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.

Ecological and evolutionary dynamics of cell-virus-virophage systems.

Microbial eukaryotes, giant viruses and virophages form a unique hyperparasitic system. Virophages are parasites of the virus transcription machinery and can interfere with virus replication, resulting in a benefit to the eukaryotic host population. Surprisingly, virophages can integrate into the genomes of their cell or virus hosts, and have been shown to reactivate during coinfection. This raises questions about the role of integration in the dynamics of cell-virus-virophage systems. We use mathematical models and computational simulations to understand the effect of virophage integration on populations of cells and viruses. We also investigate multicellularity and programmed cell-death (PCD) as potential antiviral defence strategies used by cells. We found that virophages which enter the cell independently of the host virus, such as Mavirus, are expected to integrate commonly into the genomes of their cell hosts. Our models suggest that integrations from virophages without an independent mode of entry like Sputnik, are less likely to become fixed in the cell host population. Alternatively, we found that Sputnik virophages can stably persist integrated in the virus population, as long as they do not completely inhibit virus replication. We also show that increasing virophage inhibition can stabilise oscillatory dynamics, which may explain the long-term persistence of viruses and virophages in the environment. Our results demonstrate that inhibition by virophages and multicellularity are effective antiviral strategies that may act in synergy against viral infection in microbial species.

Virophages Found in Viromes from Lake Baikal.

In this study, a previously little-studied group of viruses-virophages-was searched for and identified in the viromes of the ancient oligotrophic Lake Baikal. Virophages are small dsDNA viruses that parasitize giant viruses (e.g., Mimiviridae), which in turn affect unicellular eukaryotes. We analyzed eight viromes obtained from the deep-water areas of three basins of Lake Baikal and the shallow-water strait Maloye More in different seasons. The sequences of virophages were revealed in all viromes and were dominant after bacteriophages and algal viruses. Sixteen putative complete genomes of virophages were assembled, all of which contained four conserved genes encoding major capsid protein (MCP), minor capsid protein (mCP), maturation cysteine protease (PRO), and FtsK-HerA family DNA-packaging ATPase (ATPase). The MCP-based cluster analysis showed a sequence separation according to seasons, and a dependence on the geographical localization was not detected.

Diversity, Relationship, and Distribution of Virophages and Large Algal Viruses in Global Ocean Viromes.

Virophages are a group of small double-stranded DNA viruses that replicate and proliferate with the help of the viral factory of large host viruses. They are widely distributed in aquatic environments but are more abundant in freshwater ecosystems. Here, we mined the Global Ocean Viromes 2.0 (GOV 2.0) dataset for the diversity, distribution, and association of virophages and their potential host large viruses in marine environments. We identified 94 virophage sequences (>5 kbp in length), of which eight were complete genomes. The MCP phylogenetic tree showed that the GOV virophages were widely distributed on the global virophage tree but relatively clustered on three major branches. The gene-sharing network divided GOV virophages into 21 outliers, 2 overlaps, and 14 viral clusters, of which 4 consisted of only the GOV virophages. We also identified 45 large virus sequences, 8 of which were >100 kbp in length and possibly involved in cell-virus-virophage (C-V-v) trisome relationships. The potential eukaryotic hosts of these eight large viruses and the eight virophages with their complete genomes identified are likely to be algae, based on comparative genomic analysis. Both homologous gene and codon usage analyses support a possible interaction between a virophage (GOVv18) and a large algal virus (GOVLV1). These results indicate that diverse and novel virophages and large viruses are widespread in global marine environments, suggesting their important roles and the presence of complicated unknown C-V-v relationships in marine ecosystems.

Virophages-Known and Unknown Facts.

The paper presents virophages, which, like their host, giant viruses, are "new" infectious agents whose role in nature, including mammalian health, is important. Virophages, along with their protozoan and algal hosts, are found in fresh inland waters and oceanic and marine waters, including thermal waters and deep-sea vents, as well as in soil, plants, and in humans and animals (ruminants). Representing "superparasitism", almost all of the 39 described virophages (except Zamilon) interact negatively with giant viruses by affecting their replication and morphogenesis and their "adaptive immunity". This causes them to become regulators and, at the same time, defenders of the host of giant viruses protozoa and algae, which are organisms that determine the homeostasis of the aquatic environment. They are classified in the family Lavidaviridae with two genus (Sputnikovirus, Mavirus). However, in 2023, a proposal was presented that they should form the class Maveriviricetes, with four orders and seven families. Their specific structure, including their microsatellite (SSR-Simple Sequence Repeats) and the CVV (cell-virus-virophage, or transpovirion) system described with them, as well as their function, makes them, together with the biological features of giant viruses, form the basis for discussing the existence of a fourth domain in addition to Bacteria, Archaea, and Eukaryota. The paper also presents the hypothetical possibility of using them as a vector for vaccine antigens.

