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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.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life. © The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.

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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

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-longSulfolobus 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.

International audience; 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.

International audience; 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 homo-dimer 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 homo-dimers) 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 nonenvel-oped archaeal viruses have evolved from enveloped viruses by shedding the membrane, indicating that this trait may be relatively easily lost during virus evolution. cryo-EM | extremophiles | hyperthermophilic archaea | filamentous viruses

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., Here, the authors investigate the diversity and dynamics of the CRISPRome in the hyperthermophilic archaea of the order Sulfolobales, and find the most abundant spacers to come from mini-CRISPR arrays of archaeal viruses, which might represent a strategy for superinfection exclusion and promotion of archaeal virus speciation.

International audience; 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 superhe-lix. 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.

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 A resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence—an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins. The electron cryo-microscopy structure of Sulfolobus islandicus pili enabled the identification of SiL_2606 as the main pilin in these filaments and revealed that the pili are glycosylated, which probably explains how these structures remain soluble and stable even when cells grow at pH 3 and 80 °C.

International audience; 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.

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod-shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function. The SIRV2_Gp1 protein is highly expressed during early stages of infection and it is the only protein that is encoded twice on the viral genome. It harbours a helix-turn-helix motif and was therefore hypothesized to bind DNA. The DNA-binding behavior of SIRV2_Gp1 was characterized with electrophoretic mobility shift assays and atomic force microscopy. We provide evidence that the protein interacts with DNA and that it forms large aggregates, thereby causing extreme condensation of the DNA. Furthermore, the N-terminal domain of the protein mediates toxicity to the viral host Sulfolobus. Our findings may lead to biotechnological applications, such as the development of a toxic peptide for the containment of pathogenic bacteria, and add to our understanding of the Rudiviral infection cycle. ispartof: VIRUSES-BASEL vol:9 issue:7 ispartof: location:Switzerland status: published

Biological membranes create compartments, and are usually formed by lipid bilayers. However, in hyperthermophilic archaea that live optimally at temperatures above 80°C the membranes are monolayers which resemble fused bilayers. Many double-stranded DNA viruses which parasitize such hosts, including the filamentous virus AFV1 of Acidianus hospitalis, are enveloped with a lipid-containing membrane. Using cryo-EM, we show that the membrane in AFV1 is a ~2 nm-thick monolayer, approximately half the expected membrane thickness, formed by host membrane-derived lipids which adopt a U-shaped ‘horseshoe’ conformation. We hypothesize that this unusual viral envelope structure results from the extreme curvature of the viral capsid, as ‘horseshoe’ lipid conformations favor such curvature and host membrane lipids that permit horseshoe conformations are selectively recruited into the viral envelope. The unusual envelope found in AFV1 also has many implications for biotechnology, since this membrane can survive the most aggressive conditions involving extremes of temperature and pH. DOI: http://dx.doi.org/10.7554/eLife.26268.001, eLife digest Virtually every environment on the planet is home to some form of life, even places that, at first glance, appear to be too harsh for any organism to survive in. For example, a microscopic organism known as Acidianus hospitalis thrives in highly acidic environments that are hotter than 80°C, conditions that would kill humans and many other species. Acidianus hospitalis has many adaptations that allow it to survive in its extreme environment. For example, the membrane that surrounds its cells has a different structure to the membranes that surround the cells of most other species. Membranes are made of molecules known as lipids. Generally these lipids assemble into two distinct layers (known as a bilayer) to form the membrane. However, in A. hospitalis the membrane contains only a single layer of lipids that resembles a bilayer in which lipids in opposite layers have fused together to make longer molecules. A virus known as AFV1 is able to infect A. hospitalis. Like many other viruses, AFV1 steals part of its host cell’s membrane when it leaves the cell in search of new cells to infect. This stolen membrane helps to protect the virus from its surroundings, however, the structure of the membrane surrounding AFV1 was not known. Kasson et al. combined a technique called cryo-electron microscopy with computer simulations to study the membrane surrounding AFV1. The study shows that this membrane is only half as thick as the membrane that surrounds A. hospitalis. To make this thinner membrane, flexible lipid molecules from the A. hospitalis membrane bend into a U-shape. These findings reveal a new type of membrane structure not previously seen in the natural world. In the future, this thinner membrane could have many uses in biotechnology, such as to make probes for medical imaging in patients or to deliver drugs to specific sites in the body. Enveloped by this unusual membrane, these structures may be more resistant to the normal processes that degrade and destroy foreign materials in humans and other organisms. DOI: http://dx.doi.org/10.7554/eLife.26268.002

Encapsidation of genetic material into polyhedral particles is one of the most common structural solutions employed by viruses infecting hosts in all three domains of life. Here, we describe a new virus of hyperthermophilic archaea, Sulfolobus polyhedral virus 1 (SPV1), which condenses its circular double-stranded DNA genome in a manner not previously observed for other known viruses. The genome complexed with virion proteins is wound up sinusoidally into a spherical coil which is surrounded by an envelope and further encased by an outer polyhedral capsid apparently composed of the 20-kDa virion protein. Lipids selectively acquired from the pool of host lipids are integral constituents of the virion. None of the major virion proteins of SPV1 show similarity to structural proteins of known viruses. However, minor structural proteins, which are predicted to mediate host recognition, are shared with other hyperthermophilic archaeal viruses infecting members of the order Sulfolobales . The SPV1 genome consists of 20,222 bp and contains 45 open reading frames, only one-fifth of which could be functionally annotated. IMPORTANCE Viruses infecting hyperthermophilic archaea display a remarkable morphological diversity, often presenting architectural solutions not employed by known viruses of bacteria and eukaryotes. Here we present the isolation and characterization of Sulfolobus polyhedral virus 1, which condenses its genome into a unique spherical coil. Due to the original genomic and architectural features of SPV1, the virus should be considered a representative of a new viral family, “Portogloboviridae.”

International audience; Protein posttranslational methylation has been reported to occur in archaea, including members of the genus Sulfolobus, but has never been characterized on a proteome-wide scale. Among important Sulfolobus proteins carrying such modification are the chromatin proteins that have been described to be methylated on lysine side chains, resembling eukaryotic histones in that aspect. To get more insight into the extent of this modification and its dynamics during the different growth steps of the thermoacidophylic archaeon S. islandicus LAL14/1, we performed a global and deep proteomic analysis using a combination of high-throughput bottom-up and proteomics approaches on a single high-resolution mass spectrometer. 1,931 methylation sites on 751 proteins were found by the bottom-up analysis, with methylation sites on 526 proteins monitored throughout three cell culture growth stages: early-exponential, mid-exponential and stationary. The top-down analysis revealed 3,978 proteoforms arising from 681 proteins, including 292 methylated proteoforms, 85 of which were comprehensively characterized. Methylated proteoforms of the five chromatin proteins (Alba1, Alba2, Cren7, Sul7d1, Sul7d2) were fully characterized by a combination of bottom-up and top-down data. The top-down analysis also revealed an increase of methylation during cell growth for two chromatin proteins, which had not been evidenced by bottom-up. These results shed new light on the ubiquitous lysine methylation throughout the S. islandicus proteome. Furthermore, we found that S. islandicus proteins are frequently acetylated at the N-terminus, following the removal of the N-terminal methionine. This study highlights the great value of combining bottom-up and top-down proteomics for obtaining an unprecedented level of accuracy in detecting differentially-modified intact proteoforms. The data have been deposited to the ProteomeXchange with identifiers PXD003074 and PXD004179.

International audience; Viruses package their genetic material in diverse ways. Most known strategies include encapsulation of nucleic acids into spherical or filamentous virions with icosahedral or helical symmetry, respectively. Filamentous viruses with dsDNA genomes are currently associated exclusively with Archaea. Here, we describe a filamentous hyperthermophilic archaeal virus, Pyrobaculum filamentous virus 1 (PFV1), with a type of virion organization not previously observed in DNA viruses. The PFV1 virion, 400 ± 20 × 32 ± 3 nm, contains an envelope and an inner core consisting of two structural units: a rod-shaped helical nucleocapsid formed of two 14-kDa major virion proteins and a nucleocapsid-encompassing protein sheath composed of a single major virion protein of 18 kDa. The virion organization of PFV1 is superficially similar to that of negative-sense RNA viruses of the family Filoviridae, including Ebola virus and Marburg virus. The linear dsDNA of PFV1 carries 17,714 bp, including 60-bp-long terminal inverted repeats, and contains 39 predicted ORFs, most of which do not show similarities to sequences in public databases. PFV1 is a lytic virus that completely disrupts the host cell membrane at the end of the infection cycle.

