"Ambisense" approach for the generation of influenza A virus: vRNA and mRNA synthesis from one template

We present a system for creating influenza virus by generating viral RNA (v RNA) and mRNA from one template. Recently, a system for the generation of i nfluenza A virus entirely from cloned cDNAs was established (Neumann et ai. , 1999, Proc. Natl Acad. Sci. USA 96, 9345-9350) Cells were transfected wit h plasmids for RNA polymerase I-driven intracellular synthesis of all eight viral RNAs, and with protein expression plasmids for the synthesis of vira l structural proteins. Although this system is highly efficient in virus ge neration, the construction and cotransfection of 17 plasmids is cumbersome and may limit the use of this system to cell lines that can be transfected with high efficiencies. Synthesizing both vRNA and mRNA from one template w ould reduce the number of plasmids required for virus generation. Therefore , we generated a bidirectional transcription construct that contains cDNA e ncoding PB1 flanked by an RNA polymerase I (pol I) promoter for vRNA synthe sis and an RNA polymerase II (pol II) promoter for mRNA synthesis. The util ity of this approach is proved by the generation of virus after transfectin g the pol I/pol II-promoter-PB1 construct together with vRNA- and protein-e xpression constructs for the remaining seven segments. Because this approac h reduces the number of plasmids required for virus generation, it also red uces the work necessary for cloning, probably enhances the efficiency of vi rus generation, and expands the use of the reverse-genetics system to cell lines for which efficient cotransfection of 17 plasmids cannot be achieved. (C) 2000 Academic Press.