We then used the inducible cells to examine VLP production. Expression was induced for 36 h. The cells were then lysed and the E proteins immunoprecipitated, and the VLP in the culture media were pelleted by ultracentrifugation. Analysis by western blotting showed strong E protein expression in both WT and H244A cells, and no expression in the parent cells (Fig. 8C) . The WT cells released E protein in VLP, but VLP release from cells expressing the H244A mutant E protein was greatly reduced (Fig. 8C, -media samples) . This result is in keeping with the hypothesis that the H244A cells assemble VLP in the neutral pH environment of the ER but that VLP release is inhibited by the lack of pr protection from the low pH of the secretory pathway. To test this idea, we induced WT and H244A prM-E expression and cultured the cells in the presence of 20 mM NH 4 Cl to neutralize the acidic pH in the Golgi and TGN compartments (Fig. 8C , +NH 4 Cl lanes). The cellular expression level of either E protein was not significantly affected by NH 4 Cl treatment, and WT VLP production was similar in NH 4 Cl-treated cells and untreated cells. However, production of VLP containing the H244A mutant E protein was increased 4-7 fold in NH 4 Cl-treated cells. While H244A VLP production was still significantly decreased compared to that of WT, it was selectively rescued by NH 4 Cl treatment.