The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 105 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.
Bibliographical noteFunding Information:
For helpful discussions, we thank Anne Sadewasser (Robert Koch Institut Berlin) and Edda Klipp and Max Schelker (Humboldt-Universität zu Berlin). This work was supported by the German Ministry of Education and Research (0316180 and 0316180D, eBio: ViroSign to A.H. and T.W.), the Einstein Foundation (A-2012-140‚ single-molecule RNA to A.H. and O.S.), the German-Israeli Helmholtz Research School SignGene (to S.P.), the Deutsche Forschungsgemeinschaft (TransRegio 84 project B2 to T.W.) and the Leibniz Graduate School (to M.S.). We acknowledge support by the Open Access Publication Fund of Humboldt-Universität zu Berlin.
© 2020, The Author(s).