Plasmid-based reverse genetics has transformed influenza virus research
by enabling the production of recombinant influenza viruses from cloned
cDNA copies of viral genome segments. Reverse genetic production of
influenza viruses requires cellular expression of influenza proteins
polymerase basic 1, polymerase basic 2, polymerase acidic and
nucleoprotein which collectively allow for transcription of viral mRNA
and synthesis of new negative-sense genomic RNA, thus enabling synthesis
of all components needed to assemble infectious virus from transfected
cell lines. Given the importance of these proteins in the generation of
influenza viruses via reverse genetics, we sought to explore how
transgenic expression of mammalian-adapted PB1, PB2, PA, or NP in the
293T packaging cell line may impact the recovery of recombinant
influenza viruses. We constructed four transgenic 293T cell lines
expressing PB1, PB2, PA or NP derived from the mouse-adapted 2009
pandemic influenza A virus, UI182. Transgenic expression of UI182 PB2 in
293T cells enhanced recovery of replication-competent avian influenza
viruses generated by reverse genetics relative to levels achieved in
unmodified 293T cells. Virus recovered from PB2-expressing 293T cells
replicated with kinetics that were indistinguishable from viruses
recovered from unmodified 293T cells. Provision of UI182 PB2 protein via
transgenic expression in 293T cells resulted in enhanced viral
polymerase activity as measured by a minigenome assay, which may account
for the improved efficiency of viral packaging relative to unmodified
293T cells. Transgenic expression of mammalian-adapted PB2 in 293T cells
may serve as an important tool for enhancing influenza virus recovery in
reverse genetic systems.