REVIEWS

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 archaea is the extraordinary diversity of their
DNA viruses. Many archaeal viruses differ substantially in morphology from bacterial and eukaryotic
viruses and represent unique virus 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 life 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
Requiring high temperatures
for optimal growth.
Acidophilic
Thriving under highly acidic
conditions.
Alkaliphilic
Thriving under highly alkaline
conditions.
Halophilic
Requiring high levels of sodium
chloride for growth.
1
Department of Microbiology,
Institut Pasteur, 25 rue du Dr
Roux, Paris 75015, France.
2
Department of Biosciences,
University of Helsinki,
Helsinki 00014, Finland.
3
National Center for
Biotechnology Information,
National Library of Medicine,
Bethesda, Maryland 20894,
USA.
Correspondence to D.P. 
and M.K.
david.prangishvili@pasteur.fr;
krupovic@pasteur.fr
doi:10.1038/nrmicro.2017.125
Published online 10 Nov 2017
The year 2017 marks the 40th anniversary of the dis-
covery of archaea1. The third domain of life, which at
the time of its discovery included only a few species,
has been extensively populated in recent years with
numerous thermophilic, acidophilic, alkaliphilic, h­ alophilic,
­ ethanogenic and ammonia-oxidizing archaea.
m Virus predation is one of the primary causes of
Moreover, the exploration of microbial diversity through
culture-independent approaches has substantially
expanded our understanding of archaeal diversity and
uncovered numerous species that may represent many
new phyla and orders of the Archaea2.
Culture-independent approaches have radically
changed the perception of the typical habitats of archaea.
The original notion that archaea thrive only in environ-
ments with extreme conditions was challenged and over-
turned by the discovery of archaea in a diverse range
of terrestrial and aquatic environments and even within
and on the human body 3. Remarkably, in deep-sea eco-
systems, which constitute ~90% of the global biosphere,
the abundance of archaea is comparable to that of bac-
teria4, and archaeal species that oxidize ammonium to
nitrate (members of the phylum Thaumarchaeota) rep-
resent one of the most abundant cell types in oceans5.
The high abundance of archaea in deep-sea ecosystems
and their metabolic diversity 6 suggest that they have
a substantial impact on global nitrogen and carbon
cycles. Moreover, it has been demonstrated that deep-
sea benthic archaea have an important role in recycling
­sedimentary organic compounds7,8.
Concurrently, the abundance of archaeal viruses in
the oceans has also been documented. Metagenomic
analyses of double-stranded DNA (dsDNA) viruses in
the epipelagic zone and the mesopelagic zone of the ocean
have shown that ~10% of the most abundant viruses in
these zones are associated with archaea9. Diverse archaeal
viruses have also been detected in benthic deep-sea
­ecosystems, where their turnover is as fast as 2–3 days10.
microbial mortality, with major consequences for
global biogeochemistry. It was recently shown that in
oceanic surface sediments across 1,000–10,000 m water
depths, viral infection has a higher impact on archaea
than on bacteria and mainly affects members of the
Thaumarchaeota. Moreover, in the top 50 cm of ocean
sediment, virus-induced lysis of archaea was estimated
to account for up to one-third of the total microbial bio-
mass that is killed each year, resulting in the release of
~0.3–0.5 gigatonnes of carbon per year 10. This realiza-
tion has changed the perception of archaeal viruses from
interesting curiosities of the virosphere to prominent
players in the biosphere.
In addition to having important roles in the function-
ing of deep-sea ecosystems and global biogeochemical
cycles, archaeal viruses attract attention owing to their
remarkable diversity, unique morphologies and ability
to withstand extreme environments. Known archaeal
viruses have been isolated from terrestrial and marine
thermal environments with temperatures exceeding
80 °C and hypersaline lakes with nearly saturating con-
centrations of sodium chloride. The viruses that were iso-
lated from thermal environments infect hyperthermophilic
members of the orders Sulfolobales, Desulfurococcales
and Thermoproteales from the phylum Crenarchaeota
as well as the order Thermococcales from the phylum

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Box 1 | Metagenomics of archaeal viruses

Methanogenic
Producing methane as a
metabolic by-product in anoxic
conditions.
Benthic
Related to the ecological
region at the lowest sea level,
including the sediment surface
and some subsurface layers.
Epipelagic zone
The illuminated zone at the
surface of the sea where
enough light is available for
photosynthesis.
Mesopelagic zone
The zone close to the sea
surface in which light
penetrates but is insufficient
for photosynthesis.
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 genus 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
diversity that is associated with ubiquitous, environmentally important archaea, many of which remain uncultivated.
Most notably, over 50 genomes have been assembled for viruses, putatively called Magroviruses, associated with Marine
Group II Euryarchaeota, some of the most abundant microorganisms in ocean surface waters144,145. Magroviruses have
large genomes of ~100 kb; they are most closely related to members of the Caudovirales that infect halophilic archaea
and have similar-sized genomes12. Magroviruses encode a nearly complete DNA replication apparatus; overall,
archaeal viruses appear to follow the general trend that is observed among double-stranded DNA (dsDNA) viruses,
whereby viruses with larger genomes approach self-sufficiency for genome replication154.
Thaumarchaeota is another group of archaea that is ubiquitous in aquatic and terrestrial environments. Except for
a single putative provirus53, 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 fosmid
sequencing approaches155,156. Uncultured viruses have also been described for nano-sized archaea known as Archaeal
Richmond Mine acidophilic nanoorganisms (ARMAN)157,158. Notably, one of these viruses, ANMV1, also a member of the
Caudovirales, has been found to encode a diversity-generating retroelement, which uses mutagenic reverse transcription
and retrohoming with the potential to generate 1018 variants of the tail fibre ligand-binding domain158.
Although the uncultivated viruses that are described above remain unclassified, the International Committee on
Taxonomy of Viruses (ICTV) has recently issued a recommendation to classify viruses that are known solely by their
genome sequence159. Owing to this change, the number of recognized species of archaeal viruses is expected to
increase substantially in the future.

