title: Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase.
abstract: The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.
authors.affiliation: Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: M T Michael
authors.initials: MT
authors.lastname: Lee
authors.forename: Konrad
authors.initials: K
authors.lastname: Bishop
authors.forename: Liz
authors.initials: L
authors.lastname: Medcalf
authors.forename: Debra
authors.initials: D
authors.lastname: Elton
authors.forename: Paul
authors.initials: P
authors.lastname: Digard
authors.forename: Laurence
authors.initials: L
authors.lastname: Tiley
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009097:Multienzyme Complexes; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D009975:Orthomyxoviridae / Q000201:enzymology* / Q000235:genetics*; D011485:Protein Binding; D021122:Protein Subunits; D015347:RNA Probes / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D009097:Multienzyme Complexes; D009843:Oligoribonucleotides; C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D021122:Protein Subunits; D015347:RNA Probes; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D005914:Globins; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases