Subcellular reorganization upon phage infection reveals stepwise assembly of viral particles from membrane-associated precursors.

Viruses are obligate intracellular parasites and viral infections lead to massive host cell rearrangement to support the rapid generation of progeny. Host take-over and remodelling include formation of viral-induced compartments for viral genome replication and/or assembly. While viruses infecting bacteria, bacteriophages or phages, have been extensively characterized in vitro, the molecular mechanisms underlying the viral cycle inside the crowded cytoplasm remain unclear. Here, we investigate the spatial reorganization of SPP1-infected bacteria under near-native conditions by electron cryo tomography. The most prominent feature is the formation of a large viral DNA (vDNA) compartment from which ribosomes are excluded. In SPP1 infection, there is no membrane nor proteinaceous shell surrounding these compartments. Also, we identify novel key intermediates in virus assembly: open precursors of procapsid lattice are found at the cytoplasmic membrane in a process that requires expression of the portal protein. Next, DNA-free procapsids relocate inside the vDNA compartment where vDNA is packed in a stepwise manner. Finally, DNA-filled capsids segregate to the periphery of the compartment for assembly completion and storage. Collectively, we provide comprehensive mechanistic insights into the complete viral assembly pathway of SPP1 directly in cellula and show how specific steps are coordinated inside the reorganized bacterial cell.