Parallelized screening of virus accessory genes reveals diverse defense and counter-defense mechanisms

Genomes of bacterial and archaeal viruses are replete with fast evolving, uncharacterized accessory genes (AGs), most of which likely antagonize host defenses or other viruses. We developed a computational approach to comprehensively identify AGs in virus genomes and built a high-throughput screening platform to assay their function. We expressed a pilot set of 200 Enterobacteriophage AGs in 20 wild Escherichia coli strains and challenged them with 8 model phages. We found that multiple AGs with anti-restriction functions triggered programmed cell death (PCD) activity of a type III restriction-modification (R-M) system and a non-canonical (decoy) type I R-M system. Additionally, we identified an inhibitor of a highly evolved, single-component type IV R-M system, and AGs that interact with metabolic pathways to antagonize O-antigen barrier defenses. Our approach yields insights into the multiple levels of virus-host competition and can be rapidly deployed in various bacteria.