Review TRENDS in Microbiology Vol.13 No.11 November 2005

Viruses of hyperthermophilic Crenarchaea

David Prangishvili1 and Roger A. Garrett2

1Molecular Biology of the Gene in Extremophiles Unit, Institut Pasteur, rue Dr. Roux 25, 75724 Paris Cedex 15, France 2Danish Centre, Institute of Molecular Biology and Physiology, Copenhagen University, Sølvgade 83H, DK-1307 Copenhagen K, Denmark

Since the discovery of the Archaea – the third domain of one minor geothermal pool. For Figure 1 and Figure 3, the – by Woese and colleagues in 1977, the subsequent host range constitutes a few closely related strains of the developments in molecular and cell biology, and also crenarchaeal genus , whereas for Figure 2 genomics, have strongly reinforced the view that the host range spectrum is broader and includes repre- archaea and eukarya co-evolved, separately from bac- sentatives of the crenarchaeal genera Thermosphaera, teria, over a long time. However, when one examines Desulfurococcus, Thermophilum and Pyrobaculum the archaeal , the picture appears complex. Most (M. Ha¨ring and D. Prangishvili, unpublished data). Of viruses that are known to infect members of the the particle types observed in the three enrichment kingdom Euryarchaeota resemble bacterial viruses, cultures, w40% were shown to be infectious virions and whereas those associated with the kingdom Crenarch- could be isolated and cultured [Figure 1b ( AFV1); aeota show little resemblance to either bacterial or Figure 2c,d (virus PSV, indicated by arrows); Figure 3a eukaryal viruses. This review summarizes our current (virus ABV), 3b (virus ATV), 3c (virus ARV1), 3d (virus knowledge of this group of exceptional and highly AFV2), 3d (virus AFV3)]. diverse archaeal viruses. To date, w25 crenarchaeal viruses have been isolated from hot terrestrial habitats and characterized; these were found to infect members of the genera Sulfolobus, Acidianus, Pyrobaculum and Thermoproteus. Many of Morphological diversity these viruses have been classified into seven new families Early studies on viruses from the domain Archaea on the basis of their morphotypes and the properties of concentrated on those infecting extremely halophilic and their double-stranded (ds) DNA genomes (Table 1). Four of methanogenic members of the kingdom Euryarchaeota. the families have already been approved by the Inter- Most of these viruses are similar to head-tail bacterio- national Committee of Taxonomy of Viruses. phages in morphotype and genome organization and were assigned to the families and (reviewed in Ref. [1]). Later, the development of methods Morphotypes unique amongst viruses for cultivating aerobic and anaerobic hyperthermophiles Virions of the family are spindle-shaped led to the discovery of viruses infecting members of the with a single short tail that carries fibers, positioned at other major archaeal kingdom, the Crenarchaeota. The only one of the two similar poles, which facilitate the morphotypes of the first of these viruses to be character- attachment of virions to the host membrane. The ized were exceptional and quite unexpected (reviewed in unclassified STSV1 virus is much larger than the [2]). Subsequent screening for crenarchaeal viruses in fuselloviruses but exhibits a similar form [7].The different geographical locations, including geothermally structure of the inner core of the enveloped virions, heated springs (O808C), reinforced and extended the which apparently generates the unusual shape, is earlier results [3–5]. These studies revealed that cre- unknown. A similar uncertainty exists concerning the narchaeal viruses exhibit highly diverse morphotypes inner-core structure of the enveloped, droplet-shaped and, although some shapes (e.g. the spherical form of PSV virion of SNDV, the sole member of the .A and STIV [4,6]) are common for viruses, others are large number of densely packed thin filaments protrude extraordinary. Examples of morphotypes observed in the from the pointed end of the virion and these are likely to enrichment cultures from these habitats are illustrated in participate in cellular adsorption. SNDV is the least Figures 1–3. The most abundant particles in the enrich- studied of the isolated crenarchaeal viruses [8] and, ments exhibit linear morphotypes, which are either unfortunately, a host strain in which it could be replicated flexible or rigid (Figure 1a,b,e–g; Figure 2a,b,d; stably has not been found; therefore, the virus no longer Figure 3c–e), as well as spindle-shaped particles with exists in laboratory collections. tails of different size and form (Figure 1d,h,j; Figure 2e). The enveloped virion of ABV, the sole member of the Each figure includes virions or virion-like particles from Ampullaviridae, resembles a bottle (Figure 3a), the

Corresponding author: Garrett, R.A. ([email protected]). narrow end of which is likely to be involved in cellular Available online 8 September 2005 adsorption and in channeling of viral DNA into the host www.sciencedirect.com 0966-842X/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2005.08.013 536 Review TRENDS in Microbiology Vol.13 No.11 November 2005