Hyperthermophilic
Having an optimal growth
temperature at or above
80 °C.
Fosmid sequencing
Sequencing of large DNA
fragments cloned into a fosmid.
Mutagenic reverse
transcription and
retrohoming
Targeted replacement of a
variable repeat coding region
within a gene with a sequence
derived from reverse
transcription of a cognate
non-coding template repeat.
Hyperhalophilic
Requiring extremely high levels
of sodium chloride for growth.
Last archaeal common
ancestor
The most recent population
of organisms from which all
extant archaea have a common
descent.
Euryarchaeota, whereas viruses from saline waters infect
hyperhalophilic members of the class Halobacteria, also
from the Euryarchaeota (see Supplementary information
S1 (table) for the complete list of characterized archaeal
viruses and their taxonomic classification). In addition, a
number of uncultured archaeal viruses have been found
by using m­ etagenomics but remain unclassified (BOX 1).
All archaeal viruses that have been isolated to date
have either dsDNA or single-stranded DNA (ssDNA)
genomes. Their genomes are generally small and range
in size from 5.2 kb for the clavavirus Aeropyrum pernix
bacilliform virus 1 (APBV1)11, which is among the small-
est known dsDNA genomes, to 144 kb for Halogranum
tailed virus 1 (HGTV1), a head–tailed virus that infects
Halogranum spp.12. The dsDNA genomes are either cir-
cular or linear. Archaeal viruses with linear genomes
employ different strategies for the protection and repli-
cation of their genome ends, including covalently closed
hairpins and proteins that are covalently attached to the
termini. Considering the low stability of long RNA mol­
ecules at high temperature, at least in the extracellular
environment, it has been suggested that the absence of
RNA viruses among known archaeal viruses could be
due to the postulated hyperthermophilic lifestyle of the
last archaeal common ancestor13. Alternatively, archaeal
RNA viruses may exist but have yet to be discovered
(BOX 1). Owing to the unique features of their virions and
genomes, characterized archaeal viruses are ­classified
into 17 virus families (TABLE 1).
In this Review, we summarize the current knowledge
of archaeal virus morphotypes and structures, their
genome architectures, their mechanisms of genome
replication and virion egress and their interactions with
CRISPR–Cas systems, and we discuss their evolution
and potential origins.
Virion morphology
Archaeal viruses can be broadly divided into two cate-
gories: archaea-specific viruses that have no structural
or genetic counterparts among bacterial or eukaryotic
viruses and cosmopolitan viruses that possess struc-
tural and genetic features that are similar to those of
­bacterial and eukaryotic viruses.
Archaea-specific viruses
Nearly all known archaea-specific viruses infect hyper-
thermophiles of the phylum Crenarchaeota. They have
a range of unique morphologies that have never been