International audience

The SSPF was supported by grants from The Scottish Funding Council (references SSPF and SULSA), The Biotechnology and Biological Sciences Research Council (reference BB/S/B14450), European Union under framework 7 (reference Aeropath). The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology. Publisher PDF

Many proteins of viruses infecting hyperthermophilic Crenarchaeota have no detectable homologs in current databases, hampering our understanding of viral evolution. We used sensitive database search methods and structural modeling to show that a nucleocapsid protein (TP1) of Thermoproteus tenax virus 1 (TTV1) is a derivative of the Cas4 nuclease, a component of the CRISPR-Cas adaptive immunity system that is encoded also by several archaeal viruses. In TTV1, the Cas4 gene was split into two, with the N-terminal portion becoming TP1, and lost some of the catalytic amino acid residues, apparently resulting in the inactivation of the nuclease. To our knowledge, this is the first described case of exaptation of an enzyme for a virus capsid protein function. Reviewers This article was reviewed by Vivek Anantharaman, Christine Orengo and Mircea Podar. For complete reviews, see the Reviewers’ reports section. Electronic supplementary material The online version of this article (doi:10.1186/s13062-015-0093-2) contains supplementary material, which is available to authorized users.

A viral DNA form that survives extremes The prokaryote Sulfolobus islandicus lives at extreme temperatures (∼80°C) and acidity (pH 3). It is infected by the rudivirus SIRV2. DiMaio et al. determined the structure of the SIRV2 virus using cryo–electron microscopy to understand how the virus survives these brutal conditions. Most DNA in nature assumes a B-form shape. The virion, on the other hand, contains highly unusual A-form DNA that may help it survive adverse conditions. The viral capsid protein forms an extended α-helical structure that wraps around the viral DNA, possibly stabilizing the A-form DNA. Science , this issue p. 914

Geothermal and hypersaline environments are rich in virus-like particles, among which spindle-shaped morphotypes dominate. Currently, viruses with spindle- or lemon-shaped virions are exclusive to Archaea and belong to two distinct viral families. The larger of the two families, the Fuselloviridae , comprises tail-less, spindle-shaped viruses, which infect hosts from phylogenetically distant archaeal lineages. Sulfolobus spindle-shaped virus 1 (SSV1) is the best known member of the family and was one of the first hyperthermophilic archaeal viruses to be isolated. SSV1 is an attractive model for understanding virus-host interactions in Archaea ; however, the constituents and architecture of SSV1 particles remain only partially characterized. Here, we have conducted an extensive biochemical characterization of highly purified SSV1 virions and identified four virus-encoded structural proteins, VP1 to VP4, as well as one DNA-binding protein of cellular origin. The virion proteins VP1, VP3, and VP4 undergo posttranslational modification by glycosylation, seemingly at multiple sites. VP1 is also proteolytically processed. In addition to the viral DNA-binding protein VP2, we show that viral particles contain the Sulfolobus solfataricus chromatin protein Sso7d. Finally, we provide evidence indicating that SSV1 virions contain glycerol dibiphytanyl glycerol tetraether (GDGT) lipids, resolving a long-standing debate on the presence of lipids within SSV1 virions. A comparison of the contents of lipids isolated from the virus and its host cell suggests that GDGTs are acquired by the virus in a selective manner from the host cytoplasmic membrane, likely during progeny egress. IMPORTANCE Although spindle-shaped viruses represent one of the most prominent viral groups in Archaea , structural data on their virion constituents and architecture still are scarce. The comprehensive biochemical characterization of the hyperthermophilic virus SSV1 presented here brings novel and significant insights into the organization and architecture of spindle-shaped virions. The obtained data permit the comparison between spindle-shaped viruses residing in widely different ecological niches, improving our understanding of the adaptation of viruses with unusual morphotypes to extreme environmental conditions.

International audience; In attempts to induce putative temperate viruses, we UV-irradiated cells of the hyperthermophilic archaeon Pyrobaculum oguniense. Virus replication could not be detected; however, we observed the development of pyramidal structures with 6-fold symmetry on the cell surface. The hexagonal basis of the pyramids was continuous with the cellular cytoplasmic membrane and apparently grew via the gradual expansion of the 6 triangular lateral faces, ultimately protruding through the S-layer. When the base of these isosceles triangles reached approximately 200 nm in length, the pyramids opened like flower petals. The origin and function of these mysterious nanostructures remain unknown.

International audience; Viruses have developed a wide range of strategies to escape from the host cells in which they replicate. For egress some archaeal viruses use a pyramidal structure with sevenfold rotational symmetry. Virus-associated pyramids (VAPs) assemble in the host cell membrane from the virus-encoded protein PVAP and open at the end of the infection cycle. We characterize this unusual supramolecular assembly using a combination of genetic, biochemical, and electron microscopic techniques. By whole-cell electron cryotomography, we monitored morphological changes in virus-infected host cells. Subtomogram averaging reveals the VAP structure. By heterologous expression of PVAP in cells from all three domains of life, we demonstrate that the protein integrates indiscriminately into virtually any biological membrane, where it forms sevenfold pyramids. We identify the protein domains essential for VAP formation in PVAP truncation mutants by their ability to remodel the cell membrane. Self-assembly of PVAP into pyramids requires at least two different, in-plane and out-of-plane, protein interactions. Our findings allow us to propose a model describing how PVAP arranges to form sevenfold pyramids and suggest how this small, robust protein may be used as a general membrane-remodeling system.

Viruses with spindle-shaped virions are abundant in diverse environments. Over the years, such viruses have been isolated from a wide range of archaeal hosts. Evolutionary relationships between them remained enigmatic, however. Here, using structural proteins as markers, we define familial ties among these “dark horses” of the virosphere and segregate all spindle-shaped viruses into two distinct evolutionary lineages, corresponding to Bicaudaviridae and Fuselloviridae . Our results illuminate the utility of structure-based virus classification and bring additional order to the virosphere.

Microbial genomes encompass a sizable fraction of poorly characterized, narrowly spread fast-evolving genes. Using sensitive methods for sequences comparison and protein structure prediction, we performed a detailed comparative analysis of clusters of such genes, which we denote “dark matter islands”, in archaeal genomes. The dark matter islands comprise up to 20 % of archaeal genomes and show remarkable heterogeneity and diversity. Nevertheless, three classes of entities are common in these genomic loci: (a) integrated viral genomes and other mobile elements; (b) defense systems, and (c) secretory and other membrane-associated systems. The dark matter islands in the genome of thermophiles and mesophiles show similar general trends of gene content, but thermophiles are substantially enriched in predicted membrane proteins whereas mesophiles have a greater proportion of recognizable mobile elements. Based on this analysis, we predict the existence of several novel groups of viruses and mobile elements, previously unnoticed variants of CRISPR-Cas immune systems, and new secretory systems that might be involved in stress response, intermicrobial conflicts and biogenesis of novel, uncharacterized membrane structures. Electronic supplementary material The online version of this article (doi:10.1007/s00792-014-0672-7) contains supplementary material, which is available to authorized users.

Opinion; International audience; It has been claimed that giant DNA viruses represent a separate, fourth domain of life in addition to the domains of Bacteria, Archaea, and Eukarya. Such classification disregards fundamental differences between the two types of living entities - viruses and cells - and results in confusion and controversies in evolutionary scenarios. We highlight these problems and emphasize the importance of restricting the term 'domain' to the descendants of the last universal cellular ancestor (LUCA), based on the shared ribosome structure. We suggest tracing phylogeny of viruses along evolutionary lineages primarily defined by virion architectures and the structures of the major capsid proteins.

International audience; Two pioneers of twentieth century biology passed away during the past decade, Wolfram Zillig in April 2005 and Carl Woese in December 2012. Among several other accomplishments, Woese has been celebrated for the discovery of the domain Archaea and for establishing rRNA as the 'Rosetta Stone' of evolutionary and environmental microbiology. His work inspired many scientists in various fields of biology, and among them was Wolfram Zillig, who is credited with the discovery of several unique molecular features of archaea. In this Essay, we highlight the remarkable achievements of Woese and Zillig and consider how they have shaped the archaeal research landscape.

