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. 2001 Jul;82(Pt 7):1647-1656.
doi: 10.1099/0022-1317-82-7-1647.

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

Anita E Matusan  1 , Peter G Kelley  1 , Melinda J Pryor  1 , James C Whisstock  1 , Andrew D Davidson  1 , Peter J Wright  1
Affiliations

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

Anita E Matusan et al. J Gen Virol. 2001 Jul.

Abstract

The N-terminal one-third of the NS3 protein of Dengue virus type 2 (DEN-2) complexes with co-factor NS2B to form an active serine proteinase which cleaves the viral polyprotein. To identify sites within NS3 that may interact with NS2B, seven regions within the NS3 proteinase outside the conserved flavivirus enzyme motifs were mutated by alanine replacement. Five sites contained clusters of charged residues and were hydrophilic. Two sites were hydrophobic and highly conserved among flaviviruses. The effects of five mutations 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 effects on virus replication. The five mutant viruses showed reduced plaque size and two of the five showed significantly reduced titres. All seven mutations 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 NS2B-binding cleft. Two mutations were at the C terminus of the proteinase domain and one was solvent-exposed. The study demonstrated that charged-to-alanine mutagenesis in the viral proteinase can be used to produce growth-restricted flaviviruses that may be useful in the production of attenuated vaccine strains.

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