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The enigmatic archaeal virosphere

David Prangishvili1, Dennis H. Bamford2, Patrick Forterre1, Jaime Iranzo3, Eugene V. Koonin3 and Mart Krupovic1 Abstract | One of the most prominent features of is the extraordinary diversity of their DNA . Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic viruses and represent unique families. The distinct nature of archaeal viruses also extends to the gene composition and architectures of their genomes and the properties of the proteins that they encode. Environmental research has revealed prominent roles of archaeal viruses in influencing microbial communities in ocean ecosystems, and recent metagenomic studies have uncovered new groups of archaeal viruses that infect extremophiles and mesophiles in diverse habitats. In this Review, we summarize recent advances in our understanding of the genomic and morphological diversity of archaeal viruses and the molecular biology of their cycles and virus– host interactions, including interactions with archaeal CRISPR–Cas systems. We also examine the potential origins and evolution of archaeal viruses and discuss their place in the global virosphere.

Thermophilic The year 2017 marks the 40th anniversary of the dis- the epipelagic zone and the mesopelagic zone of the ocean Requiring high temperatures covery of archaea1. The third domain of life, which at have shown that ~10% of the most abundant viruses in for optimal growth. the time of its discovery included only a few , these zones are associated with archaea9. Diverse archaeal has been extensively populated in recent years with viruses have also been detected in benthic deep-sea Acidophilic thermophilic acidophilic alkaliphilic halophilic 10 Thriving under highly acidic numerous , , , ­ , ­ecosystems, where their turnover is as fast as 2–3 days . conditions. ­methanogenic and ammonia-oxidizing archaea. Virus predation is one of the primary causes of Moreover, the exploration of microbial diversity through microbial mortality, with major consequences for Alkaliphilic culture-independent approaches has substantially global biogeochemistry. It was recently shown that in Thriving under highly alkaline conditions. expanded our understanding of archaeal diversity and oceanic surface sediments across 1,000–10,000 m water uncovered numerous species that may represent many depths, viral infection has a higher impact on archaea Halophilic new phyla and orders of the Archaea2. than on and mainly affects members of the Requiring high levels of sodium Culture-independent approaches have radically Thaumarchaeota. Moreover, in the top 50 cm of ocean chloride for growth. changed the perception of the typical habitats of archaea. sediment, virus-induced lysis of archaea was estimated The original notion that archaea thrive only in environ- to account for up to one-third of the total microbial bio- ments with extreme conditions was challenged and over- mass that is killed each year, resulting in the release of turned by the discovery of archaea in a diverse range ~0.3–0.5 gigatonnes of carbon per year10. This realiza- of terrestrial and aquatic environments and even within tion has changed the perception of archaeal viruses from 3 1Department of Microbiology, and on the human body . Remarkably, in deep-sea eco- interesting curiosities of the virosphere to prominent Institut Pasteur, 25 rue du Dr systems, which constitute ~90% of the global biosphere, players in the biosphere. Roux, Paris 75015, France. the abundance of archaea is comparable to that of bac- In addition to having important roles in the function- 2 Department of Biosciences, teria4, and archaeal species that oxidize ammonium to ing of deep-sea ecosystems and global biogeochemical University of Helsinki, Helsinki 00014, Finland. nitrate (members of the phylum Thaumarchaeota) rep- cycles, archaeal viruses attract attention owing to their 5 3National Center for resent one of the most abundant cell types in oceans . remarkable diversity, unique morphologies and ability Biotechnology Information, The high abundance of archaea in deep-sea ecosystems to withstand extreme environments. Known archaeal National Library of Medicine, and their metabolic diversity6 suggest that they have viruses have been isolated from terrestrial and marine Bethesda, Maryland 20894, USA. a substantial impact on global nitrogen and carbon thermal environments with temperatures exceeding cycles. Moreover, it has been demonstrated that deep- 80 °C and hypersaline lakes with nearly saturating con- Correspondence to D.P. benthic and M.K. sea archaea have an important role in recycling centrations of sodium chloride. The viruses that were iso- 7,8 [email protected]; ­sedimentary organic compounds . lated from thermal environments infect hyperthermophilic [email protected] Concurrently, the abundance of archaeal viruses in members of the orders Sulfolobales, doi:10.1038/nrmicro.2017.125 the oceans has also been documented. Metagenomic and Thermoproteales from the phylum Published online 10 Nov 2017 analyses of double-stranded DNA (dsDNA) viruses in as well as the order Thermococcales from the phylum

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Box 1 | Metagenomics of archaeal viruses Many metagenomic studies have considerably expanded our perception of the global diversity of archaeal viruses. These studies were carried out on a variety of environmental samples from extreme habitats that are dominated by archaea, such as terrestrial hot springs141, hypersaline lakes140 and salt pans142, but also from various oceanic sites9,10,143–145 from which archaeal viruses had not been previously isolated. Several complete or near-complete genome sequences have been assembled for hyperthermophilic archaeal viruses of the families Rudiviridae23,141,146, Fuselloviridae147, Lipothrixviridae141, Bicaudaviridae141 and Ampullaviridae141 as well as hyperhalophilic viruses of the order Caudovirales148,149 and the Salterprovirus142. Two new genomes of uncultivated ampullaviruses are of particular value141 because the ampullavirus Acidianus bottle-shaped virus (ABV) was the sole member of the family for over a decade83. Approximately 50 complete genomes of head–tailed haloviruses have been cloned and sequenced148–150, providing the first genomic insights into viruses of Haloquadratum walsbyi, a squared archaeon that is found in hypersaline environments. These uncultivated viruses have been further investigated using metatranscriptomics, which indicated dynamic virus–host interactions in hypersaline settings151. Furthermore, several genomes have been assembled for hyperthermophilic archaeal viruses that cannot be assigned to existing families141,152, indicating that our knowledge of viral diversity in extreme geothermal environments remains limited. In particular, a positive-strand RNA virus genome that is distantly related to eukaryotic RNA viruses has been assembled from metagenomic sequences from hot springs in Yellowstone National Park, USA, that are dominated by the crenarchaeon Sulfolobus solfataricus, but the actual host of this has not been confirmed153. Perhaps the greatest advance that has been facilitated by metagenomic studies has been the discovery of the viral Methanogenic diversity that is associated with ubiquitous, environmentally important archaea, many of which remain uncultivated. Producing methane as a Most notably, over 50 genomes have been assembled for viruses, putatively called Magroviruses, associated with Marine metabolic by-product in anoxic Group II Euryarchaeota, some of the most abundant microorganisms in ocean surface waters144,145. Magroviruses have conditions. large genomes of ~100 kb; they are most closely related to members of the that infect halophilic archaea and have similar-sized genomes12. Magroviruses encode a nearly complete DNA replication apparatus; overall, Benthic archaeal viruses appear to follow the general trend that is observed among double-stranded DNA (dsDNA) viruses, Related to the ecological whereby viruses with larger genomes approach self-sufficiency for genome replication154. region at the lowest sea level, including the sediment surface Thaumarchaeota is another group of archaea that is ubiquitous in aquatic and terrestrial environments. Except for 53 and some subsurface layers. a single putative provirus , no viruses have been described for this group of archaea. However, two putative thaumarchaeal virus genomes of the order Caudovirales have been obtained through single-cell genomics and Epipelagic zone sequencing approaches155,156. Uncultured viruses have also been described for nano-sized archaea known as Archaeal The illuminated zone at the Richmond Mine acidophilic nanoorganisms (ARMAN)157,158. Notably, one of these viruses, ANMV1, also a member of the surface of the sea where Caudovirales, has been found to encode a diversity-generating retroelement, which uses mutagenic reverse transcription enough light is available for and retrohoming with the potential to generate 1018 variants of the tail fibre ligand-binding domain158. photosynthesis. Although the uncultivated viruses that are described above remain unclassified, the International Committee on of Viruses (ICTV) has recently issued a recommendation to classify viruses that are known solely by their Mesopelagic zone 159 The zone close to the sea genome sequence . Owing to this change, the number of recognized species of archaeal viruses is expected to surface in which light increase substantially in the future. penetrates but is insufficient for photosynthesis.

