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 g enome. In order to produce large amounts of the functional viral RNA polyme rase sufficient for analysis of its structure-function relationships, the c DNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under ind ependent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris, Simultaneous e xpression 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 cod ing 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, Th e kinetic properties of model template-directed RNA synthesis and the requi rements for template sequence were analyzed using the 3P complex. Furthermo re, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclud e that functional influenza virus RNA polymerase with the catalytic propert ies of a transcriptase is formed in the methylotrophic yeast P. pastoris.