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The discovery of the Archaea was a significant break-
through in the recent history of biology. Whereas cell-
ultrastructure studies had initially suggested a division
of living organisms into eukaryotes and prokaryotes,
molecular sequence analyses — pioneered by Carl Woese
in the 1970s — revealed the existence of three different
classes of ribosomal RNAs and ribosomes in cellular
organisms. This discovery led to the replacement of the
prokaryote/eukaryote dichotomy by a trinity of domains,
the Archaea, Bacteria and Eukarya
1
. Subsequently, ribos-
omal RNA sequence comparisons led to the division
of the archaeal domain into two main kingdoms, the
Crenarchaeota and the Euryarchaeota
2
.
Over the past three decades we have accrued a broad
knowledge of the biological diversity of the Archaea.
This includes an outline of their physiology, biochem-
istry and molecular biology, and many insights into
their evolutionary relationships with the Bacteria and
the Eukarya
3
. Although Archaea resemble Bacteria
in their cellular ultrastructure and genome organization,
their DNA replication, transcription and translation
machineries show many similarities to their eukaryotic
counterparts. In addition, other features seem to have
either arisen, or have been exclusively conserved, within
the archaeal domain (for example, ether-linked mem-
branes). In this context, archaeal viruses are particularly
interesting.
The first two archaeal viruses that were isolated
visually resembled bacteriophage T4 and other mem-
bers of the family Myoviridae, with icosahedral heads,
contractile helical tails and linear, double-stranded (ds)
DNA genomes
4,5
. Subsequently, a few head-tail archaeal
viruses were reported with non-contractile tails similar
to lambdoid bacteriophages of the family Siphoviridae
6,7
.
Based on these initial studies, it was inferred, albeit erro-
neously, that archaeal viruses constituted a variety of the
ubiquitous head-tail bacteriophages.
Recently however, this view has changed radically.
Electron-microscopy investigations of samples collected
from natural environments that contain predominantly
archaea, and the enrichment cultures derived therefrom,
revealed that the head-tail phenotype is rare among the
archaeal viruses (for reviews, see
REFS 8,9). In fact, cul-
tured archaeal viruses, which to date all have dsDNA
genomes, exhibit a range of virion morphotypes, most
of which have not been observed before for any dsDNA
virus. There are exceptional forms, including
fusiforms,
droplet and bottle shapes, and linear and spherical virions,
with more complex virions combining features of these
different forms. Moreover, genome-sequence analyses
have demonstrated that most of the archaeal viruses are
unrelated to other known viruses and suggest that they
might have different, and possibly multiple, evolutionary
origins
10
.
Below we present the archaeal viruses, grouped accord-
ing to the gross features of their virion morphotypes, and
describe their genomic properties and relationships with
the host cell. Moreover, the evolutionary relationships
of the archaeal viruses to those of the Bacteria and Eukarya
are discussed. The taxonomic assignments used have
either been approved, or are pending, at the International
Committee on Taxonomy of Viruses (
ICTV).
Fusiform viruses
Viruses with fusiform virions, single or two-tailed, are
common in, and exclusive to, the Archaea
11–22
. They
constitute a large fraction of the known archaeal viruses
and are associated with a broad range of hosts repre-
senting the three main phenotypes of cultured archaeal
species, the
hyperthermophiles, extreme halophiles and
anaerobic methane-producers from the Euryarchaeota
and Crenarchaeota kingdoms
(TABLE 1). Moreover,
electron-microscopy analysis revealed an abundance
of fusiform-virus-like particles in habitats where
these archaea predominate, for example in hot, acidic
springs
23–25
and in hypersaline waters
26,27
.
The isolated fusiform viruses are diverse in their
structural and genomic characteristics, and they
*Molecular Biology of the
Gene in Extremophiles Unit,
Institut Pasteur, rue du
Docteur Roux 25, F-75724
Paris Cedex 15, France.
‡
Danish Archaea Centre,
Institute of Molecular Biology,
Copenhagen University,
Sølvgade 83H, DK-1307
Copenhagen K, Denmark.
Correspondence to D.P.
e-mail: prangish@pasteur.fr
doi:10.1038/nrmicro1527
Fusiform
An organism that is spindle-
shaped: wider in the middle
and tapering towards the ends.
Hyperthermophile
An organism that has an
optimal growth temperature
above 80°C.
Extreme halophile
An organism that requires
extremely high levels of sodium
chloride for growth.
Viruses of the Archaea: a unifying view
David Prangishvili*, Patrick Forterre* and Roger A. Garrett
‡
DNA viruses of the Archaea have highly diverse and often exceptionally complex
morphotypes. Many have been isolated from geothermally heated hot environments, raising
intriguing questions about their origins, and contradicting the widespread notion of limited
biodiversity in extreme environments. Here, we provide a unifying view on archaeal viruses,
and present them as a particular assemblage that is fundamentally different in morphotype
and genome from the DNA viruses of the other two domains of life, the Bacteria and Eukarya.
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Positively supercoiled DNA
A DNA molecule in which the
number of topological links
between the two strands is
superior to the number of
turns.
Lysogen
A bacterium or archaeon that
contains a viral genome
integrated into the
chromosome.
have been classified into the Fuselloviridae family,
the proposed ‘Bicaudaviridae’ family and the genus
Salterprovirus, although some remain unclassified. Most
have a circular genome and carry an integrase gene
that can facilitate integration into host chromosomes
28
.
The exceptions are the haloarchaeal salterproviruses
Haloarcula hispanica virus 1 (
His1) and Haloarcula his-
panica virus (
His2), which have linear genomes lacking
integrase genes
11
.
The Fusellov iridae family. Known members of the
Fuselloviridae infect the hyperthermophilic crenar-
chaeon Sulfolobus
(TABLE 1), and their replication and
release leave the host cell intact. The virions, which
are in the size range 55–60 × 80–100 nm, have short
tails which are uniform in size and carry thin terminal
fibres
15,16,18–20
(FIG. 1a). The best studied is the type spe-
cies of the family, the Sulfolobus spindle-shaped virus
1 (SSV1). Its circular genome is
positively supercoiled
29
and during infection it integrates into a tRNA gene in
the host chromosome, producing a partitioned integrase
gene while the tRNA gene remains intact
28
. UV irradia-
tion or mytomycin treatment induce viral replication
and temporarily inhibit the growth of
lysogens without
causing their lysis
15
.
The transcription pattern of the SSV1 genome is
relatively simple, with most transcripts being produced
constitutively (for a review, see
REF. 30). However, follow-
ing UV irradiation, upregulation of some constitutive
transcripts is observed, together with the appearance of
a short RNA molecule that might facilitate the initiation
of DNA replication.
The ‘Bicaudaviridae’ family. The sole member
of this proposed family, the Acidianus two-tailed
virus (ATV)
14,17
, is also the only known virus of the
acidophilic, hyperthermophilic archaea that is capable
of host lysis. Its reproductive cycle has some unique
features
14,17
. Virions are extruded from host cells as
tail-less, fusiform particles, which then develop long
tails at each pointed end at temperatures above 75°C,
close to the temperature of the natural habitat of the
host
(FIG. 1b). This major, extracellular morphological
development is independent of the host cells or any
energy sources, and its molecular mechanism remains
unclear. The tails consist of tubes, which terminate in
an anchor-like structure, and contain a periodic fila-
mentous structure
(FIG. 2). One function of the elon-
gated, flexible tails might be to enhance the probability
of virion adsorption to a new host cell.
There is circumstantial evidence that the newly
discovered process of extracellular tail development of
ATV might be shared by other fusiform viruses of the
hyperthermophilic archaea. In growth cultures of
uncharacterized Acidianus species, fusiform particles
have often been observed with one or two tails, which
can differ in length
23
. This could reflect different stages
of extracellular morphogenesis.
The Salterprovirus genus. The two distantly related
viruses His1 and His2, which have been assigned to
this genus, infect strains of the extremely halophilic
genus Haloarcula of the Euryarchaeota
11,12
. Both
viruses are lytic and the linear genomes show no
sequence similarity to the circular genomes of the
other fusiform viruses. The virions of these viruses
are similar in size (44 × 67–77 nm) but, despite
pronounced morphological similarities, their major
structural proteins are not orthologous. Each of the
viruses encodes a DNA polymerase which might be
primed by proteins attached to the termini of their
linear genomes.
Table 1 | Archaeal viruses with exceptional morphologies
Family and genus Species Archaeal
kingdom
Genome
details*
References
Fusiform viruses
Fuselloviridae, Fusellovirus Sulfolobus spindle-shaped virus 1 (SSV1) Cr c, 15.5 15, 16, 18
Sulfolobus spindle-shaped virus 2 (SSV2) Cr c, 14.8 19
Sulfolobus spindle-shaped virus - Yellowstone 1 (SSV-Y1) Cr c, 16.5 20
Sulfolobus spindle-shaped virus - Kamchatka 1 (SSV-K1) Cr c, 17.4 20
Fuselloviridae, Salterprovirus Haloarcula hispanica virus 1 (His1) Eu ln, 14.5 11, 12
Haloarcula hispanica virus 2 (His2) Eu ln, 16.1 11, 12
‘Bicaudaviridae, Bicaudavirus’
‡
Acidianus two-tailed virus (ATV) Cr c, 62.7 14, 17
Unclassified Sulfolobus tengchongensis spindle-shaped virus 1 (STSV1) Cr c, 75.3 22
Pyrococcus abyssi virus 1 (PAV1) Eu c, 18.0 13
Methanococcus voltae virus-like particle (?) Eu c, 23.0 21
Bottle- and droplet-shaped viruses
‘Ampullaviridae, Ampullavirus’
‡
Acidianus bottle-shaped virus (ABV) Cr ln, 23.9 31
Guttaviridae, Guttavirus Sulfolobus neozealandicus droplet-shaped virus (SNDV) Cr c, 20.0 32
*Genome details shown are the form of the genome and the size in kb.
‡
Taxonomic proposal pending at the International Committee on Taxonomy of Viruses.
c, covalently closed circular; Cr, Crenarchaeota; Eu, Euryarchaeota; ln, linear.
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d
a
b
c
Unclassified fusiform viruses. The virion (107 ×
230 nm) of the still unclassified Sulfolobus tengchon-
gensis spindle-shaped virus 1 (
STSV1) of the hyper-
thermophilic crenarchaeon Sulfolobus is the largest
of the known fusiform viruses and has a short tail,
the length of which varies in the range 0–133 nm
22
.
Although variable tail length could result from the
extracellular tail-development process described above
for ATV, the genome shows minimal similarity in
gene content to either ATV or other fusiform viruses.
Exceptionally, it carries open reading frames (ORFs)
encoding gene products implicated in DNA modifica-
tion and has a complex pattern of DNA methylation.
A putative origin of replication has been identified,
which carries multiple AT-rich repeats.
Fusiform virus-like particles with short tails, named
Pyrococcus abyssi virus 1 (PAV1), have also been reported
to be produced by a member of the Euryarchaeota, the
hyperthermophile ‘
Pyrococcus abyssi’
13
. Although the
putative virus resembles crenarchaeal fuselloviruses in
size, morphology and genome structure, none of the
annotated encoded proteins exhibit sequence similarity
to proteins encoded by SSV1 and its relatives (C. Geslin,
personal communication). Another fusiform virus of
the Euryarchaeota might be represented by pleomorphic
particles produced by the methanogen
Methanococcus
voltae
21
, containing circular dsDNA which is also found
integrated in the host chromosome. However, owing to
the distortion of particles by purification procedures, the
report of the morphotype of these particles is contro-
versial and it is difficult to distinguish whether they are
lemon-shaped or oblate.
Bottle-shaped and droplet-shaped viruses
The virions of two other archaeal viruses, the Acidianus
bottle-shaped virus (ABV) and the Sulfolobus neozealan-
dicus droplet-shaped virus (SNDV), have morphological
features that are so unique that each of the viruses has
been assigned to a new family (
FIG. 1 c,d; TABLE 1).
The ABV virion has a complex form resembling a
bottle (230-nm long, 4–75-nm wide). The virus infects
the hyperthermophilic Acidianus genus and has been
assigned to the new family ‘Ampullaviridae’
31
. The
virion has no elements of icosahedral or helical sym-
metry
(FIG. 1c) and differs in its basic architecture from
any known virus. The envelope encases a cone-shaped
core formed by a torroidally supercoiled nucleoprotein
filament. A disc is present at the broader end, to which
20 (±2) short, thick filaments are attached
(FIG. 1c). Their
function remains unclear, while the virion seems to
adsorb to the host cell through its narrow end. As for the
genomes of salterproviruses, the ABV genome seems to
be replicated by a virus-encoded DNA polymerase that
is primed by a protein attached to the genomic termini.
The virus genome also encodes a putative RNA molecule
with notable secondary structural similarity to the RNA
molecule that has been implicated in DNA packaging of
the bacteriophage φ29 and its relatives (X. Peng, personal
communication).
The virus SNDV of the hyperthermophilic genus
Sulfolobus exhibits a complex droplet-shaped virion
(90 × 180 nm) and is the sole member of the family
Guttaviridae
32
. The droplet carries multiple long, thin
fibres that are attached at its apex, and the surface seems
to be helically ribbed
(FIG. 1d). The circular dsDNA
genome is extensively modified, probably by methylation,
and has not been sequenced.
Linear viruses
Linear particles are the main virion type found in ter-
restrial hot environments (>80°C) where crenarchaea
of the genera Sulfolobus, Acidianus and Thermoproteus
predominate
23–25,31
. All linear viruses isolated from
these environments to date infect members of these
genera. They have dsDNA genomes, a property not
previously observed for any linear virus, and there-
fore they have been classified into two new families:
the stiff, rod-like Rudiviridae
33–35
, and the flexible,
filamentous Lipothrixviridae
36–40
. Originally, the dis-
crimination of the families was based mainly on differ-
ences in virion structure and this was later supported
by comparative genomics. Nevertheless, in contrast to
members of the other archaeal viral families for which
genome sequences are available, a substantial frac-
tion of orthologous genes, including some encoding
glycosyl transferases and transcriptional regulators,
are shared by the rudiviruses and lipothrixviruses
10
.
For example, of the 45 predicted genes of the rudivirus
Sulfolobus islandicus rod-shaped virus 1 (
SIRV1), nine
share orthologues with the lipothrixvirus Sulfolobus
islandicus filamentous virus (
SIFV). These observa-
tions indicate that it is possible that the known linear
archaeal viruses share a relatively recent common
ancestor
10
. Therefore, we propose to classify the families
Figure 1 | Electron micrographs of archaeal viruses with exceptional
morphologies. a | Sulfolobus spindle-shaped virus 1 (SSV1) (inset) and its extrusion
from the host cell. b | The extracellularly developed Acidianus two tailed virus (ATV)
(inset) and its extrusion from the host cell. c | Acidianus bottle-shaped virus (ABV).
d | Sulfolobus neozealandicus droplet-shaped virus (SNDV). All images are negatively
stained with uranyl acetate, except for part b, which was platinum-shadowed. Scale
bars represent 100 nm. Parts a and d are courtesy of W. Zillig. Part b is reproduced
from
Nature REF. 14 © (2005) Macmillan Publishers Ltd. Part c is reproduced with
permission from
REF. 31 © (2005) American Society for Microbiology.
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>75°C
Rudiviridae and Lipothrixviridae into a new viral order,
the ‘Ligamenvirales’ (from the Latin ligamen, for string,
thread).
Members of the ‘Ligamenvirales’ have linear dsDNA
genomes and show similarities in their relationships
with their host cells. In contrast to archaeal viruses with
circular genomes, no integrated viral genomes, or
fragments thereof, have been detected in host chromo-
somes. Consistent with this observation, genomes of
the ligamenviruses lack an integrase gene. It seems that
these viruses persist stably in host cells and that their
replication is not induced by stress factors such as UV
irradiation or mytomycin C treatment. The only known
exception is the lytic lipothrixvirus Thermoproteus tenax
virus 1 (TTV1) of the neutrophilic hyperthermophile
genus Thermoproteus
39
.
The linear genomes of members of the ‘Ligamenvirales’
have diverse terminal repeat structures ranging from
the large (~1.5–2 kb) inverted terminal repeats (ITRs)
of the rudiviruses to the shorter (~0.5–1 kb) ITRs of the
lipothrixviruses. Many ITRs have conserved sequence
motifs including, for example, short, regularly spaced,
direct repeats in the rudiviruses and the lipothrixvirus
SIFV
34
. Remarkably, the multiple repeat pattern at the
termini of the genome of the lipothrixvirus Acidianus
filamentous virus 1 (
AFV1) resemble the telomeric ends
of the linear chromosomes of eukaryotes
37
.
The two DNA strands of the rudiviral genomes are
covalently linked, 5′-3′, generating small terminal loops
at each end
41
, however there is no evidence so far for
similar structures being present in the lipothrixviral
genomes
36
.
The Rudiviridae family. Virions belonging to this fam-
ily do not have an envelope and vary considerably in
length (the average size is 23 × 610–900 nm), with the
length proportional to the size of the linear dsDNA
genomes for the three characterized rudiviruses
33,35
(
FIG. 3a; TABLE 2). The virions contain a tube-like super-
helix formed by linear dsDNA and copies of a single,
glycosylated, basic DNA-binding protein. Plugs that are
approximately 50 nm in length are located at each end
of the tube-like structure and each carries three short
tail fibres (
FIG. 3a).
SIRV1 replicates producing head-to-head and tail-to-
tail linked replicative intermediates, which are probably
resolved by the Holliday junction resolvase that is com-
mon to all rudiviruses
34,42
. The formation of these inter-
mediates is consistent with a self-priming mechanism of
replication, similar to that proposed for some members
of a group of the larger eukaryal nucleocytoplasmic
DNA viruses (NCLDV)
43
.
Consistent with the rudiviral–host relationships
being relatively unsophisticated, in vivo studies have
demonstrated a simple transcription pattern for the viral
genomes, with few genes exhibiting temporal regula-
tion
44
. A host-encoded transcription activator was shown
to be involved in the regulation of viral transcription
45
.
The Lipothrixviridae family. The flexible, filamentous
virions of members of this family are surrounded by
envelopes containing lipids that have been acquired
from the host. They show considerable diversity in their
terminal structures, which, in combination with differ-
ing properties of their linear genomes and presumed
differing replication mechanisms, has provided a basis
for their classification into four genera.
