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.

The rotavirus (RV) genome consists of 11 double-stranded RNA segments. Sometimes, partial sequence duplication of an RNA segment leads to a rearranged RNA segment. To specify the impact of rearrangement, the replication efficiencies of human RV with rearranged segments 7, 11 or both were compared to these of the homologous human wild-type RV (wt-RV) and of the bovine wt-RV strain RF. As judged by viral growth curves, rotaviruses with a rearranged genome (r-RV) had no selective growth advantage over the homologous wt-RV. In contrast, r-RV were selected over wt-RV during competitive experiments (i.e mixed infections between r-RV and wt-RV followed by serial passages in cell culture). Moreover, when competitive experiments were performed between a human r-RV and the bovine wt-RV strain RF, which had a clear growth advantage, rearranged segments 7, 11 or both always segregated in viral progenies even when performing mixed infections at an MOI ratio of 1 r-RV to 100 wt-RV. Lastly, bovine reassortant viruses that had inherited a rearranged segment 7 from human r-RV were generated. Although substitution of wt by rearranged segment 7 did not result in any growth advantage, the rearranged segment was selected in the viral progenies resulting from mixed infections by bovine reassortant r-RV and wt-RV, even for an MOI ratio of 1 r-RV to 10⁷ wt-RV. Lack of selective growth advantage of r-RV over wt-RV in cell culture suggests a mechanism of preferential packaging of the rearranged segments over their standard counterparts in the viral progeny.

Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca²⁺ signaling. HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca²⁺ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV’s imprint on cell metabolism.

Giant DNA viruses of the phylumNucleocytoviricotaare being increasingly recognized as important regulators of natural protist populations. However, our knowledge of their infection cycles is still very limited due to a lack of cultured virus-host systems and molecular tools to study them. Here, we apply bioorthogonal noncanonical amino acid tagging (BONCAT) to pulse label the marine heterotrophic flagellateCafeteria burkhardaeduring infection with the lytic giant virus CroV. In absence of CroV, we report efficient incorporation of the L-methionine analog L-azidohomoalanine (AHA) into newly synthesized proteins of the methionine prototrophicC. burkhardae. During CroV infection, AHA was predominantly found in viral proteins, and single CroV virions were imaged with stimulated emission depletion (STED) super-resolution microscopy. CroV particles incorporated AHA with 95-100% efficiency while retaining their infectivity, which makes BONCAT/STED a powerful tool to study viral replication cycles in this ecologically relevant marine bacterivore.Significance: Giant DNA viruses are the dominant class of protist-infecting viruses, yet the vast majority of described giant virus-protist systems remain uncultured. One of the better studied cultured systems is composed of the stramenopileCafeteria burkhardae(previouslyC. roenbergensis), the giant Cafeteria roenbergensis virus (CroV) and the virophage mavirus.C. burkhardaeis a widespread marine phagotrophic protist that plays an important role in regulating bacterial populations. In addition to being grazed upon by larger zooplankton,C. burkhardaepopulations are controlled by the lytic giant virus CroV. In turn, CroV is parasitized by the virophage mavirus that increases host population survival in the presence of CroV and forms a mutualistic symbiosis with its host. Despite being of fundamental ecological and evolutionary interest, this tripartite host-virus-virophage system suffers from a lack of molecular tools. Here, we show that CroV particles can be fluorescently labeled and imaged by super-resolution microscopy. To achieve this we established robust procedures for analyzing protist and viral populations and implemented the use of bioorthogonal noncanonical amino acid tagging (BONCAT) in a marine unicellular 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 e ndogenous ma virus- l ike e lements (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.