Figure 1. Transmission electron micrographs of a variety of viruses and virus-like particles observed in a single enrichment culture established from of a sample taken from a geothermally heated, hot acidic spring (858C, pH 1.5–2.0) in Yellowstone National Park, USA. (a) A rod-shaped helical particle; inset: enlarged terminus; (b) a filamentous particle of the virus AFV1; inset: enlarged terminus, (c) a particle with ellipsoid body and two helical tails; (d) spindle-shaped particles; (e) short rod-shaped helical particles; (f) a filamentous particle with bulbous termini, both shown enlarged in the insets; (g) a filamentous particle with rounded ends, which are shown enlarged in the insets; (h) a spindle-shaped particle with a helical tail, enlarged in the inset; (i) four zipper-shaped particles; (j) four pleomorphic particles with arrow-shaped heads and tails of different length. Samples were negatively stained with 2% uranyl acetate. Bars: 200 nm (100 nm for insets). Modified with permission from Ref. [4]. cell [5]. The broad end exhibits 20 thin filaments, which genomic properties and their possible replication strat- are inserted into a disk and interconnected at the base. egies (see subsequent sections). The terminal structures at The filaments do not seem to be involved in cellular each end of a given lipothrixvirus are identical, as judged adsorption and their function remains unclear but from electron microscopy studies, although those of TTV1 intriguing. ATV, the only member of the , have not been analyzed in detail [10]. For SIFV, the body contains a lemon-shaped central structure with elongated tapers and ends in mop-like structures [11], AFV1 exhibits tails protruding from both pointed ends (Figure 3b). claw-like terminal structures, connected to the virion body Members of two viral families exhibit morphotypes by appendages [12], and AFV2 carries a complex collar similar to those of eukaryal viruses with single-stranded with two sets of filaments, resembling a bottle brush with (ss) RNA genomes. Thus, the rod-shaped non-enveloped a solid round cap at each end [13]. These terminal virions of the Rudiviridae (Figure 3c) resemble ssRNA structures are implicated in cellular adsorption for each viruses of vascular , with a body consisting of the of the lipothrixviruses and both termini seem capable of viral genome assembled with multiple copies of a single attaching to cellular receptors [12]. Most viruses adsorb DNA-binding protein [9]. However, in contrast to tobamo- directly to host cells and the mop-like terminal structures viruses, the rudiviruses have specific terminal structures, of SIFV have been shown to unfold like spiders legs before consisting of three tail fibers protruding from each end, attachment to cell membranes [11]. An exception is AFV1, which are required for adsorption on the host cell surface. in which the claw-like ends clamp onto host pili [12]. Virions of the family are spherical and The arrangement of the virion cores, which are encased by carry an envelope (Figure 2d) that encases a superhelical envelopes, differs between the lipothrixviral genera although nucleoprotein core. This structure resembles that of all carry linear dsDNA genomes. The cores of TTV1 and AFV1 ssRNA viruses of the , which infect are helical and that of TTV1 was shown to contain equimolar vertebrates. amounts of two DNA-binding proteins [10]. The arrangement Members of the have filamentous, of the SIFV core, with linear DNAwound around a zipper-like enveloped virions (Figure 1b and Figure 3d,e) and have array of a putative heterotetramer containing two proteins, been classified into four different genera (Table 1) on the resembles nucleosomes [11]. By contrast, no regular struc- basis of differences in core, terminal structures and ture was detected in the core of AFV2 [13]. www.sciencedirect.com Review TRENDS in Microbiology Vol.13 No.11 November 2005 537

Figure 2. Transmission electron micrographs of a variety of viruses and virus-like particles observed in a single enrichment culture established from a sample taken from a geothermally heated, hot spring (75–938C, pH 6.5) in Yellowstone National Park, USA. (a,b) Two types of rod-shaped particles with different termini, shown enlarged in insets; (c,d) a mixture of spherical particles of the virus PSV (shown with arrows) and rod-shaped particles; (e) pleomorphic particles with arrow-shaped heads and wide tails of different length with a specific terminal structure, shown enlarged. Samples were negatively stained with 2% uranyl acetate. Bars: 200 nm (100 nm for insets). Modified with permission from Ref. [4].

Virion components elongated tails of the virus that develop after its extrusion The number of the major protein components present in from host cells [14]. The globulovirus PSV exhibits three the virions varies from 1–2 for fuselloviruses and protein components with molecular masses of w16, 20, rudiviruses to 11 proteins for the bicaudavirus ATV. 28 kDa, the largest of which appears to form specific N-terminal sequences have been determined for many of dimers and higher aggregates [15]. The 28-kDa protein the major structural proteins and correlated with gene also contains two-thirds hydrophobic amino-acid residues, sequences. Some have been expressed and studied. For w15% of which are aromatic, and no cysteine residues, example, the 88.7-kDa protein encoded by ATV is rich in properties which are likely to contribute to high coiled-coil motifs and can generate structures that thermostability. resemble intermediate filaments, which contribute to the Basic DNA-binding proteins probably have an important role in protecting the viral DNA against degradation under the harsh environmental conditions that can involve temperatures of up to 978C and pH 2 or less. Thus, for the non-enveloped rudiviruses the single DNA-binding protein can partially protect the virion structure during autoclaving at 1208Cfor10min and it is completely disrupted only after a further 50 min [9]. Members of the Globuloviridae and of three genera of the Lipothrixviridae and STSV1 have been shown to exhibit envelopes containing host-derived lipids [7,11,12,15] (Table 1). Thin-layer chromatography reveals patterns of bands that are less complex than those of the host lipids but which also show mobility differences, suggesting that they have been modified by virus-encoded enzymes, possibly glycosyl transferases [11].Sofar,no Figure 3. Transmission electron micrographs of a variety of viruses observed in a single enrichment culture established from of a sample taken from a geothermally lipid components have been detected in the apparently heated, hot acidic spring (87–938C, pH 1.5) at Pozzuoli, Italy. They show the enveloped virions of the deltalipothrixvirus AFV2 [13] and following viruses: (a) ABV, (b) ATV, (c) ARV1, (d) AFV2 and (e) AFV3. Samples were the fusellovirus SSV1 [16]. The possible presence of lipids negatively stained with 3% uranyl acetate. Bars: 100 nm. Modified with permission from Ref. [5]. in virions of other members of the Fuselloviridae and www.sciencedirect.com 538 Review TRENDS in Microbiology Vol.13 No.11 November 2005

Table 1. Properties of the crenarchaeal viruses Family Known Origina Host Morpho- Lipids Genome Genome GCC Genome Refs species type form size (bp) content integ- (%) ration Fuselloviridae SSV1 J Sulfolobus Single- nd Circular 15465 40 C [34] tailed spindles SSV2 I Sulfolobus nd 14796 39 C [35] SSVK1 R Sulfolobus nd 17385 39 K [18] SSVRH Y Sulfolobus nd 16473 39 K [18] Rudiviridae SIRV1 I Sulfolobus Rigid rods K Linear 32308 25 K [21] SIRV2 I Sulfolobus K 35450 25 K [22] ARV1 Y Acidianus K 24655 39 K [20] Lipothrixviridae TTV1 I Thermoproteus Flexible C Linear 16000c nd [32] Alpha filaments, diverse termini Lipothrixviridae SIFV I Sulfolobus C 40852 33 K [11] Beta Lipothrixviridae TTV2 I Thermoproteus nd 16000c nd nd [10] Beta Lipothrixviridae TTV3 I Thermoproteus nd 27000c nd nd [10] Beta Lipothrixviridae AFV1 Y Acidianus C 21000 37 K [12] Gamma Lipothrixviridae AFV2 P Acidianus K 31787 36 K [13] Delta Globuloviridaed PSV Y Pyrobaculum Spherical C Linear 28337 48 K [15] TTSV1b K Thermoproteus 20933 50 nd see below Bicaudaviridaed ATV P Acidianus Two-tailed nd Circular 62730 41 C [14] spindle Guttaviridae SDNV I Sulfolobus Droplet nd Unknown nd nd K [8] Ampullaviridaed ABV P Acidianus Bottle nd Linear nd nd nd [5] Unclassified STIV Y Sulfolobus Icosahe- nd Circular 17663 36 nd [6] dral with turrets Unclassified STSV1 C Sulfolobus Large C Circular 75294 35 K [7] spindle aAbbreviations: J, Japan; I, Iceland; R, Kamchatka, Russia; Y, Yellowstone National Park, USA; P, Pozzuoli, Italy; K, Korea; C, China;. K, negative; C, positive; nd, not determined. bTTSV1 Sequence Accession Number AY722806. cApproximate genome size. dVirus families proposed but not yet approved by the International Committee for Taxonomy of Viruses.

Ampullaviridae, and other viruses, has not been investi- cells in a stable state and are not lost during continuous gated (Table 1). growth of infected cell cultures. Such a survival strategy is apparently beneficial for the virus population, helping it to Virus–host interactions avoid direct, and possibly prolonged, exposure to the harsh Hosts of all the cultured crenarchaeal viruses are conditions of the host habitat. members of the hyperthermophilic genera Sulfolobus, Only circular genomes of fuselloviruses, the bicau- Acidianus, Thermoproteus and Pyrobaculum. The first davirus ATV and STSV1 have been found to encode two genera comprise extreme acidophiles, whereas integrases and the fuselloviruses have been detected members of the last two genera are all neutrophiles and integrated within host chromosomes [7,14,17].They obligate anaerobes. Infectivity of Thermoproteus and Pyrobaculum with viruses is unaffected by exposure to generally insert within specific host tRNA genes, oxygen. For all of the hosts that grow optimally at 808Cor although exceptions have been observed in laboratory above, viral infection occurs most effectively at the optimal infection experiments [18]. Integration leaves the host growth temperature of the host (D. Prangishvili, unpub- tRNA gene intact whereas the viral integrase gene lished data), such that viruses can also be considered to be partitions into two sections. For the fusellovirus SSV1, hyperthermophiles. Little is known about either the it has been shown that virus replication can be modes of cellular entry or the assembly mechanisms of induced by ultraviolet irradiation leading to temporary the viruses. Attempts to analyze the processes of virion growth inhibition, but not lysis, of host cells [17]. adsorption by electron microscopy have, thus far, been Subsequently, virus proliferation decreases and the unsuccessful. host cells assume their earlier growth rate. The linear Viruses of acidophilic hyperthermophiles, except for genomes of crenarchaeal viruses do not encode ATV [14], are non lytic and the infected cells produce integrases and have not been detected in chromosomes virions constitutively, consistent with an equilibrium of their hosts. Moreover, it has not been shown being established between viral replication and cellular whether their replication is induced by ultraviolet multiplication. Moreover, these viruses persist in host irradiation or by other stress factors. www.sciencedirect.com Review TRENDS in Microbiology Vol.13 No.11 November 2005 539

