Elucidating the protein interaction network of one of the largest icosahedral capsids in the virosphere.

Giant viruses challenge traditional boundaries of virology with their large particle sizes, complex genomes, and unique replication strategies. Yet, despite its 750 nm diameter and incorporation of dozens of proteins, the mimivirus virion retains an icosahedral symmetry, a trait often associated with smaller viruses. The functional roles and interactions of most proteins composing such complex icosahedral particles remain elusive. Here, we dissect the spatial and functional organization of mimivirus morphogenesis by integrating bioinformatics, genetics, and interactomics. We performed protein clustering using a structure-informed approach, integrating AlphaFold models with sequence information, to classify and functionally annotate the orphan-protein-rich mimivirus proteome. To map the protein-protein interaction network during morphogenesis, we employed endogenous tagging and co-immunoprecipitation coupled to mass spectrometry. This strategy revealed distinct interaction modules associated with the virion membrane, nucleoid, and viral factory compartments. Comparative analyses with other icosahedral and non-icosahedral giant viruses uncovered conserved assembly nodes and virion-shape-specific adaptations. Our findings shed light on the global organization of mimivirus virion biogenesis and highlight the evolutionary plasticity of viral morphogenetic networks within the Nucleocytoviricota.