Recombinant Dengue Type 1 Virus NS5 Protein Expressed in Escherichia Coli Exhibits RNA-dependent RNA Polymerase Activity

Virology. 1996 Feb 15;216(2):317-25. doi: 10.1006/viro.1996.0067.

Abstract

The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • Dengue Virus / enzymology
  • Dengue Virus / genetics*
  • Escherichia coli
  • Genetic Vectors / genetics
  • Molecular Sequence Data
  • RNA Replicase / genetics
  • RNA Replicase / metabolism*
  • Rabbits
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / isolation & purification
  • Viral Nonstructural Proteins / metabolism*

Substances

  • DNA Primers
  • NS5 protein, flavivirus
  • Recombinant Fusion Proteins
  • Viral Nonstructural Proteins
  • RNA Replicase