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

Abstract The 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 Curly’s 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’s 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 . Author summary This study revealed that two polinton-like viruses (PLVs), CpV-PLV Larry and CpV-PLV Curly, rely on a previously unrecognized type of helper virus to replicate. PLVs are hyperparasites related to virophages; they infect giant viruses that themselves infect microbial hosts. Until now, all PLVs studied in the lab depended on helper viruses from a single group, the Imitervirales . We tested whether Larry and Curly followed this same pattern by infecting the freshwater alga Chrysochromulina parva with each PLV alone or together with one of two algal viruses known to infect C. parva . Neither PLV grew on its own, and neither influenced algal growth. They also failed to replicate when paired with the Imitervirales virus CpV-BQ3, showing that this known helper virus cannot support them. In contrast, both PLVs replicated when co-infecting with CpV-BQ1, a virus from the Algavirales . During this infection, CpV-BQ1’s own replication dropped, indicating a hyperparasitic interaction. These findings provide the first culture-based evidence that PLVs can use Algavirales viruses as helpers. They also show that C. parva hosts multiple, distinct layers of viral parasitism, expanding our understanding of the complexity and diversity of virus–virus interactions in microbial ecosystems.