A reverse-genetics system for Influenza A virus using T7 RNA polymerase.
The currently available reverse-genetics systems for Influenza A virus are all based on transcription of genomic RNA by RNA polymerase I, but the species specificity of this polymerase is a disadvantage. A reverse-genetics vector containing a T7 RNA polymerase promoter, hepatitis delta virus ribozyme sequence and T7 RNA polymerase terminator sequence has been developed. To achieve optimal expression in minigenome assays, it was determined that viral RNA should be inserted in this vector in the negative-sense orientation with two additional G residues downstream of the T7 RNA polymerase promoter. It was also shown that expression of the minigenome was more efficient when a T7 RNA polymerase with a nuclear-localization signal was used. By using this reverse-genetics system, recombinant influenza virus A/PR/8/34 was produced more efficiently than by using a similar polymerase I-based reverse-genetics system. Furthermore, influenza virus A/NL/219/03 could be rescued from 293T, MDCK and QT6 cells. Thus, a reverse-genetics system for the rescue of Influenza A virus has been developed, which will be useful for fundamental research and vaccine seed strain production in a variety of cell lines.
de Wit
Emmie
E
Solvay Pharmaceuticals BV, Weesp, The Netherlands.|National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Spronken
Monique I J
MIJ
Solvay Pharmaceuticals BV, Weesp, The Netherlands.|National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Vervaet
Gaby
G
National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Rimmelzwaan
Guus F
GF
National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Osterhaus
Albert D M E
ADME
National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Fouchier
Ron A M
RAM
National Influenza Center, Department of Virology and Postgraduate School of Molecular Medicine, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
D000818:Animals; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D003698:Hepatitis Delta Virus / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000235:genetics; D011401:Promoter Regions, Genetic; D011784:Quail; D012318:RNA Polymerase I / Q000378:metabolism; D016337:RNA, Catalytic / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D019913:Nuclear Localization Signals; D016337:RNA, Catalytic; D012367:RNA, Viral; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I