A billion years arms-race between viruses, virophages, and eukaryotes.

Bamfordviruses are arguably the most diverse group of viruses infecting eukaryotes. They include the Nucleocytoplasmic Large DNA viruses (NCLDVs), virophages, adenoviruses, Mavericks and Polinton-like viruses. Two main hypotheses for their origins have been proposed: the 'nuclear-escape' and 'virophage-first' hypotheses. The nuclear-escape hypothesis proposes an endogenous, Maverick-like ancestor which escaped from the nucleus and gave rise to adenoviruses and NCLDVs. In contrast, the virophage-first hypothesis proposes that NCLDVs coevolved with protovirophages; Mavericks then evolved from virophages that became endogenous, with adenoviruses escaping from the nucleus at a later stage. Here, we test the predictions made by both models and consider alternative evolutionary scenarios. We use a data set of the four core virion proteins sampled across the diversity of the lineage, together with Bayesian and maximum-likelihood hypothesis-testing methods, and estimate rooted phylogenies. We find strong evidence that adenoviruses and NCLDVs are not sister groups, and that Mavericks and Mavirus acquired the rve-integrase independently. We also found strong support for a monophyletic group of virophages (family Lavidaviridae) and a most likely root placed between virophages and the other lineages. Our observations support alternatives to the nuclear-escape scenario and a billion years evolutionary arms-race between virophages and NCLDVs.

Large-scale invasion of unicellular eukaryotic genomes by integrating DNA viruses.

Eukaryotic genomes contain a variety of endogenous viral elements (EVEs), which are mostly derived from RNA and ssDNA viruses that are no longer functional and are considered to be "genomic fossils." Genomic surveys of EVEs, however, are strongly biased toward animals and plants, whereas protists, which represent the majority of eukaryotic diversity, remain poorly represented. Here, we show that protist genomes harbor tens to thousands of diverse, ~14 to 40 kbp long dsDNA viruses. These EVEs, composed of virophages, Polinton-like viruses, and related entities, have remained hitherto hidden owing to poor sequence conservation between virus groups and their repetitive nature that precluded accurate short-read assembly. We show that long-read sequencing technology is ideal for resolving virus insertions. Many protist EVEs appear intact, and most encode integrases, which suggests that they have actively colonized hosts across the tree of eukaryotes. We also found evidence for gene expression in host transcriptomes and that closely related virophage and Polinton-like virus genomes are abundant in viral metagenomes, indicating that many EVEs are probably functional viruses.

Updated Virophage Taxonomy and Distinction from Polinton-like Viruses.

Virophages are small dsDNA viruses that hijack the machinery of giant viruses during the co-infection of a protist (i.e., microeukaryotic) host and represent an exceptional case of "hyperparasitism" in the viral world. While only a handful of virophages have been isolated, a vast diversity of virophage-like sequences have been uncovered from diverse metagenomes. Their wide ecological distribution, idiosyncratic infection and replication strategy, ability to integrate into protist and giant virus genomes and potential role in antiviral defense have made virophages a topic of broad interest. However, one limitation for further studies is the lack of clarity regarding the nomenclature and taxonomy of this group of viruses. Specifically, virophages have been linked in the literature to other "virophage-like" mobile genetic elements and viruses, including polinton-like viruses (PLVs), but there are no formal demarcation criteria and proper nomenclature for either group, i.e., virophage or PLVs. Here, as part of the ICTV Virophage Study Group, we leverage a large set of genomes gathered from published datasets as well as newly generated protist genomes to propose delineation criteria and classification methods at multiple taxonomic ranks for virophages 'sensu stricto', i.e., genomes related to the prototype isolates Sputnik and mavirus. Based on a combination of comparative genomics and phylogenetic analyses, we show that this group of virophages forms a cohesive taxon that we propose to establish at the class level and suggest a subdivision into four orders and seven families with distinctive ecogenomic features. Finally, to illustrate how the proposed delineation criteria and classification method would be used, we apply these to two recently published datasets, which we show include both virophages and other virophage-related elements. Overall, we see this proposed classification as a necessary first step to provide a robust taxonomic framework in this area of the virosphere, which will need to be expanded in the future to cover other virophage-related viruses such as PLVs.

Isolation and infection cycle of a polinton-like virus virophage in an abundant marine alga.

Virophages are small double stranded DNA (dsDNA) viruses that can only replicate in a host by co-infecting with another virus. Marine algae are commonly associated with virophage-like elements such as Polinton-like viruses (PLVs) that remain largely uncharacterized. Here we isolated a PLV that co-infects the alga Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T), a close relative of the "Phaeocystis globosa virus-virophage" genomic sequence. We name this PLV 'Gezel-14T. Gezel is phylogenetically distinct from the Lavidaviridae family where all known virophages belong. Gezel-14T co-infection decreases the fitness of its viral host by reducing burst sizes of PgV-14T, yet insufficiently to spare the cellular host population. Genomic screens show Gezel-14T-like PLVs integrated into Phaeocystis genomes, suggesting that these widespread viruses are capable of integration into cellular host genomes. This system presents an opportunity to better understand the evolution of eukaryotic dsDNA viruses as well as the complex dynamics and implications of viral parasitism.

Microsatellite signature analysis of twenty-one virophage genomes of the family Lavidaviridae.

Microsatellites or Simple Sequence Repeats (SSRs) are short motif repeat sequences constituting the most hypervariable regions of genomes. Present study extracts and analyzes the SSRs from genomes of 21 virophages. Genomic sequences were retrieved from NCBI and the microsatellite data was extracted through MISA web server. Phylogenetic analysis was performed by using MAFFT and MEGAX as per standardized protocols. The virophages have a circular/linear ds DNA genome of ~17-30 kb size. The GC% of genomes ranged from 26.8 (PSAV13) to 51.1 (PSAV12). A total of 3664 SSRs and 488 cSSR were observed with an average incidence of 174 and 23 respectively. The total SSR incidence in a genome ranged from 120 (PSAV19) to 264 (PSAV14). The cSSR (compound SSR) incidence ranged from 8 (PSAV12) to 47 (PSAV14). Mono-nucleotide repeats are the most incident microsatellites (1129 SSRs) followed by di-nucleotide (1036 SSRs) and tri-nucleotide repeats (368 SSRs). However, the same is not true for individual genomes. There are 14, 16 and 17 genomes which have no incidence of tetra-, penta- and hexa-nucleotide repeats respectively. Mono 'A' repeats having the maximum representation (average ~33 per genome) in mono-nucleotide repeats. For the di-nucleotide repeats, AT/TA motif had the highest frequency (average ~30) distantly followed by AG/GA; and CT/TC (average 5.6 & 5.5 respectively). A total of 1946 SSRs (76%) were found in the coding region. All genomes had a higher SSR density in non-coding as compared to the coding region. There are fifteen genomes which have at least one gene with no SSR. A total of 41 cSSRs with incidence across minimum of two virophages was observed. There were 12 cSSRs which had multiple presence within the same genome. The heat map of the genomes on one hand corroborates the phylogenetic tree with similar sequences (PSAV2, PSAV5, PSAV6, PSAV17 and PSAV18) being positioned together in the phylogenetic analysis while on the other hand it also highlights the diversity of the studied sequences. The conservation of cSSRs across multiple virophages highlights their potential as biomarkers.

Simultaneous Giant Virus and Virophage Quantification Using Droplet Digital PCR.

Viruses are an abundant component of aquatic systems, but their detection and quantification remain a challenge. Virophages co-replicate with giant viruses in the shared host cell, and can inhibit the production of new giant virus particles, thereby increasing the survival of the infected host population. Here, we present a protocol for Droplet Digital PCR (ddPCR) to quantify simultaneously giant virus and virophage in a mixed sample, enabling the rapid, culture-free and high throughput detection of virus and virophage. As virophage can be present as free virus particles or integrated into the virus host's genome as well as associated with organic particles, we developed a simple method that enables discrimination between free and particle-associated virophages. The latter include aggregated virophage particles as well as virophage integrated into the host genome. We used, for our experiments, a host-virus-virophage system consisting of Cafeteria burkhardae, CroV and mavirus. Our results show that ddPCR can be an efficient method to quantify virus and virophage, and we discuss potential applications of the method for studying ecological and evolutionary processes of virus and virophages.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.

