Influenza A virus uses actomyosin for genome packaging at the cell membrane

Influenza A virus encodes its genome in eight segments of viral ribonucleopropteins (vRNPs) replicated in the cell nucleus. Clustering of the eight vRNPs is thought to be a selective process promoted by negative-strand RNA interactions. Here, we visualized vRNP packaging at the plasma membrane using immuno-freeze-etching electron microscopy. After being transported to the inner surface of the plasma membrane, individual progeny vRNPs bound to actin filaments and clustered. Clustered vRNPs intermingled with actin filaments, myosin-II and the viral matrix protein M1, as shown by immunocytochemistry. Inhibition of myosin function by blebbistatin prevented proper vRNP clustering. Live-cell high-speed atomic force microscopy revealed blebbistatin reduced the budding kinetics of virions. Our findings highlight the importance of the actomyosin system in vRNP assembly and virion morphogenesis.