Hyperthermophilic Euryarchaeota, whereas viruses from saline waters infect last archaeal common ancestor13. Alternatively, archaeal Having an optimal growth hyperhalophilic members of the class Halobacteria, also RNA viruses may exist but have yet to be discovered temperature at or above from the Euryarchaeota (see Supplementary information (BOX 1). Owing to the unique features of their virions and 80 °C. S1 (table) for the complete list of characterized archaeal genomes, characterized archaeal viruses are ­classified Fosmid sequencing viruses and their taxonomic classification). In addition, a into 17 virus families (TABLE 1). Sequencing of large DNA number of uncultured archaeal viruses have been found In this Review, we summarize the current knowledge fragments cloned into a fosmid. by using metagenomics­ but remain unclassified (BOX 1). of archaeal virus morphotypes and structures, their

Mutagenic reverse All archaeal viruses that have been isolated to date genome architectures, their mechanisms of genome transcription and have either dsDNA or single-stranded DNA (ssDNA) replication and virion egress and their interactions with retrohoming genomes. Their genomes are generally small and range CRISPR–Cas systems, and we discuss their evolution Targeted replacement of a in size from 5.2 kb for the clavavirus pernix and potential origins. variable repeat coding region bacilliform virus 1 (APBV1)11, which is among the small- within a gene with a sequence derived from reverse est known dsDNA genomes, to 144 kb for Halogranum Virion morphology transcription of a cognate tailed virus 1 (HGTV1), a head–tailed virus that infects Archaeal viruses can be broadly divided into two cate- non-coding template repeat. Halogranum spp.12. The dsDNA genomes are either cir- gories: archaea-specific viruses that have no structural cular or linear. Archaeal viruses with linear genomes or genetic counterparts among bacterial or eukaryotic Hyperhalophilic Requiring extremely high levels employ different strategies for the protection and repli- viruses and cosmopolitan viruses that possess struc- of sodium chloride for growth. cation of their genome ends, including covalently closed tural and genetic features that are similar to those of hairpins and proteins that are covalently attached to the ­bacterial and eukaryotic viruses. Last archaeal common termini. Considering the low stability of long RNA mol­ ancestor ecules at high temperature, at least in the extracellular Archaea-specific viruses The most recent population of organisms from which all environment, it has been suggested that the absence of Nearly all known archaea-specific viruses infect hyper- extant archaea have a common RNA viruses among known archaeal viruses could be thermophiles of the phylum Crenarchaeota. They have descent. due to the postulated hyperthermophilic lifestyle of the a range of unique morphologies that have never been

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Capsid observed among viruses that infect bacterial or eukary- are generally highly pleomorphic and The protein shell that encloses otic cells, such as shapes that resemble bottles, spindles, have tails of different lengths at one or both pointed ends the genetic material of the droplets and coils, in addition to morphologies common­ of the spindle-shaped body18–23. For the type species of virus. in the virosphere, such as spheres and filaments. the Bicaudaviridae, Acidianus two-tailed virus (ATV), the Convergent evolution tails, which were shown to develop extracellularly, consist 24,25 The independent evolution of Viruses with unique morphologies. Arguably, archaeal of helically arranged globular subunits . similar features in species viruses with the most unusual morphologies are mem- Another unusual virion morphology is observed of different lineages. bers of the family . The champagne-­ in members of the family . Guttaviruses bottle-like shape of their virions is determined by the resemble spindles in which one of the two pointed ends extraordinary manner in which the linear dsDNA has been rounded, rendering them droplet-shaped26,27 genome is condensed by the capsid proteins into a cone- (FIG. 1a). Notably, guttaviruses share several genes in shaped inner core and encased with the lipid-containing­ common with fuselloviruses28, suggesting that the two envelope14 (FIG. 1a). The uncommon coil-like virion families of viruses are evolutionarily related. shape of members of the family is also determined by a special method of genome packing15; Viruses with common morphologies. Despite their the circular nucleoprotein filament formed from ssDNA simi­lar shape, filamentous archaeal viruses with dsDNA and capsid proteins is condensed into a rope-like struc- genomes are unrelated to filamentous bacterial and ture that is further ­condensed into a ­higher-order eukaryotic viruses. These filamentous archaeal viruses helix (FIG. 1a). belong to the families Rudiviridae, , Some of the most widespread and abundant Tristromaviridae and . The tubular, rigid archaea-specific viruses have spindle-shaped viri- and non-enveloped virions of the Rudiviridae are ons. Viruses that have this morphology belong to formed by the condensation of the linear dsDNA the or the Bicaudaviridae16 (FIG. 1a). through the binding of multiple copies of the single Members of these two families share little sequence MCP that adopts an unusual four helix-bundle fold. similarity, and their virions consist of unrelated major The ends of the viruses have three thin fibres that capsid proteins (MCPs), suggesting that their shared are involved in host cell recognition29–32 (FIG. 1a). The morphology is the result of convergent evolution. The flexible enveloped virions of Lipothrixviridae contain virions of the Fuselloviridae have a bundle of filaments a nucleoprotein core that is formed by linear dsDNA at one of the two pointed ends and show a certain and multiple copies of two MCPs, which are structur- degree of pleo­morph­icity17. The mature virions of the ally similar to each other and to the rudivirus MCP33–35.

Table 1 | Representative viruses of the Archaea Family Species Host Genome topology Refs and length (bp) Ampullaviridae Acidianus bottle-shaped virus Acidianus convivator L 23,900 14 Bicaudaviridae Acidianus two-tailed virus A. convivator C 62,730 25 Spiraviridae Aeropyrum coil-shaped virus C* 24,893 nt 15 Fuselloviridae Sulfolobus spindle-shaped virus 1 Sulfolobus shibatae C 15,465 162 Guttaviridae Sulfolobus neozealandicus Sulfolobus neozealandicus NA 26 droplet-shaped virus Aeropyrum pernix ovoid virus 1‡ A. pernix C 13,769 27 Rudiviridae Sulfolobus islandicus rod-shaped virus 2 Sulfolobus islandicus L 35,450 29 Lipothrixviridae Acidianus filamentous virus 1 Acidianus hospitalis L 21,080 78 Tristromaviridae filamentous virus 1 Pyrobaculum arsenaticum L 17,714 38 Clavaviridae Aeropyrum pernix bacilliform virus 1 A. pernix C 5,278 11 Pyrobaculum spherical virus Pyrobaculum sp. D11 L 28,337 160 Portogloboviridae Sulfolobus polyhedral virus 1 S. shibatae L 20,222 39 Halorubrum pleomorphic virus 1 Halorubrum spp. C* 7,048 nt 44 Halorubrum sodomense tailed virus 2 Halorubrum sodomense L 68,187 49 Haloarcula vallismortis tailed virus 1 Haloarcula vallismortis L 102,32 49 Haloarcula sinaiiensis tailed virus 1 Haloarcula sinaiiensis L 32,189 50 Sphaerolipoviridae Haloarcula hispanica virus SH1 Haloarcula hispanica L 30,898 163 Sulfolobus turreted icosahedral virus Sulfolobus solfataricus C 17,663 164 Listed are archaeal viruses shown in FIG. 1. All genomes — except those marked with asterisks — are double-stranded DNA. C, covalently closed circular DNA; L, linear DNA; NA, not available. *Single-stranded DNA.‡The only member of the family with a sequenced genome.

