Identification of the bacteriophage nucleus protein interaction network

Abstract

In the arms race between bacteria and bacteriophages (phages), some large-genome jumbo phages have evolved a protein shell that encloses their replicating genome to protect it against DNA-targeting immune factors. By segregating the genome from the host cytoplasm, however, the "phage nucleus" introduces the need to specifically transport mRNA and proteins through the nuclear shell, and to dock capsids on the shell for genome packaging. Here, we use proximity labeling and localization mapping to systematically identify proteins associated with the major nuclear shell protein chimallin (ChmA) and other distinctive structures assembled by these phages. We identify six uncharacterized nuclear shell-associated proteins, one of which directly interacts with self-assembled ChmA. The structure and protein-protein interaction network of this protein, which we term ChmB, suggests that it forms pores in the ChmA lattice that serve as docking sites for capsid genome packaging, and may also participate in mRNA and/or protein transport.

A phage nucleus-associated RNA-binding protein is required for jumbo phage infection

“…Confirming our earlier microscopic observations ( 13 ), we find that ectopically-expressed PhiPA3 ChmC fused to GFP localizes to the phage nuclear shell in late-stage PhiPA3 infections of P. aeruginosa ( Figure 1C ). Fluorescent microscopy is unable to determine whether ChmC is localized within the nucleus or just outside the ChmA shell, but our prior identification of ChmC through proximity-labeling with the nuclear-localized protein UvsX suggests that the protein is at least partially localized within the phage nucleus ( 13 ). We next purified GFP-tagged ChmC from P. aeruginosa cells infected with PhiPA3 and used mass spectrometry to identify associated proteins ( Figure S1 , Table S3 ).…”

“…We previously used proximity ligation to identify proteins in the nucleus-forming jumbo phage PhiPA3 that physically associate with the major nuclear shell protein ChmA or with a phage nucleus-localized protein, UvsX (gp175) ( 13 ). Through this analysis, we identified a minor nuclear shell component (ChmB) and several uncharacterized proteins with no known function ( 13 ).…”

“…We previously used proximity ligation to identify proteins in the nucleus-forming jumbo phage PhiPA3 that physically associate with the major nuclear shell protein ChmA or with a phage nucleus-localized protein, UvsX (gp175) ( 13 ). Through this analysis, we identified a minor nuclear shell component (ChmB) and several uncharacterized proteins with no known function ( 13 ). One of these proteins was PhiPA3 gp61, which is located in a highly conserved cluster of genes that includes chmA (gp53), several subunits of the phage-encoded “non-virion RNA polymerase” (nvRNAP: gp62, gp65-66, and gp67), and two additional phage nucleus-associated proteins, gp63 and gp64 ( Figure 1A ).…”

“…The requirement for translocation of mRNAs, proteins, and genomic DNA through the phage nuclear shell implies the existence of additional shell components embedded within or associated with the ChmA lattice that mediate these activities. In prior work, we used proximity labeling and localization analysis in the Chimalliviridae family phage PhiPA3 to identify proteins that physically associate with ChmA and localize to the nuclear shell ( 13 ). One of these proteins, termed ChmB, interacts directly with ChmA both in vitro and in vivo , and associates with the virion portal protein in vitro .…”

“…ChmB’s network of protein-protein interactions and its distinctive 3D structure suggest that it may form pores in the phage nuclear shell that enable the docking of pro-capsids for genome packaging. Further data showing that the expression of dominant-negative ChmB mutants compromises early steps in phage nucleus growth and maturation further suggests that ChmB may participate in mRNA and/or protein translocation through the nuclear shell ( 13 ).…”