(A) Binding affinities of fluorescently labelled M48 and M48U1 towards gp120SF162 mutants. Fluorescently labeled M48 and M48U1 were tested for their affinity towards single site-directed mutants of the SF162 gp120 protein by fluorescence polarization analysis. The fold increase in Kd is plotted relative to the WT SF162 gp120. The H105Y mutation, found in the rM48BaL virus, was also introduced in the SF162 gp120 background. (B) Sensitivity of Env-mutant pBRNL4.3 molecular clones towards miniCD4 proteins. pBRNL4.3 replication competent molecular clones, carrying the H105Y, S375R, S375N, G471R, and D474N mutations were tested for their sensitivity towards the miniCD4 in a TZM-bl based assay. Fold increase in IC50 values is given and is calculated as follows: IC50 values from the mutant pBRNL4.3 viruses divided by the IC50 value from the control wild-type SF162 pBRNL4.3 virus. >; IC50 could not be exactly quantified because maximal nontoxic levels of miniCD4 were reached. (C) Sensitivity of mutant pseudoviruses for miniCD4 proteins. The S375R mutation was introduced in the envelopes of the subtype B BaL virus, the primary subtype C VI829 virus, and the primary CRF02_AG VI1090 strain and used to produce mutant pseudoviruses. D474N was introduced in the BaL and SF162 background. Sensitivity towards the miniCD4 was evaluated in a TZM-bl based assay, and results are depicted as fold increase in IC50 values, which was calculated as described above. >; IC50 could not be exactly quantified because maximal nontoxic levels of miniCD4 were reached.