Characterization of vRNP clustering in influenza A virus-infected cells using in-situ cryo-electron tomography

Abstract text (incl. figure legends and references)

Influenza A virus has an eight-segment single-stranded RNA genome. Each genome segment is packaged into a rod-shaped vRNP complex, eight of which form a bundle containing one copy of each genome segment. During viral assembly, newly produced vRNPs subvert the recycling endosome to travel from the ER exit sites, where they phase-separate in the presence of Rab11, to the plasma membrane where budding takes place. On this route, vRNP selection and bundle formation occur, yet neither the site of clustering nor the mechanism behind are fully understood on a molecular level.To shed light on this important step in the viral replication cycle, we employed in-situ cryo-electron tomography.

To analyze the spatial distribution of vRNPs inside cryoFIB-milled cells, we established a segmentation and cluster analysis workflow. Our data show that vRNPs localize to so far uncharacterized compartments that comprise double membranes zippered together by head-to-head interactions of viral hemagglutinin, which is the main mediator of viral entry. vRNPs cluster around these membranes, with numerous points of vRNP-vRNP and vRNP-membrane interactions. Taken together, our data show a previously unknown function of HA during viral assembly in intracellular membrane remodeling and vRNP clustering.