Structural and functional characterization of an influenza virus RNA polymerase-genomic RNA complex

The replication and transcription of influenza A virus are carried out by ribonucleoproteins (RNPs) containing each genomic RNA segment associated with 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 analyzed, structurally and functionally, for the first time, polymerase-RNA template complexes obtained after replication in vivo. These complexes were generated by the cotransfection 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 negativepolarity viral RNA (vRNA) and could transcribe and replicate in vitro. The three-dimensional structure of the monomeric polymerase-vRNA complexes was similar to that of the RNP-associated polymerase and distinct from that of the polymerase devoid of template. These results suggest that the interaction with the template is sufficient to induce a significant conformation switch in the polymerase complex. Copyright © 2010, American Society for Microbiology. All Rights Reserved.
This work has been supported by grants BFU2007-60046 (J.O.) and SAF2008-00451 (O.L.) from the Spanish Ministry of Science; project RD06/0020/1001 of the Red Temática de Investigación Cooperativa en Cáncer (RTICC) (O.L.); CIBER de Enfermedades Respiratorias (J.O.), financed by the Instituto de Salud Carlos III; and the VIRHOST Program, financed by Comunidad de Madrid (J.O.). M.A.R.-C. was supported by an FPI fellowship from the Spanish Ministry of Science, and P.R.-I. was supported by a predoctoral fellowship of the Spanish Research Council. O.L.’s group is additionally supported by the Human Frontiers Science Program Organization (grant RGP39/2008), and J.O.’s group is additionally supported by the Fundación Marcelino Botin.