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The structures at the ends of filamentous virions differ long, non-contractile tails, whereas the sheath-covered among family members36; for instance, the structures of tails of myoviruses are contractile and podoviruses Acidianus filamentous virus 1 (AFV1) ends are claw-like have short tails. Members of all three families of caudo­ in appearance (FIG. 1a). The high structural similarity of viruses have been isolated from archaea49,50. Although the MCPs, as well as the fairly high numbers of homolo­ the majority of these isolates infect hyperhalophiles, gous genes (see below), indicate a common ances- head–tailed viruses have also been isolated from try of the non-enveloped Rudiviridae and enveloped methanogens­ 51. Furthermore, related proviruses have Lipothrixviridae, and the two families have ­therefore been identified in the genomes of a wide range of eury­ been classified together in the order Ligamenvirales37. archaea, including members of the classes Halobacteria, The filamentous virions of the Tristromaviridae Methanomicrobia, Archaeoglobi, Methanobacteria contain three MCPs, of which two condense the linear and Methanococci52, as well as members of the dsDNA into a helical nucleoprotein core and the third phylum Thaumarchaeota53. forms a protein sheath over this nucleoprotein core Cosmopolitan, tailless icosahedral viruses of and mediates its interaction with the lipid-containing archaea are related to bacterial and eukaryotic icosa- ­envelope of the virion. Both ends of the virion are dec- hedral viruses with capsid proteins that have a double orated with bundles of thin filaments38 (FIG. 1a). The or a single jelly-roll fold. These viruses are classified bacilliform Clavaviridae are non-enveloped and encode into two families, Turriviridae and Sphaerolipoviridae, a single MCP; the ends of the virion are asymmetric — respectively. The latter family consists of three genera, one is rounded, and the other is slightly pointed11. The of which two include viruses that infect halophilic virion does not have any terminal filaments, and it is archaea, whereas viruses from the other genus infect unclear how it interacts with the host cell (FIG. 1a). bacteria54. By contrast, turriviruses have been found to Spherical archaea-specific viruses are classified infect only crenarchaea from the genus Sulfolobus55. The into the families Globuloviridae and Portogloboviridae. overall virion organization is similar in the two groups Virions of the Globuloviridae have a lipid-­containing of viruses: the icosahedral protein capsid encases a envelope and a superhelical nucleoprotein core protein­aceous membrane vesicle that contains a dsDNA that contains linear dsDNA. In the virions of the genome, which is either linear or circular54–56. The main Portogloboviridae, the circular dsDNA genome is con- difference between viruses in the two families is that densed by capsid proteins into a spherical nucleopro- the turrivirus capsids are built from a single MCP55,57, tein coil, which forms an inner core of the virion. The whereas sphaerolipoviruses have two paralogous­ spherical inner core is surrounded by a lipid membrane MCPs54,56. and further encased by an outer icosahedral protein The morphological diversity of archaeal viruses is shell39 (FIG. 1a). remarkable, especially when compared with bacterial Viruses of the family Pleolipoviridae have envel- viruses, which have a limited range of morphologies, oped pleomorphic virions that resemble membrane despite their enormous genetic variability. The vast vesicles that are widely produced by archaea40 (FIG. 1a). morphological landscape of the archaeal virosphere These viruses lack a distinct nucleocapsid that is typi- is shaped by only a small number of species, which cal of enveloped viruses; instead, the naked genome is represent 2% of the >6,000 species of bacterial viruses packaged into a membrane vesicle that contains two that have been characterized46. Moreover, no new membrane-spanning viral proteins41,42. Remarkably, morphologies of bacterial viruses have been identified pleolipovirus particles can contain either linear dsDNA since the 1970s, despite the isolation of hundreds of or circular ssDNA or dsDNA genomes43–45. new species. By contrast, the variety of archaea-specific­ viral morphotypes is continuously expanding with the Cosmopolitan viruses description of viruses from new environments and The cosmopolitan viruses of the archaeal virosphere hosts. The evolutionary factors that drive this strik- include head–tailed viruses — viruses with icosahedral ing diversification of the archaeal virion shape are not capsids (heads) and helical appendages (tails) attached yet understood. to them — of the order Caudovirales and tailless icosa­hedral viruses of the families Sphaerolipoviridae Virion structures of archaeal viruses and Turriviridae (FIG. 1b). All of these viruses, except The remarkable morphological diversity of archaeal for turri­viruses, infect members of the phylum viruses raises important questions regarding their ori- Euryarchaeota; the biological basis for such host speci­ gins and their relationship to viruses that infect organ- ficity remains unknown and difficult to understand, isms from other domains of life. Several recent studies as some of the Euryarchaeota inhabit extreme habitats tried to address these questions by using cryo-electron

Proviruses similar to those of Crenarchaeota. microscopy (cryo‑EM) to determine the structures of Viral genomes integrated into Archaeal members of the order Caudovirales several archaeal viruses (FIGS 2,3). Structural studies have the host chromosome. are morphologically indistinguishable from head– revealed previously unexpected evolutionary relation- tailed bacterial viruses46–48. The three families of ships between viruses, which could not be predicted Jelly-roll fold the Caudovirales — Siphoviridae, Myoviridae and using genome sequence data alone, and have facilitated A structural protein fold composed of eight β‑strands Podoviridae — were originally classified according to our understanding of the molecular details that under- arranged in two antiparallel the morphology of their tails, which serve as cell rec- lie the ability of archaeal viruses to withstand extreme four-stranded β‑sheets. ognition and penetration devices. Siphoviruses have environments.

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50 Homology modelling Structures of archaeal viruses from all three families have the HK97‑like fold , a prevalent MCP topology The construction of an of the Caudovirales were determined using cryo‑EM. that is found in all tailed and in eukar- atomic-resolution model of the The capsids of the myovirus Haloarcula vallismortis yotic herpes­viruses (FIG. 2); these findings indicate that protein from its amino acid tailed virus 1 (HVTV1) and the siphovirus Halorubrum the lineage of viruses with the HK97‑like fold in their sequence and an experimental 58–61 three-dimensional structure of sodomense tailed virus 2 (HSTV2) were reconstructed MCPs spans all three domains of life . a related homologous protein. to ~10 Å resolution and the podovirus HSTV1 capsid to Cryo‑EM structures have also been determined 8.9 Å resolution49,50. The higher-resolution reconstruc- for viruses that belong to two groups of tailless archaeal tion of the HSTV1 capsid was sufficient to identify viruses with icosahedral capsids, namely, the Turriviridae the polypeptide backbone in the structure of the MCP, and Sphaerolipoviridae. The near-atomic structure of the and it validated earlier hypotheses on the evolutionary turrivirus Sulfolobus turreted icosahedral virus (STIV) relationship between viruses that infect hosts from the virion55 shows that the virion is constructed from several three domains of life. Homology modelling and sequence capsid proteins, most of which have the jelly-roll fold. The analyses52 revealed that MCPs of tailed archaeal viruses MCP has the double jelly-roll fold, which is widespread

a Archaea-specific viruses Ampullaviridae (ABV) Bicaudaviridae (ATV)

Fuselloviridae Guttaviridae Tristromaviridae Clavaviridae Globuloviridae Spiraviridae (ACV) (SSV1) (SNDV) (PFV1) (APBV1) (PSV)

Lipothrixviridae (AFV1) Rudiviridae (SIRV2) Portogloboviridae (SPV1)

Pleolipoviridae (HRPV1)

b Cosmopolitan archaeal viruses Sphaerolipoviridae Myoviridae (HSTV2) Siphoviridae (HVTV1) Podoviridae (HSTV1) (SH1) Turriviridae (STIV)

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57,62–64 A‑form in dsDNA viruses from all three domains of life , never been observed in a biological entity. However, One of the three major forms suggesting an evolutionary relationship between STIV the parameters of DNA packaging are different in the of double-stranded DNA, with and these other viruses (FIG. 2). Unlike members of the two virions: the twist of A‑form DNA in SIRV2 is a 23 Å helical diameter and Turriviridae, sphaerolipoviruses encode two MCPs, both 11.2 bp/turn, whereas in AFV1, it is 10.8 bp/turn. 11 bp per helix turn. of which have the single jelly-roll fold and form homo­ In the structures of the SIRV2 and AFV1 virions, the dimers and heterodimers that are the building blocks nucleoprotein helix is composed of asymmetric units, of the icosahedral capsid65,66 (FIG. 2). The two MCPs are each of which contains two molecules of the MCP: oriented in the capsid lattice similarly to the charac- a homodimer in the case of SIRV2 and a heterodimer in teristic orientation in viruses with MCPs that have the the case of AFV1. The helices from neighbouring protein double jelly-roll fold, such as turriviruses55. Furthermore, molecules are packed in an antiparallel, interdigitated members of Sphaerolipoviridae and Turriviridae encode arrangement and wrap around the DNA. The amino homologous genome-packaging­ ATPases and are thus terminus of the SIRV2 MCP, which is unstructured in considered to share a common ancestor67,68. solution, is folded in the virion into a helix-turn-helix The hypothesis that the unusual morphologies of structure and interacts with the DNA phosphate back- archaeal viruses reflect unknown forms of virion organ- bone through conserved polar and hydrophobic residues. ization is supported by three-dimensional reconstruc- In addition to the hydrophobic protein–­protein inter­ tions of filamentous virions of the rudivirus Sulfolobus actions across helical turns, this arrangement ensures that islandicus rod-shaped virus 2 (SIRV2) and the lipothrix- the SIRV2 DNA is completely encapsidated by protein virus AFV1 at near-atomic resolution33,69 (FIG. 3). In both and inaccessible to solvent (FIG. 3b), thereby maintaining virions, the dsDNA was found in the A‑form, which has its integrity under extreme conditions. By contrast, in the AFV1 virion, the protein–protein interactions across helical turns are absent, resulting in looser packaging ◀ Figure 1 | Electron micrographs of archaeal viruses. a | Archaea-specific viruses. Virus of the DNA, a thinner filament and greater flexibility of families and species are indicated. Acidianus filamentous virus 1 (AFV1), the inset shows the virion compared to SIRV2 (FIG. 3d). Consequently, the the terminal structure. Acidianus two-tailed virus (ATV), the arrows indicate virion tails lipid envelope of AFV1 seems to protect the viral DNA in (left), which undergo extracellular development (right). Pyrobaculum spherical virus (PSV), (FIG. 3e) the arrows indicate spherical protrusions. Negative stain with uranyl acetate, except for highly acidic environments . Aeropyrum coil-shaped virus (ACV), Halorubrum pleomorphic virus 1 (HRPV1), Sulfolobus In the structure of the AFV1 virion, the lipid enve- 69 islandicus rod-shaped virus 2 (SIRV2) and Sulfolobus polyhedral virus 1 (SPV1), which are lope is only 20–25 Å in thickness , half the thickness of in the vitreous ice. Bars, 100 nm. b | Cosmopolitan archaeal viruses. Halorubrum archaeal membranes70. The major lipids that are selec- sodomense tailed virus 2 (HSTV2), arrow and arrowhead point to the non-contracted and tively incorporated into the AFV1 membrane are contracted tails, respectively; Haloarcula hispanica virus SH1, open arrows point to the glycerol dibiphytanyl glycerol tetraethers that lack a claw-like spikes present at the five folds of the icosahedral capsid. Bars, 100 nm. cyclopentane moiety; as shown by molecular model- ABV, Acidianus bottle-shaped virus; APBV1, Aeropyrum pernix bacilliform virus 1; ling, they adopt a U‑shaped, ‘horseshoe’ conformation ATV, Acidianus two-tailed virus; HSTV1, Haloarcula sinaiiensis tailed virus 1; HVTV1, in the virion membrane (FIG. 3e,f). In addition to the Haloarcula vallismortis tailed virus 1; PFV1, Pyrobaculum filamentous virus 1; SNDV canonical lipid bilayer of the Bacteria and the Eukarya Sulfolobus neozealandicus droplet-shaped virus; SSV1, Sulfolobus spindle-shaped virus 1; STIV, Sulfolobus turreted icosahedral virus. Part a (ABV) is adapted with permission from and the archaeal lipid monolayer, the viral horseshoe American Society for Microbiology (REF. 14): Häring, M. et al. Viral diversity in hot springs membrane represents a third, previously unobserved of Pozzuoli, Italy, and characterization of a unique archaeal virus, Acidianus bottle-shaped type of biological membrane. Notably, the lipid envelope virus, from a new family, the Ampullaviridae. J. Virol. 79, 9904–9911 (2005) http://dx.doi. of the fusellovirus (SSV1) also appears to be thinner than org/10.1128/JVI.79.15.9904-9911.2005. Part a (ATV) left-panel is courtesy of R. Aramayo, the host cytoplasmic membrane and has a lipid composi- and the right-panel is adapted with permission from REF. 25, Elsevier. Part a (ACV) is tion that closely resembles that of AFV1 (REFS 69,71,72), adapted with permission from REF. 15, Proceedings of the National Academy of Sciences. suggesting that the U‑shaped conformation is a wide- Part a (APBV1) is adapted with permission from REF. 11, Elsevier. Part a (PSV) is adapted spread ­feature of archaeal virus membranes Notably, with permission from REF. 160, Elsevier. Part a (AFV1) is adapted with permission from the majority of viruses that infect hyperthermophiles REF. 78, Elsevier. Part a (PFV1) is adapted with permission from REF. 38, Proceedings of the are enveloped, suggesting that lipid membranes might National Academy of Sciences. Part a (SSV1) is adapted with permission from REF. 161, Springer. Part a (HRPV1) is adapted with permisson from the American Society for confer thermostability and provide a mechanism for 73 Microbiology (REF. 41): Pietilä, M. K. et al. Virion architecture unifies globally distributed these viruses to enter into and exit from the host cell . pleolipoviruses infecting halophilic archaea. J. Virol. 86, 5067–5079 (2012) http://dx.doi. The structures of spindle-shaped archaeal viruses org/10.1128/JVI.06915-11. Part a (SPV1) is adapted with permission from the American SSV1 and Haloarcula hispanica virus 1 (His1), which Society for Microbiology (REF. 39): Liu, Y. et al. A novel type of polyhedral viruses infecting infect hyperthermophilic-acidophilic and hyper­ hyperthermophilic archaea. J. Virol. 91, e00589-17 (2017) http://dx.doi.org/10.1128/ halophilic hosts, respectively, have also been recon- JVI.00589-17. Part a (SNDV) is adapted with permission from REF. 26, Elsevier. Part a (SIRV2) structed74,75. Owing to the inherent flexibility of SSV1 is adapted with permission from REF. 32, Biochemical Society Transactions http://dx.doi. and His1 virions, only fairly low-resolution models org/10.1042/BST20120313. Part b (HSTV1) is adapted with permission from the American of entire virions could be obtained (~32 Å and ~57 Å, Society for Microbiology (REF. 49): Pietilä, M. K. et al. Insights into head-tailed viruses respectively)76,77. Nevertheless, these models revealed infecting extremely halophilic archaea. J. Virol. 87, 3248–3260 (2013) http://dx.doi.org/ 10.1128/JVI.03397-12; and from REF. 50, Proceedings of the National Academy of interesting features of these virions; whereas the body Sciences. Part b (HSTV2) is adapted with permission from the American Society for of the His1 and SSV1 virions appears smooth and Microbiology (REF. 49): Pietilä, M. K. et al. Insights into head-tailed viruses infecting lacks major features, the terminal structures that dec- extremely halophilic archaea. J. Virol. 87, 3248–3260 (2013) http://dx.doi.org/10.1128/ orate one of the two pointed ends of the virions, and JVI.03397-12. Part b (SH1) is adapted with permission from REF. 65, Proceedings of the are presumably involved in viral attachment, display National Academy of Sciences. Part b (STIV) is courtesy of M. Young. six-fold symmetry.

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PBCV1 HCMV (’floor domain’)

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PRD1 HK97 P23-77

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Figure 2 | Major capsid proteins of cosmopolitan archaeal viruses. Homologous viral proteinsNature with Reviews the HK97 | Microbiology‑like fold, double jelly-roll (DJR) fold and single jelly-roll (SJR) fold are vertically aligned and arranged horizontally according to the domain of life to which their hosts are classified. Names of families of bacterial, archaeal and eukaryotic viruses sharing homologous capsid proteins are listed under the corresponding structures, and virus names are provided above the structural models. The structures are coloured according to the secondary structure elements: α‑helices, red; β‑strands, blue; and random coil, grey. The two major capsid proteins of bacterial and archaeal sphaerolipoviruses are boxed (dashed lines). Only the ‘floor’ domain61 of the human (HCMV) capsid protein is shown. The X‑ray structures of the capsid proteins of HCIV1 and HSTV1 are not available and are represented with homology-based models50,56. HCIV1, Haloarcula californiae icosahedral virus 1; HK97, Escherichia virus HK97; HSTV1, Haloarcula sinaiiensis tailed virus 1; NCLDV, nucleo-cytoplasmic large DNA viruses; P23‑77, Thermus virus P23‑77. PBCV1, Paramecium bursaria Chlorella virus 1; PRD1, Pseudomonas virus PRD1; STIV, Sulfolobus turreted icosahedral virus. RCSB Protein Data Bank (PDB) accession numbers for the major capsid protein structures: PBCV1, PDB entry 1J5Q; HCMV, PDB entry 5VKU; P23‑77 PDB entry 3ZMN (left) and 3ZN4 (right); PRD1 PDB entry 1HX6; HK97 PDB entry 1OHG; STIV PDB entry 1BBD.

Virus–host interactions in archaea primary stage of virus–host interactions29,78,79. This mode The morphological diversity of archaeal viruses is of interaction has been confirmed for the rudivirus matched by the range of mechanisms that are employed SIRV2, which initiates infection by binding to the tips by these viruses to interact with their hosts. In this sec- of the pilus-like appendages on Sulfolobus islandicus cells tion, we outline our current understanding of virus–host through three terminal fibres on the virion79 (FIG. 4a). interactions during archaeal viral life cycles, including The adsorption of SIRV2 is remarkably rapid: the viri- attachment and virus entry, genome replication, and ons irreversibly bind to the appendages within seconds virion morphogenesis and egress. and reach the cell surface within 1 minute (FIG. 4a). The cell surface proteins and type IV secretion proteins that Attachment and virus entry are involved in the SIRV2 entry have been identified80, Information on the early stages of infection by but the mechanisms of virion translocation and DNA archaea-specific viruses is scarce. Filamentous viruses ­injection remain unknown. that are bound through their ends to cellular appendages The spindle-shaped virions of Fuselloviridae and have been frequently observed using electron micros- Bicaudaviridae do not attach to cellular appendages. copy, which suggests that such contacts represent the Instead, they are often observed bound to cellular

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a Capsid Terminal fibres Nucleoprotein core ‘Claws’ fragments or membrane-derived vesicles, which implies that these viruses interact with receptors on the host cell surface to gain entry into cells. For a putative member Lipid envelope of the Bicaudaviridae, Sulfolobus monocaudavirus 1 b (SMV1), this primary interaction has been suggested to be followed by fusion between the virion envelope and the cell membrane21. The fusion mechanism for entry is also probably employed by members of the family Pleolipoviridae44. Similarly to bacterial viruses of the Caudovirales, the first stages of infection by archaeal head–tailed viruses appear to be mediated by the tail. For the myo- virus φCh1, which infects the haloalkaliphilic archaeon Natrialba magadii, the role of virion tail fibres in primary c adsorption has been experimentally demonstrated81. The C N N genome of φCh1 contains an invertible region that con- C tains genes that encode the tail fibre proteins separated by an invertase gene. The inversion leads to exchange of the carboxy-termini of the tail fibre proteins, thereby C creating protein variants with distinct cell surface ­adhesion specificities81. C N N Genome replication and packaging The mechanisms of genome replication have been d experimentally studied for only a small number of archaeal viruses, and in many cases, the mode of rep- lication has been inferred from the presence of genome replication-associated genes in viral genomes. Archaeal viruses of the order Caudovirales, which have the lar­ gest known genomes among archaeal viruses, encode

Figure 3 | Virion organization of filamentous viruses SIRV2 and AFV1. a | Schematic representations of e Sulfolobus islandicus rod-shaped virus 2 (SIRV2) (left) and Acidianus filamentous virus 1 (AFV1) (right). b | SIRV2 encapsidates the A‑form DNA. The left panel shows two turns of the virion nucleoprotein superhelix exposing a stretch of the viral A‑form DNA (in gold). The right panel shows a surface representation of how three dimers of the SIRV2 capsid protein bind to a stretch of the viral DNA, making it inaccessible to the solvent. c | Comparison of the homodimer of the SIRV2 capsid protein (left) with the heterodimer formed from two paralogous capsid proteins of AFV1 (right). The two monomers of SIRV2 capsid protein are shown in magenta and green, whereas the two capsid proteins of AFV1 are shown in red and yellow. d | Differences in the packing of the nucleoprotein superhelix in SIRV2 (left) and AFV1 (right). One homodimer and one heterodimer of f the capsid proteins are shown for SIRV2 and AFV1, GDGT-0 HO respectively. The proteins are coloured as in panel c. e | SIRV2 (left) and AFV1 (right) virions viewed along the long virion O O axis. Capsid proteins are shown in green. The outer ring O O surrounding the nucleoprotein core in AFV1 represents the OH lipid envelope. f | The structure of the main lipid component of the AFV1 envelope, glycerol dibiphytanyl glycerol tetraether (GDGT) lipid (GDGT‑0; top). Schematic representation (bottom left) and model (bottom right) of GDGT‑0 in the horseshoe conformation. The hydrophilic headgroups are shown in red. Part b (right-panel) is adapted with permission from REF. 33, American Association for the Advancement of Science. Part c is reproduced with permission from REF. 69, eLife. Part d (right-panel) and part e are adapted with permission from REF. 69, eLife.

