Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus

The N-terminal one-third of the NS3 protein of Dengue virus type 2 (DEN-2) complexes with cofactor NS2B to form an active serine proteinase which clea ves the viral polyprotein. To identify sites within NS3 that may interact w ith NS2B, seven regions within the NS3 proteinase outside the conserved fla vivirus enzyme motifs were mutated by alanine replacement. Five sites conta ined clusters of charged residues and were hydrophilic. Two sites were hydr ophobic and highly conserved among flaviviruses. The effects of five mutati ons on NS2B/3 processing were examined using a COS cell expression system. Four retained significant proteinase activity. Three of these mutations and two more were introduced into genomic-length cDNA and tested for their eff ects on virus replication. The five mutant viruses showed reduced plaque si ze and two of the five showed significantly reduced titres. All seven mutat ions were mapped on the X-ray crystal structure of the DEN-2 NS3 proteinase : three were located at the N terminus and two at the C terminus of the NS2 B-binding cleft. Two mutations were at the C terminus of the proteinase dom ain and one was solvent-exposed. The study demonstrated that charged-to-ala nine mutagenesis in the viral proteinase can be used to produce growth-rest ricted flaviviruses that may be useful in the production of attenuated vacc ine strains.