title: Comparison of two reconstituted systems for in vitro transcription and replication of influenza virus.
abstract: The transcription and replication of influenza RNA can be studied in vitro by the reconstitution of functional ribonucleoprotein (RNP) complex from viral core proteins including the RNA polymerase (complex of three P protein subunits) and nucleoprotein (NP), and model templates. Here, two different core protein preparations, one based on CsCl centrifugation (CS enzyme) and the other on micrococcal nuclease treatment of viral cores (MN enzyme), were compared side-by-side. Short model RNA templates and their 3'-half molecules of both viral RNA (vRNA) and complementary RNA (cRNA) senses were reconstituted with the core protein preparations in parallel, and RNA polymerase activity was tested either in the presence or absence of ApG or globin mRNA as primers. Both enzyme preparations were active in the syntheses of short vRNA and cRNA transcripts using ApG as a primer, although the synthesis of cRNA was 2-10-fold higher (depending on the template used) than the synthesis of vRNA. The MN enzyme, however, was more active per weight of total protein than the CS enzyme, probably because of its higher content of RNA polymerase. Both enzymes failed to show primer-independent synthesis of vRNA. The differences observed in the synthesis of short transcripts using globin mRNA as a primer are discussed.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, U.K.
authors.forename: B L
authors.initials: BL
authors.lastname: Seong
authors.forename: M
authors.initials: M
authors.lastname: Kobayashi
authors.forename: K
authors.initials: K
authors.lastname: Nagata
authors.forename: G G
authors.initials: GG
authors.lastname: Brownlee
authors.forename: A
authors.initials: A
authors.lastname: Ishihama
mesh_terms: D001483:Base Sequence; D002499:Centrifugation, Density Gradient / Q000379:methods; D002586:Cesium; D002712:Chlorides; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004533:Egtazic Acid / Q000494:pharmacology; D005914:Globins / Q000235:genetics; D006018:Glycols; D009980:Influenza A virus / Q000235:genetics*; D046911:Macromolecular Substances; D008836:Micrococcal Nuclease / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication / Q000235:genetics*
chemical_list: D002712:Chlorides; D006018:Glycols; D046911:Macromolecular Substances; D012333:RNA, Messenger; D012367:RNA, Viral; D012261:Ribonucleoproteins; D002586:Cesium; D004533:Egtazic Acid; D005914:Globins; D012321:DNA-Directed RNA Polymerases; D008836:Micrococcal Nuclease; C028019:cesium chloride