The dengue viral protease is a heterodimer of nonstructural protein 2B (NS2B) and NS3. The amino-terminal third of NS3 contains four regions of limited sequence homology to trypsin-like serine proteases, including the three active center residues. Two of these four regions in NS3 ("homology boxes" 3 and 4) contain residues predicted to be important for substrate binding (aa 1604, 1605, 1625, 1627 and 1628) based on analogy with the structure of the trypsin P1 specificity pocket. We performed a mutagenesis study of some of the residues in homology boxes 3 and 4 of dengue virus type 2 in order to characterize their requirement for protease activity and to identify mutants with partial cleavage defects. Initially, 46 mutations were analyzed for their effect on intramolecular cleavage at the NS2B-NS3 junction. Most mutations in box 4, including those in aa 1625, 1627, and 1628, abolished protease activity. This is consistent with the hypothesis that box 4 forms part of the substrate-binding pocket. In contrast, many of the mutations in box 3, including those in aa 1604 and 1605, retained significant protease activity, even wild-type activity in a few cases. Surprisingly, asp 1604, predicted to contact a positively charged residue at the cleavage site, could be changed to lys or arg with significant residual activity. Analogous mutations in trypsin abolish cleavage. Other mutations in box 3, clustered around the catalytic serine, abolished protease activity. This confirmed that some residues in box 3 are important for protease function, but is inconsistent with the proposed role for aa 1604 and 1605 in substrate binding. Control mutations outside of the homology boxes were well-tolerated. More recently, these 46 mutations were also analyzed for their effects on intermolecular cleavage at the NS4B-NS5 junction. In general, for both wild-type and mutant constructs, cleavage in trans was somewhat more efficient than cleavage in cis. However, the relative order of the effects of the mutations on protease activity in cis or in trans were the same, e.g. mutations which severely diminished protease activity in cis were also severely defective in trans. Work is underway to introduce some of the mutations in NS3 which permit significant residual protease activity back into the dengue genome, in an effort to confirm the observed effect on protease function and possibly to attenuate the virus. As a first step toward this goal, two silent mutations were successfully introduced into the DEN2 infectious clone to create unique restriction sites which flank the mutagenized region, which will facilitate the introduction of the NS3 mutations into the genome. Publications: Valle, R. P. C., and B. Falgout. Mutagenesis of the NS3 protease of dengue virus type 2. 1998. J. Virol. 72:624-632.