The virion of the α lipothrixvirus TTV1 (400 nm
× 38 nm) has a lipid bilayer envelope which encases a
helical core consisting of DNA covered by multimers of
two DNA-binding proteins
39
. The β lipothrixvirus SIFV
(1,950 nm × 24 nm) differs in that the virion termini
taper, ending in mop-like structures to which six tail
fibres are attached
36
. This structure unfolds like a spi-
der’s legs, before attaching to receptors on the host cell
membrane. For the γ lipothrixvirus AFV1 (900 × 24 nm),
the virion termini carry exceptional claw-like structures
(FIG. 3b), which clamp onto viral receptors located on host
cell pili and maintain a firm contact
37
. The virion of the
δ lipothrixvirus AFV2 (1100 × 24 nm) has a complex
collar at the termini with two sets of attached filaments,
resembling a bottle brush
38
(FIG. 3c).
Spherical viruses
The known species of archaeal spherical viruses exhibit
virions of two main types (
FIG. 3 d,e; TABLE 2). The
spherical virion (diameter, 100 nm) of the Pyrobaculum
spherical virus (
PSV), a member of the proposed family
‘Globuloviridae’, is enveloped and contains multimers
of a 33-kDa protein
46
(FIG. 3d). The lipid-containing
envelope encases a nucleoprotein core which has a
superhelical arrangement. PSV infects species of the
anaerobic and hyperthermophilic crenarchaeal genera
Pyrobaculum and Thermoproteus. The linear genome
carries 190-bp ITRs, and encodes a putative viroid-
like RNA molecule of unknown function (X. Peng,
personal communication). The Thermoproteus tenax
spherical virus 1 (
TTSV1), with similar morphological
Figure 2 | Extracellular tail development of Acidianus two-tailed virus (ATV).
Cryo-electron micrographs of tailless and two-tailed virions. The lower panels show
sections through the three-dimensional reconstructions of different portions of the
negatively stained tails obtained by electron tomography. Scale bars represent 50 nm.
Modified with permission from
REF.17 © (2006) Elsevier.
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a
b
d
e
c
Mesophilic
An organism that grows best in
a temperature range between
20°C and 45°C.
and genomic properties to PSV, should probably also be
assigned to this family
47
.
The two other known spherical viruses, Sulfolobus
turreted icosahedral virus (
STIV)
48,49
and Haloarcula
hispanica virus (
SH1) of the haloarchaeal genera
Haloarcula and Halorubrum
50,51
, reveal some mor-
phological similarities. The virions of both are non-
tailed icosahedra with an internal lipid layer
(FIG. 3e).
Therefore, they share an architectural principle with
virions of the bacterial Tectiv iridae family. Moreover,
the crystal structure of the 37-kDa major capsid pro-
tein of STIV is closely similar to those of the major
capsid proteins of the bacterial tectivirus PRD1 and
the eukaryal phycodnavirus Paramecium bursaria
Chlorella virus 1 (
PBCV1), suggesting a common
ancestry, although the protein sequences show no sig-
nificant similarity
52
. Image reconstruction of the STIV
virion revealed a unique virus architecture including
complex, turret-like projections extending from each
of the vertices
48
.
SH1 is a lytic virus with a high burst-size of approxi-
mately 200 virus particles per cell, and the release mech-
anism is based on cell disruption
51
. By contrast, STIV is
non-lytic and persists stably in the host cell. In line with
differences in morphology and virus-host interactions,
the genomes of the two viruses reveal no evidence of
homologous gene content. Furthermore, although the
STIV genome is circular, that of SH1 is linear, carrying
309-bp ITRs.
Head-tail viruses
In addition to the icosahedral viruses, archaea repli-
cate another virion morphotype that is common to
bacterial viruses. These are the head-tail phages, with
non-enveloped virions carrying icosahedral heads and
helical tails
(FIG. 4). All are associated with the kingdom
Euryarchaeota and they exclusively infect extreme halo-
philes or methanogens that are either
mesophilic or mod-
erately thermophilic. Sixteen head-tail phages have been
reported, which exhibit diverse sizes of heads, tails and
linear dsDNA genomes, and they have been assigned to
the bacteriophage families Myoviridae and Siphoviridae
(for reviews, see
REFS 8,10,53). Most head-tail phages
have not been studied beyond a basic description and
are currently unavailable in laboratory collections. The
best characterized are two pairs of closely related myo-
viruses, ΦH
54–57
and ΦCh1 (REFS 58–60), and HF1 and
HF2
(REFS 61–64), which infect haloarchaea, and the
siphovirus ΨM1 and its deletion mutant ΨM2, which
infect Methanothermobacter
65–67
(TABLE 3). In addition to
their phage-like virion structures, these viruses resem-
ble dsDNA bacteriophages in their genome content (see
below), and in possessing mosaic genomes that have
undergone extensive genetic exchange
68,69
.
Although so far viral diversity has been studied
in only a few archaea-rich habitats, it is becoming
increasingly clear that head-tail particles might be
a rare virion morphotype in environments where
archaea dominate. For example, in samples from high-
temperature hydrothermal sites, many morphotypes
are observed but rarely head-tail forms
23–25,70
. Moreover,
in hypersaline waters where haloarchaea predominate,
spindle-shaped, spherical and star-shaped virus-like
particles are most common
26,27
. Therefore, the abun-
dance of head-tail haloarchaeal viruses among those
isolated almost certainly reflects a major bias in the
approaches used for their detection and isolation.
These were based mainly on their ability to induce host-
cell lysis and produce plaques on lawns of a limited
diversity of host cells
53
.
The ecology of the archaeal viruses
Despite the widespread presence of archaea on our planet,
specific screening for archaeal viruses so far has only been
done in extreme hydrothermal and hypersaline environ-
ments. The available results suggest that the composition
of viral communities reflects that of their hosts and is
similar at different geographical locations with compa-
rable environmental conditions. Therefore, overlapping
subsets of known morphotypes of hyperthermophilic
viruses have been observed at geothermal environments
in Iceland, Eastern Russia (the Kamchatka peninsula), the
Naples region of Italy and Yellowstone National Park in
the USA, and in deep-sea hydrothermal vents
23–25, 31,70
.
Viruses from the same family which have been isolated
from different geographical sites and infect closely related
hosts often show strong sequence conservation. Therefore,
approximately half of the genes of PSV and TTSV1, which
have been assigned to the ‘Globuloviridae’ with origins in
the USA and Indonesia, respectively, are orthologous
10
. A
comparable degree of similarity occurs for members of the
Figure 3 | Electron micrographs of linear and spherical viruses of the archaea. The
figure shows: a | Sulfolobus islandicus rod-shaped virus 1 (SIRV1); b | Acidianus filamentous
virus 1 (AFV1); and c | Acidianus filamentous virus 2 (AFV2) (with terminal structures
shown in insets); d | Pyrobaculum spherical virus (PSV); e | Haloarcula hispanica virus
(SH1). All negatively stained with uranyl acetate. Scale bars represent 100 nm in main
images, and 50 nm in insets. Part a is courtesy of W. Zillig. Part b is reproduced with
permission from
REF.37 © (2003) Elsevier. Part c is reproduced with permission from
REF 38 © (2005) American Society for Microbiology. Part d is reproduced with
permission from
REF.46 © (2004) Elsevier. Part e is reproduced with permission from
REF.51 © (2005) Elsevier.
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Ribbon-helix-helix
(RHH). A structural motif
consisting of four helices in an
open array of two hairpins.
Helix-turn-helix
(HTH). A structural motif
common in DNA-binding
proteins; typically the second
helix fits into the DNA major
groove.
Fuselloviridae isolated on three different continents
20,71
.
These genomes contain a fairly conserved region, or
conserved sets of genes, and a more variable region
19,20
.
Some of this genetic diversity might arise by intergenomic
recombination between related species.
The high similarity in the gene content of viral spe-
cies from the same family that are separated by large
geographical distances is in contrast to the minimal
similarity observed for species from different families
that coexist in the same local microbial community.
The most extreme example of this is provided by four
viruses that were isolated from an acidic hot spring
(pH2, 85°C) at Pozzuoli, near Naples, Italy. The rod-
shaped ARV1, filamentous AFV2, bottle-shaped ABV
and two-tailed ATV, representing four different viral
families, coexist in the same habitat and can even
replicate in the same host strains. Despite this, these
viruses show no detectable signs of intergenomic
genetic exchange.
Acidothermophilic aquatic environments, from
which the most unusual viral morphotypes have been
isolated, contain a concentration of viruses that is sig-
nificantly lower than the concentration observed in
other analysed ecosystems
72,73
. This could be caused by
several factors including, for example, the limited stabil-
ity of virions at high temperatures and low pH, or by the
capacity of most known viruses from such environments
to persist stably in the host cell rather than cause lysis.
The latter strategy apparently reduces the possibility of
direct exposure of a virus to the harsh environmental
conditions. ATV, the only known lytic virus from an
acidic hot spring, finalizes its replication cycle extra-
cellularly under the environmental conditions that are
favourable for host growth, and this might contribute to
its survival strategy.
Archaeal virus genomics
The genes of archaeal viruses generally yield few sig-
nificant matches to sequences in the public sequence
databases
10
. The exceptions are the several homolo-
gous matches between genes of the euryarchaeal
head-tail viruses ΦCh1, ΨM1/2, HF1 and HF2,
and those of the head-tail bacteriophages from the
families Myoviridae and Siphoviridae
58,63,64,67
. These
viruses carry homologous genes encoding, for exam-
ple, proteins involved in capsid and tail formation and
virion assembly, as well as transcriptional regulators,
ATPases and nucleases.
For non-head-tail viruses, most predicted gene
products lack recognizable functions and homologues
in extant databases, other than in closely related
archaeal viruses. Identified functions are confined
to a few proteins involved in glycosylation (glycosyl-
transferases
74
), DNA replication, recombination and
integration (a Holliday junction resolvase
42
, DNA
polymerase (
REF.11 and X. Peng, personal communica-
tion), RecB endonuclease and integrase
28
), nucleotide
metabolism (a dUTPase
75
and thymidylate synthase),
diverse transcriptional regulatory proteins, mainly
of the
ribbon-helix-helix (RHH) and helix-turn-helix
(HTH) types, and functionally diverse ATPases. In a
recent re-evaluation and comparative study of all the
annotated genomes of crenarchaeal viruses, working
close to the limits of statistical significance, several
additional sequence matches were identified, in par-
ticular matches to small transcriptional regulatory
proteins. The functional subgroups of, for example,
AAA+ ATPases (ATPases associated with various cel-
lular activities), and DNA replication enzymes, were
classified in more detail
10
. Nevertheless, the current
uniqueness of most gene products of these viruses
Table 2 | Archaeal linear and spherical viruses
Family and genus Species Archaeal
kingdom
Genome
details*
References
Linear viruses
Rudiviridae, Rudivirus Sulfolobus islandicus rod-shaped virus 1 (SIRV1) Cr ln, 32.3 33
Sulfolobus islandicus rod-shaped virus 1 (SIRV2) Cr ln, 35.5 33
Acidianus rod-shaped virus 1 (ARV1) Cr ln, 24.7 35
Lipothrixviridae, α-lipothrixvirus
Thermoproteus tenax virus 1 (TTV1)
§
Cr ln, 15.9 39, 40
Lipothrixviridae, β-lipothrixvirus
Sulfolobus islandicus filamentous virus (SIFV) Cr ln, 40.9 36
Thermoproteus tenax virus 2 (TTV2)
§
Cr ND 39
Thermoproteus tenax virus 3 (TT3)
§
Cr ND 39
Lipothrixviridae, γ-lipothrixvirus
Acidianus filamentous virus 1 (AFV1) Cr ln, 21.1 37
Lipothrixviridae, δ-lipotrixvirus
‡
Acidianus filamentous virus 2 (AFV2) Cr ln, 31.8 38
Spherical viruses
‘Globuloviridae’
‡
, ‘Globulovirus’ Pyrobaculum spherical virus (PSV) Cr ln, 28.3 46
Thermoproteus tenax spherical virus 1 (TTSV1) Cr ln, 20.9 47
Unclassified Haloarcula hispanica virus (SH1) Eu ln, 30.9 50, 51
Sulfolobus turreted icosahedral virus (STIV) Cr ln, 17.7 48
*Genome details show the form of the genome and the size in kb.
‡
Currently not available in laboratory collections.
§
Taxonomic proposal pending at the
International Committee on Taxonomy of Viruses. c, covalently closed circular; Cr, Crenarchaeota; Eu, Euryarchaeota; ln, linear; ND, not determined.
