Definition of the minimal viral components required for the initiation of unprimed RNA synthesis by influenza virus RNA polymerase

Michael T Lee, Konrad Bishop, Liz Medcalf, Debra Elton, Paul Digard, Laurence Tiley

Research output: Contribution to journalArticlepeer-review


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.
Original languageEnglish
Pages (from-to)429-38
Number of pages10
JournalNucleic Acids Research
Publication statusPublished - Jan 2002


  • Binding Sites
  • Chromatography, Affinity
  • DNA-Directed RNA Polymerases/genetics
  • DNA-Directed RNA Polymerases/isolation & purification
  • DNA-Directed RNA Polymerases/metabolism
  • Electrophoretic Mobility Shift Assay
  • Enzymes, Immobilized/genetics
  • Enzymes, Immobilized/metabolism
  • Genome, Viral
  • Globins/genetics
  • HeLa Cells
  • Humans
  • Multienzyme Complexes
  • Oligoribonucleotides/genetics
  • Oligoribonucleotides/metabolism
  • Orthomyxoviridae/enzymology
  • Orthomyxoviridae/genetics
  • Protein Binding
  • Protein Subunits
  • RNA Probes/genetics
  • RNA Probes/metabolism
  • RNA Replicase/chemistry
  • RNA Replicase/genetics
  • RNA Replicase/isolation & purification
  • RNA Replicase/metabolism
  • RNA, Messenger/biosynthesis
  • RNA, Messenger/genetics
  • RNA, Viral/biosynthesis
  • RNA, Viral/genetics
  • RNA-Binding Proteins/genetics
  • RNA-Binding Proteins/isolation & purification
  • RNA-Binding Proteins/metabolism
  • Recombinant Proteins/metabolism
  • Templates, Genetic
  • Transcription, Genetic/genetics
  • Viral Proteins/genetics
  • Viral Proteins/isolation & purification
  • Viral Proteins/metabolism

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