Expression of functional influenza virus RNA polymerase in the methylotrophic yeast Pichia pastoris.
Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni(2+)-agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris.
Hwang
J S
JS
Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan.
Yamada
K
K
Honda
A
A
Nakade
K
K
Ishihama
A
A
D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D012333:RNA, Messenger; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004279:DNA, Viral; C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases