Jumping DNA polymerases in bacteriophages.

Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. Here we identify four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage replication. We hypothesize that DNAP swapping is driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication.

Mriyaviruses: small relatives of giant viruses.

The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCPs) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name "Mriyaviricetes" (after Ukrainian "mriya," dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family "Gamadviridae." The previously characterized members of these families, yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism. IMPORTANCE: The origin of giant viruses of eukaryotes that belong to the phylum Nucleocytoviricota is not thoroughly understood and remains a matter of major interest and debate. Here, we combine metagenome database searches with extensive protein sequence and structure analysis to describe a distinct group of viruses with comparatively small genomes of 35-45 kilobases that appear to comprise a distinct class within the phylum Nucleocytoviricota that we provisionally named "Mriyaviricetes." Mriyaviruses appear to be the closest identified relatives of the ancestors of the Nucleocytoviricota. Analysis of proteins encoded in mriyavirus genomes suggests that they replicate their genome via the rolling circle mechanism that is unusual among viruses with double-stranded DNA genomes and so far not described for members of Nucleocytoviricota.

Mriyaviruses: Small Relatives of Giant Viruses.

The phylum Nucleocytoviricota consists of large and giant viruses that range in genome size from about 100 kilobases (kb) to more than 2.5 megabases. Here, using metagenome mining followed by extensive phylogenomic analysis and protein structure comparison, we delineate a distinct group of viruses with double-stranded (ds) DNA genomes in the range of 35-45 kb that appear to be related to the Nucleocytoviricota. In phylogenetic trees of the conserved double jelly-roll major capsid proteins (MCP) and DNA packaging ATPases, these viruses do not show affinity to any particular branch of the Nucleocytoviricota and accordingly would comprise a class which we propose to name "Mriyaviricetes" (after Ukrainian Mriya, dream). Structural comparison of the MCP suggests that, among the extant virus lineages, mriyaviruses are the closest one to the ancestor of the Nucleocytoviricota. In the phylogenetic trees, mriyaviruses split into two well-separated branches, the family Yaraviridae and proposed new family "Gamadviridae". The previously characterized members of these families, Yaravirus and Pleurochrysis sp. endemic viruses, infect amoeba and haptophytes, respectively. The genomes of the rest of the mriyaviruses were assembled from metagenomes from diverse environments, suggesting that mriyaviruses infect various unicellular eukaryotes. Mriyaviruses lack DNA polymerase, which is encoded by all other members of the Nucleocytoviricota, and RNA polymerase subunits encoded by all cytoplasmic viruses among the Nucleocytoviricota, suggesting that they replicate in the host cell nuclei. All mriyaviruses encode a HUH superfamily endonuclease that is likely to be essential for the initiation of virus DNA replication via the rolling circle mechanism.

Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.

TnpB proteins are RNA-guided nucleases that are broadly associated with IS200/605 family transposons in prokaryotes. TnpB homologs, named Fanzors, have been detected in genomes of some eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. We searched genomes of diverse eukaryotes and their viruses for TnpB homologs and identified numerous putative RNA-guided nucleases that are often associated with various transposases, suggesting they are encoded in mobile genetic elements. Reconstruction of the evolution of these nucleases, which we rename Horizontally-transferred Eukaryotic RNA-guided Mobile Element Systems (HERMES), revealed multiple acquisitions of TnpBs by eukaryotes and subsequent diversification. In their adaptation and spread in eukaryotes, HERMES proteins acquired nuclear localization signals, and genes captured introns, indicating extensive, long term adaptation to functioning in eukaryotic cells. Biochemical and cellular evidence show that HERMES employ non-coding RNAs encoded adjacent to the nuclease for RNA-guided cleavage of double-stranded DNA. HERMES nucleases contain a re-arranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that HERMES can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.

Structural atlas of a human gut crassvirus.

CrAssphage and related viruses of the order Crassvirales (hereafter referred to as crassviruses) were originally discovered by cross-assembly of metagenomic sequences. They are the most abundant viruses in the human gut, are found in the majority of individual gut viromes, and account for up to 95% of the viral sequences in some individuals1-4. Crassviruses are likely to have major roles in shaping the composition and functionality of the human microbiome, but the structures and roles of most of the virally encoded proteins are unknown, with only generic predictions resulting from bioinformatic analyses4,5. Here we present a cryo-electron microscopy reconstruction of Bacteroides intestinalis virus ΦcrAss0016, providing the structural basis for the functional assignment of most of its virion proteins. The muzzle protein forms an assembly about 1 MDa in size at the end of the tail and exhibits a previously unknown fold that we designate the 'crass fold', that is likely to serve as a gatekeeper that controls the ejection of cargos. In addition to packing the approximately 103 kb of virus DNA, the ΦcrAss001 virion has extensive storage space for virally encoded cargo proteins in the capsid and, unusually, within the tail. One of the cargo proteins is present in both the capsid and the tail, suggesting a general mechanism for protein ejection, which involves partial unfolding of proteins during their extrusion through the tail. These findings provide a structural basis for understanding the mechanisms of assembly and infection of these highly abundant crassviruses.

