Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a.

Expression of dengue virus gene products involves specific proteolytic cleavages of a precursor polyprotein. To study the flanking sequences required for expression of the dengue virus nonstructural glycoprotein NS1, we constructed a series of recombinant vaccinia viruses that contain the coding sequence for NS1 in combination with various lengths of upstream and downstream sequences. The NS1 products expressed by these viruses in infected CV-1 cells were immune precipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show that the 24-residue hydrophobic sequence preceding NS1 was necessary and sufficient for the production of glycosylated NS1 and that this sequence was cleaved from NS1 in the absence of most dengue virus proteins. This finding is consistent with previous proposals that this hydrophobic sequence serves as an N-terminal signal sequence that is cleaved by signal peptidase. The cleavage between the C terminus of NS1 and the downstream protein NS2a occurred when the complete NS2a was present. Recombinant viruses containing NS1 plus 15 or 49% of NS2a produced proteins larger than authentic NS1, indicating that the cleavage between NS1 and NS2a had not occurred. Failure of cleavage was not corrected by coinfection with a recombinant virus capable of cleavage. These results suggest that NS2a may be a cis-acting protease that cleaves itself from NS1, or NS2a may provide sequences for recognition by a specific cellular protease that cleaves at the NS1-NS2a junction.