biostudies-literatureRigou SEuropean Research CouncilChemistry Biology Health (CBH) Graduate School of University Grenoble Alpes grantAgence Nationale de la Recherche grant ProFIe2515074122https://www.ebi.ac.uk/biostudies/studies/S-EPMC12415211biostudies-literatureUnknown122(35)Despite sharing fewer than 10 core genes, the hyperdiverse <i>Nucleocytoviricota</i> phylum (ranging from poxviruses to giant viruses) universally assembles viral factories (VFs) resembling biomolecular condensates. Regardless, it is unclear how these viruses achieve such a level of functional conservation without clear conserved genetic information. We demonstrate that the VFs produced by amoeba-infecting viruses have liquid-like properties and identify a conserved molecular grammar governing viral factory scaffold protein: charge-patterned intrinsically disordered regions that drive phase separation independently of sequence homology. This grammar predicts functional scaffold proteins across the 15 viral families, revealing evolutionary constraints invisible to sequence or structural anaProceedings of the National Academy of Sciences of the United States of AmericaEvolutionarily conserved grammar rules viral factories of amoeba-infecting members of the hyperdiverse &lt;i&gt;Nucleocytoviricota&lt;/i&gt; phylum.PMC12415211ANR-10-INBS-08832601101160452ANR-17-EURE-0003Mayer LBelmudes LCoute YRigou SJeudy SLartigue ADanner LGiry COlivero-Deibe NTrabelsi FLegendre MBerois MAbergel CMoreno ABLe Guenno HBisio HSchmitt AfalseEvolutionarily conserved grammar rules viral factories of amoeba-infecting members of the hyperdiverse &lt;i&gt;Nucleocytoviricota&lt;/i&gt; phylum.Despite sharing fewer than 10 core genes, the hyperdiverse <i>Nucleocytoviricota</i> phylum (ranging from poxviruses to giant viruses) universally assembles viral factories (VFs) resembling biomolecular condensates. Regardless, it is unclear how these viruses achieve such a level of functional conservation without clear conserved genetic information. We demonstrate that the VFs produced by amoeba-infecting viruses have liquid-like properties and identify a conserved molecular grammar governing viral factory scaffold protein: charge-patterned intrinsically disordered regions that drive phase separation independently of sequence homology. This grammar predicts functional scaffold proteins across the 15 viral families, revealing evolutionary constraints invisible to sequence or structural ana2025-01-01T00:00:00Z2025 Sep2026-07-16T21:55:26.754Z2026-07-10T03:15:35.343ZS-EPMC124152114086465210.1073/pnas.2515074122