“Ambisense” Approach for the Generation of Influenza A Virus: vRNA and mRNA Synthesis from One Template

Abstract

We present a system for creating influenza virus by generating viral RNA (vRNA) and mRNA from one template. Recently, a system for the generation of influenza A virus entirely from cloned cDNAs was established (Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9345–9350). Cells were transfected with plasmids for RNA polymerase I-driven intracellular synthesis of all eight viral RNAs, and with protein expression plasmids for the synthesis of viral structural proteins. Although this system is highly efficient in virus generation, the construction and cotransfection of 17 plasmids is cumbersome and may limit the use of this system to cell lines that can be transfected with high efficiencies. Synthesizing both vRNA and mRNA from one template would reduce the number of plasmids required for virus generation. Therefore, we generated a bidirectional transcription construct that contains cDNA encoding PB1 flanked by an RNA polymerase I (pol I) promoter for vRNA synthesis and an RNA polymerase II (pol II) promoter for mRNA synthesis. The utility of this approach is proved by the generation of virus after transfecting the pol I/pol II-promoter–PB1 construct together with vRNA- and protein-expression constructs for the remaining seven segments. Because this approach reduces the number of plasmids required for virus generation, it also reduces the work necessary for cloning, probably enhances the efficiency of virus generation, and expands the use of the reverse-genetics system to cell lines for which efficient cotransfection of 17 plasmids cannot be achieved.