Ebola viral factories are biomolecular condensates with specialized assembly dynamics for viral RNA synthesis.

A hallmark of Ebola virus (EBOV) infection is the formation of membrane-less organelles termed viral factories (VFs). Accumulation of viral proteins drives the formation of VFs, which offer a specialized compartment for viral replication. Multiple EBOV proteins are present inside VFs, but the involvement of individual viral components in VF assembly is unclear. Key to VF function is the recruitment of EBOV polymerase L, which, in complex with viral cofactor VP35, mediates replication and transcription of viral RNAs. How VFs spatially accommodate and control EBOV RNA synthesis remains elusive. Here, we detected both connected network-like and spherical Ebola VFs during infection, suggesting that spinodal decomposition and nucleation-and-growth both contribute to VF assembly. Using fluorescence recovery after photobleaching with live cell imaging, we measured highly dynamic molecular exchange within Ebola VFs reconstituted with mixtures of two viral proteins, NP and VP35. Co-expression of EBOV L partitions L into VFs but immobilizes a fraction of VP35, which likely results from L forming an arrested network pattern with interconnected foci inside VFs. We further introduced a surrogate viral genome into reconstituted VFs to allow L-directed viral RNA synthesis, from which we observed an intriguing link between the spacing of interconnected L-foci and a functional switch of viral RNA synthesis. We examined the same reconstituted VFs with electron microscopy and observed electron-dense materials in the cytoplasm, which resemble arrested L-network patterns observed with light microscopy. EBOV polymerase complex (L-VP35) localizes to select sites at the boundary of electron-dense materials, indicating viral RNA synthesis could initiate at multicomponent phase boundaries and benefit from the directional flux of viral proteins. In summary, our work provides an unprecedented view of EBOV propagation, which exploits fundamental rules of phase transition to regulate virus biogenesis.