The Discovery of a New Mimivirus Isolate in Association with Virophage-Transpoviron Elements in Brazil Highlights the Main Genomic and Evolutionary Features of This Tripartite System.

Mimiviruses are giant viruses of amoeba that can be found in association with virophages. These satellite-like viruses are dependent on the mimivirus viral factory to replicate. Mimiviruses can also be associated with linear DNA molecules called transpovirons. Transpovirons and virophages are important drivers of giant virus evolution although they are still poorly studied elements. Here, we describe the isolation and genomic characterization of a mimivirus/virophage/transpoviron tripartite system from Brazil. We analyzed transmission electron microscopy images and performed genome sequencing and assembly, gene annotation, and phylogenetic analysis. Our data confirm the isolation of a lineage A mimivirus (1.2 Mb/1012 ORFs), called mimivirus argentum, and a sputnik virophage (18,880 bp/20 ORFs). We also detected a third sequence corresponding to a transpoviron from clade A (6365 bp/6 ORFs) that presents small terminal inverted repeats (77 nt). The main genomic features of mimivirus argentum and of its virophage/transpoviron elements corroborates with what is described for other known elements. This highlights that this triple genomic and biological interaction may be ancient and well-conserved. The results expand the basic knowledge about unique and little-known elements and pave the way to future studies that might contribute to a better understanding of this tripartite relationship.

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.

Virophages: association with human diseases and their predicted role as virus killers.

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.

Polinton-like viruses are abundant in aquatic ecosystems.

BACKGROUND: Polintons are large mobile genetic elements found in the genomes of eukaryotic organisms that are considered the ancient ancestors of most eukaryotic dsDNA viruses. Originally considered as transposons, they have been found to encode virus capsid genes, suggesting they may actually be integrated viruses; however, an extracellular form has yet to be detected. Recently, circa 25 Polinton-like viruses have been discovered in environmental metagenomes and algal genomes, which shared distantly related genes to both Polintons and virophages (Lavidaviridae). These entities could be the first members of a major class of ancient eukaryotic viruses; however, owing to the lack of available genomes for analysis, information on their global diversity, evolutionary relationships, eukaryotic hosts, and status as free virus particles is limited. RESULTS: Here, we analysed the metaviromes of an alpine lake to show that Polinton-like virus genome sequences are abundant in the water column. We identify major capsid protein genes belonging to 82 new Polinton-like viruses and use these to interrogate publicly available metagenomic datasets, identifying 543 genomes and a further 16 integrated into eukaryotic genomes. Using an analysis of shared gene content and major capsid protein phylogeny, we define large groups of Polinton-like viruses and link them to diverse eukaryotic hosts, including a new group of viruses, which possess all the core genes of virophages and infect oomycetes and Chrysophyceae. CONCLUSIONS: Our study increased the number of known Polinton-like viruses by 25-fold, identifying five major new groups of eukaryotic viruses, which until now have been hidden in metagenomic datasets. The large enrichment (> 100-fold) of Polinton-like virus sequences in the virus-sized fraction of this alpine lake and the fact that their viral major capsid proteins are found in eukaryotic host transcriptomes support the hypothesis that Polintons in unicellular eukaryotes are viruses. In summary, our data reveals a diverse assemblage of globally distributed viruses, associated with a wide range of unicellular eukaryotic hosts. We anticipate that the methods we have developed for Polinton-like virus detection and the database of over 20,000 genes we present will allow for continued discovery and analysis of these new viral groups. Video abstract.

The Virophage Family Lavidaviridae.

Double-stranded (ds) DNA viruses of the family Lavidaviridae, commonly known as virophages, are a fascinating group of eukaryotic viruses that depend on a coinfecting giant dsDNA virus of the Mimiviridae for their propagation. Instead of replicating in the nucleus, virophages multiply in the cytoplasmic virion factory of a coinfecting giant virus inside a phototrophic or heterotrophic protistal host cell. Virophages are parasites of giant viruses and can inhibit their replication, which may lead to increased survival rates of the infected host cell population. The genomes of virophages are 17-33 kilobase pairs (kbp) long and encode 16-34 proteins. Genetic signatures of virophages can be found in metagenomic datasets from various saltwater and freshwater environments around the planet. Most virophages share a set of conserved genes that code for a major and a minor capsid protein, a cysteine protease, a genome-packaging ATPase, and a superfamily 3 helicase, although the genomes are otherwise diverse and variable. Lavidaviruses share genes with other mobile genetic elements, suggesting that horizontal gene transfer and recombination have been major forces in shaping these viral genomes. Integrases are occasionally found in virophage genomes and enable these DNA viruses to persist as provirophages in the chromosomes of their viral and cellular hosts. As we watch the genetic diversity of this new viral family unfold through metagenomics, additional isolates are still lacking and critical questions regarding their infection cycle, host range, and ecology remain to be answered.

