Silencing Transcription from an Influenza Reverse Genetics Plasmid in Enhances Gene Stability.

Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into . Using GFP as a reporter, we demonstrate that expresses the target genes in the RG plasmid at low levels. Incorporating operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in . Viruses rescued from the operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in helps to maintain the correct influenza gene sequence and that the operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between operators can be used for reducing DNA segment instability in any plasmid that is propagated in which express the Lac repressor.