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Capsid
The protein shell that encloses
the genetic material of the
virus.
Convergent evolution
The independent evolution of
similar features in species
of different lineages.
observed among viruses that infect bacterial or eukary- Bicaudaviridae are generally highly pleomorphic and
otic cells, such as shapes that resemble bottles, spindles,
have tails of different lengths at one or both pointed ends
droplets and coils, in addition to morphologies c­ ommon of the spindle-shaped body 18–23. For the type species of
in the virosphere, such as spheres and filaments. the Bicaudaviridae, Acidianus two-tailed virus (ATV), the
tails, which were shown to develop extracellularly, consist
Viruses with unique morphologies. Arguably, archaeal of helically arranged globular subunits24,25.
viruses with the most unusual morphologies are mem- Another unusual virion morphology is observed
bers of the family Ampullaviridae. The champagne-­ in members of the family Guttaviridae. 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 Spiraviridae is also
determined by a special method of genome packing 15; 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 ­c ondensed into a ­higher-order eukaryotic viruses. These filamentous archaeal viruses
helix (FIG. 1a). belong to the families Rudiviridae, Lipothrixviridae,
Some of the most widespread and abundant Tristromaviridae and Clavaviridae. 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 Fuselloviridae or the Bicaudaviridae 16 (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 pleo­morph­icity 17. 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 Aeropyrum pernix 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 Pyrobaculum filamentous virus 1 Pyrobaculum arsenaticum L 17,714 38
Clavaviridae Aeropyrum pernix bacilliform virus 1 A. pernix C 5,278 11
Globuloviridae Pyrobaculum spherical virus Pyrobaculum sp. D11 L 28,337 160
Portogloboviridae Sulfolobus polyhedral virus 1 S. shibatae L 20,222 39
Pleolipoviridae Halorubrum pleomorphic virus 1 Halorubrum spp. C* 7,048 nt 44
Myoviridae Halorubrum sodomense tailed virus 2 Halorubrum sodomense L 68,187 49
Siphoviridae Haloarcula vallismortis tailed virus 1 Haloarcula vallismortis L 102,32 49
Podoviridae Haloarcula sinaiiensis tailed virus 1 Haloarcula sinaiiensis L 32,189 50
Sphaerolipoviridae Haloarcula hispanica virus SH1 Haloarcula hispanica L 30,898 163
Turriviridae 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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Proviruses
Viral genomes integrated into
the host chromosome.
Jelly-roll fold
A structural protein fold
composed of eight β‑strands
arranged in two antiparallel
four-stranded β‑sheets.
The structures at the ends of filamentous virions differ
among family members36; for instance, the structures of
Acidianus filamentous virus 1 (AFV1) ends are claw-like
in appearance (FIG. 1a). The high structural similarity of
the MCPs, as well as the fairly high numbers of homolo­
gous genes (see below), indicate a common ances-
try of the non-enveloped Rudiviridae and enveloped
Lipothrixviridae, and the two families have ­therefore
been classified together in the order Ligamenvirales 37.
The filamentous virions of the Tristromaviridae
contain three MCPs, of which two condense the linear
dsDNA into a helical nucleoprotein core and the third
forms a protein sheath over this nucleoprotein core
and mediates its interaction with the lipid-containing
­envelope of the virion. Both ends of the virion are dec-
orated with bundles of thin filaments38 (FIG. 1a). The
bacilliform Clavaviridae are non-enveloped and encode
a single MCP; the ends of the virion are asymmetric —
one is rounded, and the other is slightly pointed11. The
virion does not have any terminal filaments, and it is
unclear how it interacts with the host cell (FIG. 1a).
Spherical archaea-specific viruses are classified
into the families Globuloviridae and Portogloboviridae.
Virions of the Globuloviridae have a lipid-­containing
envelope and a superhelical nucleoprotein core
that contains linear dsDNA. In the virions of the
Portogloboviridae, the circular dsDNA genome is con-
densed by capsid proteins into a spherical nucleopro-
tein coil, which forms an inner core of the virion. The
spherical inner core is surrounded by a lipid membrane
and further encased by an outer icosahedral protein
shell39 (FIG. 1a).
Viruses of the family Pleolipoviridae have envel-
oped pleomorphic virions that resemble membrane
vesicles that are widely produced by archaea40 (FIG. 1a).
These viruses lack a distinct nucleocapsid that is typi-
cal of enveloped viruses; instead, the naked genome is
packaged into a membrane vesicle that contains two
membrane-spanning viral proteins41,42. Remarkably,
pleolipovirus particles can contain either linear dsDNA
or circular ssDNA or dsDNA genomes43–45.
Cosmopolitan viruses
The cosmopolitan viruses of the archaeal virosphere
include head–tailed viruses — viruses with icosahedral
capsids (heads) and helical appendages (tails) attached
to them — of the order Caudovirales and tailless
icosa­hedral viruses of the families Sphaerolipoviridae
and Turriviridae (FIG. 1b). All of these viruses, except
for turri­v iruses, infect members of the phylum
Euryarchaeota; the biological basis for such host speci­
ficity remains unknown and difficult to understand,
as some of the Euryarchaeota inhabit extreme habitats
similar to those of Crenarchaeota.
Archaeal members of the order Caudovirales
are morphologically indistinguishable from head–
tailed bacterial viruses 46–48. The three families of
the Caudovirales — Siphoviridae, Myoviridae and
Podoviridae — were originally classified according to
the morphology of their tails, which serve as cell rec-
ognition and penetration devices. Siphoviruses have
long, non-contractile tails, whereas the sheath-covered
tails of myoviruses are contractile and podoviruses
have short tails. Members of all three families of caudo­
viruses have been isolated from archaea49,50. Although
the majority of these isolates infect hyperhalophiles,
head–tailed viruses have also been isolated from
methano­gens51. Furthermore, related proviruses have
been identified in the genomes of a wide range of eury­
archaea, including members of the classes Halobacteria,
Methanomicrobia, Archaeoglobi, Methanobacteria
and Methanococci 52 , as well as members of the
phylum Thaumarchaeota53.
Cosmopolitan, tailless icosahedral viruses of
archaea are related to bacterial and eukaryotic icosa-
hedral viruses with capsid proteins that have a double
or a single jelly-roll fold. These viruses are classified
into two families, Turriviridae and Sphaerolipoviridae,
respectively. The latter family consists of three genera,
of which two include viruses that infect halophilic
archaea, whereas viruses from the other genus infect
bacteria54. By contrast, turriviruses have been found to
infect only crenarchaea from the genus Sulfolobus 55. The
overall virion organization is similar in the two groups
of viruses: the icosahedral protein capsid encases a
protein­aceous membrane vesicle that contains a dsDNA
genome, which is either linear or circular 54–56. The main
difference between viruses in the two families is that
the turrivirus capsids are built from a single MCP55,57,
whereas sphaerolipoviruses have two p ­ aralogous
MCPs54,56.
The morphological diversity of archaeal viruses is
remarkable, especially when compared with bacterial
viruses, which have a limited range of morphologies,
despite their enormous genetic variability. The vast
morphological landscape of the archaeal virosphere
is shaped by only a small number of species, which
represent 2% of the >6,000 species of bacterial viruses
that have been characterized 46. Moreover, no new
morphologies of bacterial viruses have been identified
since the 1970s, despite the isolation of hundreds of
new species. By contrast, the variety of archaea-­specific
viral morphotypes is continuously expanding with the
description of viruses from new environments and
hosts. The evolutionary factors that drive this strik-
ing diversification of the archaeal virion shape are not
yet understood.
Virion structures of archaeal viruses
The remarkable morphological diversity of archaeal
viruses raises important questions regarding their ori-
gins and their relationship to viruses that infect organ-
isms from other domains of life. Several recent studies
tried to address these questions by using cryo-electron
microscopy (cryo‑EM) to determine the structures of
several archaeal viruses (FIGS 2,3). Structural studies have
revealed previously unexpected evolutionary relation-
ships between viruses, which could not be predicted
using genome sequence data alone, and have facilitated
our understanding of the molecular details that under-
lie the ability of archaeal viruses to withstand extreme
environments.

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Homology modelling
The construction of an
atomic-resolution model of the
protein from its amino acid
sequence and an experimental
three-dimensional structure of
a related homologous protein.
Structures of archaeal viruses from all three families
of the Caudovirales were determined using cryo‑EM.
The capsids of the myovirus Haloarcula vallismortis
tailed virus 1 (HVTV1) and the siphovirus Halorubrum
sodomense tailed virus 2 (HSTV2) were reconstructed
to ~10 Å resolution and the podovirus HSTV1 capsid to
8.9 Å resolution49,50. The higher-resolution reconstruc-
tion of the HSTV1 capsid was sufficient to identify
the polypeptide backbone in the structure of the MCP,
and it validated earlier hypotheses on the evolutionary
relationship between viruses that infect hosts from the
three domains of life. Homology modelling and sequence
analyses52 revealed that MCPs of tailed archaeal viruses
have the HK97‑like fold50, a prevalent MCP topology
that is found in all tailed bacteriophages and in eukar-
yotic herpes­viruses (FIG. 2); these findings indicate that
the lineage of viruses with the HK97‑like fold in their
MCPs spans all three domains of life58–61.
Cryo‑EM structures have also been determined
for viruses that belong to two groups of tailless archaeal
viruses with icosahedral capsids, namely, the Turriviridae
and Sphaerolipoviridae. The near-atomic structure of the
turrivirus Sulfolobus turreted icosahedral virus (STIV)
virion55 shows that the virion is constructed from several
capsid proteins, most of which have the jelly-roll fold. The
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)

