Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

If You Use a Screen Reader

This content is available through Read Online (Free) program, which relies on page scans. Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.

A DNA Transfection System for Generation of Influenza a Virus from Eight Plasmids

Erich Hoffmann, Gabriele Neumann, Yoshihiro Kawaoka, Gerd Hobom and Robert G. Webster
Proceedings of the National Academy of Sciences of the United States of America
Vol. 97, No. 11 (May 23, 2000), pp. 6108-6113
Stable URL: http://www.jstor.org/stable/122589
Page Count: 6
  • Read Online (Free)
  • Subscribe ($19.50)
  • Cite this Item
Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
A DNA Transfection System for Generation of Influenza a Virus from Eight Plasmids
Preview not available

Abstract

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 × 105 and 2 × 107 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.

Page Thumbnails

  • Thumbnail: Page 
6108
    6108
  • Thumbnail: Page 
6109
    6109
  • Thumbnail: Page 
6110
    6110
  • Thumbnail: Page 
6111
    6111
  • Thumbnail: Page 
6112
    6112
  • Thumbnail: Page 
6113
    6113