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.

Varidnaviruses in the Human Gut: A Major Expansion of the Order Vinavirales.

Bacteriophages play key roles in the dynamics of the human microbiome. By far the most abundant components of the human gut virome are tailed bacteriophages of the realm Duplodnaviria, in particular, crAss-like phages. However, apart from duplodnaviruses, the gut virome has not been dissected in detail. Here we report a comprehensive census of a minor component of the gut virome, the tailless bacteriophages of the realm Varidnaviria. Tailless phages are primarily represented in the gut by prophages, that are mostly integrated in genomes of Alphaproteobacteria and Verrucomicrobia and belong to the order Vinavirales, which currently consists of the families Corticoviridae and Autolykiviridae. Phylogenetic analysis of the major capsid proteins (MCP) suggests that at least three new families should be established within Vinavirales to accommodate the diversity of prophages from the human gut virome. Previously, only the MCP and packaging ATPase genes were reported as conserved core genes of Vinavirales. Here we report an extended core set of 12 proteins, including MCP, packaging ATPase, and previously undetected lysis enzymes, that are shared by most of these viruses. We further demonstrate that replication system components are frequently replaced in the genomes of Vinavirales, suggestive of selective pressure for escape from yet unknown host defenses or avoidance of incompatibility with coinfecting related viruses. The results of this analysis show that, in a sharp contrast to marine viromes, varidnaviruses are a minor component of the human gut virome. Moreover, they are primarily represented by prophages, as indicated by the analysis of the flanking genes, suggesting that there are few, if any, lytic varidnavirus infections in the gut at any given time. These findings complement the existing knowledge of the human gut virome by exploring a group of viruses that has been virtually overlooked in previous work.

Three families of Asgard archaeal viruses identified in metagenome-assembled genomes.

Asgardarchaeota harbour many eukaryotic signature proteins and are widely considered to represent the closest archaeal relatives of eukaryotes. Whether similarities between Asgard archaea and eukaryotes extend to their viromes remains unknown. Here we present 20 metagenome-assembled genomes of Asgardarchaeota from deep-sea sediments of the basin off the Shimokita Peninsula, Japan. By combining a CRISPR spacer search of metagenomic sequences with phylogenomic analysis, we identify three family-level groups of viruses associated with Asgard archaea. The first group, verdandiviruses, includes tailed viruses of the class Caudoviricetes (realm Duplodnaviria); the second, skuldviruses, consists of viruses with predicted icosahedral capsids of the realm Varidnaviria; and the third group, wyrdviruses, is related to spindle-shaped viruses previously identified in other archaea. More than 90% of the proteins encoded by these viruses of Asgard archaea show no sequence similarity to proteins encoded by other known viruses. Nevertheless, all three proposed families consist of viruses typical of prokaryotes, providing no indication of specific evolutionary relationships between viruses infecting Asgard archaea and eukaryotes. Verdandiviruses and skuldviruses are likely to be lytic, whereas wyrdviruses potentially establish chronic infection and are released without host cell lysis. All three groups of viruses are predicted to play important roles in controlling Asgard archaea populations in deep-sea ecosystems.

Conservation and Evolution of the Sporulation Gene Set in Diverse Members of the Firmicutes.

The current classification of the phylum Firmicutes (new name, Bacillota) features eight distinct classes, six of which include known spore-forming bacteria. In Bacillus subtilis, sporulation involves up to 500 genes, many of which do not have orthologs in other bacilli and/or clostridia. Previous studies identified about 60 sporulation genes of B. subtilis that were shared by all spore-forming members of the Firmicutes. These genes are referred to as the sporulation core or signature, although many of these are also found in genomes of nonsporeformers. Using an expanded set of 180 firmicute genomes from 160 genera, including 76 spore-forming species, we investigated the conservation of the sporulation genes, in particular seeking to identify lineages that lack some of the genes from the conserved sporulation core. The results of this analysis confirmed that many small acid-soluble spore proteins (SASPs), spore coat proteins, and germination proteins, which were previously characterized in bacilli, are missing in spore-forming members of Clostridia and other classes of Firmicutes. A particularly dramatic loss of sporulation genes was observed in the spore-forming members of the families Planococcaceae and Erysipelotrichaceae. Fifteen species from diverse lineages were found to carry skin (sigK-interrupting) elements of different sizes that all encoded SpoIVCA-like recombinases but did not share any other genes. Phylogenetic trees built from concatenated alignments of sporulation proteins and ribosomal proteins showed similar topology, indicating an early origin and subsequent vertical inheritance of the sporulation genes. IMPORTANCE Many members of the phylum Firmicutes (Bacillota) are capable of producing endospores, which enhance the survival of important Gram-positive pathogens that cause such diseases as anthrax, botulism, colitis, gas gangrene, and tetanus. We show that the core set of sporulation genes, defined previously through genome comparisons of several bacilli and clostridia, is conserved in a wide variety of sporeformers from several distinct lineages of Firmicutes. We also detected widespread loss of sporulation genes in many organisms, particularly within the families Planococcaceae and Erysipelotrichaceae. Members of these families, such as Lysinibacillus sphaericus and Clostridium innocuum, could be excellent model organisms for studying sporulation mechanisms, such as engulfment, formation of the spore coat, and spore germination.

Ancient Gene Capture and Recent Gene Loss Shape the Evolution of Orthopoxvirus-Host Interaction Genes.

The survival of viruses depends on their ability to resist host defenses and, of all animal virus families, the poxviruses have the most antidefense genes. Orthopoxviruses (ORPV), a genus within the subfamily Chordopoxvirinae, infect diverse mammals and include one of the most devastating human pathogens, the now eradicated smallpox virus. ORPV encode ∼200 genes, of which roughly half are directly involved in virus genome replication and expression as well as virion morphogenesis. The remaining ∼100 "accessory" genes are responsible for virus-host interactions, particularly counter-defense of innate immunity. Complete sequences are currently available for several hundred ORPV genomes isolated from a variety of mammalian hosts, providing a rich resource for comparative genomics and reconstruction of ORPV evolution. To identify the provenance and evolutionary trends of the ORPV accessory genes, we constructed clusters including the orthologs of these genes from all chordopoxviruses. Most of the accessory genes were captured in three major waves early in chordopoxvirus evolution, prior to the divergence of ORPV and the sister genus Centapoxvirus from their common ancestor. The capture of these genes from the host was followed by extensive gene duplication, yielding several paralogous gene families. In addition, nine genes were gained during the evolution of ORPV themselves. In contrast, nearly every accessory gene was lost, some on multiple, independent occasions in numerous lineages of ORPV, so that no ORPV retains them all. A variety of functional interactions could be inferred from examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently. IMPORTANCE Orthopoxviruses (ORPV) include smallpox (variola) virus, one of the most devastating human pathogens, and vaccinia virus, comprising the vaccine used for smallpox eradication. Among roughly 200 ORPV genes, about half are essential for genome replication and expression as well as virion morphogenesis, whereas the remaining half consists of accessory genes counteracting the host immune response. We reannotated the accessory genes of ORPV, predicting the functions of uncharacterized genes, and reconstructed the history of their gain and loss during the evolution of ORPV. Most of the accessory genes were acquired in three major waves antedating the origin of ORPV from chordopoxviruses. The evolution of ORPV themselves was dominated by gene loss, with numerous genes lost at the base of each major group of ORPV. Examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently during ORPV evolution allows prediction of different types of functional interactions.

Thousands of previously unknown phages discovered in whole-community human gut metagenomes.

