Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories

Ebola virus (EBOV) infection induces formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and from which diverse viral biogenesis arises. Key to viral factory function is recruitment of the EBOV polymerase, a multifunctional machine that mediates replication and expression of the viral RNA genome. Here we show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal dynamics of exchange that likely facilitates viral replication. We report that EBOV viral factories display either droplet-like or network-like morphology, which could be influenced by multivalent intermolecular interactions between viral proteins. Within the viral factory, we found EBOV polymerase is not uniformly distributed, but instead clusters into foci. The distance between these foci increases when viral replication is enabled. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence and regulate viral biogenesis.