Morphological analysis of the behaviour of influenza A virus genome vRNPs (ribonucleoproteins) in host cells by advanced immuno-freeze-etch electron microscopy

Influenza A virus (IAV) encodes its genome in eight segments of viral ribonucleoproteins (vRNPs), complexes of viral RNAs and nucleoproteins (NPs), which are replicated extensively in the infected (host) cell. However, the behaviour of progeny vRNPs after replication in host cells, particularly the process of selective packaging (assembling) of eight distinct vRNPs for forming a core of progeny virion and the host cell factors contributing to it, remain unclear. Accordingly, immuno-freeze-etching electron microscopy (immuno-FE), combined with either unroofing or cryosection techniques, was employed to visualise the localisation and packaging processes of progeny vRNPs in host cells. In the cytoplasm, anti-vRNP antibody binding sites (i.e., vRNPs) were mainly localized on membrane-like structures that were irregularly distributed. Notably, little clustering of vRNPs was observed in these regions. Immuno-FE combined with unroofing techniques enabled us to observe the cytoplasmic side surface of the plasma membrane of infected cells in three dimensions for the first time. Numerous progeny vRNPs were transported to and accumulated there. When zoomed in, the vRNPs bound to actin filaments and formed clusters of various shapes and sizes. Myosin was also detected along actin filaments by antibody labelling. The movement of actin filaments just below the plasma membrane was confirmed by atomic force live cell imaging (AFM). Hence, vRNPs bound to actin filaments are also likely to be moved. Indeed, blocking myosin activity with blebbistatin inhibited both the movement of actin filament and the proper clustering of vRNPs. The exact mechanism responsible for the mutual recognition between progeny vRNPs is not yet fully understood, but these findings suggest that actin filament motility is involved in the selective packaging of eight distinct vRNPs.