Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: Effects on enzyme activity and virus replication

The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstr uctural protein specified by the virus and is known to possess multiple enz ymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latt er region has seven conserved helicase motifs found in all members of the f amily Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. Th e effects of 10 mutations on ATPase and RNA helicase activity were examined . Residues at four sites within enzyme motifs I, II, and VI were substitute d, and six sites outside motifs were altered by clustered charged-to-alanin e mutagenesis. The mutations were also tested for their effects on virus re plication by incorporation into genomic-length cDNA. Two mutations, both in motif I (G198A and K199A) abolished both ATPase and helicase activity. Two further mutations, one in motif VI (R457A,R458A) and the other a clustered charged-to-alanine substitution at R(376)KNGK(380), abolished helicase act ivity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replicati on. The remaining six mutations resulted in various levels of enzyme activi ties, and four permitted virus replication. For the two nonreplicating viru ses encoding clustered changes at R184KR186 and D(436)GEE(439), we propose that the substituted residues are surface located and that the viruses are defective through altered interaction of NS3 with other components of the v iral replication complex. Two of the replicating viruses displayed a temper ature-sensitive phenotype. One contained a clustered mutation at D334EE336 and grew too poorly for further characterization. However, virus with an M2 83F substitution in motif II was examined in a temperature shift experiment (33 to 37 degreesC) and showed reduced RNA synthesis at the higher tempera ture.