Reconstitution of influenza virus RNA polymerase from three subunits expressed using recombinant baculovirus system.
Influenza virus RNA polymerase catalyzes multiple step reactions in transcription and replication of the genome RNA. The core enzyme is composed of each one of the three P proteins, PB1, PB2 and PA (Honda et al. (1990) J. Biochem. 107, 624-628). For detailed analysis of the role of each P protein and of the functional domains on each P polypeptide, we expressed individual P proteins in cultured insect cells after infection with recombinant baculoviruses. PB1 and PB2 accumulated in cell nuclei whereas PA stayed in cytoplasm. Both the PB1 and PB2 proteins were purified from aggregates in the respective nuclear extract, and the PA was partially purified from the cytoplasm. RNA polymerase was reconstituted by mixing the three P proteins in a urea solution and then dialyzing against a reconstitution buffer. The reconstituted enzyme was able to transcribe model RNA templates. Minus-sense RNA was a better template than plus-sense RNA.
Kobayashi
M
M
Department of Molecular Genetics, National Institute of Genetics, Shizuoka, Japan.
Tuchiya
K
K
Nagata
K
K
Ishihama
A
A
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D009975:Orthomyxoviridae / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases