Virophage-like viruses replicate with a candidate phycodnavirus of C. parva

TThe discovery of virophages and polinton-like viruses (PLVs) sparked studies of their hyperparasitic relationships with their associated giant viruses and hosts. To date, every cultured virophage and PLV depends on an Imitervirales helper virus. The freshwater alga Chrysochromulina parva is infected by viruses in the Imitervirales and Algavirales, and recent work showed that the PLV, CpV-PLV Moe, replicates via co-infection with the Imitervirales host CpV-BQ3. The other known PLVs, CpV PLV Larry and CpV PLV Curly, were subsequently isolated alongside CpV-BQ1, a putative Algavirales virus, raising the possibility that they depend on an Algavirales helper. To determine the nature of Larry and Curlys hyperparasitic relationships, infection experiments were conducted with each PLV and either CpV-BQ3 or CpV-BQ1. These experiments demonstrated that that neither Larry nor Curly replicated when introduced to C. parva alone, nor during mixed infections that included the Imitervirales virus BQ3. In contrast, the Algavirales virus CpV-BQ1 supported replication of both PLVs, with each increasing roughly 1000-fold over the course of infection while CpV-BQ1 replication was simultaneously reduced up to 100-fold. Together, these findings provide the first culture based evidence that PLV replication can be supported by an Algavirales helper and demonstrate that C. parva supports at least two distinct hyperparasitic relationships spanning different viral orders within the class Megaviricetes.