Extracellular cytochrome nanowires appear to be ubiquitous in prokaryotes.

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

Spindle-shaped archaeal viruses evolved from rod-shaped ancestors to package a larger genome.

Spindle- or lemon-shaped viruses infect archaea in diverse environments. Due to the highly pleomorphic nature of these virions, which can be found with cylindrical tails emanating from the spindle-shaped body, structural studies of these capsids have been challenging. We have determined the atomic structure of the capsid of Sulfolobus monocaudavirus 1, a virus that infects hosts living in nearly boiling acid. A highly hydrophobic protein, likely integrated into the host membrane before the virions assemble, forms 7 strands that slide past each other in both the tails and the spindle body. We observe the discrete steps that occur as the tail tubes expand, and these are due to highly conserved quasiequivalent interactions with neighboring subunits maintained despite significant diameter changes. Our results show how helical assemblies can vary their diameters, becoming nearly spherical to package a larger genome and suggest how all spindle-shaped viruses have evolved from archaeal rod-like viruses.

Mechanical tomography of an archaeal lemon-shaped virus reveals membrane-like fluidity of the capsid and liquid nucleoprotein cargo.

Archaeal lemon-shaped viruses have unique helical capsids composed of highly hydrophobic protein strands which can slide past each other resulting in remarkable morphological reorganization. Here, using atomic force microscopy, we explore the biomechanical properties of the lemon-shaped virions of Sulfolobus monocaudavirus 1 (SMV1), a double-stranded DNA virus which infects hyperthermophilic (~80 °C) and acidophilic (pH ~ 2) archaea. Our results reveal that SMV1 virions are extremely soft and withstand repeated extensive deformations, reaching remarkable strains of 80% during multiple cycles of consecutive mechanical assaults, yet showing scarce traces of disruption. SMV1 virions can reversibly collapse wall-to-wall, reducing their volume by ~90%. Beyond revealing the exceptional malleability of the SMV1 protein shell, our data also suggest a fluid-like nucleoprotein cargo which can flow inside the capsid, resisting and accommodating mechanical deformations without further alteration. Our experiments suggest a packing fraction of the virus core to be as low as 11%, with the amount of the accessory proteins almost four times exceeding that of the viral genome. Our findings indicate that SMV1 protein capsid displays biomechanical properties of lipid membranes, which is not found among protein capsids of other viruses. The remarkable malleability and fluidity of the SMV1 virions are likely necessary for the structural transformations during the infection and adaptation to extreme environmental conditions.

The evolution of archaeal flagellar filaments.

Flagellar motility has independently arisen three times during evolution: in bacteria, archaea, and eukaryotes. In prokaryotes, the supercoiled flagellar filaments are composed largely of a single protein, bacterial or archaeal flagellin, although these two proteins are not homologous, while in eukaryotes, the flagellum contains hundreds of proteins. Archaeal flagellin and archaeal type IV pilin are homologous, but how archaeal flagellar filaments (AFFs) and archaeal type IV pili (AT4Ps) diverged is not understood, in part, due to the paucity of structures for AFFs and AT4Ps. Despite having similar structures, AFFs supercoil, while AT4Ps do not, and supercoiling is essential for the function of AFFs. We used cryo-electron microscopy to determine the atomic structure of two additional AT4Ps and reanalyzed previous structures. We find that all AFFs have a prominent 10-strand packing, while AT4Ps show a striking structural diversity in their subunit packing. A clear distinction between all AFF and all AT4P structures involves the extension of the N-terminal α-helix with polar residues in the AFFs. Additionally, we characterize a flagellar-like AT4P from Pyrobaculum calidifontis with filament and subunit structure similar to that of AFFs which can be viewed as an evolutionary link, showing how the structural diversity of AT4Ps likely allowed for an AT4P to evolve into a supercoiling AFF.

Archaeal bundling pili of Pyrobaculum calidifontis reveal similarities between archaeal and bacterial biofilms.

While biofilms formed by bacteria have received great attention due to their importance in pathogenesis, much less research has been focused on the biofilms formed by archaea. It has been known that extracellular filaments in archaea, such as type IV pili, hami, and cannulae, play a part in the formation of archaeal biofilms. We have used cryo-electron microscopy to determine the atomic structure of a previously uncharacterized class of archaeal surface filaments from hyperthermophilic Pyrobaculum calidifontis. These filaments, which we call archaeal bundling pili (ABP), assemble into highly ordered bipolar bundles. The bipolar nature of these bundles most likely arises from the association of filaments from at least two different cells. The component protein, AbpA, shows homology, both at the sequence and structural level, to the bacterial protein TasA, a major component of the extracellular matrix in bacterial biofilms, contributing to biofilm stability. We show that AbpA forms very stable filaments in a manner similar to the donor-strand exchange of bacterial TasA fibers and chaperone-usher pathway pili where a ß-strand from one subunit is incorporated into a ß-sheet of the next subunit. Our results reveal likely mechanistic similarities and evolutionary connection between bacterial and archaeal biofilms, and suggest that there could be many other archaeal surface filaments that are as yet uncharacterized.

Virus-induced cell gigantism and asymmetric cell division in archaea.

Archaeal viruses represent one of the most mysterious parts of the global virosphere, with many virus groups sharing no evolutionary relationship to viruses of bacteria or eukaryotes. How these viruses interact with their hosts remains largely unexplored. Here we show that nonlytic lemon-shaped virus STSV2 interferes with the cell cycle control of its host, hyperthermophilic and acidophilic archaeon Sulfolobus islandicus, arresting the cell cycle in the S phase. STSV2 infection leads to transcriptional repression of the cell division machinery, which is homologous to the eukaryotic endosomal sorting complexes required for transport (ESCRT) system. The infected cells grow up to 20-fold larger in size, have 8,000-fold larger volume compared to noninfected cells, and accumulate massive amounts of viral and cellular DNA. Whereas noninfected Sulfolobus cells divide symmetrically by binary fission, the STSV2-infected cells undergo asymmetric division, whereby giant cells release normal-sized cells by budding, resembling the division of budding yeast. Reinfection of the normal-sized cells produces a new generation of giant cells. If the CRISPR-Cas system is present, the giant cells acquire virus-derived spacers and terminate the virus spread, whereas in its absence, the cycle continues, suggesting that CRISPR-Cas is the primary defense system in Sulfolobus against STSV2. Collectively, our results show how an archaeal virus manipulates the cell cycle, transforming the cell into a giant virion-producing factory.

DeepTracer-ID: De novo protein identification from cryo-EM maps.

The recent revolution in cryo-electron microscopy (cryo-EM) has made it possible to determine macromolecular structures directly from cell extracts. However, identifying the correct protein from the cryo-EM map is still challenging and often needs additional sequence information from other techniques, such as tandem mass spectrometry and/or bioinformatics. Here, we present DeepTracer-ID, a server-based approach to identify the candidate protein in a user-provided organism de novo from a cryo-EM map, without the need for additional information. Our method first uses DeepTracer to generate a protein backbone model that best represents the cryo-EM map, and this model is then searched against the library of AlphaFold2 predictions for all proteins in the given organism. This method is highly accurate and robust for high-resolution cryo-EM maps: in all 13 experimental maps tested blindly, DeepTracer-ID identified the correct proteins as the top candidates. Eight of the maps were of known structures, while the other five unpublished maps were validated by prior protein annotation and careful inspection of the model refined into the map. The program also showed promising results for both homomeric and heteromeric protein complexes. This platform is possible because of the recent breakthroughs in large-scale three-dimensional protein structure prediction.

Spindle-shaped viruses infect marine ammonia-oxidizing thaumarchaea.

Ammonia-oxidizing archaea (AOA) from the phylum Thaumarchaeota are ubiquitous in marine ecosystems and play a prominent role in carbon and nitrogen cycling. Previous studies have suggested that, like all microbes, thaumarchaea are infected by viruses and that viral predation has a profound impact on thaumarchaeal functioning and mortality, thereby regulating global biogeochemical cycles. However, not a single virus capable of infecting thaumarchaea has been reported thus far. Here we describe the isolation and characterization of three Nitrosopumilus spindle-shaped viruses (NSVs) that infect AOA and are distinct from other known marine viruses. Although NSVs have a narrow host range, they efficiently infect autochthonous Nitrosopumilus strains and display high rates of adsorption to their host cells. The NSVs have linear double-stranded DNA genomes of ∼28 kb that do not display appreciable sequence similarity to genomes of other known archaeal or bacterial viruses and could be considered as representatives of a new virus family, the "Thaspiviridae." Upon infection, NSV replication leads to inhibition of AOA growth, accompanied by severe reduction in the rate of ammonia oxidation and nitrite reduction. Nevertheless, unlike in the case of lytic bacteriophages, NSV propagation is not associated with detectable degradation of the host chromosome or a decrease in cell counts. The broad distribution of NSVs in AOA-dominated marine environments suggests that NSV predation might regulate the diversity and dynamics of AOA communities. Collectively, our results shed light on the diversity, evolution, and potential impact of the virosphere associated with ecologically important mesophilic 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.

