The dynamics, mechanism, and interactions of defective RNA cheater viruses

The RNA bacteriophage MS2 is one of the smallest and fastest evolving entities on Earth. During experimental evolution of the phage, we unexpectedly observed the parallel emergence of a point deletion mutation that leads to a frameshift in two of the four phage open reading frames. We hypothesized that the deletion mutant is a “cheater” phage (also known as a defective interfering particle), defined here as a phage unable to replicate on its own, yet bearing a replicative fitness advantage over the wild-type (WT) phage during co-infection. We substantiated our hypothesis through single plaque sequencing, trans-complementation, real-time PCR measurements, and long read MinION sequencing. Surprisingly our results revealed the existence of another synonymous mutation cheater, which was found to be mutually exclusive of the deletion cheater. Continued evolution of the experimene revealed the demise of the deletion cheater and takeover of the synonymous cheater. A mathematical model based on game theory inferred that while one cheater is expected to reach an equilibrium with WT, two cheaters may create an antagonistic interaction that eventually leads to the pattern observed in the experiments. While there is growing interest in using defective interfering particles as therapeutic strategies, here we show that the evolutionary dynamics of such cheater viruses may be highly unexpected.