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ab
von Willebrand factor A
motif
A structural motif that has
been implicated in the
formation of diverse types of
specific protein–protein
interaction and cell adhesion.
was verified, strengthening the idea that crenarchaeal
viruses are unrelated to any other viruses
10
. These
general properties and conclusions also extend to the
non head-tail euryarchaeal viruses, including the fusi-
form viruses His1, His2 and PAV, and the spherical
virus SH1, which have been sequenced and analysed
more recently (
REFS 11,50 and C. Geslin, personal
communication).
Few homologous genes are shared between
members of different virus families (except for the
‘Ligamenviridae’) and there is no evidence so far for
intergenomic recombination occurring between virus
families. Nevertheless, comparative studies on fusello-
virus genomes suggest that they carry one region in
which the gene content varies
19,20,76
, and evidence for
intergenomic recombination has recently been observed
among genomes of the lipothrixviruses (G. Vestergaard,
personal communication).
Exceptionally, the bicaudavirus ATV carries four
transposase genes, possibly present as insertion
sequence (IS) elements, which are similar in sequence to
the transposase genes in Sulfolobus spp. chromosomes
and conjugative plasmids
17
, suggesting that some interg-
enomic exchange has occurred
(FIG. 5a). ATV is excep-
tional in that it encodes six large proteins, in the size
range of 600–1,940 amino acids, which are rich in repeat
structures, with some exhibiting extensive predicted
coiled-coil regions, as well as regions of low complexity
sequence, one of which carries a
Von Willebrand factor
A motif
. At least some of these proteins seem to con-
tribute to the extracellular development of the tail-like
appendages
14,17
.
Cellular defence or regulatory mechanisms
Almost all archaeal genomes contain large clusters
of regularly spaced repeat elements (known as short
regularly spaced repeats (SRSR) or clustered regularly
interspaced short palindrome repeats (CRISPR)
77
) that
can constitute more than 1% of the whole genome
78
.
Much smaller clusters are found in about 50% of
bacterial genomes
78
. For a given archaeon cluster,
although the sequence of the repeats and size of
the spacer sequences tend to be highly conserved, the
spacer sequences themselves are generally unique
78
.
Initially, evidence was presented for the clusters
resembling centromere structures and being involved
in chromosomal segregation or plasmid partitioning
77
.
Although this hypothesis has not been refuted, recent
evidence from the same authors demonstrated that a
few of the spacer sequences present in these clusters
are highly similar to sequences found in the genomes
of archaeal rudiviruses or conjugative plasmids
79
. This
result was reinforced on the completion of the ATV
viral genome sequence, which revealed nine posi-
tive viral matches with spacer sequences in clusters
of the
Sulfolobus solfataricus P2 genome
78
(FIG. 5). As
the repeat clusters produce larger transcripts that are
probably processed into smaller fragments approach-
ing the size of the spacer sequences, they can poten-
tially target and inactivate viral messenger RNAs or
genes
78,80,81
. Therefore, it has been suggested that the
spacer sequences might provide the basis for a cellular
defence mechanism against future invasion by related
viruses or plasmids
79
. Bioinformatics evidence suggests
that the group of genes, designated cas for CRISPR-
associated, are physically associated and co-functional
with some clusters
82
. They might be involved in adding
new repeat-spacer units to the clusters by interact-
ing with the flanking sequences that adjoin actively
Figure 4 | Electron micrographs of head-tail viruses of
archaea and bacteria. a | The haloarchaeal virus φH1.
b | The bacteriophage P2. Both are negatively stained with
uranyl acetate. Scale bars represent 100 nm. Part a is
courtesy of W. Zillig. Part b is reproduced with permission
from http://www.biochem.wisc.edu/inman/empics.
Table 3 | Archaeal head-tail viruses*
Family and genus Species Host Archaeal
kingdom
Genome
details
‡
References
Myoviridae, ‘φH-like viruses’ φH
Halobacterium salinarum Eu ln, 59.0 54–56
φCh1
Natrialba magadii Eu ln, 58.5 58,60
Unassigned species in the
family
HF1 Haloferax volcanii and
Halobacterium salinarum
Eu ln, 75.9 61,63
HF2 Halorubrum coriense Eu ln, 77.7 61–64
Siphoviridae, ‘ ψM1-like viruses’ ψM1/2
Methanothermobacter marburgensis Eu ln, 30.4/26.1 65–67
*Only those with sequenced genomes are listed.
‡
Genome details show the form of the genome and the size in kb. Eu, Euryarchaeota; ln, linear.
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61
192
545
326b
618
892 (2×)
710
529
145
+145 +326b–618 –127 –61
127
ATV
62,730 bp
a
b
24bp identical repeats 41bp non-ATV spacers Spacers matching sequences
from corresponding ATV ORFs
Small interfering RNA
Non-coding RNAs (around 22
nucleotides long) derived from
the processing of long double-
stranded RNA during RNA
interference. They direct the
destruction of mRNA targets
that have the same sequence.
Micro RNA
A short (21–22-nucleotide)
RNA silencing trigger that is
processed from short stem–
loop precursors that are
encoded in the genomes of
metazoans and certain viruses.
growing clusters, possibly involving a retrotransposi-
tion mechanism
78,83
. The whole apparatus could be
evolutionarily related to the eukaryotic
small interfering
RNA
(siRNA) and micro RNA (miRNA) systems
83
.
Unresolved questions include why the clusters
are so extensive in archaea and why so many spacer
matches to ATV are found? One possible explana-
tion for the latter is that whereas most non-head-tail
archaeal viruses enjoy a benign relationship with
the host, and persist stably in the cell at a low copy
number without causing its lysis, a high level of ATV
replication results in host lysis and so perhaps the lytic
ATV induces a stronger host response.
Some insight into how archaeal viruses overcome cel-
lular defence mechanisms of the host is provided by the
rudivirus SIRV1. When passed through diverse, closely
related host strains of Sulfolobus islandicus, SIRV1 under-
goes major genomic changes, including transpositions,
duplications and deletions, all concentrated in six main
regions of the viral genome and often resulting in an
alteration in gene size
84
. Many of the changes are caused
by insertions or deletions of 12 bp, although the mecha-
nism by which this occurs is still not fully understood
84
.
However, the changes seem to reflect the adaptation of
the virus to the new host and could be a reaction to the
RNAs being produced from spacers of the chromosomal
repeat clusters.
Another mechanism of adaptation to cellular
defences might be exemplified by the protein TPX,
which is expressed from the α lipothrixvirus TTV1.
The genome carries two low-complexity sequence
regions consisting of the repetitive sequence CCNACN
(where N is any nucleotide) one of which is located
within the tpx gene and the other in a non-coding
region. Frequent recombination seems to occur
between these two regions, which generates multiple
variants of the TPX protein in the cell
85
.
Evolutionary considerations
Despite a modest number of isolated species, the
morphological diversity of the dsDNA viruses of
the Archaea strongly surpasses that of the numerous
known species of dsDNA viruses of the Bacteria
86
.
According to the recent report of the ICTV, 96% of
the 452 known species of bacterial dsDNA viruses are
non-enveloped head-tail bacteriophages, assigned to
three families of the order Caudovirales (Myoviridae,
Siphoviridae and Podoviridae), whereas only about
4% are non-enveloped tailless icosahedra or envel-
oped pleomorphic spheres
87
. As discussed above, in
addition to the Caudovirales and tailless icosahedral
viruses, archaea replicate a plethora of viruses with
morphotypes that are not encountered among dsDNA
viruses of either bacteria or eukarya
(FIG. 6). The diver-
sity of archaeal viruses in hot habitats is especially
striking compared with the relative uniformity of the
viral landscape in aquatic environments at moderate
and low temperatures, which is dominated by head-
tail viruses
88
. The origin and nature of this biodiver-
sity raises intriguing evolutionary questions. Possibly,
such diversity was once common in all environments
but was later reduced by the successful expansion of
bacteria and their phages in biotopes with moderate
and low temperatures, whereas hot environments still
remain a refuge for multiple unusual viral forms.
The occurrence of the Caudovirales among
archaeal viruses was earlier interpreted as evidence
for these viruses predating the divergence of the
Archaea and Bacteria, as it was believed that viruses
were unable to spread across domain boundaries
owing to the pronounced differences in the molecu-
lar biology and lifestyles of the two domains
89
.
However, it is remarkable that, in known cases,
caudoviruses infect archaea carrying a high percent-
age of genes of bacterial origin, in particular certain
mesophilic or moderately thermophilic haloarchaea
or methanoarchaea
90,91
. Apparently, these bacte-
rial genes have adapted successfully to an archaeal
cellular context. Furthermore, extremely halophilic
Figure 5 | Genome structure of Acidianus two-tailed virus (ATV). a | Map of the
circular ATV genome with open reading frames (ORFs) represented by arrows and
labelled according to the number of amino acids in the predicted protein. The ORFs
are colour coded as follows: grey, have homologues in other crenarchaeal viral
genomes; green, have homologues in archaeal conjugative plasmids; blue,
transposases; purple, genes for structural proteins. For the numbered ORFs,
fragments of identical, or near identical, sequences are present as CRISPR spacers
in the chromosome of Sulfolobus solfataricus P2. b | Repeat-spacer elements from a
section of the 96-repeat-spacer CRISPR cluster ‘d’ of S. solfataricus P2. The colour-
coded elements are: yellow, 24-bp identical repeats; grey, ~41-bp non-ATV spacers;
red, spacers matching sequences from corresponding ATV ORFs (+ and – indicate
the same and opposite direction, respectively, relative to the ORF mRNA). Part a is
modified with permission from
REF. 17 © (2006) Elsevier. Part b is modified with
permission from
REF.78.
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Myoviridae
Salterprovirus
Unclassified (Fuselloviridae?)
Fuselloviridae
Nimaviridae
Baculoviridae
Ascoviridae
Polydnaviridae
Herpesviridae
Adenoviridae
Polyomaviridae, Papillomaviridae
Poxviridae
Asfarviridae
Mimiviridae
Iridoviridae
Phycodnaviridae
STIV
SH1
Rudiviridae
Lipothrixviridae
Guttaviridae
‘Globuloviridae’
‘Ampullaviridae’
‘Bicaudaviridae’
Siphoviridae
Myoviridae
Siphoviridae
Podoviridae
Plasmaviridae
Corticoviridae
Tectiviridae
Bacteria Archaea
?
Eukarya
Euryarchaea Crenarchaea
Last universal common
ancestor
The progenitor from which all
current life is thought to have
evolved.
bacteria have been discovered, which could have
facilitated the adaptation of bacteriophages to rep-
licate in the haloarchaea
92
. Therefore, it now seems
likely that the Caudovirales first entered archaea by
interdomain spreading. Such an origin is also consist-
ent with archaeal caudaviruses carrying many genes
with homologues in either bacterial chromosomes or
plasmids.
Assuming that archaeal Caudovirales do, indeed,
originate from bacteria, then we are faced with the
intriguing perspective that each of the three domains
of life was originally characterized by a unique set of
associated dsDNA viruses
(FIG. 6). Could the associa-
tion of a specific set of viruses with each domain mean
that the old preconception of viruses originating from
their hosts is correct? Probably not, because there are
no apparent exclusive evolutionary links between the
basic viral components, the capsid and replication
apparatus found in one domain and the cellular com-
ponents of that domain. On the contrary, recent struc-
tural studies have shown that some viruses present in
different domains could have evolved from entities (or
a common gene pool) that predated the divergence of
the domains
93
. For instance, the icosahedral archaeal
viruses STIV and SH1 share a basic architectural
principle and/or a capsid protein with similar struc-
tures (demonstrated for STIV) with several bacte-
rial (Tectiviridae) and eukaryotic (Phycodnaviridae,
Adenoviridae) icosahedral viruses
52
.
One possible explanation for the existence of three
different ‘virospheres’, each associated with a specific
domain, is that these virospheres were selected when
the domains first arose. Therefore, the first evolving
organisms of each separate domain could have already
been infected by different subsets of viruses from the
ancestral virosphere, which predated the
last universal
common ancestor
, LUCA. Although the descendants
of LUCA might have rapidly expanded on the planet,
the lineages leading to the three ancestors and their
associated viruses might well have originated later in
distinct areas and remained geographically isolated for
a long time period. Therefore, viruses might already
have been specialized to co-evolve with cells of one
domain and were unable to efficiently infect cells of
another domain when the cells started to share the
same environment. The mechanism and the tim-
ing of the speciation of the three domains remains
unknown, however, a recent hypothesis suggests that
DNA viruses could have played a direct role in their
formation by introducing DNA genomes into ancient
RNA cells
(BOX 1). Such a model would be compatible
with each domain being associated with its own viral
landscape if one assumes that three ancestral RNA cells
were infected by three different sets of DNA viruses at
the time of the RNA–DNA transition
(BOX 1).
The present lack of known archaeal RNA viruses
also raises an intriguing evolutionary issue. The strate-
gies recently used to detect and isolate archaeal viruses,
Figure 6 | The families of dsDNA viruses associated with the three domains of life. The virus families listed are
approved by the International Committee on Taxonomy of viruses, and the schematic representation of virions (not drawn
to scale) are presented as in
REF. 87. Proposed families are shown in inverted commas. SH1, Haloarcula hispanica virus 1;
STIV, Sulfolobus turreted icosahedral virus.
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E
LUCA
EV
Eukarya
Eukaryotic viruses
BBacteria
Bacterial
viruses
Archaeal
viruses
Archaea
BV
A
AV
?
Modern
virosphere
Ancient virosphere
Ancestors of
modern viruses
Modern
virosphere
at least from hydrothermal environments
72
, might have
been expected to yield RNA viruses, although one can-
not yet exclude a methodological problem. A possible
explanation for their absence could be that they were
already absent from the virosphere associated with the
common archaeal ancestor. Given that RNA is much
more labile than DNA at high temperature, this infer-
ence would concur with recent phylogenetic analyses
based on the available sequences of the translation
and transcription apparatus, which indicate that the
archaeal ancestor was a hyperthermophile
94
.
The discovery and exploration of the fascinating
archaeal DNA viruses has paralleled, and contributed
Box 1 | The origins of viruses
Three hypotheses have been
proposed to explain the origin
of viruses: (
a) they originated
in a pre-cellular world (the
‘virus first’ hypothesis); (
b)
they originated by a reduction
from parasitic cells; or (
c) they
originated from fragments of
cellular genetic material that
escaped from cellular control
and became parasites (the
escape hypothesis). Originally,
these hypotheses were
proposed in the framework of
the prokaryote/eukaryote
dichotomy. Just as the
erroneous concept of the
prokaryotes became the
paradigm for considering
bacterial evolution, the
escape hypothesis became the
paradigm for explaining the origin of viruses.
In its classical version, the escape hypothesis maintained that bacteriophages originated from bacterial genomes and
eukaryotic viruses from eukaryotic genomes. Amazingly, although most archaeal viruses are completely unrelated to
bacterial viruses, they are still classified as ‘bacteriophages’ in this outdated framework. For example, archaeal viruses are
illustrated under the heading “Families and genera infecting bacteria” in the latest edition of Virus Taxonomy:
Classification and Nomenclature of Viruses
(REF. 87). This occurs presumably because the archaeal domain is still not
recognized by some biologists.
The problem of virus origin remained deadlocked until recently, when progress in the molecular description
of viral proteins caused many scientists to realize that viruses form a world of their own, and that it is futile to
continue to speculate on their origin in the framework of the discredited prokaryote/eukaryote dichotomy.
The discovery that viruses which are associated with different domains can share similar, and apparently
homologous, features strongly suggests that viruses are ancient and that they predated the last universal
cellular ancestor (LUCA)
93,95
.
The early hypotheses for viral origin have now been re-evaluated in the context of this new framework
95
.
Currently, the main debate is between those who suggest a long period of acellular evolution (up to the actual
emergence of archaea and bacteria) and those who favour an early appearance of cells. Those who suggest the
former have revived the virus-first hypothesis, hypothesis (
a) above. For instance, Koonin and Martin recently
suggested that viruses emerged from an assemblage of self-replicating elements thriving in a hydrothermal vent,
using inorganic compartments as their hosts
96
. For some of those who favour an early emergence of cellular
organisms, viruses are considered to have originated from RNA–protein-based cells, either by reduction from
parasitic RNA cells or from genetic material that escaped from the genomes of these cells (variants of hypotheses (
b)
and (
c) above)
95
. A major question mark is the evolutionary relationship between DNA viruses and RNA viruses: did
DNA viruses originate from RNA viruses or from primitive DNA cells, or both
95,97
? In one hypothesis, DNA itself is
considered a viral invention, that is, DNA first appeared in viruses and was later transferred to cells
98
. In this model,
three such independent transfers could have initiated the three modern DNA lineages and the modern virospheres
associated with them
99
.
The figure shows a model of the formation of the three modern virospheres, coincident with the formation of the
three domains of life.
to, a recent upsurge of interest in the evolution of
viruses in general
(BOX 1). Although the known
archaeal viruses reveal an exceptional degree of diver-
sity with regard to both morphotypes and genomes,
this might still be an underestimation as they have
only been isolated from a limited number of host spe-
cies and taxa (mainly from the orders Sulfolobales and
Halobacteriales). Therefore, screening for virus–host
systems in other phylogenetic taxa of the archaeal
domain is an important research priority that prom-
ises to provide new insights into important questions
concerning the nature and origin of the modern
virosphere.
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Acknowledgements
We would like to thank all the members of our laboratories who
have contributed to this work, and to thank colleagues who
have made their unpublished results available to us.
Competing interests statement
The authors declare no competing financial interests.
DATABASES
The following terms in this article are linked online to:
Entrez Genome: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=genome
AFV1 | His1 | His2 | PBCV1 | PSV | SH1 | SIFV | SIRV1 | STIV |
STSV1 | TTSV1
Entrez Genome Project:
http://www.ncbi.nlm.nih.gov/
entrez/query.fcgi?db=genomeprj
Methanococcus voltae | Pyrococcus abyssi | Sulfolobus
solfataricus P2
FURTHER INFORMATION
David Prangishvili’s homepage: http://www.pasteur.fr/
recherche/unites/bmge-archaea/david%20Prangishvili/pran
gishvili%20personal%20page.htm
ICTV: http://www.ncbi.nlm.nih.gov/ICTVdb/index.htm
The Sulfolobus Database: http://www.sulfolobus.org/cbin/
mutagen.pl
Access to this links box is available online.
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