BACKGROUND: Double-stranded DNA bacteriophages (dsDNA phages) play pivotal roles in structuring human gut microbiomes; yet, the gut virome is far from being fully characterized, and additional groups of phages, including highly abundant ones, continue to be discovered by metagenome mining. A multilevel framework for taxonomic classification of viruses was recently adopted, facilitating the classification of phages into evolutionary informative taxonomic units based on hallmark genes. Together with advanced approaches for sequence assembly and powerful methods of sequence analysis, this revised framework offers the opportunity to discover and classify unknown phage taxa in the human gut. RESULTS: A search of human gut metagenomes for circular contigs encoding phage hallmark genes resulted in the identification of 3738 apparently complete phage genomes that represent 451 putative genera. Several of these phage genera are only distantly related to previously identified phages and are likely to found new families. Two of the candidate families, "Flandersviridae" and "Quimbyviridae", include some of the most common and abundant members of the human gut virome that infect Bacteroides, Parabacteroides, and Prevotella. The third proposed family, "Gratiaviridae," consists of less abundant phages that are distantly related to the families Autographiviridae, Drexlerviridae, and Chaseviridae. Analysis of CRISPR spacers indicates that phages of all three putative families infect bacteria of the phylum Bacteroidetes. Comparative genomic analysis of the three candidate phage families revealed features without precedent in phage genomes. Some "Quimbyviridae" phages possess Diversity-Generating Retroelements (DGRs) that generate hypervariable target genes nested within defense-related genes, whereas the previously known targets of phage-encoded DGRs are structural genes. Several "Flandersviridae" phages encode enzymes of the isoprenoid pathway, a lipid biosynthesis pathway that so far has not been known to be manipulated by phages. The "Gratiaviridae" phages encode a HipA-family protein kinase and glycosyltransferase, suggesting these phages modify the host cell wall, preventing superinfection by other phages. Hundreds of phages in these three and other families are shown to encode catalases and iron-sequestering enzymes that can be predicted to enhance cellular tolerance to reactive oxygen species. CONCLUSIONS: Analysis of phage genomes identified in whole-community human gut metagenomes resulted in the delineation of at least three new candidate families of Caudovirales and revealed diverse putative mechanisms underlying phage-host interactions in the human gut. Addition of these phylogenetically classified, diverse, and distinct phages to public databases will facilitate taxonomic decomposition and functional characterization of human gut viromes. Video abstract.

Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features.

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.

Structure and function of virion RNA polymerase of a crAss-like phage.

CrAss-like phages are a recently described expansive group of viruses that includes the most abundant virus in the human gut1-3. The genomes of all crAss-like phages encode a large virion-packaged protein2,4 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)5. Here, using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited state, which probably occurs before virion packaging. This conformation is attained with the help of a cleft-blocking domain that interacts with the active site and occupies the cavity in which the RNA-DNA hybrid binds. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs that are involved in RNA interference6,7, although most of the phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of the protein fold space. Considering this structural similarity, we propose that eukaryal RNA interference polymerases have their origins in phage, which parallels the emergence of the mitochondrial transcription apparatus8.

The crAss-like Phage Group: How Metagenomics Reshaped the Human Virome.

Metagenomics is currently the primary means for identifying new viruses. One of the most impactful metagenomic discoveries is that of crAssphage, the most abundant human-associated virus that is found in about 50% of human gut viromes where it can comprise up to 90% of the virus sequences. Although initial genome analysis of crAssphage failed to detect related phages, or functionally annotate most of the genes, subsequent reanalysis with powerful computational methods and larger databases led to the identification of an expansive group of crAss-like phages. The functions of most crAssphage proteins were predicted, including unusual ones such as giant RNA polymerase polyproteins. The host range of the crAss-like phages consists of various members of the bacterial phylum Bacteroidetes as demonstrated by CRISPR spacer analysis and by analysis of genes acquired by phages from the hosts. New metagenomic studies vastly expanded the crAss-like phage group and demonstrated its global spread and ancient association with primates. The first members of the crAss-like group was recently isolated and shown to infect the bacterium Bacteroides intestinales. Characterization of this phage validated the predicted podovirus-like virion structure and the identity of the major capsid protein and other predicted virion proteins, including three RNA polymerase subunits.

Global Organization and Proposed Megataxonomy of the Virus World.

Viruses and mobile genetic elements are molecular parasites or symbionts that coevolve with nearly all forms of cellular life. The route of virus replication and protein expression is determined by the viral genome type. Comparison of these routes led to the classification of viruses into seven "Baltimore classes" (BCs) that define the major features of virus reproduction. However, recent phylogenomic studies identified multiple evolutionary connections among viruses within each of the BCs as well as between different classes. Due to the modular organization of virus genomes, these relationships defy simple representation as lines of descent but rather form complex networks. Phylogenetic analyses of virus hallmark genes combined with analyses of gene-sharing networks show that replication modules of five BCs (three classes of RNA viruses and two classes of reverse-transcribing viruses) evolved from a common ancestor that encoded an RNA-directed RNA polymerase or a reverse transcriptase. Bona fide viruses evolved from this ancestor on multiple, independent occasions via the recruitment of distinct cellular proteins as capsid subunits and other structural components of virions. The single-stranded DNA (ssDNA) viruses are a polyphyletic class, with different groups evolving by recombination between rolling-circle-replicating plasmids, which contributed the replication protein, and positive-sense RNA viruses, which contributed the capsid protein. The double-stranded DNA (dsDNA) viruses are distributed among several large monophyletic groups and arose via the combination of distinct structural modules with equally diverse replication modules. Phylogenomic analyses reveal the finer structure of evolutionary connections among RNA viruses and reverse-transcribing viruses, ssDNA viruses, and large subsets of dsDNA viruses. Taken together, these analyses allow us to outline the global organization of the virus world. Here, we describe the key aspects of this organization and propose a comprehensive hierarchical taxonomy of viruses.

