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Unidirectional RNA polymerase I–polymerase II transcription system for the generation of influenza A virus from eight plasmids

Department of Virology and Molecular Biology, St Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA¹
Department of Pathology, University of Tennessee, Memphis, TN, USA²

Author for correspondence: Robert Webster (at St Jude Children’s Research Hospital). Fax +1 901 523 2622. e-mail robert.webster{at}stjude.org

Recently, we developed a system for the generation of influenza A virus by cotransfecting only eight plasmids from which negative-sense vRNA and positive-sense mRNA are expressed (Hoffmann et al., Proceedings of the National Academy of Sciences, USA 97, 6108–6113, 2000). Here we report the establishment of a different transcription system for the expression of virus-like RNAs, 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) resulted in the generation of infectious influenza A virus, albeit with a lower yield than the bidirectional system. Our approach of producing either vRNA and mRNA or cRNA and mRNA intracellularly from a minimum set of plasmids should be useful for the establishment or optimization of reverse genetics systems for other RNA viruses.

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