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a 1

2 3

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1 4 UV 2 Cytoplasm 3 Appendages

5a Cell membrane VAP 4b 6a 4a 5b

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Figure 4 | Life cycles of lytic and temperate archaeal viruses. a | Steps of the interaction of the rudivirus Sulfolobus islandicus rod-shaped virus 2 (SIRV2) with the host cell: step 1 and step 2: progression of theNature virion (green) Reviews from | Microbiology the tip of a cellular appendage towards the cell surface; step 3: disassembly of the virion at the cell surface and delivery of the viral genome into the cell interior; step 4: replication of the viral genome in a discrete focus in the cell cytoplasm; step 5a: assembly of the virions, arranged into bundles containing ~50 viral particles; step 5b: concomitantly with virion assembly, pyramidal structures form on the cell surface, perforating the S‑layer; step 6a: disintegration of the virion bundles into separate, mature virions; and step 6b: opening of the pyramidal structures, allowing virion egress. b | Steps of the induction of the temperate fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1): step 1: UV irradiation leads to reactivation of the SSV1 provirus (orange), which is integrated in the chromosome of its host Sulfolobus shibatae (black circle); step 2: replication of the SSV1 genome; step 3: expression of viral proteins and formation of the viral nucleoprotein (red); step 4a and step 4b: budding of the viral nucleoprotein through the cytoplasmic membrane containing viral capsid proteins (green), resulting in formation of elongated virus-like particles; and step 5: maturation of the elongated particles into characteristic spindle-shaped virions and their detachment from the cytoplasmic membrane. The scheme is complemented with the tomographic surface-rendered volumes of SSV1 particles at different stages of budding. Red, putative nucleoprotein. Scale bars, 200 nm. The 3D models in part a are reproduced with permission from REF. 103, Proceedings of the National Academy of Sciences. The 3D models in part b are reproduced with permission from American Society for Microbiology (REF. 71): Quemin, E. R. et al. Eukaryotic-like virus budding in Archaea. mBio 7, Invertible region e01439‑16 (2016) http://dx.doi.org/10.1128/mBio.01439-16. A genome region that can excise and reintegrate into the same genome in inverted orientation. some or even most components of the DNA replication respective hosts on several independent occasions82. Protein-primed DNA machinery, including DNA polymerases, proliferat- Viruses with small genomes (10–20 kb) tend to encode polymerases ing cell nuclear antigen (PCNA; a DNA sliding clamp either protein-primed­ DNA polymerases74,83 or rolling-­circle DNA polymerases capable of ­replication 40,84 the protein-primed initiation that acts as a processivity factor for DNA polymerase), endo­nucleases (RCRE) , key proteins 85,86 step of DNA elongation. archaeo-eukaryotic primases and replicative helicases. for ssDNA replication in all three domains of life . Viruses with medium-sized genomes (20–50 kb) typ- It should be noted, however, that for many archaea-­ Rolling-circle replication ically encode only essential proteins that recruit the specific viruses, DNA replication proteins have not The model of unidirectional host DNA replication machinery. For example, the been found in their genomes, implying that these viruses DNA replication that can rapidly synthesize multiple replicative minichromosome-maintenance (MCM) rely primarily on the host DNA replication machinery copies of circular ssDNA complex (a heli­case component of the DNA replication or employ heretofore uncharacterized mechanisms of molecules. fork) has been acquired by different viruses from their genome replication. One such unique mechanism that

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appears to rely on recombination for both initiation and virion of the fusellovirus SSV1 acquires its envelope­ termination of genome replication was observed for the during budding of the nucleoprotein inner core lipothrixvirus AFV1, although the genes that facilitate through the cytoplasmic membrane of the host cell71 this mechanism are currently unknown87. (FIG. 4b). Virions first emerge as elongated particles that Most efforts in understanding genome replication are attached to the cell membrane and subsequently during archaeal virus infection have been directed undergo maturation into characteristic spindle-shaped towards the rudivirus SIRV2. Similar to some bacterial particles. Virus egress mechanisms strongly resemble viruses, SIRV2 DNA synthesis is confined to a region the budding of enveloped eukaryotic viruses such as near the periphery of the infected cells88. Although viral HIV, influenza virus and Ebola virus. Moreover, the DNA synthesis is performed by one of the four paralo­ formation of a constricted ‘neck’ that facilitates viral gous host DNA polymerases, namely, DNA polymer- budding is indistinguishable in appearance for SSV1 ase 1 (Dpo1), recruitment of the replication machinery and eukary­otic viruses. Many eukaryotic enveloped appears to be orchestrated by viral proteins. One of viruses use components of the endosomal sorting com- the early-expressed viral DNA-binding proteins, gp1 plexes required for transport (ESCRT) machinery for (REF. 89), interacts with the host-encoded sliding clamp budding94. The ESCRT pathway catalyses membrane PCNA and presumably recruits it for the assembly of fission events that are required for the abscission stage the replisome on the viral DNA90. Furthermore, SIRV2 of cytokinesis and for vesicle biogenesis95. The pres- encodes several proteins that are thought to be involved ence of homologues of the eukaryotic ESCRT proteins in DNA replication, recombination and repair, some of in the Sulfolobus spp., where they appear to perform which have been experimentally characterized. These similar functions96, suggests that the budding of SSV1 proteins include a Holliday junction resolvase, a ssDNA-­ and enveloped ­eukaryotic viruses may rely on similar binding protein, a ssDNA-annealing ATPase, a Cas4‑like cellular functions. ssDNA nuclease and a Rep protein that is homologous The morphogenesis of Bicaudaviridae virions is also to RCRE32. It was suggested that SIRV2 could employ likely to take place at the host cell surface and could a combination of strand-displacement, rolling-circle and be coupled to ESCRT-dependent virus budding97. The strand-coupled genome replication mechanisms, which virion morphogenesis of the type species of the family, generate multimeric and highly branched intermedi- ATV, is not completed at the cell surface: after being ates reaching >1,200 kb in size (~34 genome units)91. released, the spindle-shaped virions develop long However, whether all of these mechanisms occur dur- protrusions from the two pointed ends. High tem- ing each cycle of viral DNA replication and the specific perature, close to that of the natural environment, is proteins that are involved remain unclear. a prerequi­site for this morphological transformation, Genome packaging has not been studied in detail which is accompanied by contraction of the ‘spindle’ for any archaeal virus. However, based on compara­ and the longitudinal association of globular subunits of tive genomics, archaeal viruses of the Caudovirales unknown identity into helical, hollow tubes24. Virions order employ virion assembly, maturation and genome of SMV1, one of the several putative family members, packaging strategies similar to those that are used by can also develop tails in the extracellular environment21. bacterial viruses of the order. In particular, all archaeal However, for other bicaudaviruses, such as Sulfolobus caudoviruses encode homologues of the large terminase tengchongensis spindle-shaped viruses 1 and 2 (STSV1 subunit, an enzyme that packages the viral DNA into and STSV2), no extracellular stage of virion morpho- preassembled, empty capsids and subsequently processes genesis has been observed; once released from the cell, the concatameric viral DNA into single-genome-length the virion morphology does not appear to change20. units52. Similarly, members of the Turriviridae and By contrast, all stages of Rudiviridae and Turriviridae Sphaerolipoviridae families encode A32‑like packaging virion morphogenesis seem to occur in the cytoplasm ATPases of the FtsK–HerA superfamily, which is typical of the host cell. Mature virions exit the cells via a spe- of dsDNA viruses with MCPs that have double or single cial gateway structure that has a seven-fold symmetry, 68 Holliday junction resolvase jelly-roll folds . Moreover, the DNA packaging enzymes known as the virus-associated pyramid (VAP). The A highly specialized struc- of two turriviruses have been shown to have ATPase VAP develops on the surface of infected cells and pro- ture-selective endonuclease activity in vitro, and the proteins have been identified in trudes through the surface layer (S‑layer), and it opens as that cleaves four-way DNA virions92,93, as is also the case for bacterial viruses with the ‘flower petals’ at the end of the infection cycle98,99 (FIG. 4a). intermediates that can form 68 during DNA replication. homologous capsid proteins , emphasizing the evolu- The VAP consists of multiple copies of a 10 kDa viral tionary connection between these bacterial and archaeal protein100–102. The protein has a transmembrane domain Strand-displacement viruses. Thus, related viruses that infect hosts from dif- that facilitates its insertion into cellular membranes and Of genome replication, ferent domains of life encode homologous proteins for can form pyramidal structures in all tested biological involving the displacement of 103 a downstream DNA strand virion formation and genome packaging, whereas pro- membranes from all three domains of life . Pyramidal encountered during DNA teins that are involved in genome replication either are structures, albeit of six-fold symmetry, have been replication. unique or are recruited from the respective hosts. observed on the surface of uncultivated Pyrobaculum spp. cells that are infected with an unidentified filamen- Strand-coupled genome Virion morphogenesis and egress tous virus104 as well as on the surface of Pyrobaculum replication 105 The model of DNA replication The morphogenesis of pleomorphic virions of the oguniense , suggesting that the VAP-based virus egress that couples leading-strand Pleolipoviridae and of the spindle-shaped virions of mechanism is more common among crenarchaeal and lagging-strand synthesis. the Fuselloviridae is coupled with virion egress. The viruses than previously­ thought.

