Nucleocytoviricota viral factories are transient organelles made by phase separation

Phase separation is a common mechanism utilized by viruses to achieve replication, host manipulation and virion morphogenesis. The newly defined phylum Nucleocytoviricota encompass ubiquitous and diverse viruses including Poxviridae, the climate-modulating Emiliania huxleyi virus and the previously termed Nucleocytoplasmic large DNA viruses (NCLDV). Cytoplasmic members of this phylum form viral factories but their nature remains unknow. Here we show that these viral factories are formed by phase separation. We demonstrate that mimivirus viral factories are formed by multilayered phase separation using at least two scaffold proteins. We also generate a pipeline to bioinformatically identify putative scaffold proteins in all other Nucleocytoviricota despite major primary sequence variability. Such predictions were based on a conserved molecular grammar governed by electrostatic interactions. Scaffold candidates were validated for the family Marseilleviridae and highlighted a role of H5 as a scaffold protein in poxviruses. Finally, we provide a repertoire of client proteins of the nucleus-like viral factory of mimivirus and demonstrate important sub-compartmentalization of functions including the central dogma. Overall, we reveal a new mechanism for the acquisition of nuclear-like functions entirely based on phase separation and re-classified phylum Nucleocytoviricota viral factories as biomolecular condensates.