Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.

Shijian Zhang (1) , Leiyun Weng (1) , Liqing Geng (1) , Jinlan Wang (1) , Jingling Zhou (1) , Vincent Deubel (1, 2) , Philippe Buchy (2) , Tetsuya Toyoda (1)

1 LMVI - Key Laboratory of Molecular Virology & Immunology
2 Institut Pasteur du Cambodge

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.

Domaines: Microbiologie et Parasitologie

Shijian Zhang, Leiyun Weng, Liqing Geng, Jinlan Wang, Jingling Zhou, et al.. Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.. Biochemical and Biophysical Research Communications, 2010, 391 (1), pp.570-4. ⟨10.1016/j.bbrc.2009.11.100⟩. ⟨pasteur-00624863⟩