title: Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication.
abstract: The protein NS3 of Dengue virus type 2 (DEN-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the N-terminal region and an NTPase-helicase in the remaining 70% of the protein. The latter region has seven conserved helicase motifs found in all members of the family Flaviviridae. DEN-2 NS3 lacking the proteinase region was synthesized as a fusion protein with glutathione S-transferase in Escherichia coli. The effects of 10 mutations on ATPase and RNA helicase activity were examined. Residues at four sites within enzyme motifs I, II, and VI were substituted, and six sites outside motifs were altered by clustered charged-to-alanine mutagenesis. The mutations were also tested for their effects on virus replication 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 activity only. No virus was detected for any mutation which prevented helicase activity, demonstrating the requirement of this enzyme for virus replication. The remaining six mutations resulted in various levels of enzyme activities, and four permitted virus replication. For the two nonreplicating viruses encoding clustered changes at R(184)KR(186) 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 viral replication complex. Two of the replicating viruses displayed a temperature-sensitive phenotype. One contained a clustered mutation at D(334)EE(336) and grew too poorly for further characterization. However, virus with an M283F substitution in motif II was examined in a temperature shift experiment (33 to 37 degrees C) and showed reduced RNA synthesis at the higher temperature.
authors.affiliation: Department of Microbiology, Monash University, Clayton, Victoria 3168, Australia.
authors.forename: A E
authors.initials: AE
authors.lastname: Matusan
authors.forename: M J
authors.initials: MJ
authors.lastname: Pryor
authors.forename: A D
authors.initials: AD
authors.lastname: Davidson
authors.forename: P J
authors.initials: PJ
authors.lastname: Wright
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D003716:Dengue Virus / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D020365:RNA Helicases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases; D013696:Temperature; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000251:Adenosine Triphosphatases; D000409:Alanine; D000956:Antigens, Viral; D005982:Glutathione Transferase; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases