The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5' end of the PB2 segment

Virology. 2005 Oct 10;341(1):34-46. doi: 10.1016/j.virol.2005.06.040.

Abstract

We generated recombinant A/WSN/33 influenza A viruses expressing a PB2 protein fused to a Flag epitope at the N- (Flag-PB2) or C-terminus (PB2-Flag), which replicated efficiently and proved to be stable upon serial passage in vitro on MDCK cells. Rescue of PB2-Flag viruses required that the 5' end of the PB2 segment was kept identical to the wild-type beyond the 34 noncoding terminal nucleotides. This feature was achieved by a duplication of the 109 last nucleotides encoding PB2 between the Flag sequence and the 5'NCR. In PB2 mini-genomes rescue experiments, both the 5' and 3' coding ends of the PB2 segment were found to promote the incorporation of mini-genomes into virions. However, the presence of the Flag sequence at the junction between the 3'NCR and the coding sequence did not prevent the rescue of Flag-PB2 viruses. Our observations define requirements that may be useful for the purpose of engineering influenza RNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • DNA, Viral / genetics
  • Dogs
  • Humans
  • Influenza A virus / genetics*
  • Influenza A virus / physiology*
  • Recombinant Fusion Proteins / genetics
  • Recombination, Genetic
  • Viral Proteins / genetics*
  • Virus Assembly

Substances

  • DNA, Viral
  • PB2 protein, influenza virus
  • Recombinant Fusion Proteins
  • Viral Proteins