Resa-Infante P, Recuero-Checa MA, Zamarreño N, Llorca O & Ortín J.
Replication and transcription of influenza A virus is carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated to nucleoprotein monomers and the heterotrimeric polymerase complex. These RNPs are responsible for virus transcription and replication in the infected cell nucleus. Here we have expressed, purified and analysed structurally and functionally for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by co-transfection of plasmids expressing the polymerase subunits and a genomic plasmid expressing a minimal template of positive- or negative-polarity. Their generation in vivo was strictly dependent on the polymerase activity, they contained mainly negative-polarity viral RNA and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to the RNP-associated polymerase and distinct from the polymerase devoid of template. These results suggest that interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex.