Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into 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 capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

The PDF file includes: Figs. S1 to S7 Table S1 Legends for movies S1 to S4 Other Supplementary Material for this manuscript includes the following: Movies S1 to S4 Fig. S1.Fluorescent micrographs of uninfected control cells.The cells were grown on agarose pads, incubated at 30°C for 3 hours, and were induced by indicated arabinose concentration to express fluorescent protein fusions.Membranes were stained with FM 4-64 (red) and DNA with DAPI (blue).Cover slips were put on immediately before the microcopy and images were then collected.PA3-gp12, PA3-gp136, PA3-gp164, PA3-gp165, PA3-gp166, PA3-gp167 appeared uniformly distributed throughout the cells in the absence of infection whereas PA3-gp11 formed small foci.Scale bar, 1 micron.

Fig. S2 .
Fig. S2.Fluorescent micrographs of 201Phi2-1 infected P. chlororaphis cells expressing major capsid (gp200)-mCherry and internal head protein (gp246)-GFP showing phage maturation of phage 201Phi2-1 in vivo.(A, B).At late time point after DNA packaging, extranuclear staining structures we term "Phage Bouquet" (orange arrows) appear adjacent to the phage nucleus (nu).Major capsid (gp200, red) and the internal head protein (gp246, green) colocalize on the structure suggesting that the structures are comprised of the DNA packaged-mature capsids.Scale bar, 1 micron.The region inside the dashed box in (A) is magnified in (B) to more clearly show the colocalization of major capsid and the internal head protein on the phage bouquet.DAPI staining is shown in white in the top row panel A, in blue in the bottom row of panel A, and in white and blue in panel B. Scale bar, 0.5 micron.

Fig. S3 .
Fig. S3.Fluorescent micrographs of PA3 infected P. aeruginosa cells expressing fluorescent protein; GFP and mCherry.The cells were grown on agarose pads and were induced by 0.2% arabinose to express the fluorescent protein prior to infection.At late time point, phage bouquets were assembled and partially excluded fluorescent proteins suggesting that the physiology inside phage bouquet is distinct from cell cytoplasm.Scale bar, 1 micron.

Fig. S4 .
Fig.S4.Time-series of fluorescent micrographs of PA3 infected P. aeruginosa cells expressing major capsid (gp136)-mCherry and tail sheath (gp165)-GFP.The cells were grown on agarose pads and were induced by 0.05% arabinose to express fluorescent proteins.High titer phage PA3 lysates were added on top of the pad to initiate infection.Time-series throughout infection were imaged and collected.By 45 mpi, capsids assemble, package DNA at the phage nucleus, detach and localize in the cytoplasm.At this time point, capsids (red foci) did not appear to contain assembled tails (pink arrows).After 45 mpi, more capsids were incorporated into phage bouquets where tails localized inside and were surrounded by capsids (orange arrows).Scale bar, 1 micron.

Fig. S6 .
Fig. S6.Pipeline showing the methodolgy to obtain ribosome average from the tomogram shown in Figure 4. 3-dimensional template matching (TM) was performed in Warp (50) and the extracted ribosomes were passed through 3D classification and 3D refinement operations in RELION (51) and further refinement in M (52).Scale bar for the tomogram: 200 nm.

Fig. S7 .
Fig. S7.Fluorescent micrographs of PA3 infected P. aeruginosa cells expressing wildtype GFP-PhuZ with either tail sheath (gp165)-mCherry (A) or major capsid (gp136)-mCherry (B) at late infection.The cells were grown on agarose pads and were induced by 0.025% arabinose to express the fluorescent proteins.High titer phage PA3 lysates were added on top of the pad to initiate infection.In the presence wildtype PhuZ filament, phage bouquets were located adjacent to the phage nucleus.Major capsids and tail sheath were localized and associated with the phage bouquets.Scale bar, 1 micron.