Conserved jumbo phage factors required for protein import into a phage nucleus

Bacteriophages use diverse mechanisms to evade anti-phage defenses systems. {Phi}KZ-like jumbo phages assemble a proteinaceous nucleus-like compartment that excludes antagonistic host nucleases, while internalizing DNA replication and transcription machinery1,2,3,4. The phage factors required for protein import and the mechanisms of selectivity remain unknown, however. Here, we uncover an import system composed of proteins highly conserved across nucleus-forming phages, together with additional cargo-specific contributors. Using a genetic selection that forces the phage to decrease or abolish import of specific proteins, we determine that the import of five different phage nuclear-localized proteins (Nlp) all require distinct interfaces of the same factor, Imp1 (gp69). Imp1 forms discrete puncta in the phage nuclear periphery likely in complex with a direct interactor Imp6 (gp67), a conserved protein encoded nearby. The import of some proteins, including a host topoisomerase (TopA), additionally require Imp3 (gp59), a factor required for proper Imp1 function. Three additional phage proteins (Imp2, Imp4, Imp5) are also required for the import of two queried nuclear cargos, perhaps acting as specific adaptors. We therefore propose a core import system including Imp1, Imp3, and Imp6 with the highly selective Imp1 protein licensing transport through a protein lattice.