Abstract
Bacteriophage T4 is a model for tailed viruses, the most abundant biological entities on Earth. During virion assembly, the DNA genome is tightly packed inside the head, which then attaches to tail via a portal-neck connector. Keeping this pressurized head leak-proof during these transactions is a challenge because even a few basepairs leakage could disrupt assembly, but the mechanisms are not understood. Here, we solved the structures of in vitro-assembled portal-neck complexes and found a double "genome-gate" in the neck that plugs the packaged head. The first gate is formed by gp14 hexamer, while the second, unexpectedly, consists of a host component, Hfq hexamer. Furthermore, Hfq stabilizes the neck and facilitate its correct docking onto the head. Thus, a pre-assembled, gp13/gp14/Hfq neck complex awaits genome packaging completion, triggers portal conformational changes and packaging motor ejection, which exposes binding sites for the neck to securely seal the pressurized head.