Nature Reviews | Microbiology

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A‑form in dsDNA viruses from all three domains of life57,62–64,
One of the three major forms suggesting an evolutionary relationship between STIV
of double-stranded DNA, with and these other viruses (FIG. 2). Unlike members of the
a 23 Å helical diameter and Turriviridae, sphaerolipoviruses encode two MCPs, both
11 bp per helix turn.
of which have the single jelly-roll fold and form homo­
dimers and heterodimers that are the building blocks
of the icosahedral capsid65,66 (FIG. 2). The two MCPs are
oriented in the capsid lattice similarly to the charac-
teristic orientation in viruses with MCPs that have the
double jelly-roll fold, such as turriviruses55. Furthermore,
members of Sphaerolipoviridae and Turriviridae encode
homologous genome-­packaging ATPases and are thus
considered to share a common ancestor 67,68.
The hypothesis that the unusual morphologies of
archaeal viruses reflect unknown forms of virion organ-
ization is supported by three-dimensional reconstruc-
tions of filamentous virions of the rudivirus Sulfolobus
islandicus rod-shaped virus 2 (SIRV2) and the lipothrix-
virus AFV1 at near-atomic resolution33,69 (FIG. 3). In both
virions, the dsDNA was found in the A‑form, which has
◀ Figure 1 | Electron micrographs of archaeal viruses. a | Archaea-specific viruses. Virus
families and species are indicated. Acidianus filamentous virus 1 (AFV1), the inset shows
the terminal structure. Acidianus two-tailed virus (ATV), the arrows indicate virion tails
(left), which undergo extracellular development (right). Pyrobaculum spherical virus (PSV),
the arrows indicate spherical protrusions. Negative stain with uranyl acetate, except for
Aeropyrum coil-shaped virus (ACV), Halorubrum pleomorphic virus 1 (HRPV1), Sulfolobus
islandicus rod-shaped virus 2 (SIRV2) and Sulfolobus polyhedral virus 1 (SPV1), which are
in the vitreous ice. Bars, 100 nm. b | Cosmopolitan archaeal viruses. Halorubrum
sodomense tailed virus 2 (HSTV2), arrow and arrowhead point to the non-contracted and
contracted tails, respectively; Haloarcula hispanica virus SH1, open arrows point to the
claw-like spikes present at the five folds of the icosahedral capsid. Bars, 100 nm.
ABV, Acidianus bottle-shaped virus; APBV1, Aeropyrum pernix bacilliform virus 1;
ATV, Acidianus two-tailed virus; HSTV1, Haloarcula sinaiiensis tailed virus 1; HVTV1,
Haloarcula vallismortis tailed virus 1; PFV1, Pyrobaculum filamentous virus 1; SNDV
Sulfolobus neozealandicus droplet-shaped virus; SSV1, Sulfolobus spindle-shaped virus 1;
STIV, Sulfolobus turreted icosahedral virus. Part a (ABV) is adapted with permission from
American Society for Microbiology (REF. 14): Häring, M. et al. Viral diversity in hot springs
of Pozzuoli, Italy, and characterization of a unique archaeal virus, Acidianus bottle-shaped
virus, from a new family, the Ampullaviridae. J. Virol. 79, 9904–9911 (2005) http://dx.doi.
org/10.1128/JVI.79.15.9904-9911.2005. Part a (ATV) left-panel is courtesy of R. Aramayo,
and the right-panel is adapted with permission from REF. 25, Elsevier. Part a (ACV) is
adapted with permission from REF. 15, Proceedings of the National Academy of Sciences.
Part a (APBV1) is adapted with permission from REF. 11, Elsevier. Part a (PSV) is adapted
with permission from REF. 160, Elsevier. Part a (AFV1) is adapted with permission from
REF. 78, Elsevier. Part a (PFV1) is adapted with permission from REF. 38, Proceedings of the
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
Microbiology (REF. 41): Pietilä, M. K. et al. Virion architecture unifies globally distributed
pleolipoviruses infecting halophilic archaea. J. Virol. 86, 5067–5079 (2012) http://dx.doi.
org/10.1128/JVI.06915-11. Part a (SPV1) is adapted with permission from the American
Society for Microbiology (REF. 39): Liu, Y. et al. A novel type of polyhedral viruses infecting
hyperthermophilic archaea. J. Virol. 91, e00589-17 (2017) http://dx.doi.org/10.1128/
JVI.00589-17. Part a (SNDV) is adapted with permission from REF. 26, Elsevier. Part a (SIRV2)
is adapted with permission from REF. 32, Biochemical Society Transactions http://dx.doi.
org/10.1042/BST20120313. Part b (HSTV1) is adapted with permission from the American
Society for Microbiology (REF. 49): Pietilä, M. K. et al. Insights into head-tailed viruses
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
Sciences. Part b (HSTV2) is adapted with permission from the American Society for
Microbiology (REF. 49): Pietilä, M. K. et al. Insights into head-tailed viruses infecting
extremely halophilic archaea. J. Virol. 87, 3248–3260 (2013) http://dx.doi.org/10.1128/
JVI.03397-12. Part b (SH1) is adapted with permission from REF. 65, Proceedings of the
National Academy of Sciences. Part b (STIV) is courtesy of M. Young.
never been observed in a biological entity. However,
the parameters of DNA packaging are different in the
two virions: the twist of A‑form DNA in SIRV2 is
11.2 bp/turn, whereas in AFV1, it is 10.8 bp/turn.
In the structures of the SIRV2 and AFV1 virions, the
nucleoprotein helix is composed of asymmetric units,
each of which contains two molecules of the MCP:
a homodimer in the case of SIRV2 and a heterodimer in
the case of AFV1. The helices from neighbouring protein
molecules are packed in an antiparallel, interdigitated
arrangement and wrap around the DNA. The amino
terminus of the SIRV2 MCP, which is unstructured in
solution, is folded in the virion into a helix-turn-helix
structure and interacts with the DNA phosphate back-
bone through conserved polar and hydrophobic residues.
In addition to the hydrophobic protein–­protein inter­
actions across helical turns, this arrangement ensures that
the SIRV2 DNA is completely encapsidated by protein
and inaccessible to solvent (FIG. 3b), thereby maintaining
its integrity under extreme conditions. By contrast, in
the AFV1 virion, the protein–protein interactions across
helical turns are absent, resulting in looser packaging
of the DNA, a thinner filament and greater flexibility of
the virion compared to SIRV2 (FIG. 3d). Consequently, the
lipid envelope of AFV1 seems to protect the viral DNA in
highly acidic environments (FIG. 3e).
In the structure of the AFV1 virion, the lipid enve-
lope is only 20–25 Å in thickness69, half the thickness of
archaeal membranes70. The major lipids that are selec-
tively incorporated into the AFV1 membrane  are
glycerol dibiphytanyl glycerol tetraethers that lack a
cyclopentane moiety; as shown by molecular model-
ling, they adopt a U‑shaped, ‘horseshoe’ conformation
in the virion membrane (FIG. 3e,f). In addition to the
canonical lipid bilayer of the Bacteria and the Eukarya
and the archaeal lipid monolayer, the viral horseshoe
membrane represents a third, previously unobserved
type of biological membrane. Notably, the lipid envelope
of the fusellovirus (SSV1) also appears to be thinner than
the host cytoplasmic membrane and has a lipid composi-
tion that closely resembles that of AFV1 (REFS 69,71,72),
suggesting that the U‑shaped conformation is a wide-
spread ­feature of archaeal virus membranes Notably,
the majority of viruses that infect hyperthermophiles
are enveloped, suggesting that lipid membranes might
confer thermostability and provide a mechanism for
these viruses to enter into and exit from the host cell73.
The structures of spindle-shaped archaeal viruses
SSV1 and Haloarcula hispanica virus 1 (His1), which
infect hyperthermophilic-acidophilic and hyper­
halophilic hosts, respectively, have also been recon-
structed74,75. Owing to the inherent flexibility of SSV1
and His1 virions, only fairly low-resolution models
of entire virions could be obtained (~32 Å and ~57 Å,
respectively)76,77. Nevertheless, these models revealed
interesting features of these virions; whereas the body
of the His1 and SSV1 virions appears smooth and
lacks major features, the terminal structures that dec-
orate one of the two pointed ends of the virions, and
are presumably involved in viral attachment, display
six-fold symmetry.

