Figure 3.

Reversions restore gp120 folding. HeLa cells were infected with VVT7 and transfected with plasmids encoding wt gp120, mut, revertant (R1, R2, or R3) or two additional mutants FG1 and FG2. Cells were pulse labeled for 5 min and chased for the indicated times. Cells were lysed and gp120 was immunoprecipitated from lysates. Immunoprecipitates were deglycosylated with endoglycosidase H and analyzed by either reducing or nonreducing 7.5% SDS-PAGE. Folding intermediates (IT), the native form (NT), the reduced state from which the signal peptide was cleaved off (Rc) or not (Ru) are indicated. In addition, secreted gp120 was immunoprecipitated from the culture supernatant after 8 h of chase and directly analyzed by reducing SDS-PAGE (bottom). Percentages of NT = native (nonreduced), cleaved = Rc (reduced), and secreted gp120 are given below lanes of chase samples. Percentage of secretion is relative to wt.