Archaeal host cells infected by Sulfolobus turreted icosahedral virus (STIV) and Sulfolobus islandicus rod-shaped virus 2 (SIRV2) produce unusual pyramid-like structures on the cell surface prior to virus-induced cell lysis. This viral lysis process is distinct from known viral lysis processes associated with bacterial or eukaryal viruses. The STIV protein C92 and the SIRV2 protein 98 are the only viral proteins required for the formation of the pyramid lysis structures of STIV and SIRV2, respectively. Since SIRV2 and STIV have fundamentally different morphotypes and genome sequences, it is surprising that they share this lysis system. In this study, we have constructed a collection of C92/P98 chimeric proteins and tested their abilities, both in the context of virus replication and alone, to form pyramid lysis structures in S. solfataricus . The results of this study illustrate that these proteins are functionally homologous when expressed as individual chimeric proteins but not when expressed in the context of complete STIV infection.

International audience; While the basal transcription machinery in archaea is eukaryal-like, transcription factors in archaea and their viruses are usually related to bacterial transcription factors. Nevertheless, some of these organisms show predicted classical zinc fingers motifs of the C2H2 type, which are almost exclusively found in proteins of eukaryotes and most often associated with transcription regulators. In this work, we focused on the protein AFV1p06 from the hyperthermophilic archaeal virus AFV1. The sequence of the protein consists of the classical eukaryotic C2H2 motif with the fourth histidine coordinating zinc missing, as well as of N- and C-terminal extensions. We showed that the protein AFV1p06 binds zinc and solved its solution structure by NMR. AFV1p06 displays a zinc finger fold with a novel structure extension and disordered N- and C-termini. Structure calculations show that a glutamic acid residue that coordinates zinc replaces the fourth histidine of the C2H2 motif. Electromobility gel shift assays indicate that the protein binds to DNA with different affinities depending on the DNA sequence. AFV1p06 is the first experimentally characterised archaeal zinc finger protein with a DNA binding activity. The AFV1p06 protein family has homologues in diverse viruses of hyperthermophilic archaea. A phylogenetic analysis points out a common origin of archaeal and eukaryotic C2H2 zinc fingers.

Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea . It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo .

The structural and functional analysis of the protein AvtR encoded by Acidianus filamentous virus 6 (AFV6), which infects the archaeal genus Acidianus , revealed its unusual structure and involvement in transcriptional regulation of several viral genes. The crystal structure of AvtR (100 amino acids) at 2.6-Å resolution shows that it is constituted of a repeated ribbon-helix-helix (RHH) motif, which is found in a large family of bacterial transcriptional regulators. The known RHH proteins form dimers that interact with DNA using their ribbon to create a central β-sheet. The repeated RHH motifs of AvtR superpose well on such dimers, but its central sheet contains an extra strand, suggesting either conformational changes or a different mode of DNA binding. Systematic evolution of ligands by exponential enrichment (SELEX) experiments combined with systematic mutational and computational analysis of the predicted site revealed 8 potential AvtR targets in the AFV6 genome. Two of these targets were studied in detail, and the complex role of AvtR in the transcriptional regulation of viral genes was established. Repressing transcription from its own gene, gp29 , AvtR can also act as an activator of another gene, gp30 . Its binding sites are distant from both genes' TATA boxes, and the mechanism of AvtR-dependent regulation appears to include protein oligomerization starting from the protein's initial binding sites. Many RHH transcriptional regulators of archaeal viruses could share this regulatory mechanism.

Known viruses build their particles using a restricted number of redundant structural solutions. Here, we describe the Aeropyrum coil-shaped virus (ACV), of the hyperthermophilic archaeon Aeropyrum pernix , with a virion architecture not previously observed in the viral world. The nonenveloped, hollow, cylindrical virion is formed from a coiling fiber, which consists of two intertwining halves of a single circular nucleoprotein. The virus ACV is also exceptional for its genomic properties. It is the only virus with a single-stranded (ss) DNA genome among the known hyperthermophilic archaeal viruses. Moreover, the size of its circular genome, 24,893 nt, is double that of the largest known ssDNA genome, suggesting an efficient solution for keeping ssDNA intact at 90–95 °C, the optimal temperature range of A. pernix growth. The genome content of ACV is in line with its unique morphology and confirms that ACV is not closely related to any known virus.

By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix , and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae .

The genomes of two Sulfolobus islandicus strains obtained from Icelandic solfataras were sequenced and analyzed. Strain REY15A is a host for a versatile genetic toolbox. It exhibits a genome of minimal size, is stable genetically, and is easy to grow and manipulate. Strain HVE10/4 shows a broad host range for exceptional crenarchaeal viruses and conjugative plasmids and was selected for studying their life cycles and host interactions. The genomes of strains REY15A and HVE10/4 are 2.5 and 2.7 Mb, respectively, and each genome carries a variable region of 0.5 to 0.7 Mb where major differences in gene content and gene order occur. These include gene clusters involved in specific metabolic pathways, multiple copies of VapBC antitoxin-toxin gene pairs, and in strain HVE10/4, a 50-kb region rich in glycosyl transferase genes. The variable region also contains most of the insertion sequence (IS) elements and high proportions of the orphan orfB elements and SMN1 miniature inverted-repeat transposable elements (MITEs), as well as the clustered regular interspaced short palindromic repeat (CRISPR)-based immune systems, which are complex and diverse in both strains, consistent with them having been mobilized both intra- and intercellularly. In contrast, the remainder of the genomes are highly conserved in their protein and RNA gene syntenies, closely resembling those of other S. islandicus and Sulfolobus solfataricus strains, and they exhibit only minor remnants of a few genetic elements, mainly conjugative plasmids, which have integrated at a few tRNA genes lacking introns. This provides a possible rationale for the presence of the introns.

The role of viruses at different stages of the origin of life has recently been reconsidered. It appears that viruses may have accompanied the earliest forms of life, allowing the transition from an RNA to a DNA world and possibly being involved in the shaping of tree of life in the three domains that we know presently. In addition, a large variety of viruses has been recently identified in extreme environments, hosted by extremophilic microorganisms, in ecosystems considered as analogues to those of the early Earth. The earliest traces of life were preserved by the precipitation of silica on organic structures. The study of the in situ and experimental fossilisation of microorganisms allows better understanding of the fossilisation processes and helps identification of traces of life in ancient rocks. In a continuation of these studies, we present the results of the first experimental fossilisation by silica of viruses from extremophilic Archaea (SIRV2 – Sulfolobus islandicus Virus 2, TPV1 – Thermococcus prieurii virus 1, and PAV1 – Pyrococcus abyssi virus 1). Our results confirm that viruses can be fossilised, with silica precipitating on the different viral structures (proteins, envelope) over several months. However differences in the silicification process were noticed, depending on the viral structure and composition. The fossilisation mechanism is similar to that of the fossilisation of microorganisms. This study thus suggests that viral remains or traces could be preserved in the rock record although their identification may be challenging due to the small size of the viral particles.

SUMMARY Prokaryotes, bacteria and archaea, are the most abundant cellular organisms among those sharing the planet Earth with human beings (among others). However, numerous ecological studies have revealed that it is actually prokaryotic viruses that predominate on our planet and outnumber their hosts by at least an order of magnitude. An understanding of how this viral domain is organized and what are the mechanisms governing its evolution is therefore of great interest and importance. The vast majority of characterized prokaryotic viruses belong to the order Caudovirales, double-stranded DNA (dsDNA) bacteriophages with tails. Consequently, these viruses have been studied (and reviewed) extensively from both genomic and functional perspectives. However, albeit numerous, tailed phages represent only a minor fraction of the prokaryotic virus diversity. Therefore, the knowledge which has been generated for this viral system does not offer a comprehensive view of the prokaryotic virosphere. In this review, we discuss all families of bacterial and archaeal viruses that contain more than one characterized member and for which evolutionary conclusions can be attempted by use of comparative genomic analysis. We focus on the molecular mechanisms of their genome evolution as well as on the relationships between different viral groups and plasmids. It becomes clear that evolutionary mechanisms shaping the genomes of prokaryotic viruses vary between different families and depend on the type of the nucleic acid, characteristics of the virion structure, as well as the mode of the life cycle. We also point out that horizontal gene transfer is not equally prevalent in different virus families and is not uniformly unrestricted for diverse viral functions.

International audience; Recently a unique mechanism of virion release was discovered in Archaea, different from lysis and egress systems of bacterial and eukaryotic viruses. It involves formation of pyramidal structures on the host cell surface that rupture the S-layer and by opening outwards, create apertures through which mature virions escape the cell. Here we present results of a protein analysis of Sulfolobus islandicus cells infected with the rudivirus SIRV2, which enable us to postulate SIRV2-encoded protein P98 as the major constituent of these exceptional cellular ultrastructures.

