Studies of in vitro processing of the dengue virus polyprotein were continued. Such studies are justified by the following rationale: (1) Flavivirus morphogenesis occurs at the ER membrane. Therefore, this event can conceivably be studied in vitro. (2) The flavivirus polyprotein can be thought of as a polytopic membrane protein which is internally cleaved. Principles governing the association of the polyprotein with membranes have broader implications for understanding the assembly of proteins into membranes in general (3) The phenotype(s) """"""""Ess"""""""" of some mutations potentially useful for vaccine development when incorporated into infectious RNA prepared from full-length dengue cDNA can be tested in vitro. Flavivirus structural proteins capsid (C), pre-membrane (prM), and envelope (E) are in that order cleaved from the amino-terminus of the flavivirus polyprotein co-translationally by the host cell enzyme, signalase. prM and E each have a hydrophobic carboxy-terminus 40 to 47 amino acids in length assymetrically interrupted by a conserved hydrophilic residue or residues. In dengue virus type 4 cDNA, the hydrophobic carboxy-terminus of prM spans residues 243 to 279 of the polyprotein, with an interrupting Arg at position 264. It is predicted that hydrophobic residues 243 to 263 constitute a transmembrane segment for prM and that hydrophobic residues 265-279 constitute a signal for E. The requirement for the conserved interrupting hydrophilic residue is not understood. We studied the processing of prM, E, and NS1 translated from RNA transcripts prepared from dengue virus type 4 cDNA. Mutations were introduced into sequences, encoding the hydrophobic carboxy-terminus of prM, and the phenotype of those mutations were compared to that of the wild-type (wt) sequence. The results of this study permit the establishment of a model for understanding the processing of the flavivirus structural proteins and the mechanisms by which their final membrane orientation is attained.
