Generation of a Recombinant Mimivirus by Homologous Recombination Using Direct Transfection of a PCR Amplicon

Giant viruses are large double-stranded DNA viruses encoding hundreds of genes. These genes are considered to reprogram the host cell machinery, which, in turn, modulates the global nutrient cycles in the environment. However, the functions of giant-virus genes are largely unknown due to the limited molecular biology tools. Recently, reverse-genetics systems have been developed in giant viruses infecting a free-living amoeba, Acanthamoeba castellanii. One of the methods relies on homologous recombination by plasmid transfection, but plasmid construction is generally laborious. In this study, we developed a modified method to generate a recombinant giant virus, mimivirus, with direct transfection of a PCR amplicon into host cells, a widely used approach in budding yeast and other organisms. Consequently, we successfully generated a recombinant mimivirus with a marker gene. Our results demonstrated that a bacterium-free method could be applied to giant viruses to generate recombinants, accelerating further experimental studies related to these viruses.