Processing of the dengue virus polyprotein is initiated by cleavage of the capsid protein from the premembrane protein (pM) at a site downstream from the pM signal sequence presumably recognized by the enzyme signal peptidase. Cleavage of pM from the N-terminus of the envelope glycoprotein (E) also appeared to be mediated by signal peptidase. Requirement for autoproteolytic activity of the dengue capsid protein was not detected. In order to extend these observations RNA transcripts prepared from wild type (wt) and mutant dengue DNA encoding the N-terminal 1,040 or 691 amino acids of the polyprotein were translated in vitro in rabbit reticulocyte or wheat germ extracts in the presence or absence of added dog pancreatic microsomal membranes. Results have not been interpretable due to internal initiation of translation at multiple sites and/or failure of commercially purchased membranes to mediate cleavage. Capping of RNA transcripts and immune- precipitation of cell-free translation products with rabbit antibodies to peptides representing the N-termini respectively of capsid, pM, and E currently under study may solve these problems. In addition, five mutations were cloned into a 4kb fragment of dengue DNA encoding capsid, pM, E, NS1, and NS2A present in the vector pSC11 to facilitate recombination into vaccinia virus. These mutations included: (1) a deletion of a trypsin-like potential cleavage site (Arg-Lys-Arg; residues 97-99) which precedes the pM signal; (2) a substitution mutation of the pM signal in which residues 109 and 111 have been converted to hydrophilic Lys and Arg, respectively; (3) deletion of the upstream hydrophobic segment in pM (246-263); (4) alteration of Arg (residue 264) to Phe-Phe-Val; (5) deletion of the downstream hydrophobic segment in pM (265-279). Analysis of phenotypes of mutant vaccinia virus recombinants is not complete. Mutation (2) appears to abrogate capsid-pM cleavage; mutation (3) results in partial inhibition of pM-E cleavage. Mutation (5) completely inhibits pM-E cleavage. Further studies of these mutants and of the fate of capsid in vaccinia virus and dengue virus infected cells are in progress.