The properties of double-stranded DNA (dsDNA) viruses that infect Crenarchaea living in acidic hot springs (pH 1.5 to 3 and 75 to 95°C) are radically different from those of the viruses that infect Bacteria and Eukarya. Not only are the shapes of these viruses distinct from those of all other known viruses, but ∼80% of their open reading frames (ORFs) do not share any sequence similarity with ORFs of other viruses or of cellular life forms apart from other archaeal viruses (44). Seven novel viral families have been created to categorize their unique characteristics: the spindle-shaped Fuselloviridae, the filamentous Rudiviridae and Lipothrixviridae, the bottle-shaped Ampullaviridae, the droplet-shaped Guttaviridae, the spherical Globuloviridae, the two-tailed Bicaudaviridae, and the unclassified Sulfolobus turreted icosahedral virus (STIV) (43). The study of archaeal viruses is still in its infancy compared to that of eukaryal and bacterial viruses, and little is known regarding crenarchaeal virus life cycles, virus-host relationships, genetics, or biochemistry. Further studies of these viruses are expected to provide genetic, biochemical, and evolutionary insight into their crenarchaeal hosts and the requirements for life in the harsh environments. Transcription cycles of the fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) (16) and the rudiviruses Sulfolobus islandicus rod-shaped virus 1 and 2 (SIRV1 and SIRV2) (24) have been analyzed. Also, recent results concerning the lytic viruses STIV and SIRV2 shed new light on their replication cycle and interaction with their hosts (7, 9, 38). Because structures generally are much better conserved than sequences, structural studies have aimed at uncovering functional and evolutionary relationships that are not apparent from the primary sequence. To date, 11 protein structures from crenarchaeal viruses have been reported (18, 19, 22, 23, 25-30, 33). Most of these proteins share structural similarity with proteins of known function: three winged-helix proteins are likely involved in transcriptional regulation (two from the fusellovirus SSV1 [27, 33] and one from STIV [28]); one glycosyltransferase from STIV displays the GT-A fold (30); one adaptor protein from SSV1 is similar to the repressor of primer (ROP) of Escherichia coli (26); and the major coat protein of STIV has revealed the first evolutionary relationship spanning the three domains of life (25). The structures of the highly conserved ORFs among these viruses, ORF109 of lipothrixvirus AFV3 (22) and ORFB116 from STIV (29), suggest that they are DNA-binding proteins that function in transcriptional regulation. Filamentous viruses, the most abundant morphotype in these extreme environments, form the new viral order Ligamenvirales, which is divided into the Rudiviridae and Lipothrixviridae families (43). Lipothrixviruses (Acidianus filamentous virus 1 to 9, Thermoproteus tenax virus 1 to 3, and Sulfolobus islandicus filamentous virus) (8, 43), which were the first enveloped filamentous viruses with linear dsDNA genomes discovered, infect acidophilic and hyperthermophilic Crenarchaea. They are classified into α, β, γ, and δ genera based on their genomic properties and on the diversity of their terminal appendages, which are involved in host cell recognition. AFV1 is a γ-lipothrixvirus isolated from an acidic hot spring in Yellowstone National Park, where the temperature is above 85°C and the pH below 3 (6). The linear, double-stranded 20.8-kb DNA genome of AFV1 encodes 40 putative ORFs, 32% of which are homologous to viral ORFs from the lipothrixvirus SIFV and the rudiviruses SIRV1 and SIRV2. The predicted products generally are too dissimilar to the sequences in the public databases to allow functional assignment; only two glycosyltransferases, two CopG-like proteins, and one transcription regulator have been detected (6). It is highly unlikely that all of the encoded proteins consist entirely of unique protein folds serving novel functions. Therefore, to get insight into the biology of the Lipothrixviridae, we performed crystallographic studies of the AFV1 proteome. Although the solved structures of AFV1-102 (23) and the homologs AFV1-99 (19) and SIFV-014 (18) have not revealed any structural homologs, they have suggested that the proteins are involved in protein-protein interaction and are minor structural components, respectively. Here, we report the crystal structure of AFV1-157 and its biochemical characterization. AFV1-157 is a 157-residue protein with one homolog in the fusellovirus Sulfolobus spindle-shaped virus Ragged Hills (SSVRH). It has a novel α+β fold that remotely resembles the nucleotidyltransferase topology. We demonstrated that (i) AFV1-157 exhibits in vitro nuclease activity that degrades linear dsDNA, and (ii) the E86 residue is essential for the nuclease activity.