title: Biochemical and kinetic analysis of the influenza virus RNA polymerase purified from insect cells.
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.
authors.affiliation: 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.
authors.forename: Shijian
authors.initials: S
authors.lastname: Zhang
authors.forename: Leiyun
authors.initials: L
authors.lastname: Weng
authors.forename: Liqing
authors.initials: L
authors.lastname: Geng
authors.forename: Jinlan
authors.initials: J
authors.lastname: Wang
authors.forename: Jingling
authors.initials: J
authors.lastname: Zhou
authors.forename: Vincent
authors.initials: V
authors.lastname: Deubel
authors.forename: Philippe
authors.initials: P
authors.lastname: Buchy
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Toyoda
mesh_terms: 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
chemical_list: 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