Exploring the Archaeal Virosphere by Metagenomics.

During the past decade, environmental research has demonstrated that archaea are abundant and widespread in nature and play important ecological roles at a global scale. Currently, however, the majority of archaeal lineages cannot be cultivated under laboratory conditions and are known exclusively or nearly exclusively through metagenomics. A similar trend extends to the archaeal virosphere, where isolated representatives are available for a handful of model archaeal virus-host systems. Viral metagenomics provides an alternative way to circumvent the limitations of culture-based virus discovery and offers insight into the diversity, distribution, and environmental impact of uncultured archaeal viruses. Presently, metagenomics approaches have been successfully applied to explore the viromes associated with various lineages of extremophilic and mesophilic archaea, including Asgard archaea (Asgardarchaeota), ANME-1 archaea (Methanophagales), thaumarchaea (Nitrososphaeria), altiarchaea (Altiarchaeota), and marine group II archaea (Poseidoniales). Here, we provide an overview of methods widely used in archaeal virus metagenomics, covering metavirome preparation, genome annotation, phylogenetic and phylogenomic analyses, and archaeal host assignment. We hope that this summary will contribute to further exploration and characterization of the enigmatic archaeal virome lurking in diverse environments.

Diversity, taxonomy, and evolution of archaeal viruses of the class Caudoviricetes.

The archaeal tailed viruses (arTV), evolutionarily related to tailed double-stranded DNA (dsDNA) bacteriophages of the class Caudoviricetes, represent the most common isolates infecting halophilic archaea. Only a handful of these viruses have been genomically characterized, limiting our appreciation of their ecological impacts and evolution. Here, we present 37 new genomes of haloarchaeal tailed virus isolates, more than doubling the current number of sequenced arTVs. Analysis of all 63 available complete genomes of arTVs, which we propose to classify into 14 new families and 3 orders, suggests ancient divergence of archaeal and bacterial tailed viruses and points to an extensive sharing of genes involved in DNA metabolism and counterdefense mechanisms, illuminating common strategies of virus-host interactions with tailed bacteriophages. Coupling of the comparative genomics with the host range analysis on a broad panel of haloarchaeal species uncovered 4 distinct groups of viral tail fiber adhesins controlling the host range expansion. The survey of metagenomes using viral hallmark genes suggests that the global architecture of the arTV community is shaped through recurrent transfers between different biomes, including hypersaline, marine, and anoxic environments.

Egress of archaeal viruses.

Viruses of Archaea, arguably the most mysterious part of the virosphere due to their unique morphotypes and genome contents, exploit diverse mechanisms for releasing virus progeny from the host cell. These include virus release as a result of the enzymatic degradation of the cell wall or budding through it, common for viruses of Bacteria and Eukarya, as well as a unique mechanism of virus egress through small polygonal perforations on the cell surface. The process of the formation of these perforations includes the development of pyramidal structures on the membrane of the infected cell, which gradually grow by the expansion of their faces and eventually open like flower petals. This mechanism of virion release is operating exclusively in cells of hyperthermophilic hosts from the phylum Crenarchaeota, which are encased solely by a layer of surface proteins, S-layer. The review focuses on recent developments in understanding structural and biochemical details of all three types of egress mechanisms of archaeal viruses. TAKE AWAYS: Many archaeal viruses exit the host via polygonal perforations on the cell membrane. The molecular mechanism of exit via specific apertures is unique for archaeal viruses. Some enveloped archaeal viruses exploit the budding mechanism for egress.

Structural basis of RNA polymerase inhibition by viral and host factors.

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

Bacterial Viruses Subcommittee and Archaeal Viruses Subcommittee of the ICTV: update of taxonomy changes in 2021.

In this article, we - the Bacterial Viruses Subcommittee and the Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV) - summarise the results of our activities for the period March 2020 - March 2021. We report the division of the former Bacterial and Archaeal Viruses Subcommittee in two separate Subcommittees, welcome new members, a new Subcommittee Chair and Vice Chair, and give an overview of the new taxa that were proposed in 2020, approved by the Executive Committee and ratified by vote in 2021. In particular, a new realm, three orders, 15 families, 31 subfamilies, 734 genera and 1845 species were newly created or redefined (moved/promoted).

A filamentous archaeal virus is enveloped inside the cell and released through pyramidal portals.

The majority of viruses infecting hyperthermophilic archaea display unique virion architectures and are evolutionarily unrelated to viruses of bacteria and eukaryotes. The lack of relationships to other known viruses suggests that the mechanisms of virus-host interaction in Archaea are also likely to be distinct. To gain insights into archaeal virus-host interactions, we studied the life cycle of the enveloped, ∼2-µm-long Sulfolobus islandicus filamentous virus (SIFV), a member of the family Lipothrixviridae infecting a hyperthermophilic and acidophilic archaeon Saccharolobus islandicus LAL14/1. Using dual-axis electron tomography and convolutional neural network analysis, we characterize the life cycle of SIFV and show that the virions, which are nearly two times longer than the host cell diameter, are assembled in the cell cytoplasm, forming twisted virion bundles organized on a nonperfect hexagonal lattice. Remarkably, our results indicate that envelopment of the helical nucleocapsids takes place inside the cell rather than by budding as in the case of most other known enveloped viruses. The mature virions are released from the cell through large (up to 220 nm in diameter), six-sided pyramidal portals, which are built from multiple copies of a single 89-amino-acid-long viral protein gp43. The overexpression of this protein in Escherichia coli leads to pyramid formation in the bacterial membrane. Collectively, our results provide insights into the assembly and release of enveloped filamentous viruses and illuminate the evolution of virus-host interactions in Archaea.

New insights into the diversity and evolution of the archaeal mobilome from three complete genomes of Saccharolobus shibatae.

Saccharolobus (formerly Sulfolobus) shibatae B12, isolated from a hot spring in Beppu, Japan in 1982, is one of the first hyperthermophilic and acidophilic archaeal species to be discovered. It serves as a natural host to the extensively studied spindle-shaped virus SSV1, a prototype of the Fuselloviridae family. Two additional Sa. shibatae strains, BEU9 and S38A, sensitive to viruses of the families Lipothrixviridae and Portogloboviridae, respectively, have been isolated more recently. However, none of the strains has been fully sequenced, limiting their utility for studies on archaeal biology and virus-host interactions. Here, we present the complete genome sequences of all three Sa. shibatae strains and explore the rich diversity of their integrated mobile genetic elements (MGE), including transposable insertion sequences, integrative and conjugative elements, plasmids, and viruses, some of which were also detected in the extrachromosomal form. Analysis of related MGEs in other Sulfolobales species and patterns of CRISPR spacer targeting revealed a complex network of MGE distributions, involving horizontal spread and relatively frequent host switching by MGEs over large phylogenetic distances, involving species of the genera Saccharolobus, Sulfurisphaera and Acidianus. Furthermore, we characterize a remarkable case of a virus-to-plasmid transition, whereby a fusellovirus has lost the genes encoding for the capsid proteins, while retaining the replication module, effectively becoming a plasmid.

ICTV Virus Taxonomy Profile: Portogloboviridae.

Portogloboviridae is a family of viruses with circular, double-stranded DNA genomes of about 20 kbp. Their icosahedral virions have a diameter of 87 nm, and consist of an outer protein shell, an inner lipid layer and a nucleoprotein core wound up into a spherical coil. Portogloboviruses infect hyperthermophilic archaea of the genus Saccharolobus, order Sulfolobales and are presumably nonlytic. Portogloboviruses encode mini-CRISPR arrays which they use to compete against other co-infecting viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Portogloboviridae, which is available at ictv.global/report/portogloboviridae.

Adnaviria: a New Realm for Archaeal Filamentous Viruses with Linear A-Form Double-Stranded DNA Genomes.

The International Committee on Taxonomy of Viruses (ICTV) has recently adopted a comprehensive, hierarchical system of virus taxa. The highest ranks in this hierarchy are realms, each of which is considered monophyletic but apparently originated independently of other realms. Here, we announce the creation of a new realm, Adnaviria, which unifies archaeal filamentous viruses with linear A-form double-stranded DNA genomes and characteristic major capsid proteins unrelated to those encoded by other known viruses.

Microbial Diversity and Phage-Host Interactions in the Georgian Coastal Area of the Black Sea Revealed by Whole Genome Metagenomic Sequencing.