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).

Double autoinhibition mechanism of signal transduction ATPases with numerous domains (STAND) with a tetratricopeptide repeat sensor.

Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.

Virus evolution toward limited dependence on nonessential functions of the host: the case of bacteriophage SPP1.

UNLABELLED: All viruses are obligate intracellular parasites and depend on certain host cell functions for multiplication. However, the extent of such dependence and the exact nature of the functions provided by the host cell remain poorly understood. Here, we investigated if nonessential Bacillus subtilis genes are necessary for multiplication of bacteriophage SPP1. Screening of a collection of 2,514 single-gene knockouts of nonessential B. subtilis genes yielded only a few genes necessary for efficient SPP1 propagation. Among these were genes belonging to the yuk operon, which codes for the Esat-6-like secretion system, including the SPP1 receptor protein YueB. In addition, we found that SPP1 multiplication was negatively affected by the absence of two other genes, putB and efp. The gene efp encodes elongation factor P, which enhances ribosome activity by alleviating translational stalling during the synthesis of polyproline-containing proteins. PutB is an enzyme involved in the proline degradation pathway that is required for infection in the post-exponential growth phase of B. subtilis, when the bacterium undergoes a complex genetic reprogramming. The putB knockout shortens significantly the window of opportunity for SPP1 infection during the host cell life cycle. This window is a critical parameter for competitive phage multiplication in the soil environment, where B. subtilis rarely meets conditions for exponential growth. Our results in combination with those reported for other virus-host systems suggest that bacterial viruses have evolved toward limited dependence on nonessential host functions. IMPORTANCE: A successful viral infection largely depends on the ability of the virus to hijack cellular machineries and to redirect the flow of building blocks and energy resources toward viral progeny production. However, the specific virus-host interactions underlying this fundamental transformation are poorly understood. Here, we report on the first systematic analysis of virus-host cross talk during bacteriophage infection in Gram-positive bacteria. We show that lytic bacteriophage SPP1 is remarkably independent of nonessential genes of its host, Bacillus subtilis, with only a few cellular genes being necessary for efficient phage propagation. We hypothesize that such limited dependence of the virus on its host results from a constant "evolutionary arms race" and might be much more widespread than currently thought.

Extracellular membrane vesicles harbouring viral genomes.

Cells from the three domains of life produce extracellular membrane vesicles (MVs), suggesting that MV production is a fundamental aspect of cellular physiology. We have recently shown that MVs produced by the hyperthermophilic archaeon Thermococcus kodakaraensis can be used as vehicles to transfer exogenous recombinant plasmid DNA from cell to cell. Here, we show that Thermococcus nautilus, which harbours three plasmids, pTN1, pTN2 and pTN3, produces MVs, and that some of them selectively incorporate pTN1 and pTN3. Interestingly, pTN3 represents the genome of a defective virus, which encodes signature proteins common to a large group of viruses infecting hosts from all three cellular domains. However, preparations of MVs produced by T. nautilus have a protein composition similar to that of classical MVs from Thermococcales and do not contain the viral major capsid protein encoded by pTN3. Our results suggest that MVs can serve as vehicles for the intercellular transport of viral genomes and facilitate recombination between viral, plasmid and/or cellular chromosomes in the absence of viral infection.

Two distinct archaeal type IV pili structures formed by proteins with identical sequence.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.

Two dramatically distinct archaeal type IV pili structures formed by the same pilin.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from relatively small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Using cryo-electron microscopy (cryo-EM), we determined atomic structures of two dramatically different T4P from Saccharolobus islandicus REY15A. Unexpectedly, both pili were assembled from the same pilin protein but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same protein exists in three different conformations. The three conformations involve different orientations of the outer immunoglobulin (Ig)-like domains, mediated by a very flexible linker, and all three of these conformations are very different from the single conformation found in the mono-pilus. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations, formally similar to what has been shown for outer domains in bacterial flagellar filaments, despite lack of homology between bacterial flagella and archaeal T4P. Interestingly, both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. However, the expression of the ATPase and TadC genes was significantly different under the conditions yielding mono- and tri-pili. While archaeal T4P are homologs of archaeal flagellar filaments, our results show that in contrast to the rigid supercoil that the flagellar filaments must adopt to serve as helical propellers, archaeal T4P are likely to have fewer constraints on their structure and enjoy more internal degrees of freedom.

Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery.

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been 'domesticated', that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.

Extracellular membrane vesicles and nanotubes in Archaea.

Membrane-bound extracellular vesicles (EVs) are secreted by cells from all three domains of life and their implication in various biological processes is increasingly recognized. In this review, we summarize the current knowledge on archaeal EVs and nanotubes, and emphasize their biological significance. In archaea, the EVs and nanotubes have been largely studied in representative species from the phyla Crenarchaeota and Euryarchaeota. The archaeal EVs have been linked to several physiological processes such as detoxification, biomineralization and transport of biological molecules, including chromosomal, viral or plasmid DNA, thereby taking part in genome evolution and adaptation through horizontal gene transfer. The biological significance of archaeal nanotubes is yet to be demonstrated, although they could participate in EV biogenesis or exchange of cellular contents. We also discuss the biological mechanisms leading to EV/nanotube biogenesis in Archaea. It has been recently demonstrated that, similar to eukaryotes, EV budding in crenarchaea depends on the ESCRT machinery, whereas the mechanism of EV budding in euryarchaeal lineages, which lack the ESCRT-III homologues, remains unknown.

Archaeal extracellular vesicles are produced in an ESCRT-dependent manner and promote gene transfer and nutrient cycling in extreme environments.

Membrane-bound extracellular vesicles (EVs), secreted by cells from all three domains of life, transport various molecules and act as agents of intercellular communication in diverse environments. Here we demonstrate that EVs produced by a hyperthermophilic and acidophilic archaeon Sulfolobus islandicus carry not only a diverse proteome, enriched in membrane proteins, but also chromosomal and plasmid DNA, and can transfer this DNA to recipient cells. Furthermore, we show that EVs can support the heterotrophic growth of Sulfolobus in minimal medium, implicating EVs in carbon and nitrogen fluxes in extreme environments. Finally, our results indicate that, similar to eukaryotes, production of EVs in S. islandicus depends on the archaeal ESCRT machinery. We find that all components of the ESCRT apparatus are encapsidated into EVs. Using synchronized S. islandicus cultures, we show that EV production is linked to cell division and appears to be triggered by increased expression of ESCRT proteins during this cell cycle phase. Using a CRISPR-based knockdown system, we show that archaeal ESCRT-III and AAA+ ATPase Vps4 are required for EV production, whereas archaea-specific component CdvA appears to be dispensable. In particular, the active EV production appears to coincide with the expression patterns of ESCRT-III-1 and ESCRT-III-2, rather than ESCRT-III, suggesting a prime role of these proteins in EV budding. Collectively, our results suggest that ESCRT-mediated EV biogenesis has deep evolutionary roots, likely predating the divergence of eukaryotes and archaea, and that EVs play an important role in horizontal gene transfer and nutrient cycling in extreme environments.

Phospholipids act as secondary receptor during the entry of the enveloped, double-stranded RNA bacteriophage phi6.

Bacteriophage phi6 is the type member of the family Cystoviridae and infects Gram-negative Pseudomonas syringae cells. The virion consists of a protein-rich lipid envelope enclosing a nucleocapsid. The nucleocapsid covers the icosahedral polymerase complex that encloses the double-stranded RNA genome. Here, we demonstrate that nucleocapsid surface protein P8 is the single nucleocapsid component interacting with the cytoplasmic membrane. This interaction takes place between P8 and phospholipid. Based on this and previous studies, we propose a model where the periplasmic nucleocapsid interacts with the phospholipid head groups and, when the membrane voltage exceeds the threshold of 110 mV, this interaction drives the nucleocapsid through the cytoplasmic membrane, resulting in an intracellular vesicle containing the nucleocapsid.

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.