These data are consistent with the idea that, similar to WT E, the mutant protein is assembled with prM into VLP in the ER. The membrane insertion and trimerization activity of H244A suggest that the full-length mutant protein would be fusion-active on such VLP once they are transported from the neutral pH of the ER to the low pH of the Golgi and TGN [31] . Thus, the decreased release of H244A VLP and its partial rescue by neutralization of the exocytic pathway support a critical role for pr in protecting DENV from exocytic low pH, and suggest that virus/VLP fuses in the TGN in the absence of pr-E interaction. Rescue of H244A VLP production by NH 4 Cl was clearly incomplete. This may be due to complex aspects of both virus and cell, such as direct effects of the H244A mutation on particle assembly in the ER, or difficulties in blocking fusion of a virus with the relatively high pH threshold of DENV.