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 minigenomes rescue experiments, both the 5′ and 3′ coding ends of the PB2 segment were found to promote the incorporation of minigenomes 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.