Competition between social cheater viruses is driven by mechanistically different cheating strategies.

Cheater viruses, also known as defective interfering viruses, cannot replicate on their own yet replicate faster than the wild type upon coinfection. While there is growing interest in using cheaters as antiviral therapeutics, the mechanisms underlying cheating have been rarely explored. During experimental evolution of MS2 phage, we observed the parallel emergence of two independent cheater mutants. The first, a point deletion mutant, lacked polymerase activity but was advantageous in viral packaging. The second synonymous mutant cheater displayed a completely different cheating mechanism, involving an altered RNA structure. Continued evolution revealed the demise of the deletion cheater and rise of the synonymous cheater. A mathematical model inferred that while a single cheater is expected to reach an equilibrium with the wild type, cheater demise arises from antagonistic interactions between coinfecting cheaters. These findings highlight layers of parasitism: viruses parasitizing cells, cheaters parasitizing intact viruses, and cheaters may parasitize other cheaters.