Fig. 4.

TM1 modified Env is functional and maintains broadly neutralizing antibody epitopes. (A) A cell-cell fusion assay was performed by infecting (MOI = 1) HIV-permissive TZM-bl cells with recombinant VACV vectors encoding HIV-1 Envs with WT or TM1 mutated CTs or a control VACV encoding no recombinant gene. Cells were fixed at 18 h and syncytia were visualized by Giemsa stain. (B, C) HEK 293T cells were transduced with recombinant VACV vectors encoding (B) the HIV-1 89.6 or (C) 89.6 N7 (N₁₉₇Q) Envs with WT or TM1 mutated CTs, and cells were stained (N = 3) under non-permeabilizing conditions with the indicated mAbs or a CD4-IgG fusion protein. Fold changes in MFI (TM1:WT) for each mAb were normalized to the fold change in 2G12 stain to account for differences in surface expression. No significant fold differences (p > 0.05) relative to parental Env (dashed line) were noted using one-sample t-test on log-transformed data with Bonferroni correction.