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.

Characterization of Blf4, an Archaeal Lytic Virus Targeting a Member of the Methanomicrobiales.

Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.

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).

ICTV Virus Taxonomy Profile: Thaspiviridae 2021.

Members of the family Thaspiviridae have linear dsDNA genomes of 27 to 29 kbp and are the first viruses known to infect mesophilic ammonia-oxidizing archaea of the phylum Thaumarchaeota. The spindle-shaped virions of Nitrosopumilus spindle-shaped virus 1 possess short tails at one pole and measure 64±3 nm in diameter and 112±6 nm in length. This morphology is similar to that of members of the families Fuselloviridae and Halspiviridae. Virus replication is not lytic but leads to growth inhibition of the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Thaspiviridae, which is available at ictv.global/report/thaspiviridae.

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.

ICTV Virus Taxonomy Profile: Ovaliviridae.

Ovaliviridae is a family of enveloped viruses with a linear dsDNA genome. The virions are ellipsoidal, and contain a multi-layered spool-like capsid. The viral genome is presumably replicated through protein priming by a putative DNA polymerase encoded by the virus. Progeny virions are released through hexagonal openings resulting from the rupture of virus-associated pyramids formed on the surface of infected cells. The only known host is a hyperthermophilic archaeon of the genus Sulfolobus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Ovaliviridae, which is available at ictv.global/report/ovaliviridae.

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.

Pleomorphic archaeal viruses: the family Pleolipoviridae is expanding by seven new species.

Established in 2016, the family Pleolipoviridae comprises globally distributed archaeal viruses that produce pleomorphic particles. Pseudo-spherical enveloped virions of pleolipoviruses are membrane vesicles carrying a nucleic acid cargo. The cargo can be either a single-stranded or double-stranded DNA molecule, making this group the first family introduced in the 10th Report on Virus Taxonomy including both single-stranded and double-stranded DNA viruses. The length of the genomes is approximately 7-17 kilobase pairs, or kilonucleotides in the case of single-stranded molecules. The genomes are circular single-stranded DNA, circular double-stranded DNA, or linear double-stranded DNA molecules. Currently, eight virus species and seven proposed species are classified in three genera: Alphapleolipovirus (five species), Betapleolipovirus (nine species), and Gammapleolipovirus (one species). Here, we summarize the updated taxonomy of the family Pleolipoviridae to reflect recent advances in this field, with the focus on seven newly proposed species in the genus Betapleolipovirus: Betapleolipovirus HHPV3, HHPV4, HRPV9, HRPV10, HRPV11, HRPV12, and SNJ2.

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.

Discovery and Characterization of Thermoproteus Spherical Piliferous Virus 1: a Spherical Archaeal Virus Decorated with Unusual Filaments.

We describe the discovery of an archaeal virus, one that infects archaea, tentatively named Thermoproteus spherical piliferous virus 1 (TSPV1), which was purified from a Thermoproteales host isolated from a hot spring in Yellowstone National Park (USA). TSPV1 packages an 18.65-kb linear double-stranded DNA (dsDNA) genome with 31 open reading frames (ORFs), whose predicted gene products show little homology to proteins with known functions. A comparison of virus particle morphologies and gene content demonstrates that TSPV1 is a new member of the Globuloviridae family of archaeal viruses. However, unlike other Globuloviridae members, TSPV1 has numerous highly unusual filaments decorating its surface, which can extend hundreds of nanometers from the virion. To our knowledge, similar filaments have not been observed in any other archaeal virus. The filaments are remarkably stable, remaining intact across a broad range of temperature and pH values, and they are resistant to chemical denaturation and proteolysis. A major component of the filaments is a glycosylated 35-kDa TSPV1 protein (TSPV1 GP24). The filament protein lacks detectable homology to structurally or functionally characterized proteins. We propose, given the low host cell densities of hot spring environments, that the TSPV1 filaments serve to increase the probability of virus attachment and entry into host cells.IMPORTANCE High-temperature environments have proven to be an important source for the discovery of new archaeal viruses with unusual particle morphologies and gene content. Our isolation of Thermoproteus spherical piliferous virus 1 (TSPV1), with numerous filaments extending from the virion surface, expands our understanding of viral diversity and provides new insight into viral replication in high-temperature environments.

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.

An Uncultivated Virus Infecting a Nanoarchaeal Parasite in the Hot Springs of Yellowstone National Park.

The Nanoarchaeota are small cells with reduced genomes that are found attached to and dependent on a second archaeal cell for their growth and replication. Initially found in marine hydrothermal environments and subsequently in terrestrial geothermal hot springs, the Nanoarchaeota species that have been described are obligate ectobionts, each with a different host species. However, no viruses had been described that infect the Nanoarchaeota. Here, we identify a virus infecting Nanoarchaeota by the use of a combination of viral metagenomic and bioinformatic approaches. This virus, tentatively named Nanoarchaeota Virus 1 (NAV1), consists of a 35.6-kb circular DNA genome coding for 52 proteins. We further demonstrate that this virus is broadly distributed among Yellowstone National Park hot springs. NAV1 is one of the first examples of a virus infecting a single-celled organism that is itself an ectobiont of another single-celled organism.IMPORTANCE Here, we present evidence of the first virus found to infect Nanoarchaeota, a symbiotic archaean found in acidic hot springs of Yellowstone National Park, USA. Using culture-independent techniques, we provide the genome sequence and identify the archaeal host species of a novel virus, NAV1. NAV1 is the first example of a virus infecting an archaeal species that is itself an obligate symbiont and dependent on a second host organism for growth and cellular replication. On the basis of annotation of the NAV1 genome, we propose that this virus is the founding member of a new viral family, further demonstrating the remarkable genetic diversity of archaeal viruses.

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.

Cryptic inoviruses revealed as pervasive in bacteria and archaea across Earth's biomes.

Bacteriophages from the Inoviridae family (inoviruses) are characterized by their unique morphology, genome content and infection cycle. One of the most striking features of inoviruses is their ability to establish a chronic infection whereby the viral genome resides within the cell in either an exclusively episomal state or integrated into the host chromosome and virions are continuously released without killing the host. To date, a relatively small number of inovirus isolates have been extensively studied, either for biotechnological applications, such as phage display, or because of their effect on the toxicity of known bacterial pathogens including Vibrio cholerae and Neisseria meningitidis. Here, we show that the current 56 members of the Inoviridae family represent a minute fraction of a highly diverse group of inoviruses. Using a machine learning approach leveraging a combination of marker gene and genome features, we identified 10,295 inovirus-like sequences from microbial genomes and metagenomes. Collectively, our results call for reclassification of the current Inoviridae family into a viral order including six distinct proposed families associated with nearly all bacterial phyla across virtually every ecosystem. Putative inoviruses were also detected in several archaeal genomes, suggesting that, collectively, members of this supergroup infect hosts across the domains Bacteria and Archaea. Finally, we identified an expansive diversity of inovirus-encoded toxin-antitoxin and gene expression modulation systems, alongside evidence of both synergistic (CRISPR evasion) and antagonistic (superinfection exclusion) interactions with co-infecting viruses, which we experimentally validated in a Pseudomonas model. Capturing this previously obscured component of the global virosphere may spark new avenues for microbial manipulation approaches and innovative biotechnological applications.

Novel haloarchaeal viruses from Lake Retba infecting Haloferax and Halorubrum species.