A New Family of DNA Viruses Causing Disease in Crustaceans from Diverse Aquatic Biomes.

Panulirus argus virus 1 (PaV1) is the only known virus infecting the Caribbean spiny lobster (Panulirus argus) from the Caribbean Sea. Recently, related viruses, Dikerogammarus haemobaphes virus 1 (DhV1) and Carcinus maenas virus 1 (CmV1), have been detected in the demon shrimp (Dikerogammarus haemobaphes) and the European shore crab (Carcinus maenas), respectively, from sites in the United Kingdom. The virion morphology of these crustacean viruses is similar to that of iridoviruses. However, unlike iridoviruses and other nucleocytoplasmic large DNA viruses (NCLDVs), these viruses complete their morphogenesis in the host cell nucleus rather than in the cytoplasm. To date, these crustacean viruses have remained unclassified due to a lack of genomic data. Using an Illumina MiSeq sequencer, we sequenced the complete genomes of PaV1, CmV1, and DhV1. Comparative genome analysis shows that these crustacean virus genomes encode the 10 hallmark proteins previously described for the NCLDVs of eukaryotes, strongly suggesting that they are members of this group. With a size range of 70 to 74 kb, these are the smallest NCLDV genomes identified to date. Extensive gene loss, divergence of gene sequences, and the accumulation of low-complexity sequences reflect the extreme degradation of the genomes of these "minimal" NCLDVs rather than any direct relationship with the NCLDV ancestor. Phylogenomic analysis supports the classification of these crustacean viruses as a distinct family, "Mininucleoviridae," within the pitho-irido-Marseille branch of the NCLDVs.IMPORTANCE Recent genomic and metagenomic studies have led to a dramatic expansion of the known diversity of nucleocytoplasmic large DNA viruses (NCLDVs) of eukaryotes, which include giant viruses of protists and important pathogens of vertebrates, such as poxviruses. However, the characterization of viruses from nonmodel hosts still lags behind. We sequenced the complete genomes of three viruses infecting crustaceans, the Caribbean spiny lobster, demon shrimp, and European shore crab. These viruses have the smallest genomes among the known NCLDVs, with losses of many core genes, some of which are shared with iridoviruses. The deterioration of the transcription apparatus is compatible with microscopic and ultrastructural observations indicating that these viruses replicate in the nucleus of infected cells rather than in the cytoplasm. Phylogenomic analysis indicates that these viruses are sufficiently distinct from all other NCLDVs to justify the creation of a separate family, for which we propose the name "Mininucleoviridae" (i.e., small viruses reproducing in the cell nucleus).

Evolution of a major virion protein of the giant pandoraviruses from an inactivated bacterial glycoside hydrolase.

The diverse viruses in the phylum Nucleocytoviricota (also known as NLCDVs, Nucleo-cytoplasmic Large DNA Viruses) typically possess large icosahedral virions. However, in several families of Nucleocytoviricota, the icosahedral capsid was replaced by irregular particle shapes, most notably, the amphora-like virions of pandoraviruses and pithoviruses, the largest known virus particles in the entire virosphere. Pandoraviruses appear to be the most highly derived viruses in this phylum because their evolution involved not only the change in the virion shape, but also, the actual loss of the gene encoding double-jelly roll major capsid protein (DJR MCP), the main building block of icosahedral capsids in this virus assemblage. Instead, pandoravirus virions are built of unrelated abundant proteins. Here we show that the second most abundant virion protein of pandoraviruses, major virion protein 2 (MVP2), evolved from an inactivated derivative of a bacterial glycoside hydrolase of the GH16 family. The ancestral form of MVP2 was apparently acquired early in the evolution of the Nucleocytoviricota, to become a minor virion protein. After a duplication in the common ancestor of pandoraviruses and molliviruses, one of the paralogs displaces DJR MCP in pandoraviruses, conceivably, opening the way for a major increase in the size of the virion and the genome. Exaptation of a carbohydrate-binding protein for the function of the MVP is a general trend in virus evolution and might underlie the transformation of the virion shape in other groups of the Nucleocytoviricota as well.

Unique structure and function of viral rhodopsins.

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.