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PBCV1 HCMV (’floor domain’)

Eukarya

Adenoviridae, Lavidaviridae, Herpesviridae

NCLDV, Polintons

PRD1 HK97
P23-77

Bacteria

Myoviridae, Podoviridae,
Sphaerolipoviridae Corticoviridae, Tectiviridae Siphoviridae

STIV HSTV1
HCIV1

Archaea

Myoviridae, Podoviridae,
Sphaerolipoviridae Turriviridae Siphoviridae

SJR fold DJR fold HK97 fold

Figure 2 | Major capsid proteins of cosmopolitan archaeal viruses. Homologous viral proteins with
Nature the HK97‑like
Reviews fold,
| Microbiology
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 cytomegalovirus (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
matched by the range of mechanisms that are employed
by these viruses to interact with their hosts. In this sec-
tion, we outline our current understanding of virus–host
interactions during archaeal viral life cycles, including
attachment and virus entry, genome replication, and
virion morphogenesis and egress.
Attachment and virus entry
Information on the early stages of infection by
archaea-specific viruses is scarce. Filamentous viruses
that are bound through their ends to cellular appendages
have been frequently observed using electron micros-
copy, which suggests that such contacts represent the
of interaction has been confirmed for the rudivirus
SIRV2, which initiates infection by binding to the tips
of the pilus-like appendages on Sulfolobus islandicus cells
through three terminal fibres on the virion79 (FIG. 4a).
The adsorption of SIRV2 is remarkably rapid: the viri-
ons irreversibly bind to the appendages within seconds
and reach the cell surface within 1 minute (FIG. 4a). The
cell surface proteins and type IV secretion proteins that
are involved in the SIRV2 entry have been identified80,
but the mechanisms of virion translocation and DNA
­injection remain unknown.
The spindle-shaped virions of Fuselloviridae and
Bicaudaviridae do not attach to cellular appendages.
Instead, they are often observed bound to cellular

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 REVIEWS

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
creating protein variants with distinct cell surface
C
­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 respectively. The proteins are coloured as in panel c. e | SIRV2
HO
(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.

Nature Reviews | Microbiology

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a 1

2
3

S-layer
b

4 1
UV 2
Cytoplasm
3
Appendages

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

6b

Invertible region
A genome region that can
excise and reintegrate into the
same genome in inverted
orientation.
Protein-primed DNA
polymerases
DNA polymerases capable of
the protein-primed initiation
step of DNA elongation.
Rolling-circle replication
The model of unidirectional
DNA replication that can
rapidly synthesize multiple
copies of circular ssDNA
molecules.
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) from the tip of a
Reviews | Microbiology
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,
e01439‑16 (2016) http://dx.doi.org/10.1128/mBio.01439-16.
some or even most components of the DNA replication respective hosts on several independent occasions82.
machinery, including DNA polymerases, proliferat- Viruses with small genomes (10–20 kb) tend to encode
ing cell nuclear antigen (PCNA; a DNA sliding clamp either p­ rotein-primed DNA polymerases74,83 or rolling-­circle
that acts as a processivity factor for DNA polymerase), ­replication endo­nucleases (RCRE) 40,84, key proteins
archaeo-eukaryotic primases and replicative helicases. for ssDNA replication in all three domains of life85,86.
Viruses with medium-sized genomes (20–50 kb) typ- It should be noted, however, that for many archaea-­
ically encode only essential proteins that recruit the specific viruses, DNA replication proteins have not
host DNA replication machinery. For example, the been found in their genomes, implying that these viruses
replicative minichromosome-maintenance (MCM) rely primarily on the host DNA replication machinery
complex (a heli­case component of the DNA replication or employ heretofore uncharacterized mechanisms of
fork) has been acquired by different viruses from their genome replication. One such unique mechanism that

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Holliday junction resolvase
A highly specialized struc-
ture-selective endonuclease
that cleaves four-way DNA
intermediates that can form
during DNA replication.
Strand-displacement
Of genome replication,
involving the displacement of
a downstream DNA strand
encountered during DNA
replication.
Strand-coupled genome
replication
The model of DNA replication
that couples leading-strand
and lagging-strand synthesis.
appears to rely on recombination for both initiation and
termination of genome replication was observed for the
lipothrixvirus AFV1, although the genes that facilitate
this mechanism are currently unknown87.
Most efforts in understanding genome replication
during archaeal virus infection have been directed
towards the rudivirus SIRV2. Similar to some bacterial
viruses, SIRV2 DNA synthesis is confined to a region
near the periphery of the infected cells88. Although viral
DNA synthesis is performed by one of the four paralo­
gous host DNA polymerases, namely, DNA polymer-
ase 1 (Dpo1), recruitment of the replication machinery
appears to be orchestrated by viral proteins. One of
the early-expressed viral DNA-binding proteins, gp1
(REF. 89), interacts with the host-encoded sliding clamp
PCNA and presumably recruits it for the assembly of
the replisome on the viral DNA90. Furthermore, SIRV2
encodes several proteins that are thought to be involved
in DNA replication, recombination and repair, some of
which have been experimentally characterized. These
proteins include a Holliday junction resolvase, a ssDNA-­
binding protein, a ssDNA-annealing ATPase, a Cas4‑like
ssDNA nuclease and a Rep protein that is homologous
to RCRE32. It was suggested that SIRV2 could employ
a combination of strand-displacement, rolling-circle and
strand-coupled genome replication mechanisms, which
generate multimeric and highly branched intermedi-
ates reaching >1,200 kb in size (~34 genome units)91.
However, whether all of these mechanisms occur dur-
ing each cycle of viral DNA replication and the specific
proteins that are involved remain unclear.
Genome packaging has not been studied in detail
for any archaeal virus. However, based on compara­
tive genomics, archaeal viruses of the Caudovirales
order employ virion assembly, maturation and genome
packaging strategies similar to those that are used by
bacterial viruses of the order. In particular, all archaeal
caudoviruses encode homologues of the large terminase
subunit, an enzyme that packages the viral DNA into
preassembled, empty capsids and subsequently processes
the concatameric viral DNA into single-genome-length
units52. Similarly, members of the Turriviridae and
Sphaerolipoviridae families encode A32‑like packaging
ATPases of the FtsK–HerA superfamily, which is typical
of dsDNA viruses with MCPs that have double or single
jelly-roll folds68. Moreover, the DNA packaging enzymes
of two turriviruses have been shown to have ATPase
activity in vitro, and the proteins have been identified in
virions92,93, as is also the case for bacterial viruses with the
homologous capsid proteins68, emphasizing the evolu-
tionary connection between these bacterial and archaeal
viruses. Thus, related viruses that infect hosts from dif-
ferent domains of life encode homologous proteins for
virion formation and genome packaging, whereas pro-
teins that are involved in genome replication either are
unique or are recruited from the respective hosts.
Virion morphogenesis and egress
The morphogenesis of pleomorphic virions of the
Pleolipoviridae and of the spindle-shaped virions of
the Fuselloviridae is coupled with virion egress. The
virion of the fusellovirus SSV1 acquires its e­ nvelope
during budding of the nucleoprotein inner core
through the cytoplasmic membrane of the host cell71
(FIG. 4b). Virions first emerge as elongated particles that
are attached to the cell membrane and subsequently
undergo maturation into characteristic spindle-shaped
particles. Virus egress mechanisms strongly resemble
the budding of enveloped eukaryotic viruses such as
HIV, influenza virus and Ebola virus. Moreover, the
formation of a constricted ‘neck’ that facilitates viral
budding is indistinguishable in appearance for SSV1
and eukary­otic viruses. Many eukaryotic enveloped
viruses use components of the endosomal sorting com-
plexes required for transport (ESCRT) machinery for
budding 94. The ESCRT pathway catalyses membrane
fission events that are required for the abscission stage
of cytokinesis and for vesicle biogenesis95. The pres-
ence of homologues of the eukaryotic ESCRT proteins
in the Sulfolobus spp., where they appear to perform
similar functions96, suggests that the budding of SSV1
and enveloped ­eukaryotic viruses may rely on similar
cellular functions.
The morphogenesis of Bicaudaviridae virions is also
likely to take place at the host cell surface and could
be coupled to ESCRT-dependent virus budding 97. The
virion morphogenesis of the type species of the family,
ATV, is not completed at the cell surface: after being
released, the spindle-shaped virions develop long
protrusions from the two pointed ends. High tem-
perature, close to that of the natural environment, is
a prerequi­site for this morphological transformation,
which is accompanied by contraction of the ‘spindle’
and the longitudinal association of globular subunits of
unknown identity into helical, hollow tubes24. Virions
of SMV1, one of the several putative family members,
can also develop tails in the extracellular environment 21.
However, for other bicaudaviruses, such as Sulfolobus
tengchongensis spindle-shaped viruses 1 and 2 (STSV1
and STSV2), no extracellular stage of virion morpho-
genesis has been observed; once released from the cell,
the virion morphology does not appear to change20.
By contrast, all stages of Rudiviridae and Turriviridae
virion morphogenesis seem to occur in the cytoplasm
of the host cell. Mature virions exit the cells via a spe-
cial gateway structure that has a seven-fold symmetry,
known as the virus-associated pyramid (VAP). The
VAP develops on the surface of infected cells and pro-
trudes through the surface layer (S‑layer), and it opens as
‘flower petals’ at the end of the infection cycle98,99 (FIG. 4a).
The VAP consists of multiple copies of a 10 kDa viral
protein100–102. The protein has a transmembrane domain
that facilitates its insertion into cellular membranes and
can form pyramidal structures in all tested biological
membranes from all three domains of life103. Pyramidal
structures, albeit of six-fold symmetry, have been
observed on the surface of uncultivated Pyrobaculum
spp. cells that are infected with an unidentified filamen-
tous virus104 as well as on the surface of Pyrobaculum
oguniense105, suggesting that the VAP-based virus egress
mechanism is more common among crenarchaeal
viruses than p ­ reviously thought.

