Unidirectional RNA polymerase I-polymerase II transcription system for the generation 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 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.
Hoffmann
Erich
E
Department of Virology and Molecular Biology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA1.
Webster
Robert G
RG
Department of Pathology, University of Tennessee, Memphis, TN, USA2.|Department of Virology and Molecular Biology, St Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794, USA1.
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D018920:Coculture Techniques; D004285:Dogs; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D009980:Influenza A virus / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012318:RNA Polymerase I / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D019065:Virus Assembly
D012333:RNA, Messenger; D012367:RNA, Viral; D012319:RNA Polymerase II; D012318:RNA Polymerase I