Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase.
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.
Lee
M T Michael
MT
Centre for Veterinary Science, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
Bishop
Konrad
K
Medcalf
Liz
L
Elton
Debra
D
Digard
Paul
P
Tiley
Laurence
L
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*
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