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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., eLife digest 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.

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., Measurement(s)DNA • mitochondrial_DNA • sequence_assembly • sequence feature annotationTechnology Type(s)DNA sequencing • genome assembly • sequence annotationSample Characteristic - OrganismCafeteria roenbergensisSample Characteristic - Environmentmarine water bodySample Characteristic - LocationNorthwest Atlantic Ocean • Carribean Sea • Southeast Pacific Ocean • North East Pacific Ocean Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11419098

International audience; Chronic infection with hepatitis C virus (HCV) is one of the main causes of hepatocellular carcinoma. However, the molecular mechanisms linking the infection to cancer development remain poorly understood. Here we used HCV-infected cells and liver biopsies to study how HCV modulates the glutaminolysis pathway, which is known to play an important role in cellular energetics, stress defense and neoplastic transformation. Transcript levels of glutaminolytic factors were quantified in Huh7.5 cells or primary human hepatocytes infected with the JFH1 HCV strain as well as in biopsies of chronic HCV patients. Nutrient deprivation, biochemical analysis and metabolite quantification were performed with JFH1 infected Huh7.5 cells. Furthermore, shRNA vectors and small molecule inhibitors were used to investigate the dependence of HCV replication on metabolic changes. We show that HCV modulates the transcript levels of key enzymes of the glutamine metabolism in vitro and in liver biopsies of chronic HCV patients. Consistently, HCV infection increases glutamine utilization and dependence. We finally show that inhibiting glutamine metabolism attenuates HCV infection and the oxidative stress associated with HCV infection. Conclusions Our data suggest that HCV establishes glutamine dependence, which is required for viral replication. Importantly, glutamine addiction is also a hallmark of tumor cells. While HCV induces glutaminolysis to create an environment favorable for viral replication, it predisposes the cell to transformation. Glutaminolytic enzymes may be interesting therapeutic targets for prevention of hepatocarcinogenesis in chronic hepatitis C. This article is protected by copyright. All rights reserved

This protocol allows to remove bacteria from cultures of the marine heterotrophic nanoflagellate Cafeteria roenbergensis without affecting flagellate viability. Bacterial removal is essential to reduce contamination and background signal in subsequent microscopy analysis, protein extraction, DNA extraction or other applications. With some changes, this protocol could be adapted to different types of protists.

Viruses are obligate intracellular parasites and thus cannot replicate outside a living cell. Whereas a susceptible and permissive host cell is sufficient for most viruses to produce infectious progeny, some viruses such as satellite viruses require two different biological entities for their replication. In contrast to most satellite viruses with small RNA-based genomes, the lavidaviruses (or virophages) that replicate in eukaryotic microbes (protists) are comparable to adenoviruses in terms of particle size and genetic complexity. Lavidaviruses only replicate during a co-infection with a giant DNA virus (GV; see, e.g., [1]), and the first described virophage, Sputnik, was serendipitously discovered during a search for new GVs [2].

Fluorescence microscopy is essential to understand,between others, the cell organization and the life cycle of viruses that infect them. This protocol describes the steps neccesary to fix and permeabilize Cafeteria roenbergensis cells allowing its staining with antibodies or chemical reactions as Click-IT reactions.