UC San Diego

Upon infection of host cells, jumbo Pseudomonas phages 201Φ2-1, ΦKZ, and ΦPA3 assemble a “phage nucleus” that encloses replicating viral DNA. The proteinaceous compartment segregates both phage and bacterial host proteins based on their function. Fluorescent microscopy indicates that proteins related to DNA transcription and translation, including phage homologs of RNA polymerase and DNA helicase as well as the bacterial DNA topoisomerase I, are localized inside the phage nucleus. The host ribosomes and other proteins related to translation are excluded from the nucleus along with phage-encoded metabolic enzymes. Each phage also encodes a three-stranded tubulin, PhuZ, that forms a bipolar spindle array in the cell. PhuZ pushes the phage nucleus from one cell pole to midcell then maintains the centered position. After capsids are assembled on the bacterial cell membrane, they traffic along PhuZ filaments to the phage nucleus and dock on the nuclear shell for DNA packaging. Following encapsidation of DNA, filled capsid particles migrate away from the nucleus to complete maturation in the cytoplasm. With genomes ranging from 280-316kb, proportionately large capsid sizes, and relatively small burst sizes, these phages have likely evolved this complicated system to reproduce more efficiently. In addition, the nuclear shell may protect viral DNA from host defenses. It is currently unknown how protein sorting occurs in this structure. However, one notable quirk of the ΦKZ infection system may eventually provide insight into the transport system. One variant of GFP, specifically GFPmut1, is transported into the ΦKZ phage nucleus. Furthermore, any protein fused to GFPmut1 on either the C or N terminus will also be transported into the phage nucleus. Although it is still unclear exactly why this happens, we have been able to utilize this for selective transport into the ΦKZ nucleus. GFPmut1 can be used as a tool for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.