Minimum molecular architectures for transcription and replication of the influenza virus

For in vitro analysis of transcription and replication of influenza virus, use of the purified RNA polymerase is critical, because contaminating cellular oligonucleotides serve as primers for transcription by the vRNA polymerase and contaminating cellular polymerases and nucleases interfere with the detection of RNA synthesis activity by the vRNA polymerase. The initial success of the expression of recombinant RNA polymerase was achieved by using vaccina virus vector (27,28). The expressed RNA polymerase is functional in vivo as detected by expression of reporter RNA under the control of influenza virus promoters (35, 36). This system is, however, not useful for large scale purification of the influenza virus RNA polymerase for in vitro studies. To achieve a high yield of the RNA polymerase, we have developed an expression system of the influenza virus RNA polymerase in methlotrophic yeast Pichia pastoris (30) but it was difficult to remove trace amounts of cellular nucleases from the RNA polymerase. We then used the simultaneous expression system of all three P proteins in insect cells after coinfection with three species of the recombinant baculovirus, each coding for the PB1, PB2, or PA protein (29). The assembled 3P complex contained nearly equal amounts of the three P proteins, supporting the concept that the core RNA polymerase is composed of one molecule each of the three P proteins (13).

The results herein described are apparently inconsistent with the observation that the expression of not only PB1, PA but also PB2 is required for transcription of both plus- and minus-strand model RNAs (36). The simultaneous expression does not necessarily indicate the direct involvement of PB2 in the catalytic function of RNA replication, but may be required for formation of the functional 3P complex and/or increase in the metabolic stability of the 3P complex. During preparation of this manuscript, Lee et al. (38) reported that all three P proteins are required for the initiation of unprimed RNA synthesis in vitro using affinity-purified 3P complex through paramagnetic bead-bound 5’ –vRNA oligonucleotide probe, but they failed to affinity-purify the 2P complex supposedly because of weak binding of PA-PB1 to the vRNA probe. With use of such crude cell extracts, it is generally difficult to detect the low level of specific RNA synthesis dependent on the externally added vRNA template.