Unidirectional RNA polymerase I-polymerase II transcription system for thegeneration of influenza A virus from eight plasmids

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and posit ive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108-6113, 2000). Here we report the establish ment of a different transcription system for the expression of virus-like R NAs, allowing the intracellular synthesis of noncapped positive-sense cRNA and 5'-capped mRNA from one template. Cotransfection of eight RNA pol I-pol II tandem promoter plasmids containing the cDNA of A/WSN/33 (H1N1) resulte d in the generation of infectious influenza A virus, albeit with a lower yi eld than the bidirectional system. Our approach of producing either vRNA an d mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids shou ld be useful for the establishment or optimization of reverse genetics syst ems for other RNA viruses.