Giant DNA viruses infect a wide range of protists and can be considered important mortality agents of marine and freshwater phyto- and zooplankton. These viruses encode hundreds of proteins and replicate in cytoplasmic factories that serve as centers for viral gene expression, DNA replication, and virion assembly. Due to their complexity, giant virus factories can be parasitized by a class of smaller DNA viruses called virophages. Even though virophages encode their own DNA replication and capsid proteins, they strictly depend for their propagation on a co-infecting giant virus. It is presumed that virophages utilize the giant virus-encoded transcription machinery for gene expression. By parasitizing the giant virus factory, virophages inhibit the production of new giant virus particles, which, in turn, increases survival of the host cell population. Therefore, protist populations that are susceptible to infection by giant viruses will gain a selective advantage in the presence of virophages.
Here we show that the mavirus virophage is able to integrate into the nuclear genome of Cafeteria roenbergensis, a ubiquitous marine bacterivore. Endogenous virophages are structurally and genetically similar to the eukaryotic Maverick/Polinton DNA transposons. Integrated mavirus genes are transcriptionally silent, unless the cell is infected with the lytic giant virus CroV. In the presence of CroV, the endogenous mavirus resumes gene expression and genome replication, which results in the production of infectious mavirus particles. These reactivated virophage particles are then able to protect host cell populations from CroV-induced lysis. Our results suggest that virophage protection may play a significant role in regulating natural protist populations.