis), cell populations with higher or lower degree of heterotrimer conformations can be formed and are used in our model to predict binding patterns. (C) Experimental binding data. 19b (circles) and 1-79 (triangles) binding to 293-T cells expressing the indicated heterotrimers at varying ratios (indicated on the x axis) was studied by flow cytometry. Data of three independent experiments are shown. The highest MFI measured in each experiment was set as the maximum fluorescence level (100%), and measured MFI signals were normalized to the percentage of maximum fluorescence (% max). Dotted lines correspond to the minimum and maximum fluorescence levels (O adn 100%). Unprocessed MFI data for mAbs 19b, 1-79, and 2G12 (to monitor total Env expression) are depicted in Fig. S2.