title: Expression of a foreign protein by influenza A virus.
abstract: In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT). The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein. The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences. The resultant gene produces a polyprotein which is proteolytically cleaved to release both CAT and NA. The intramolecular cleavage occurs at the C terminus of the 2A sequence between a glycine-proline dipeptide motif such that the released NA protein has an additional N-terminal proline residue. The transfectant virus is stable upon passage in tissue culture. CAT activity is expressed at high levels in cell culture supernatants and in the allantoic fluid of infected eggs. Since the chimeric segment must maintain the heterologous reading frame to retain viability, the virus stability is dependent upon concomitant synthesis of the heterologous protein. This design may be particularly appropriate for utilization of influenza virus as a mammalian expression vector.
authors.affiliation: Department of Microbiology, Mount Sinai School of Medicine, New York, New York 10029.
authors.forename: N
authors.initials: N
authors.lastname: Percy
authors.forename: W S
authors.initials: WS
authors.lastname: Barclay
authors.forename: A
authors.initials: A
authors.lastname: García-Sastre
authors.forename: P
authors.initials: P
authors.lastname: Palese
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D004285:Dogs; D005822:Genetic Vectors; D006868:Hydrolysis; D009980:Influenza A virus / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D015500:Chloramphenicol O-Acetyltransferase