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: 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.

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.

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.

Diversity, evolution, and classification of virophages uncovered through global metagenomics.

BACKGROUND: Virophages are small viruses with double-stranded DNA genomes that replicate along with giant viruses and co-infect eukaryotic cells. Due to the paucity of virophage reference genomes, a collective understanding of the global virophage diversity, distribution, and evolution is lacking. RESULTS: Here we screened a public collection of over 14,000 metagenomes using the virophage-specific major capsid protein (MCP) as "bait." We identified 44,221 assembled virophage sequences, of which 328 represent high-quality (complete or near-complete) genomes from diverse habitats including the human gut, plant rhizosphere, and terrestrial subsurface. Comparative genomic analysis confirmed the presence of four core genes in a conserved block. We used these genes to establish a revised virophage classification including 27 clades with consistent genome length, gene content, and habitat distribution. Moreover, for eight high-quality virophage genomes, we computationally predicted putative eukaryotic virus hosts. CONCLUSION: Overall, our approach has increased the number of known virophage genomes by 10-fold and revealed patterns of genome evolution and global virophage distribution. We anticipate that the expanded diversity presented here will provide the backbone for further virophage studies.

Virophages of Giant Viruses: An Update at Eleven.

The last decade has been marked by two eminent discoveries that have changed our perception of the virology field: The discovery of giant viruses and a distinct new class of viral agents that parasitize their viral factories, the virophages. Coculture and metagenomics have actively contributed to the expansion of the virophage family by isolating dozens of new members. This increase in the body of data on virophage not only revealed the diversity of the virophage group, but also the relevant ecological impact of these small viruses and their potential role in the dynamics of the microbial network. In addition, the isolation of virophages has led us to discover previously unknown features displayed by their host viruses and cells. In this review, we present an update of all the knowledge on the isolation, biology, genomics, and morphological features of the virophages, a decade after the discovery of their first member, the Sputnik virophage. We discuss their parasitic lifestyle as bona fide viruses of the giant virus factories, genetic parasites of their genomes, and then their role as a key component or target for some host defense mechanisms during the tripartite virophage-giant virus-host cell interaction. We also present the latest advances regarding their origin, classification, and definition that have been widely discussed.

Characteristics of virophages and giant viruses.

Five years after being discovered in 2003, some giant viruses were demonstrated to play a role of the hosts for virophages, their parasites, setting out a novel and yet unknown regulatory mechanism of the giant viruses presence in an aqueous. So far, 20 virophages have been registered and 13 of them have been described as a metagenomic material, which indirectly impacts the number of single- and multi-cell organisms, the environment where giant viruses replicate.

Amoebae, Giant Viruses, and Virophages Make Up a Complex, Multilayered Threesome.

Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories.

The Expanding Family of Virophages.

Virophages replicate with giant viruses in the same eukaryotic cells. They are a major component of the specific mobilome of mimiviruses. Since their discovery in 2008, five other representatives have been isolated, 18 new genomes have been described, two of which being nearly completely sequenced, and they have been classified in a new viral family, Lavidaviridae. Virophages are small viruses with approximately 35-74 nm large icosahedral capsids and 17-29 kbp large double-stranded DNA genomes with 16-34 genes, among which a very small set is shared with giant viruses. Virophages have been isolated or detected in various locations and in a broad range of habitats worldwide, including the deep ocean and inland. Humans, therefore, could be commonly exposed to virophages, although currently limited evidence exists of their presence in humans based on serology and metagenomics. The distribution of virophages, the consequences of their infection and the interactions with their giant viral hosts within eukaryotic cells deserve further research.

Metagenomics Reveals a Novel Virophage Population in a Tibetan Mountain Lake.

Virophages are parasites of giant viruses that infect eukaryotic organisms and may affect the ecology of inland water ecosystems. Despite the potential ecological impact, limited information is available on the distribution, diversity, and hosts of virophages in ecosystems. Metagenomics revealed that virophages were widely distributed in inland waters with various environmental characteristics including salinity and nutrient availability. A novel virophage population was overrepresented in a planktonic microbial community of the Tibetan mountain lake, Lake Qinghai. Our study identified coccolithophores and coccolithovirus-like phycodnaviruses in the same community, which may serve as eukaryotic and viral hosts of the virophage population, respectively.