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Journal of Virology, May 2000, p. 4074-4084, Vol. 74, No. 9
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Jung-Shan Hwang,1 Kazunori Yamada,2 Ayae Honda,1,3 Kohji Nakade,2 and Akira Ishihama1,*

Department of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540,1 Mitsubishi Chemical Co., Yokohama Research Center, Aoba-ku, Yokohama 227-8502,2 and Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012,3 Japan

Received 26 July 1999/Accepted 4 February 2000

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 Ni2+-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.

* Corresponding author. Mailing address: National Institute of Genetics, Department of Molecular Genetics, Mishima, Shizuoka 411-8540, Japan. Phone: 81-559-81-6741. Fax: 81-559-81-6746. E-mail: aishiham{at}lab.nig.ac.jp.

Journal of Virology, May 2000, p. 4074-4084, Vol. 74, No. 9
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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