Expression, purification, and characterization of the RNA 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3

Virology. 2002 Jul 20;299(1):122-32. doi: 10.1006/viro.2002.1504.

Abstract

Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5' end. The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host. NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5'-RNA triphosphatase activity, suggesting its role in the synthesis of the 5'-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified protein has 5'-RNA triphosphatase activity that cleaves the gamma-phosphate moiety of the 5'-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside triphosphatase and 5'-RNA triphosphatase activities of NS3 share a common active site.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acid Anhydride Hydrolases / genetics
  • Acid Anhydride Hydrolases / isolation & purification
  • Acid Anhydride Hydrolases / metabolism*
  • Adenosine Triphosphate / pharmacology
  • Binding Sites
  • Dengue Virus / enzymology*
  • Enzyme Activation / drug effects
  • Escherichia coli / genetics
  • Magnesium Chloride / pharmacology
  • Mutation
  • Potassium Chloride / pharmacology
  • Protein Biosynthesis
  • RNA Helicases
  • RNA, Viral / genetics
  • Recombinant Proteins / metabolism
  • Serine Endopeptidases
  • Substrate Specificity
  • Time Factors
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / isolation & purification
  • Viral Nonstructural Proteins / metabolism*

Substances

  • NS3 protein, flavivirus
  • RNA, Viral
  • Recombinant Proteins
  • Viral Nonstructural Proteins
  • Magnesium Chloride
  • Potassium Chloride
  • Adenosine Triphosphate
  • Serine Endopeptidases
  • Acid Anhydride Hydrolases
  • RNA triphosphatase
  • RNA Helicases