Summary Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages were long assumed to assemble randomly throughout the host cell cytoplasm. Here we visualized the subcellular location of viral particles formed during replication of Pseudomonas nucleus-forming jumbo phages and discovered that they assemble a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to the capsids, and these particles accumulate to form bouquets at specific subcellular locations. In these bouquets, the viral particles are arranged in a spherical pattern with tails oriented inward and the heads outwards. Localized at fixed distances on either side of the phage nucleus, bouquets grow in size and number over time as new phage particles are added. In the presence of mutations that cause the phage nucleus to be mispositioned away from its typical position at the midcell, bouquets still localize at the same fixed distance from the nucleus, suggesting an active mechanism for their formation and positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phage, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.
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