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The enveloped Tristromaviridae virions also appear conserved genes in archaeal viral genomes encode pro- to mature in the cytoplasm of the host cell. For exam- teins that are involved in virus–host interactions, for ple, at the late stages of the viral life cycle, the host cells example, in counteracting host defence systems, such are densely packed with the Pyrobaculum filamentous as CRISPR–Cas systems (see below). virus 1 (PFV1) virions (>100 per cell), and their release Structural and comparative genomics studies coincides with cell lysis38. The mechanism of intra­ clearly indicate that cosmopolitan archaeal viruses cellular acquisition of the lipid envelope by the virions share common ancestry with specific groups of bac- remains unclear. Release of the archaeal members of the terial viruses (Sphaerolipoviridae) or bacterial and order Caudovirales also occurs by cell lysis. Notably, the eukaryotic viruses­ (Turriviridae and Caudovirales)28,67. siphovirus ψM2, which infects Methanothermobacter By contrast, archaea-specific viruses have no counter- marburgensis, encodes pseudomurein endoisopeptidase parts or even strong phylogenetic relationships with (PeiP)51, which may be analogous to the endolysin com- bacteriophages or eukaryotic viruses, raising impor- ponent of the holin–endolysin lysis systems that are tant questions regarding their origins. Recently, the widely encoded by tailed bacteriophages106. Although evolutionary relationships between all dsDNA viruses PeiP mediates lysis of M. marburgensis cells in vitro107, were examined using the bipartite network approach, its participation in virion release or, alternatively, virus which traces connections between viral genomes entry has not been investigated in vivo. through shared gene families113. This analysis grouped Despite considerable gains in knowledge in recent all bacterial and eukaryotic viruses and the cosmo­ years, our understanding of virus–host interactions in politan archaeal viruses into four distinct supermodules, archaea remains incomplete. In particular, the molecular whereas archaea-specific viruses remained largely dis- mechanisms and, in many cases, the key genes that are connected from the global dsDNA virosphere67, despite involved in different stages of archaeal viral life cycles occasional ‘gene sharing’ with the other modules. The remain unknown. Future research is required to fill these subsequent detailed dissection of the archaea-specific gaps in our understanding and to provide novel insights virus network revealed strong modularity, with six dis- into virus–host co-evolution­ and molecular details of tinct modules, each including one or two virus families, the infection process under extreme environmental­ and four additional virus families disconnected from conditions. the rest of the archaeal virus network28. Compared to simi­lar networks of eukaryotic and bacterial viruses, the Genomics and evolutionary relationships archaea-specific viruses are sparsely connected within At the onset of archaeal virus research, the information the network, with only a few shared gene families, most that could be gained from archaeal virus genomes was notably the ribbon–helix–helix (RHH) DNA-binding limited owing to the high number of uncharacterized proteins and glycosyltransferases, shared by different and unique genes. The expansion of genomic data- network modules (FIG. 5). In these cases, the prevalence bases enabled the power of comparative genomics to of the two gene families in viral genomes most likely be applied to evolutionary studies of archaeal viruses. results from their independent acquisition from hosts In this section, we outline the current understanding and/or horizontal gene transfer between viruses. The of the evolutionary relationships between different lack of strong connectivity among the modules sug- groups of archaeal viruses and with bacteriophages, gests that most of the archaea-specific virus groups are eukaryotic viruses and non-viral mobile genetic evolutionarily distinct and have evolved independently elements (MGEs). of one another. The order , which The unique nature of viruses that infect hyper­ includes two fairly distant but clearly related families of Pseudomurein thermo­philic Crenarchaeota is limited not only to archaea-specific viruses and forms one of the modules endoisopeptidase their morphology but also to the content of their in the bipartite network, is the only notable exception (PeiP). An enzyme that cleaves genomes, with ~90% of the genes lacking detect­ discovered so far. pseudomurein cell-wall sacculi of the methanogens. able homologues (except those from other cren­ From whence did the archaea-specific viruses archaeal viruses) in existing sequence databases28,108. originate,­ and when did this happen? Why do they Endolysin The genomes of some archaeal viruses do not have a only infect archaea? There are at least two non-­ A type of peptidoglycan- single gene with a functionally characterized homo- mutually exclusive explanations. Some of the archaea-­ hydrolysing enzyme produced logue108. As the tertiary structure of proteins is more specific virus groups could have emerged during the by many bacterial viruses structural genomics towards the end of the conserved than genetic sequences, early stages of cellular evolution and been retained lytic cycle. has been employed to uncover potential functions and in the Archaea but lost in the domains Bacteria and the provenance of viral genes109. The crystal structures Eukarya114. Other archaeal virus groups could have Structural genomics of many archaeal viral proteins have been resolved; evolved concomitantly with the Archaea or, even The description of the three‑dimensional structure however, many of these proteins adopt unusual, novel more recently, within specific archaeal lineages. The of every protein encoded by folds, providing limited insight into the function and observed limited gene sharing between different groups a given genome. origin of these proteins110,111. Unexpectedly, in the case of archaea-specific viruses seems to make the latter pos- of the fusello­virus SSV1, almost half of the viral genes, sibility particularly plausible. The large diversity of the Supermodules many of which are functionally uncharacterized, are not archaea-specific viruses and the lack of evolutionary Clusters of modules of tightly connected genomes joined essential for infectivity and could be knocked out with- connections between different groups sharply contrast 112 through higher-level shared out obvious phenotypic effects . An intriguing possi- with the extensive exchange of genes among bacterio- genes. bility is that many of the uncharacterized and poorly phages that also extends to the cosmopolitan archaeal

