Bacteriophages use diverse mechanisms to evade antiphage defence systems. ΦKZ-like jumbo phages assemble a proteinaceous, nucleus-like compartment that excludes antagonistic host nucleases and also internalizes DNA replication and transcription machinery1–4. The phage factors required for protein import and the mechanisms of selectivity remain unknown, however. Here we uncover an import system comprising 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 the import of specific proteins, we determine that the importation of five different phage nuclear-localized proteins requires distinct interfaces of the same factor, Imp1 (gp69). Imp1 localizes early to the nascent phage nucleus and forms discrete puncta in the mature phage nuclear periphery, probably in complex with direct interactor Imp6 (gp67), a conserved protein encoded in the same locus. The import of certain proteins, including a host topoisomerase, additionally requires Imp3 (gp59), a conserved factor necessary for proper Imp1 function. Three additional non-conserved phage proteins (Imp2 and Imp4/Imp5) are required for the import of two queried nuclear cargos (nuclear-localized protein 1 and host topoisomerase, respectively), perhaps acting as specific adaptors. We therefore propose a core import system that includes Imp1, Imp3 and Imp6, with multiple interfaces of Imp1 licensing transport through a protein lattice.
