FIG. 4.

All of the H8 down mutations can be partially suppressed by two additional mutations, Q227R and D243Y, and rescued by chlorpromazine. (A) Titers of H8 down mutant viruses with (▪) or without () the suppressor mutations Q227R and D243Y. Mouse NIH 3T3 fibroblast cells were exposed to 10-fold serial dilutions of virus stocks.Titers were calculated based on an endpoint dilution (n = 4). (B) Western blot analysis of virion containing mutant envelope proteins. Proteins were separated by sodium dodecyl sulfate-8% polyacrylamide gel. Membranes were cut into two parts at 45 kDa. The top part was probed with anti-SU antisera, and the bottom portion was reacted with anti-CA antisera. The experiment was performed twice; a representative is shown. (C) XC cells were incubated on ice for 30 min with 10-fold serial dilutions of virus stocks in BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid)-buffered medium (pH 7.4) containing Polybrene (20 μg/ml; Sigma) and then shifted to 37°C for another 30 min. Cells were rinsed twice to remove unbound virus and then incubated with (▪) or without () 0.4 mM chlorpromazine (pH 7.4) for 1 min. Chlorpromazine was immediately removed, and the cells were washed twice again. After 40 h, cells were stained with X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) for transduction of β-galactosidase activity, and the infectious titers were calculated from the endpoint dilution (n = 4). The experiments were performed at least twice. A representative is shown. No infection of XC cells by particles lacking Env was observed in the presence of chlorpromazine.