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

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

Keywords

  • 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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