Virophages are double-stranded DNA viruses that hyperparasitize giant viruses infecting unicellular eukaryotes. Parasitization by virophages often reduces the replication of giant viruses, thereby modulating microbial communities in the environment. However, the molecular mechanisms underlying the tripartite relationship are largely unknown due to methodological limitations. In the present study, we developed a reverse-genetics system for a Sputnik virophage that parasitizes the amoeba-infecting giant virus, mimivirus. We demonstrated that transfection of genomic DNA could recover infectious virophage particles. Transfection of genomic DNA synthesized by circular polymerase extension reaction (CPER) also resulted in the recovery of infectious viruses. As a proof of concept, we successfully modified two Sputnik genes by transfecting CPER-assembled mutant genomic DNA. Collectively, our reverse-genetics system provides a framework for assessing the functional importance of Sputnik genes and should facilitate future genetic studies of virophages.
Chen, J., Ogata, H., Hikida, H.
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