Proteolytic processing of the polyprotein encoded by the positive strand RNA viral genome of dengue virus (as well as other flaviviruses) is a prerequisite for viral gene expression. Recent evidence from our Section indicates that a minimum length of 8 amino acids at the NS1 C-terminus preceding the NS1-NS2A junction is required for cleavage to take place. This study was initiated to analyze the amino acid sequence motif that is optimal cleavage of the NS1-NS2A junction during processing of this region of the viral protein. For this purpose, several amino acid substitutions, at position -1 (Gly or Ala), -2 (Thr), or -3 (Val) of the NS1-NS2A junction of the dengue 4 sequence were introduced and the resulting mutant NS1-NS2A was expressed by a recombinant vaccinia virus. Analysis of mutant sequences expressed by a vaccinia virus recombinant indicated that substitution of Ala at position -1, or Val at -3, yielded an uncleaved NS1-NS2A fusion protein suggesting that the amino acids at positions -1 and -3, that are strictly conserved among flaviviruses, are optimal for cleavage of this junction. On the other hand, replacement of Thr at position -2, a position that is not conserved among flaviviruses, had only a slight to moderate negative effect on cleavage. Variation in amino acid sequence at position +1 occurs among flaviviruses. Nonetheless, substitutions at this site resulted in 42% to 90% reduction in cleavage. Work is in progress to introduce amino acid substitutions at other positions (-4 to -8) to extend our analysis of the sequence motif at the NS1-NS2A junction. Also, construction of full length dengue cDNA containing mutations at strategic NS1-NS2A cleavage sites is underway. Mutant viruses recovered from transfected cells will be evaluated with respect to viral growth in cultured cells and virulence in an infected experimental animal host. Ultimately, this analysis may lead to isolation of attenuated dengue virus mutants that have the potential for use in a live virus vaccine for humans.