FIG. 6.

Disruption of disulfide linkage between gp70 and Pr15E occurs through a thiol-disulfide exchange reaction within the Env complex. Four parallel cultures of BHK-21 cells were infected with recSFV expressing the amphotropic Mo-MLV env gene. Two of these were labeled for 15 min with l-[³⁵S]cysteine and chased for 120 min, whereas the others were not labeled. At the end of the chase, all the cultures were put on ice. One culture of labeled cells and one culture of nonlabeled cells were incubated twice for 1 min with PBS–20 mM NEM and then washed four times in PBS lacking NEM. The remaining cell cultures were washed with PBS lacking NEM. Thereafter, all the cultures were lysed in the absence of NEM. The cell lysates were then incubated for 15 h at 4°C in the following (1:1) mixtures: lysate of labeled, alkylated cells plus lysis buffer lacking NEM (lane 1); lysate of labeled, alkylated cells plus lysate of nonlabeled, nonalkylated cells (lane 2); lysate of labeled, nonalkylated cells plus lysis buffer lacking NEM (lane 3); and lysate of labeled, nonalkylated cells plus lysate of nonlabeled, alkylated cells (lane 4). As a control to check the amount of gp70-S-S-Pr15E at the time of mixing, we diluted lysates of labeled, alkylated cells (lane 5) and labeled, nonalkylated cells (lane 6) with lysis buffer containing NEM to block further disruption of the complexes during incubation. The Env protein was thereafter immunoprecipitated with the polyclonal anti-MLV serum and analyzed under nonreducing conditions in an SDS–12% polyacrylamide gel.