title: A plasmid-based reverse genetics system for influenza A virus.
abstract: A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York 10029, USA.
authors.forename: S
authors.initials: S
authors.lastname: Pleschka
authors.forename: R
authors.initials: R
authors.lastname: Jaskunas
authors.forename: O G
authors.initials: OG
authors.lastname: Engelhardt
authors.forename: T
authors.initials: T
authors.lastname: Zürcher
authors.forename: P
authors.initials: P
authors.lastname: Palese
authors.forename: A
authors.initials: A
authors.lastname: García-Sastre
mesh_terms: D001483:Base Sequence; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics*; D012261:Ribonucleoproteins / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D012367:RNA, Viral; D012261:Ribonucleoproteins