A virophage cross-species infection through mutant selection represses giant virus propagation, promoting host cell survival.

Virus adaptation to new hosts is a major cause of infectious disease emergence. This mechanism has been intensively studied in the context of zoonotic virus spillover, due to its impact on global health. However, it remains unclear for virophages, parasites of giant viruses and potential regulators of microbial communities. Here, we present, for the first time to our knowledge, evidence of cross-species infection of a virophage. We demonstrated that challenging the native population of Guarani virophage with two previously unidentified giant viruses, previously nonpermissive to this virophage, allows the selection of a mutant genotype able to infect these giant viruses. We were able to characterize the potential genetic determinant (deletion) carried by the virophage with the expanded-host range. Our study also highlights the relevant biological impact of this host adaptation by demonstrating that coinfection with the mixture containing the mutant virophage abolishes giant virus production and rescues the host cell population from lysis.

Novel Cell-Virus-Virophage Tripartite Infection Systems Discovered in the Freshwater Lake Dishui Lake in Shanghai, China.

Virophages are small parasitic double-stranded DNA (dsDNA) viruses of giant dsDNA viruses infecting unicellular eukaryotes. Except for a few isolated virophages characterized by parasitization mechanisms, features of virophages discovered in metagenomic data sets remain largely unknown. Here, the complete genomes of seven virophages (26.6 to 31.5 kbp) and four large DNA viruses (190.4 to 392.5 kbp) that coexist in the freshwater lake Dishui Lake, Shanghai, China, have been identified based on environmental metagenomic investigation. Both genomic and phylogenetic analyses indicate that Dishui Lake virophages (DSLVs) are closely related to each other and to other lake virophages, and Dishui Lake large DNA viruses are affiliated with the micro-green alga-infecting Prasinovirus of the Phycodnaviridae (named Dishui Lake phycodnaviruses [DSLPVs]) and protist (protozoan and alga)-infecting Mimiviridae (named Dishui Lake large alga virus [DSLLAV]). The DSLVs possess more genes with closer homology to that of large alga viruses than to that of giant protozoan viruses. Furthermore, the DSLVs are strongly associated with large green alga viruses, including DSLPV4 and DSLLAV1, based on codon usage as well as oligonucleotide frequency and correlation analyses. Surprisingly, a nonhomologous CRISPR-Cas like system is found in DSLLAV1, which appears to protect DSLLAV1 from the parasitization of DSLV5 and DSLV8. These results suggest that novel cell-virus-virophage (CVv) tripartite infection systems of green algae, large green alga virus (Phycodnaviridae- and Mimiviridae-related), and virophage exist in Dishui Lake, which will contribute to further deep investigations of the evolutionary interaction of virophages and large alga viruses as well as of the essential roles that the CVv plays in the ecology of algae.IMPORTANCE Virophages are small parasitizing viruses of large/giant viruses. To our knowledge, the few isolated virophages all parasitize giant protozoan viruses (Mimiviridae) for propagation and form a tripartite infection system with hosts, here named the cell-virus-virophage (CVv) system. However, the CVv system remains largely unknown in environmental metagenomic data sets. In this study, we systematically investigated the metagenomic data set from the freshwater lake Dishui Lake, Shanghai, China. Consequently, four novel large alga viruses and seven virophages were discovered to coexist in Dishui Lake. Surprisingly, a novel CVv tripartite infection system comprising green algae, large green alga viruses (Phycodnaviridae- and Mimiviridae-related), and virophages was identified based on genetic link, genomic signature, and CRISPR system analyses. Meanwhile, a nonhomologous CRISPR-like system was found in Dishui Lake large alga viruses, which appears to protect the virus host from the infection of Dishui Lake virophages (DSLVs). These findings are critical to give insight into the potential significance of CVv in global evolution and ecology.