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Pseudomurein
endoisopeptidase
(PeiP). An enzyme that cleaves
pseudomurein cell-wall sacculi
of the methanogens.
Endolysin
A type of peptidoglycan-
hydrolysing enzyme produced
by many bacterial viruses
towards the end of the
lytic cycle.
Structural genomics
The description of the
three‑dimensional structure
of every protein encoded by
a given genome.
Supermodules
Clusters of modules of tightly
connected genomes joined
through higher-level shared
genes.
The enveloped Tristromaviridae virions also appear
to mature in the cytoplasm of the host cell. For exam-
ple, at the late stages of the viral life cycle, the host cells
are densely packed with the Pyrobaculum filamentous
virus 1 (PFV1) virions (>100 per cell), and their release
coincides with cell lysis38. The mechanism of intra­
cellular acquisition of the lipid envelope by the virions
remains unclear. Release of the archaeal members of the
order Caudovirales also occurs by cell lysis. Notably, the
siphovirus ψM2, which infects Methanothermobacter
marburgensis, encodes pseudomurein endoisopeptidase
(PeiP)51, which may be analogous to the endolysin com-
ponent of the holin–endolysin lysis systems that are
widely encoded by tailed bacteriophages106. Although
PeiP mediates lysis of M. marburgensis cells in vitro107,
its participation in virion release or, alternatively, virus
entry has not been investigated in vivo.
Despite considerable gains in knowledge in recent
years, our understanding of virus–host interactions in
archaea remains incomplete. In particular, the molecular
mechanisms and, in many cases, the key genes that are
involved in different stages of archaeal viral life cycles
remain unknown. Future research is required to fill these
gaps in our understanding and to provide novel insights
into virus–host co-­evolution and molecular details of
the infection process under extreme e­ nvironmental
conditions.
Genomics and evolutionary relationships
At the onset of archaeal virus research, the information
that could be gained from archaeal virus genomes was
limited owing to the high number of uncharacterized
and unique genes. The expansion of genomic data-
bases enabled the power of comparative genomics to
be applied to evolutionary studies of archaeal viruses.
In this section, we outline the current understanding
of the evolutionary relationships between different
groups of archaeal viruses and with bacteriophages,
eukaryotic viruses and non-viral mobile genetic
elements (MGEs).
The unique nature of viruses that infect hyper­
thermo­philic Crenarchaeota is limited not only to
their morphology but also to the content of their
genomes, with ~90% of the genes lacking detect­
able homologues (except those from other cren­
archaeal viruses) in existing sequence databases28,108.
The genomes of some archaeal viruses do not have a
single gene with a functionally characterized homo-
logue108. As the tertiary structure of proteins is more
conserved than genetic sequences, structural genomics
has been employed to uncover potential functions and
the provenance of viral genes109. The crystal structures
of many archaeal viral proteins have been resolved;
however, many of these proteins adopt unusual, novel
folds, providing limited insight into the function and
origin of these proteins110,111. Unexpectedly, in the case
of the fusello­virus SSV1, almost half of the viral genes,
many of which are functionally uncharacterized, are not
essential for infectivity and could be knocked out with-
out obvious phenotypic effects112. An intriguing possi-
bility is that many of the uncharacterized and poorly
conserved genes in archaeal viral genomes encode pro-
teins that are involved in virus–host interactions, for
example, in counteracting host defence systems, such
as CRISPR–Cas systems (see below).
Structural and comparative genomics studies
clearly indicate that cosmopolitan archaeal viruses
share common ancestry with specific groups of bac-
terial viruses (Sphaerolipoviridae) or bacterial and
eukary­otic viruses (Turriviridae and Caudovirales)28,67.
By contrast, archaea-specific viruses have no counter-
parts or even strong phylogenetic relationships with
bacteriophages or eukaryotic viruses, raising impor-
tant questions regarding their origins. Recently, the
evolutionary relationships between all dsDNA viruses
were examined using the bipartite network approach,
which traces connections between viral genomes
through shared gene families113. This analysis grouped
all bacterial and eukaryotic viruses and the cosmo­
politan archaeal viruses into four distinct supermodules,
whereas archaea-specific viruses remained largely dis-
connected from the global dsDNA virosphere67, despite
occasional ‘gene sharing’ with the other modules. The
subsequent detailed dissection of the archaea-specific
virus network revealed strong modularity, with six dis-
tinct modules, each including one or two virus families,
and four additional virus families disconnected from
the rest of the archaeal virus network28. Compared to
simi­lar networks of eukaryotic and bacterial viruses, the
archaea-specific viruses are sparsely connected within
the network, with only a few shared gene families, most
notably the ribbon–helix–helix (RHH) DNA-binding
proteins and glycosyltransferases, shared by different
network modules (FIG. 5). In these cases, the prevalence
of the two gene families in viral genomes most likely
results from their independent acquisition from hosts
and/or horizontal gene transfer between viruses. The
lack of strong connectivity among the modules sug-
gests that most of the archaea-specific virus groups are
evolutionarily distinct and have evolved independently
of one another. The order Ligamenvirales, which
includes two fairly distant but clearly related families of
archaea-specific viruses and forms one of the modules
in the bipartite network, is the only notable exception
discovered so far.
From whence did the archaea-specific viruses
origi­nate, and when did this happen? Why do they
only infect archaea? There are at least two non-­
mutually exclusive explanations. Some of the archaea-­
specific virus groups could have emerged during the
early stages of cellular evolution and been retained
in the Archaea but lost in the domains Bacteria and
Eukarya114. Other archaeal virus groups could have
evolved concomitantly with the Archaea or, even
more recently, within specific archaeal lineages. The
observed limited gene sharing between different groups
of archaea-specific viruses seems to make the latter pos-
sibility particularly plausible. The large diversity of the
archaea-specific viruses and the lack of evolutionary
connections between different groups sharply contrast
with the extensive exchange of genes among bacterio-
phages that also extends to the cosmopolitan archaeal

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Archaeal virosphere encode replication initiation endonucleases homolo-

Non-viral mobilome
Ligamenvirales Clavaviridae
Tristromaviridae
Ampullaviridae
GTase Caudovirales
Bicaudaviridae
RHH Turriviridae
DnaA
Fuselloviridae
Sphaerolipoviridae
Guttaviridae
Spiraviridae
Globuloviridae
Portogloboviridae
Pleolipoviridae
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
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.
These modules can be interpreted as sharing common evolutionary history. Families
outside grey circles form separate, single-family modules. Viruses from the same module
share numerous genes (not shown in the figure), but gene sharing across modules is
limited to three widespread genes, namely, glycosyltransferase of the GT‑B superfamily
(GTase), ribbon–helix–helix-domain-containing protein (RHH) and an AAA+ superfamily
ATPase homologous to bacterial DnaA. Bidirectional arrows indicate the connections
between some families of archaeal viruses and capsidless (non-viral) mobile genetic
elements that are discussed in the text.
viruses28,67,113,115. The biological basis of this disjointed
organization of the archaea-specific virosphere remains
enigmatic but could be clarified by further studies of
archaeal virus–host interactions and a comprehensive
characterization of archaeal virus diversity.
Notably, several modules in the gene-sharing net-
work of archaeal viruses also include non-viral, cap-
sidless MGEs 28, namely, plasmids and casposons
(a recently discovered group of integrative MGEs that
appear to have given rise to the CRISPR–Cas adaptation
machinery responsible for the acquisition of new spa­
cers from the invading viruses and plasmids)116,117. In all
these cases, viruses and non-viral MGEs share genes
that encode major genome replication proteins. In par-
ticular, family 1 casposons encode protein-primed DNA
polymerases closely related to those found in ampulla­
viruses, the salterprovirus His1 and gamma­pleo­lipo­
virus His2 (REF.  116), whereas alphapleolipoviruses
gous to the corresponding proteins encoded by small
plasmids that replicate through rolling-circle repli-
cation, such as the Archaeoglobus profundus plasmid
pGS5 (REF. 118) and the Thermococcus prieurii plas-
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
plasmids121. Thus, the replication modules of many
archaea-specific viruses could be derived from non-­
viral MGEs. Combining such modules with the archi-
tectural modules that provide viral structural genes
could have resulted in the emergence of the new viral
lineages. The origins of the major virion proteins are
unknown for the majority of archaea-specific viruses.
However, many of these proteins have fairly simple
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
comprise two highly hydrophobic α-helixes16. Thus, it is
possible that both of these proteins could evolve de novo
into virion components in the context of archaeal virus
genomes. Moreover, it was recently found that one of
the major nucleocapsid proteins of Thermoproteus
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.
Archaeal viruses and CRISPR–Cas
CRISPR–Cas systems provide an RNA-based mech-
anism to defend against invasive genetic elements
in prokaryotes. The archaeal hyperthermophiles are
hosts to multiple groups of archaea-specific viruses
with unique morphotypes and gene complements
(see above), and they also universally encompass
CRISPR–Cas systems, often multiple loci of different
types within the same genomes 124. The ubiquity of
CRISPR–Cas systems in archaeal hyperthermophiles
contrasts with their relative rarity among bacteria,
<40% of which encode a CRISPR–Cas system, accord-
ing to recent estimates124,125, and implies that CRISPR–
Cas defence might have, to a large extent, shaped the
evolution of the viral genomes. The presence of many
genes that counteract host defences is a common fea-
ture of viral genomes, as exemplified by the numerous
proteins that antagonize the immune system in animal
poxviruses126 and herpesviruses127 or RNAi suppres-
sors in plant RNA viruses128. Moreover, multiple anti-
CRISPR proteins with specificity for different types and
subtypes of CRISPR–Cas systems have been identified
and studied in several bacteriophages129–131. Similar to
these viruses, archaeal viruses are expected to have
evolved anti-CRISPR counter defence strategies, but
they have so far not been identified. As many of the
proteins that are encoded by archaeal viruses are small,
poorly conserved and lack identifiable domains with
known activities, similar to counter defence proteins
in general and anti-CRISPR proteins in particular,

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

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Acknowledgements
D.P. is supported by l’Agence Nationale de la Recherche
(France) project EXAVIR and by the European Union’s Horizon
2020 research and innovation programme under grant
agreement 685778, project VIRUS‑X. D.H.B. was supported
by the Academy Professors programme, Academy of Finland
funding grants 283072 and 255342. P.F. is supported by the
European Research Council under the European Union’s
Seventh Framework Program (FP/2007-2013)/Project
EVOMOBIL — ERC Grant Agreement no. 340440. J.I. and
E.V.K. are supported by intramural funds of the US
Department of Health and Human Services (to the National
Library of Medicine). M.K. is supported by l’Agence Nationale
de la Recherche (France) project ENVIRA.
Author contributions
D.P., M.K., D.H.B. and E.V.K. researched data for the article.
D.P., M.K., P.F., E.V.K. and J.I. substantially contributed to
discussion of content. D.P., M.K. and E.V.K. wrote the article.
D.P., M.K. and E.V.K. reviewed and edited the manuscript
before submission.
Competing interests statement
The authors declare no competing interests.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.
DATABASES
RCSB Protein Data Bank:
http://www.rcsb.org/pdb/home/home.do
1J5Q | 5VKU | 3ZMN | 3ZN4 | 1HX6 | 1OHG | 1BBD
SUPPLEMENTARY INFORMATION
See online article: S1 (table)
ALL LINKS ARE ACTIVE IN THE ONLINE PDF

NATURE REVIEWS | MICROBIOLOGY VOLUME 15 | DECEMBER 2017 | 739

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