Viruses have the greatest abundance and highest genetic diversity in marine ecosystems. The interactions between viruses and their hosts is one of the hot spots of marine ecology. Besides their important role in various ecosystems, viruses, especially bacteriophages and their gene pool, are of enormous interest for the development of new gene products with high innovation value. Various studies have been conducted in diverse ecosystems to understand microbial diversity and phage-host interactions; however, the Black Sea, especially the Eastern coastal area, remains among the least studied ecosystems in this regard. This study was aimed at to fill this gap by analyzing microbial diversity and bacteriophage-host interactions in the waters of Eastern Black Sea using a metagenomic approach. To this end, prokaryotic and viral metagenomic DNA from two sampling sites, Poti and Gonio, were sequenced on the Illumina Miseq platform and taxonomic and functional profiles of the metagenomes were obtained using various bioinformatics tools. Our metagenomics analyses allowed us to identify the microbial communities, with Proteobacteria, Cyanobacteria, Actinibacteria, and Firmicutes found to be the most dominant bacterial phyla and Synechococcus and Candidatus Pelagibacter phages found to be the most dominant viral groups in the Black Sea. As minor groups, putative phages specific to human pathogens were identified in the metagenomes. We also characterized interactions between the phages and prokaryotic communities by determining clustered regularly interspaced short palindromic repeats (CRISPR), prophage-like sequences, and integrase/excisionase sequences in the metagenomes, along with identification of putative horizontally transferred genes in the viral contigs. In addition, in the viral contig sequences related to peptidoglycan lytic activity were identified as well. This is the first study on phage and prokaryote diversity and their interactions in the Eastern coastal area of the Black Sea using a metagenomic approach.

Structures of filamentous viruses infecting hyperthermophilic archaea explain DNA stabilization in extreme environments.

Living organisms expend metabolic energy to repair and maintain their genomes, while viruses protect their genetic material by completely passive means. We have used cryo-electron microscopy (cryo-EM) to solve the atomic structures of two filamentous double-stranded DNA viruses that infect archaeal hosts living in nearly boiling acid: Saccharolobus solfataricus rod-shaped virus 1 (SSRV1), at 2.8-Å resolution, and Sulfolobus islandicus filamentous virus (SIFV), at 4.0-Å resolution. The SIFV nucleocapsid is formed by a heterodimer of two homologous proteins and is membrane enveloped, while SSRV1 has a nucleocapsid formed by a homodimer and is not enveloped. In both, the capsid proteins wrap around the DNA and maintain it in an A-form. We suggest that the A-form is due to both a nonspecific desolvation of the DNA by the protein, and a specific coordination of the DNA phosphate groups by positively charged residues. We extend these observations by comparisons with four other archaeal filamentous viruses whose structures we have previously determined, and show that all 10 capsid proteins (from four heterodimers and two homodimers) have obvious structural homology while sequence similarity can be nonexistent. This arises from most capsid residues not being under any strong selective pressure. The inability to detect homology at the sequence level arises from the sampling of viruses in this part of the biosphere being extremely sparse. Comparative structural and genomic analyses suggest that nonenveloped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution.

The structures of two archaeal type IV pili illuminate evolutionary relationships.

We have determined the cryo-electron microscopic (cryo-EM) structures of two archaeal type IV pili (T4P), from Pyrobaculum arsenaticum and Saccharolobus solfataricus, at 3.8 Å and 3.4 Å resolution, respectively. This triples the number of high resolution archaeal T4P structures, and allows us to pinpoint the evolutionary divergence of bacterial T4P, archaeal T4P and archaeal flagellar filaments. We suggest that extensive glycosylation previously observed in T4P of Sulfolobus islandicus is a response to an acidic environment, as at even higher temperatures in a neutral environment much less glycosylation is present for Pyrobaculum than for Sulfolobus and Saccharolobus pili. Consequently, the Pyrobaculum filaments do not display the remarkable stability of the Sulfolobus filaments in vitro. We identify the Saccharolobus and Pyrobaculum T4P as host receptors recognized by rudivirus SSRV1 and tristromavirus PFV2, respectively. Our results illuminate the evolutionary relationships among bacterial and archaeal T4P filaments and provide insights into archaeal virus-host interactions.

Structure of a filamentous virus uncovers familial ties within the archaeal virosphere.

Viruses infecting hyperthermophilic archaea represent one of the most enigmatic parts of the global virome, with viruses from different families showing no genomic relatedness to each other or to viruses of bacteria and eukaryotes. Tristromaviruses, which build enveloped filamentous virions and infect hyperthermophilic neutrophiles of the order Thermoproteales, represent one such enigmatic virus families. They do not share genes with viruses from other families and have been believed to represent an evolutionarily independent virus lineage. A cryo-electron microscopic reconstruction of the tristromavirus Pyrobaculum filamentous virus 2 at 3.4 Å resolution shows that the virion is constructed from two paralogous major capsid proteins (MCP) which transform the linear dsDNA genome of the virus into A-form by tightly wrapping around it. Unexpectedly, the two MCP are homologous to the capsid proteins of other filamentous archaeal viruses, uncovering a deep evolutionary relationship within the archaeal virosphere.

