Cryo-EM structures of bacteriophage T4 portal-neck assembly intermediates reveal a viral genome retention mechanism

Bacteriophage T4 has long served as an extraordinary model for tailed phages. During virion assembly, the viral DNA genome is tightly packed into the head, to which tail attaches via a portal-neck connector. Keeping this highly pressurized head leak-proof during these transactions is a challenge, yet the mechanisms remain poorly understood. Here we show that T4 seals its DNA-filled capsid using a double “genome-gate” mechanism. By reconstituting portal (gp20)–neck (gp13/gp14) assembly intermediates in vitro and determining their structures, we find that the gp14 hexamer forms a primary gate that closes the portal-neck opening. This gate is reinforced by a second gate formed by the host protein Hfq, which is hijacked by the virus as an accessory structural component. Hfq also stabilizes neck assembly and prevents its mis-assembly with portal. These studies define a viral genome retention mechanism in which a pre-assembled gp13/gp14/Hfq neck complex seals the pressurized, genome-filled capsid during virion maturation. After genome packaging, tailed bacteriophages must seal the pressurized head to prevent DNA leakage. Here, the authors show that the T4 phage assembles a neck complex with closed double genome-gate by co-opting a host protein Hfq that plugs the head and retains the genome inside.