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 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.
Matusan
Anita E
AE
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
Kelley
Peter G
PG
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
Pryor
Melinda J
MJ
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
Whisstock
James C
JC
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
Davidson
Andrew D
AD
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
Wright
Peter J
PJ
Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia.
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D003716:Dengue Virus / Q000201:enzymology* / Q000254:growth & development; D010450:Endopeptidases / Q000235:genetics*; D008958:Models, Molecular; D017354:Point Mutation; D011485:Protein Binding; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D010948:Viral Plaque Assay; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000409:Alanine; D010450:Endopeptidases; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C080102:NS2B protein, flavivirus; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases