Nucleocytoviricota viral factories are transient organelles made by phase separation
Phase separation is a widespread mechanism in viral processes, mediating replication, host manipulation and virion morphogenesis. The phylum Nucleocytoviricota encompasses diverse and ubiquitous viruses, including Poxviridae, the climate-modulating Emiliania huxleyi virus and other so-called Giant Viruses. Cytoplasmic members of this phylum form viral factories but their nature has remained unresolved. Here, we demonstrate that these viral factories are formed by liquid-liquid phase separation. We prove that mimivirus viral factories are formed by multilayered phase separation, orchestrated by at least two scaffold proteins. To extend these findings across the phylum Nucleocytoviricota, we developed a bioinformatic pipeline to predict scaffold proteins based on a conserved molecular grammar, despite major primary sequence variability. Scaffold candidates were validated in Marseilleviridae and Poxviridae, highlighting a role of H5 as a scaffold protein in the vaccinia virus. Finally, we provide a repertoire of client proteins of the nucleus-like viral factory of mimivirus and demonstrate important sub-compartmentalization of functions, including those related to the central dogma. Overall, we reveal a new mechanism for an organelle to deploy nuclear-like functions entirely based on phase separation and re-classified phylum Nucleocytoviricota viral factories as biomolecular condensates.