FIG. 6.

Mutations within the hr2 of SU-A that result in reduced Tva binding also result in reduced binding of mc8C5-4. (A to C) Df-1 cells were transfected in duplicate with pCB6-EnvA DNA coding for either wild-type or hr2 mutant glycoprotein (sM5, sM12, sM20, sM21, and sM28 [8, 46]) or with pCB6-EnvC. At 24 h posttransfection, cells were prepared for immunofluorescence. For whole-cell staining and control of transfection efficiency (A and C), Env in fixed cells was detected with rb-anti-A-tail or rb-anti-C-tail, respectively. Binding of mc8C5-4 to cell surface-expressed Env (B and C), was performed with unfixed cells on ice. (B) Individual cells from each field have been enlarged for better viewing. (D) Df-1 cells mock transfected or transfected with pCB6-EnvA wild type (wt) or hr2 mutants were analyzed by FACS after incubation of unfixed cells on ice with mc8C5-4 ascites, followed by rb-anti-mouse Alexa488, and PFA fixation. Binding affinity of mc8C5-4 is reflected in the mean fluorescence values of the gated populations, which were corrected for background fluorescence of nontransfected cells, and are plotted as relative mean fluorescence values compared to wild-type EnvA.