Studies of the processing of the flavivirus structural proteins, pre- membrane (prM) and envelope (E), previously reported (Project Z01 AI 00501-05), were concluded. prM contains a structurally conserved 35- to 40- amino-acid predominantly hydro- phobic domain at its carboxy- terminus which determines the arrest of translocation of prM and the re- initiation of translocation of the polyprotein, as well as the signalase-mediated cleavage at the prM-E site. Hydrophobicity within this domain is assymmetrically interrupted by a conserved Arg residue associated with a short amphipathic segment. We studied the topogenic functions of the 37-amino-acid carboxy-terminus of the dengue virus type 4 (DEN4) prM (residues 243-279 of the DEN4 polyprotein). The 20-amino- acid hydrophobic domain upstream from the conserved Arg (residue 264 in the DEN4 polyprotein) was predicted to constitute the stop- transfer/transmembrane domain for prM (the tm segment). Hydrophobic residues downstream from conserved Arg-264 were predicted to constitute the signal peptide for E. Mutations resulting in the deletion of hydrophobic domains and/or of conserved Arg-264 and the associated amphipathic domain were introduced into DEN4 cDNA. Glycosylation, cleavage at the prM-E and E-NS1 sites, translocation, and membrane integration of wild-type and mutant polypeptides were assessed in vitro. Mutational analysis confirmed that hydrophobic residues 265-279 function as E signal peptide within the larger hydrophobic domain. However, these residues were also necessary but not sufficient for arrest of the translocation of prM and its stable membrane integration, since upstream hydrophobic residues 247-262 were also required for these events to occur. Thus, the topogenic sequences in prM appeared to comprise a novel cleavable signal-anchor (SA) domain not previously described. Results also showed that conserved Arg-264 and the associated amphipathic segment were not required for proper processing of prM and E.
