A DNA transfection system for generation of influenza A virus from eight plasmids

Proceedings of the National Academy of Sciences of the United States of America - Tập 97 Số 11 - Trang 6108-6113 - 2000
Erich Hoffmann1, Gabriele Neumann1, Yoshihiro Kawaoka1, Gerd Hobom1, Robert G. Webster1
1Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794; Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin–Madison, 2015 Linden Drive West, Madison, WI 53706; Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan; Institut für Mikro- und Molekularbiologie, Frankfurter Strasse 107, 35392 Giessen, Germany; and Department of Pathology, University of Tennessee,...

Tóm tắt

We have developed an eight-plasmid DNA transfection system for the rescue of infectious influenza A virus from cloned cDNA. In this plasmid-based expression system, viral cDNA is inserted between the RNA polymerase I (pol I) promoter and terminator sequences. This entire pol I transcription unit is flanked by an RNA polymerase II (pol II) promoter and a polyadenylation site. The orientation of the two transcription units allows the synthesis of negative-sense viral RNA and positive-sense mRNA from one viral cDNA template. This pol I–pol II system starts with the initiation of transcription of the two cellular RNA polymerase enzymes from their own promoters, presumably in different compartments of the nucleus. The interaction of all molecules derived from the cellular and viral transcription and translation machinery results in the generation of infectious influenza A virus. The utility of this system is proved by the recovery of the two influenza A viruses: A/WSN/33 (H1N1) and A/Teal/HK/W312/97 (H6N1). Seventy-two hours after the transfection of eight expression plasmids into cocultured 293T and MDCK cells, the virus yield in the supernatant of the transfected cells was between 2 × 10 5 and 2 × 10 7 infectious viruses per milliliter. We also used this eight-plasmid system for the generation of single and quadruple reassortant viruses between A/Teal/HK/W312/97 (H6N1) and A/WSN/33 (H1N1). Because the pol I–pol II system facilitates the design and recovery of both recombinant and reassortant influenza A viruses, it may also be applicable to the recovery of other RNA viruses entirely from cloned cDNA.

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Proceedings of the National Academy of Sciences of the United States of America

Tập 97 Số 11

6108-6113

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