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.
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.
Dos Santos Afonso
Emmanuel
E
Unité de Génétique Moléculaire des Virus Respiratoires, URA CNRS 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 PARIS Cedex 15, France.
Escriou
Nicolas
N
Leclercq
India
I
van der Werf
Sylvie
S
Naffakh
Nadia
N
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics*; D019065:Virus Assembly
D004279:DNA, Viral; C502893:PB2 protein, influenza virus; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins