Endogenous virophages are active and mitigate giant virus infection in the marine protist Cafeteria burkhardae.

Endogenous viral elements (EVEs) are common genetic passengers in various protists. Some EVEs represent viral fossils, whereas others are still active. The marine heterotrophic flagellate Cafeteria burkhardae contains several EVE types related to the virophage mavirus, a small DNA virus that parasitizes the lytic giant virus CroV. We hypothesized that endogenous virophages may act as an antiviral defense system in protists, but no protective effect of virophages in wild host populations has been shown so far. Here, we tested the activity of virophage EVEs and studied their impact on giant virus replication. We found that endogenous mavirus-like elements (EMALEs) from globally distributed Cafeteria populations produced infectious virus particles specifically in response to CroV infection. However, reactivation was stochastic, often inefficient, and poorly reproducible. Interestingly, only one of eight EMALE types responded to CroV infection, implying that other EMALEs may be linked to different giant viruses. We isolated and cloned several reactivated virophages and characterized their particles, genomes, and infection dynamics. All tested virophages inhibited the production of CroV during coinfection, thereby preventing lysis of the host cultures in a dose-dependent manner. Comparative genomics of different C. burkhardae strains revealed that inducible EMALEs are common and are not linked to specific geographic locations. We demonstrate that naturally occurring virophage EVEs reactivate upon giant virus infection, thus providing a striking example that eukaryotic EVEs can become active under specific conditions. Moreover, our results support the hypothesis that virophages can act as an adaptive antiviral defense system in protists.

Virophages and retrotransposons colonize the genomes of a heterotrophic flagellate.

Virophages can parasitize giant DNA viruses and may provide adaptive anti-giant virus defense in unicellular eukaryotes. Under laboratory conditions, the virophage mavirus integrates into the nuclear genome of the marine flagellate Cafeteria burkhardae and reactivates upon superinfection with the giant virus CroV. In natural systems, however, the prevalence and diversity of host-virophage associations has not been systematically explored. Here, we report dozens of integrated virophages in four globally sampled C. burkhardae strains that constitute up to 2% of their host genomes. These endogenous mavirus-like elements (EMALEs) separated into eight types based on GC-content, nucleotide similarity, and coding potential and carried diverse promoter motifs implicating interactions with different giant viruses. Between host strains, some EMALE insertion loci were conserved indicating ancient integration events, whereas the majority of insertion sites were unique to a given host strain suggesting that EMALEs are active and mobile. Furthermore, we uncovered a unique association between EMALEs and a group of tyrosine recombinase retrotransposons, revealing yet another layer of parasitism in this nested microbial system. Our findings show that virophages are widespread and dynamic in wild Cafeteria populations, supporting their potential role in antiviral defense in protists.

Viruses exist in all ecosystems in vast numbers and infect many organisms. Some of them are harmful but others can protect the organisms they infect. For example, a group of viruses called virophages protect microscopic sea creatures called plankton from deadly infections by so-called giant viruses. In fact, virophages need plankton infected with giant viruses to survive because they use enzymes from the giant viruses to turn on their own genes. A virophage called mavirus integrates its genes into the DNA of a type of plankton called Cafeteria. It lays dormant in the DNA until a giant virus called CroV infects the plankton. This suggests that the mavirus may be a built-in defense against CroV infections and laboratory studies seem to confirm this. But whether wild Cafeteria also use these defenses is unknown. Hackl et al. show that virophages are common in the DNA of wild Cafeteria and that the two appear to have a mutually beneficial relationship. In the experiments, the researchers sequenced the genomes of four Cafeteria populations from the Atlantic and Pacific Oceans and looked for virophages in their DNA. Each of the four Cafeteria genomes contained dozens of virophages, which suggests that virophages are important to these plankton. This included several relatives of the mavirus and seven new virophages. Virophage genes were often interrupted by so called jumping genes, which may take advantage of the virophages the way the virophages use giant viruses to meet their own needs. The experiments show that virophages often co-exist with marine plankton from around the world and these relationships are likely beneficial. In fact, the experiments suggest that the virophages may have played an important role in the evolution of these plankton. Further studies may help scientists learn more about virus ecology and how viruses have shaped the evolution of other creatures.

Four high-quality draft genome assemblies of the marine heterotrophic nanoflagellate Cafeteria roenbergensis.

The heterotrophic stramenopile Cafeteria roenbergensis is a globally distributed marine bacterivorous protist. This unicellular flagellate is host to the giant DNA virus CroV and the virophage mavirus. We sequenced the genomes of four cultured C. roenbergensis strains and generated 23.53 Gb of Illumina MiSeq data (99-282 × coverage per strain) and 5.09 Gb of PacBio RSII data (13-45 × coverage). Using the Canu assembler and customized curation procedures, we obtained high-quality draft genome assemblies with a total length of 34-36 Mbp per strain and contig N50 lengths of 148 kbp to 464 kbp. The C. roenbergensis genome has a GC content of ~70%, a repeat content of ~28%, and is predicted to contain approximately 7857-8483 protein-coding genes based on a combination of de novo, homology-based and transcriptome-supported annotation. These first high-quality genome assemblies of a bicosoecid fill an important gap in sequenced stramenopile representatives and enable a more detailed evolutionary analysis of heterotrophic protists.