Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.
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.
Zhang
Shijian
S
Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Key Laboratory of Molecular Virology & Immunology, Chinese Academy of Sciences, 411 Hefei Road, 200025 Shanghai, PR China.
Weng
Leiyun
L
Geng
Liqing
L
Wang
Jinlan
J
Zhou
Jingling
J
Deubel
Vincent
V
Buchy
Philippe
P
Toyoda
Tetsuya
T
D000818:Animals; D002478:Cells, Cultured; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D007313:Insecta / Q000166:cytology; D007700:Kinetics; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D014764:Viral Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase