Processing of flavivirus structural glycoproteins: stable membrane insertion of premembrane requires the envelope signal peptide.

The flavivirus structural proteins capsid (C), premembrane (prM), and envelope (E) are cleaved in that order from the N-terminus of the polyprotein by the ER intralumenal enzyme signal peptidase. The prM-E and E-NS1 junctions contain hydrophobic domains with both transmembrane and signal function. These domains reside at the C-termini of prM and E, respectively, after cleavage. We studied the functions of the 37-amino-acid C-terminus of the dengue virus type 4 (DEN4) prM (amino acids 243-279 of the DEN4 polyprotein) in the processing of prM and E. Hydrophobicity in this domain is interrupted by a conserved Arg residue (Arg-264) within a short amphipathic segment. Hydrophobic amino acids upstream from Arg-264 (aa 243-263) were presumed to constitute the membrane anchor for prM (the "tm" segment). Previous results had suggested that sequences downstream from Arg-264 (aa 265-279) constitute the E signal peptide. RNA transcripts prepared from wild-type (wt) and deletion-mutant DEN4 cDNAs encoding the prM signal peptide, prM, E, and the N-terminus of the nonstructural glycoprotein, NS1, were translated in rabbit reticulocyte lysate in the presence of microsomes. Processing of wt prM and E in vitro appeared to mimic processing occurring during flavivirus infection. Analysis of mutants confirmed the localization of the E signal peptide within residues 265 to 279. However, deletions within either the E signal peptide or the tm segment resulted in a defect in both membrane insertion of prM and cleavage of the prM-E junction. Membrane anchoring of prM appeared to be a two-step process requiring function of both the tm segment and the E signal peptide, and fully efficient prM-E cleavage was also dependent upon the integrity of both hydrophobic domains. We propose a model for the processing of the flavivirus structural glycoproteins based on these results.