New virus isolates from Italian hydrothermal environments underscore the biogeographic pattern in archaeal virus communities.

Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.

Taxonomy of prokaryotic viruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

ICTV Virus Taxonomy Profile: Spiraviridae.

The family Spiraviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped, hollow, cylindrical virions are formed from a coiling fibre that consists of two intertwining halves of a single circular nucleoprotein filament. A short appendage protrudes from each end of the cylindrical virion. The genome is circular, positive-sense, single-stranded DNA of 24 893 nucleotides. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Spiraviridae, which is available at ictv.global/report/spiraviridae.

Structure and assembly of archaeal viruses.

Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among bacteriophages or viruses of eukaryotes. However, recent environmental studies have shown that archaeal viruses are widespread also in moderate ecosystems, where they play an important ecological role by influencing the turnover of microbial communities, with a global impact on the carbon and nitrogen cycles. In this review, we summarize recent advances in understanding the molecular details of virion organization and assembly of archaeal viruses. We start by briefly introducing the 20 officially recognized families of archaeal viruses and then outline the similarities and differences of archaeal virus assembly with the morphogenesis pathways used by bacterial and eukaryotic viruses, and discuss the evolutionary implications of these observations. Generally, the assembly of the icosahedral archaeal viruses closely follows the mechanisms employed by evolutionarily related bacterial and eukaryotic viruses with the HK97 fold and double jelly-roll major capsid proteins, emphasizing the overall conservation of these pathways over billions of years of evolution. By contrast, archaea-specific viruses employ unique virion assembly mechanisms. We also highlight some of the molecular adaptations underlying the stability of archaeal viruses in extreme environments. Despite considerable progress during the past few years, the archaeal virosphere continues to represent one of the least studied parts of the global virome, with many molecular features awaiting to be discovered and characterized.

Virus-borne mini-CRISPR arrays are involved in interviral conflicts.

CRISPR-Cas immunity is at the forefront of antivirus defense in bacteria and archaea and specifically targets viruses carrying protospacers matching the spacers catalogued in the CRISPR arrays. Here, we perform deep sequencing of the CRISPRome-all spacers contained in a microbiome-associated with hyperthermophilic archaea of the order Sulfolobales recovered directly from an environmental sample and from enrichment cultures established in the laboratory. The 25 million CRISPR spacers sequenced from a single sampling site dwarf the diversity of spacers from all available Sulfolobales isolates and display complex temporal dynamics. Comparison of closely related virus strains shows that CRISPR targeting drives virus genome evolution. Furthermore, we show that some archaeal viruses carry mini-CRISPR arrays with 1-2 spacers and preceded by leader sequences but devoid of cas genes. Closely related viruses present in the same population carry spacers against each other. Targeting by these virus-borne spacers represents a distinct mechanism of heterotypic superinfection exclusion and appears to promote archaeal virus speciation.

A packing for A-form DNA in an icosahedral virus.

Studies on viruses infecting archaea living in the most extreme environments continue to show a remarkable diversity of structures, suggesting that the sampling continues to be very sparse. We have used electron cryo-microscopy to study at 3.7-Å resolution the structure of the Sulfolobus polyhedral virus 1 (SPV1), which was originally isolated from a hot, acidic spring in Beppu, Japan. The 2 capsid proteins with variant single jelly-roll folds form pentamers and hexamers which assemble into a T = 43 icosahedral shell. In contrast to tailed icosahedral double-stranded DNA (dsDNA) viruses infecting bacteria and archaea, and herpesviruses infecting animals and humans, where naked DNA is packed under very high pressure due to the repulsion between adjacent layers of DNA, the circular dsDNA in SPV1 is fully covered with a viral protein forming a nucleoprotein filament with attractive interactions between layers. Most strikingly, we have been able to show that the DNA is in an A-form, as it is in the filamentous viruses infecting hyperthermophilic acidophiles. Previous studies have suggested that DNA is in the B-form in bacteriophages, and our study is a direct visualization of the structure of DNA in an icosahedral virus.

ICTV Virus Taxonomy Profile: Clavaviridae.

The family Clavaviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped rigid virions are rod-shaped, with dimensions of about 143×16 nm, and have terminal cap structures, one of which is pointed and carries short fibres, while the other is rounded. The virion displays helical symmetry and is constructed from a single major α-helical protein, which is heavily glycosylated, and several minor capsid proteins. The 5278 bp, circular, double-stranded DNA genome of Aeropyrum pernix bacilliform virus 1 is packed inside the virion as a left-handed superhelix. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Clavaviridae, which is available at www.ictv.global/report/clavaviridae.