he phage nucleus synergizes with an anti-defense protein to resist bacterial immunity

Chimallivirus bacteriophages enclose their replicating genomes in a protein-based compartment termed the phage nucleus. While the phage nucleus segregates phage DNA from host immune proteins, it is not known if additional factors are required to protect against DNA-targeting host defenses. Here, we identify a chimallivirus-encoded DarG2-like antitoxin that localizes to the phage nucleus and provides protection against phage-targeting DarTG2 toxin-antitoxin systems. This protein, which we term AdfM (Anti-darT factor Macro), contains a macro domain and removes DarT2-mediated ADP-ribose modifications from DNA. In the absence of AdfM, DarTG2 restricts chimalliviruses by blocking virion formation despite DarT2 being largely excluded from the phage nucleus. Increasing the nuclear concentration of DarT2, while decreasing the nuclear concentration of AdfM, reduces phage replication. These results show that the phage nucleus is insufficient to completely protect the chimallivirus genome from host defenses, rather it is one component of a multilayered counter-defense strategy.