The diversity of archaeal viruses is severely undersampled compared with that of viruses infecting bacteria and eukaryotes, limiting our understanding on their evolution and environmental impacts. Here, we describe the isolation and characterization of four new viruses infecting halophilic archaea from the saline Lake Retba, located close to Dakar on the coast of Senegal. Three of the viruses, HRPV10, HRPV11 and HRPV12, have enveloped pleomorphic virions and should belong to the family Pleolipoviridae, whereas the forth virus, HFTV1, has an icosahedral capsid and a long non-contractile tail, typical of bacterial and archaeal members of the order Caudovirales. Comparative genomic and phylogenomic analyses place HRPV10, HRPV11 and HRPV12 into the genus Betapleolipovirus, whereas HFTV1 appears to be most closely related to the unclassified Halorubrum virus HRTV-4. Differently from HRTV-4, HFTV1 encodes host-derived minichromosome maintenance helicase and PCNA homologues, which are likely to orchestrate its genome replication. HFTV1, the first archaeal virus isolated on a Haloferax strain, could also infect Halorubrum sp., albeit with an eightfold lower efficiency, whereas pleolipoviruses nearly exclusively infected autochthonous Halorubrum strains. Mapping of the metagenomic sequences from this environment to the genomes of isolated haloarchaeal viruses showed that these known viruses are underrepresented in the available viromes.

New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures.

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.

Novel Caudovirales associated with Marine Group I Thaumarchaeota assembled from metagenomes.

Marine Group I (MGI) Thaumarchaeota are some of the most abundant microorganisms in the deep ocean and responsible for much of the ammonia oxidation occurring in this environment. In this work, we present 35 sequences assembled from metagenomic samples of the first uncultivated Caudovirales viruses associated with Thaumarchaeota, which we designated marthavirus. Most of the sequences were obtained from cellular metagenomes confirming that they represent an important tool to study environmental viral communities due to cells retrieved while undergoing viral lysis. Metagenomic recruitment showed that this viral population is formed by very divergent entities with high intrapopulation homogeneity. However, metatranscriptomic analyses revealed the same differential expression profile with the capsid as major transcript, indicative of viruses during the lytic cycle. The cobalamine biosynthesis gene cobS, an auxiliary metabolic gene, was also highly expressed during the infection. These analyses expand our understanding of the global diversity of archaeal viruses.

Characterization of the lytic archaeal virus Drs3 infecting Methanobacterium formicicum.

Viruses are ubiquitous in the biosphere and greatly affect the hosts they infect. It is generally accepted that members of every microbial taxon are susceptible to at least one virus, and a plethora of bacterial viruses are known. In contrast, knowledge of the archaeal virosphere is still limited. Here, a novel lytic archaeal virus is described, designated "Drs3", as well as its host, Methanobacterium formicicum strain Khl10. This hydrogenotrophic methanogenic archaeon and its virus were isolated from the anaerobic digester of an experimental biogas plant in Germany. The tailed virus has an icosahedral head with a diameter of approximately 60 nm and a long non-contractile tail of approximately 230 nm. These structural observations suggest that the new isolate belongs to the family Siphoviridae, but it could not be assigned to an existing genus. Lysis of the host Khl10 was observed 40-44 h after infection. Lysis of the type strain Methanobacterium formicicum DSMZ 1535 was not observed in the presence of Drs3, pointing towards resistance in the type strain or a rather narrow host range of this newly isolated archaeal virus. The complete 37-kb linear dsDNA genome of Drs3 contains 39 open reading frames, only 12 of which show similarity to genes with predicted functions.

Extremely halophilic pleomorphic archaeal virus HRPV9 extends the diversity of pleolipoviruses with integrases.

Certain pleomorphic archaeal viruses are highly infectious even at saturated salt. These viruses belong to the genus Betapleolipovirus of the recently described archaeal virus family Pleolipoviridae. Pleolipoviruses comprise single-stranded or double-stranded, circular or linear DNA genomes that share countless homologues among various archaeal genetic elements. Here we describe a new extremely halophilic betapleolipovirus, Halorubrum pleomorphic virus 9 (HRPV9), which has an integrase gene. We also identified new genes encoding minor pleolipoviral structural proteins. The studies on HRPV9 enhance our knowledge on pleolipoviruses, especially their reciprocal relatedness and relation to certain archaeal plasmids, proviruses and membrane vesicles.