A phage satellite manipulates the viral DNA packaging motor to inhibit phage and promote satellite spread.

ICP1, a lytic bacteriophage of Vibrio cholerae, is parasitized by phage satellites, PLEs, which hijack ICP1 proteins for their own horizontal spread. PLEs' dependence on ICP1's DNA replication machinery and virion components results in inhibition of ICP1's lifecycle. PLEs are expected to depend on ICP1 factors for genome packaging, but the mechanism(s) PLEs use to inhibit ICP1 genome packaging is currently unknown. Here, we identify and characterize Gpi, PLE's indiscriminate genome packaging inhibitor. Gpi binds to ICP1's large terminase (TerL), the packaging motor, and blocks genome packaging. To overcome Gpi's negative effect on TerL, a component PLE also requires, PLE uses two genome packaging specifiers, GpsA and GpsB, that specifically allow packaging of PLE genomes. Surprisingly, PLE also uses mimicry of ICP1's pac site as a backup strategy to ensure genome packaging. PLE's pac site mimicry, however, is only sufficient if PLE can inhibit ICP1 at other stages of its lifecycle, suggesting an advantage to maintaining Gpi, GpsA and GpsB. Collectively, these results provide mechanistic insights into another stage of ICP1's lifecycle that is inhibited by PLE, which is currently the most inhibitory of the documented phage satellites. More broadly, Gpi represents the first satellite-encoded inhibitor of a phage TerL.