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Review
. 1999 Aug 3;96(16):8804-6.
doi: 10.1073/pnas.96.16.8804.

Reverse genetics of negative-strand RNA viruses: closing the circle

A Pekosz  1 , B He, R A Lamb
Affiliations
Review

Reverse genetics of negative-strand RNA viruses: closing the circle

A Pekosz et al. Proc Natl Acad Sci U S A. .
No abstract available

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Figures

Figure 1
Figure 1
Methods used to recover negative-strand RNA viruses from plasmid DNA. Several methodologies have been used to rescue negative-strand RNA viruses from plasmid-derived DNA. The initial replacement of individual RNA segments of influenza virus involved either (i) the in vitro reconstitution of RNPs or (ii) the in vivo assembly of RNPs after transfection of a cell with plasmids that use pol II promoters driving the expression of the PA, PB1, PB2, and NP proteins, and pol I promoters and terminators controlling viral genome synthesis. In either case, transfection was followed by infection with a helper influenza virus. Nonsegmented negative-strand virus rescue involves the transfection of plasmids encoding the viral P, N, and L proteins (and sometimes other viral proteins depending on the virus), as well as the viral antigenome, all under control of the bacteriophage T7 RNAP promoter. The T7 RNAP is provided by either infection with vv-T7, a recombinant vaccinia virus that expresses T7 RNAP or by transfecting into cell lines that stably express the protein. Bunyavirus rescue requires the transfection of plasmids encoding the three RNA segments in the antigenomic sense, along with three plasmids encoding the viral proteins, all under control of the T7 RNAP promoter. Influenza virus rescue entirely from plasmid DNA involves the transfection of plasmids encoding each of the eight RNA segments (under control of the pol I promoter and terminator) and plasmids encoding the four proteins that make up the polymerase complex (under control of the pol II promoter).
See this image and copyright information in PMC

Comment on

  • Generation of influenza A viruses entirely from cloned cDNAs.
    Neumann G, Watanabe T, Ito H, Watanabe S, Goto H, Gao P, Hughes M, Perez DR, Donis R, Hoffmann E, Hobom G, Kawaoka Y. Neumann G, et al. Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9345-50. doi: 10.1073/pnas.96.16.9345. Proc Natl Acad Sci U S A. 1999. PMID: 10430945 Free PMC article.

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