Genetic manipulation of a giant virus-associated virophage

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. Significance statementVirophages are viruses that hyperparasitize giant viruses, which infect unicellular eukaryotes and have extremely large particles and genomes. Giant viruses modulate microbial communities not only by killing their hosts but also by altering host cellular functions. Virophages modulate the replication of giant viruses, thereby driving ecosystem dynamics. Previous studies have demonstrated their widespread distribution through isolation and metagenomic analyses. However, the functions of most virophage genes remain unknown. Due to the lack of genetic tools, the molecular mechanisms underlying the interactions between virophages and giant viruses remain largely elusive. Here, we established a virophage reverse-genetics system based on circular polymerase extension reaction. Our results demonstrate that the system can dissect virophage gene functions and will accelerate virophage genetics.