Biochemical and Kinetic Analysis of the Influenza Virus RNA Polymerase Purified From Insect Cells

Biochem Biophys Res Commun. 2010 Jan 1;391(1):570-4. doi: 10.1016/j.bbrc.2009.11.100. Epub 2009 Nov 20.

Abstract

The influenza virus RNA polymerase (RdRp) was purified from insect cells (around 0.2mg/l). The RdRp catalyzed all the biochemical reactions of influenza virus transcription and replication in vitro; dinucleotide ApG and globin mRNA-primed transcription, de novo initiation (replication), and polyadenylation. The optimal Mg concentration, pH and temperature were 8mM, 8.0 and 25 degrees C, respectively, which were slightly different from those measured for RdRp of virions. This system is a single-round transcription system. K(m) (microM) were 10.74+/-0.26 (GTP), 33.22+/-3.37 (ATP), 28.93+/-0.48 (CTP) and 22.01+/-1.48 (UTP), and V(max) (fmol nucleotide/pmol RdRp/min) were 2.40+/-0.032 (GTP), 1.95+/-0.17 (ATP), 2.07+/-0.17 (CTP), and 1.52+/-0.38 (UTP), which agreed with high mutation of influenza viruses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Humans
  • Influenza A Virus, H1N1 Subtype / enzymology*
  • Influenza A Virus, H1N1 Subtype / genetics
  • Influenza A Virus, H1N1 Subtype / isolation & purification
  • Influenza A Virus, H5N1 Subtype / enzymology*
  • Influenza A Virus, H5N1 Subtype / genetics
  • Influenza A Virus, H5N1 Subtype / isolation & purification
  • Insecta / cytology
  • Kinetics
  • RNA Replicase / biosynthesis
  • RNA Replicase / chemistry*
  • RNA Replicase / isolation & purification
  • Viral Proteins / biosynthesis
  • Viral Proteins / chemistry*
  • Viral Proteins / isolation & purification

Substances

  • PA protein, influenza viruses
  • PB2 protein, Influenzavirus A
  • Viral Proteins
  • influenza virus polymerase basic protein 1
  • RNA Replicase