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Archaeal virosphere encode replication initiation endonucleases homolo- Non-viral mobilome gous to the corresponding proteins encoded by small that replicate through rolling-circle repli- cation, such as the Archaeoglobus profundus pGS5 (REF. 118) and the Thermococcus prieurii plas- Ligamenvirales Clavaviridae mid pTP2 (REF. 119). In some archaeal viruses, for exam- ple, spindle-shaped Pyrococcus abyssi virus 1 (PAV1)120, nearly half of the viral genome shares homology with Tristromaviridae plasmids121. Thus, the replication modules of many archaea-specific viruses could be derived from non-­ viral MGEs. Combining such modules with the archi- Ampullaviridae tectural modules that provide viral structural genes GTase Caudovirales could have resulted in the emergence of the new viral lineages. The origins of the major virion proteins are Bicaudaviridae unknown for the majority of archaea-specific viruses. RHH Turriviridae However, many of these proteins have fairly simple DnaA Fuselloviridae folds that are widespread in cellular proteomes62. For example, the major virion proteins of rudiviruses and bicaudaviruses have distinct helical-bundle folds33,122, whereas those of spindle-shaped viruses presumably Sphaerolipoviridae comprise two highly hydrophobic α-helixes16. Thus, it is Guttaviridae possible that both of these proteins could evolve de novo into virion components in the context of archaeal virus Spiraviridae Globuloviridae genomes. Moreover, it was recently found that one of the major nucleocapsid proteins of Thermoproteus Portogloboviridae Pleolipoviridae tenax virus 1, a member of the family Tristromaviridae, was exapted from a truncated and inactivated archaeal Cas4‑like endonuclease123. The similar exaptation of cel- lular proteins to function as major virion components appears to be a recurrent theme in virus evolution62. Figure 5 | Schematic representation of the gene-sharing network among different Nature Reviews | Microbiology families of archaeal viruses. Viral families that share genes are grouped in modules that Archaeal viruses and CRISPR–Cas are highlighted in grey. Network modules were identified by applying a community detection algorithm to the bipartite network of gene sharing in the archaeal virosphere27. CRISPR–Cas systems provide an RNA-based mech- These modules can be interpreted as sharing common evolutionary history. Families anism to defend against invasive genetic elements outside grey circles form separate, single-family modules. Viruses from the same module in prokaryotes. The archaeal hyperthermophiles are share numerous genes (not shown in the figure), but gene sharing across modules is hosts to multiple groups of archaea-specific viruses limited to three widespread genes, namely, glycosyltransferase of the GT‑B superfamily with unique morphotypes and gene complements (GTase), ribbon–helix–helix-domain-containing protein (RHH) and an AAA+ superfamily (see above), and they also universally encompass ATPase homologous to bacterial DnaA. Bidirectional arrows indicate the connections CRISPR–Cas systems, often multiple loci of different between some families of archaeal viruses and capsidless (non-viral) mobile genetic types within the same genomes124. The ubiquity of elements that are discussed in the text. CRISPR–Cas systems in archaeal hyperthermophiles contrasts with their relative rarity among bacteria, <40% of which encode a CRISPR–Cas system, accord- viruses28,67,113,115. The biological basis of this disjointed ing to recent estimates124,125, and implies that CRISPR– organization of the archaea-specific virosphere remains Cas defence might have, to a large extent, shaped the enigmatic but could be clarified by further studies of evolution of the viral genomes. The presence of many archaeal virus–host interactions and a comprehensive genes that counteract host defences is a common fea- characterization of archaeal virus diversity. ture of viral genomes, as exemplified by the numerous Notably, several modules in the gene-sharing net- proteins that antagonize the immune system in work of archaeal viruses also include non-viral, cap- poxviruses126 and herpesviruses127 or RNAi suppres- sidless MGEs28, namely, plasmids and casposons sors in RNA viruses128. Moreover, multiple anti- (a recently discovered group of integrative MGEs that CRISPR proteins with specificity for different types and appear to have given rise to the CRISPR–Cas adaptation subtypes of CRISPR–Cas systems have been identified machinery responsible for the acquisition of new spa­ and studied in several bacteriophages129–131. Similar to cers from the invading viruses and plasmids)116,117. In all these viruses, archaeal viruses are expected to have these cases, viruses and non-viral MGEs share genes evolved anti-CRISPR counter defence strategies, but that encode major genome replication proteins. In par- they have so far not been identified. As many of the ticular, family 1 casposons encode protein-primed DNA proteins that are encoded by archaeal viruses are small, polymerases closely related to those found in ampulla­ poorly conserved and lack identifiable domains with viruses, the His1 and gamma­pleo­lipo­ known activities, similar to counter defence proteins virus His2 (REF. 116), whereas alphapleolipoviruses in general and anti-CRISPR proteins in particular,

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Mesophiles there are many candidates to screen. The search for Conclusions and outlook Organisms that grow best anti-CRISPR proteins in archaeal viruses is undoubt- Viruses of the archaea have remarkable virion architec- in moderate temperature, edly a promising research direction for understand- tures, genome structures, genes and modes of interaction typically between 20 and ing CRISPR–Cas immunity and counter defences with their hosts. Recent structural and functional studies 45 °C. in archaea. have further expanded the repertoire of unique structures CRISPR spacers It remains unclear why CRISPR–Cas systems are and mechanisms found in this part of the virosphere Short fragments of viral DNA ubiquitous in archaeal hyperthermophiles. One poten- through unexpected discoveries, such as the A‑form DNA from previous exposure to tial explanation is that the comparatively low mutation in virions, a new type of membrane in enveloped viruses the virus, inserted between repetitive sequences of the rate of viruses that propagate in extreme environments, and the VAPs. Understanding the biological and evolu- CRISPR–Cas system. combined with the lower effective population sizes of tionary basis of these unusual features is a major challenge hyperthermophiles compared to mesophiles, leads to for future research that will require the development of Protospacers fairly low virus diversity, which would maximize the new model systems for studying virus–host interactions Fragments of invading mobile benefits of immune memory; that is, the emergence in archaea. Concomitant metagenomic studies have led genetic element from which 132,133 CRISPR spacers are derived. of virus escape mutants is expected to be slower . to the discovery of new groups of archaeal viruses. Some Simply put, owing to the limited opportunity for virus of these, such as the magroviruses, are highly abundant in Primed adaptation escape, the CRISPR–Cas systems of hyperthermophiles the environment but remained unknown for a long period A process in which an existing appear to be more efficient in ‘remembering’ past of time because their hosts have not been cultivated. spacer against a foreign DNA promotes rapid and efficient infections and controlling the respective viruses than Moreover, concurrent environmental and geochemical acquisition of additional those of mesophiles. A recent comprehensive survey of research has revealed that archaeal viruses are important spacers from the same the CRISPR spacers in bacterial and archaeal genomes players in the biogeochemistry of the biosphere. foreign DNA. identi­fied a low proportion of protospacers with per- Despite substantial progress in the field of archaeal fect complementarity in both hyperthermophilic and virology, a comprehensive understanding of the global mesophilic archaea (~1% in hyperthermophilic archaea diversity of archaeal viruses is still lacking. Indeed, the compared to the mean value of ~7% among all bacte- viruses that have been characterized thus far infect only a rial and archaeal phyla). As expected, the majority of limited number of host taxa and have been isolated from the protospacers mapped to archaeal virus genomes134. specific environments. Future research should focus on The low proportion of spacers with a detectable proto- the isolation of new archaeal virus–host systems from a spacer in the viral or plasmid genome could be due to broader range of ecosystems. Of particular interest are the ability of different CRISPR–Cas systems, particu- viruses that infect currently uncultured archaea, many larly type I‑A and type III systems that are common in of which are abundant in the environment and have archaea124, to utilize spacers with multiple mismatches major roles in the biosphere. It is likely that advances for inter­ference and primed adaptation135–138. However, in this direction will be achieved by combining culture-­ the existence of uncharacterized, local viromes could dependent and culture-independent high-throughput be a complementary explanation for the scarcity of approaches, such as metagenomics, metaproteomics spacers that could be matched to known archaeal virus and single-cell genomics. This research is expected to genomes. In agreement with this hypothesis, it has been provide an unbiased view of the distribution, abundance shown that CRISPR arrays in archaeal communities and diversity of archaeal viruses, allowing more-­accurate are enriched in spacers that better align to local viral comparisons between bacterial and archaeal viruses genomes than foreign viral genomes139,140. and informing construction of evolutionary scenarios. Given the high prevalence of CRISPR–Cas systems in Another promising line of inquiry, boosted by the devel- archaea, especially hyperthermophiles, future detailed opment of diverse tools of molecular biology, genetics studies of the co-evolution of archaeal viruses with their and microscopy, should focus on deciphering the molec- hosts and the strategies that are exploited by archaeal ular mechanisms that underlie virus–host interactions. viruses to overcome CRISPR immunity could provide This research should yield crucial insights into the evo- valuable insights into the mechanisms and evolution of lutionary history of viruses, their adaptation to extreme prokaryotic adaptive immunity. ­environments and their co‑evolution with their hosts.

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