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

Abstract

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.

A cytoskeletal vortex drives phage nucleus rotation during jumbo phage replication in E. coli

“…Between 40 and 50 mpi, the small bouquets expanded and acquired internal capsids, but only the exterior ring of capsids was stained with DAPI in MG1655, as discussed above. This time course resembles the trafficking of capsids from the membrane to the nuclear shell as described for the nucleus-forming Pseudomonas phages (Chaikeeratisak et al, 2019) and the assembly of bouquets of DNA-filled capsids in those phages (Chaikeeratisak et al, 2022). Time-lapse microscopy of the capsid fusion without any dyes present confirmed the temporal progression of capsid localization from membrane or cytosol, to the periphery of the nucleus, and then to the adjacent bouquet(s) (Figure S6E; Video S7).…”

“…These observations suggest that Goslar capsids are organized into spherically shaped formations with the tails packed together as observed in the Pseudomonas phages (Chaikeeratisak et al, 2022), which deform as they enlarge, filling the cell with capsids in the center, which was not previously observed for the Pseudomonas phages (Figure 6D). To further investigate this model and better understand the stages of capsid localization, we simultaneously visualized capsid-GFP and DAPI staining at various times post infection (30, 40, 50, 70, and 90 min) (Figure 6E).…”

“…At late stages of infection, Goslar virus particles accumulate in large spherical structures similar to the ones recently described in the nucleus-forming Pseudomonas phages and termed bouquets (Chaikeeratisak et al, 2022). In phage bouquets, virions are arranged in a sphere with capsids on the outside and tails facing inward.…”

“…The non-virion multi-subunit RNA polymerase is expressed during infection and transcribes late genes ( Ceyssens et al, 2014 ; Orekhova et al, 2019 ; Sokolova et al, 2020 ; Yakunina et al, 2015 ). In addition, we recently described the organization of maturing virion particles into phage bouquets ( Chaikeeratisak et al, 2022 ). These nearly spherical structures are formed by viral particles undergoing final assembly with tails pointing inward, resembling a bouquet of flowers.…”

Type III CRISPR–Cas provides resistance against nucleus-forming jumbo phages via abortive infection

“…Jumbo phages are abundant, defined as having genomes >200 kb and some form a proteinaceous ‘nucleus’ inside the bacterium that encloses phage DNA for replication and transcription (Chaikeeratisak et al, 2017a; Chaikeeratisak et al, 2017b; Malone et al ., 2020). During virion assembly, phage DNA is directly loaded into phage capsids docked on the outside of the nucleus, which then dissociate, attach to phage tails and exit the cell upon lysis (Chaikeeratisak et al, 2022; Chaikeeratisak et al ., 2017a; Chaikeeratisak et al ., 2017b; Nieweglowska et al, 2022). Therefore, the proteinaceous nucleus provides a physical barrier to exclude bacterial defences from targeting phage DNA (Malone et al ., 2020; Mendoza et al ., 2020).…”