Genome structure Gene function The uniqueness and diversity of the viral morphotypes is Sequence similarities between genes of the different reflected in their genomic properties. Each of the crenarchaeal viral families are generally limited and crenarchaeal viruses investigated so far carries a dsDNA most predicted genes only yield good matches with those genome. The circular genomes of the fuselloviruses, of other members of the same family [24]. Most viral bicaudavirus ATV and STSV1 occur in the size range genomescodeonbothDNAstrands,althoughthe 15–75 kb (Table 1). All known rudiviruses, lipothrix- globuloviruses are exceptional, with coding almost exclu- viruses and globuloviruses contain linear genomes falling sively on one strand [15]. in the smaller size range 25–45 kb and some of them carry Few genes have been assigned functions. Likely major large inverted terminal repeats (ITR). Whether any of coat proteins have been identified for some viruses. Holliday these assume a circular form during their cellular life junction resolvases, essential for genome replication, are cycle is unknown. For viral families with several known encoded by the rudiviruses [25]. Glycosyl transferases are members – primarily the fuselloviruses, which are produced by most of the viruses and integrases are encoded extremely abundant in nature [19], and lipothrixviruses in the circular genomes. dUTPases and/or thymidylate of the beta-genus – one region of the genome that carries synthases are encoded by rudiviruses and STSV1 and the genes for the viral structural proteins is highly probably help to maintain a low dUTP:dTTP ratio and conserved in gene content and order, whereas the thereby minimize mis-incorporation of uracil into DNA remainder of the genome is more variable [18] [7,20]. A few putative DNA-binding proteins and transcrip- (G. Vestergaard, R.A. Garrett and D. Prangishvili, tional regulatory proteins have also been identified. Few unpublished data). gene products target or influence the translational appar- Modifications have been detected in two of the viral atus, exceptions being the intron-containing tRNALys genomes. SNDV carries a dam-like N(6) methylation of encoded in AFV2 and the tRNA guanine transglycosylases adenine residues in GATC sequences [8], whereas the present in several viruses [13] (G. Vestergaard, R.A. Garrett DNA of STSV1, which encodes three putative modifying and D. Prangishvili, unpublished data). enzymes, undergoes methylation of specific cytosine In general, crenarchaeal viral genes yield significant residues within the sequence CCGG. Preliminary evi- sequence matches primarily with genes from other dence suggests the cytosine methylations are absent from crenarchaeal viruses. Few significant matches are the host chromosomal DNA [7]. obtained with either bacterial or eukaryal genes. A possible exception was the observation of several matches, albeit at low significance levels, between rudiviral genes Genomic termini and those of eukaryal pox and Chlorella viruses, which Most of the linear genomes carry ITRs. For the elongated could relate to their employing a similar mechanism of virions, this is consistent with the symmetrical structures genome replication [22] (see subsequent sections). There is of their ends (Figure 3c–e). However, for some viruses the also evidence that a 37-kDa capsid protein of STIV can terminal repeats and genomic termini are only partially generate a tertiary and quaternary structure similar to characterized, predominantly because of the difficulties that of capsid proteins of bacterial and viruses, involved in obtaining complete sequences. Generally the despite the lack of significant sequence similarity [6]. terminal regions are not represented in shotgun clone libraries and they also tend to give low yields using DNA DNA replication amplification procedures [12,20]. Nevertheless, some of Genomic replication mechanisms have not been studied these genomes have been sequenced completely (Table 1). experimentally for any crenarchaeal viruses and they They include three rudiviral genomes each exhibiting a remain largely unknown, as do the protein components, large ITR (600–2000 bp) [20–22], the globulovirus PSV, host- or viral-encoded, which facilitate these processes. which carries a 190 bp ITR [15], and the lipothrixvirus Nevertheless, the viral genome organizations and gene AFV1, which has multiple short direct repeats near the contents provide some clues. For example, the large circular termini, with an 11 G-C bp at the termini. The terminal genome of STSV1 exhibits a single putative origin of regions of AFV1 also carry larger direct and inverted replication containing multiple, imperfect, ACT-rich repeats [12]. repeats [7]. Moreover, the diverse structures of the linear Some genomic termini are covalently modified genomes suggest that different modes of replication occur for [11,15,21,23]. For example, in the rudiviruses SIRV1 and these viruses. For example, a short terminal repeat at each SIRV2 the two DNA strands are covalently linked at the end of the lipothrixvirus AFV1 is preceded by a region of ends, generating a 4 bp loop that is susceptible to Bal31 w300 bp consisting of many direct repeats of the pentanu- endonuclease [21,23]. Similar structures might occur at cleotide TTGTT, or close variants thereof, which the ends of the PSV viral genome [15]. resemble telomeric ends of eukaryal chromosomes Although no proteins seem to be covalently attached to [12]. This raises the possibility of a primitive telomeric the DNA termini of SIRV1 and SIRV2 [21], they might be mechanism operating in AFV1 replication. Some bound to the lipothrixviral genomic termini. Evidence for lipothrixviral genomes exhibit a repeat-rich, internal, this derives from the demonstration that restriction non-protein-coding region. For example, AFV2 carries fragments containing the termini of the genomes of a 1008 bp element bordered by an ITR (GTCACTGA- TTV1 and AFV1 were hydrophobic and produced low CATAATA) that is rich in repeat sequences [13]. Such molar yields on phenol extraction [10,12]. regions could constitute internal origins of replication. www.sciencedirect.com 540 Review TRENDS in Microbiology Vol.13 No.11 November 2005

The observation of head-to-head and tail-to-tail linked regulation of transposase activity [31] and some of the replicative intermediates in cells infected with the viruses carry IS elements, including the bicaudavirus ATV rudivirus SIRV1 suggested a self-priming mechanism of [14]. Thus, host- or virus-encoded untranslated RNAs might replication, similar to that proposed for large eukaryotic be involved in the regulation of viral gene expression. Some dsDNA viruses, including poxviruses [21,22,26]. Consist- of the viral genomes, many of which are highly ACT-rich ent with this proposal are the similarities in the structures (Table 1), contain local GCC-rich regions that could encode of linear genomes of these archaeal and eukaryal viruses, stable RNAs but no systematic transcriptional studies have including long ITRs and covalently closed termini. More- yet been undertaken on these regions. over, each of these viruses encodes a Holliday junction resolvase, which is likely to resolve Holliday junctions Genome variation formed by the replicative intermediates. Different viruses show different degrees of genome The large ITRs of the rudiviruses are likely to have a stability. Sequencing of clone libraries of some genomes, common function in viral genome replication [20–22]: they including those of the rudiviruses SIRV2 and ARV1, all carry repeat sequence motifs that could function as showed no evidence of sequence heterogeneities even in signals for replication, including the tandem direct genomic regions where the clone sequence coverage was repeats TTTTTTTGC located near the genomic termini 10–15-fold. Other genomes show local regions with a few of the SIRV1 genome. Moreover, all of the rudiviral ITRs point mutations or the odd duplication. For example, PSV contain the internal repeat sequence AATTTAGGAATT- clones revealed 34 point mutations, one-third of which TAGGAATTT, located 100–150 bp from the genomic were concentrated within one short intergenic region, and termini. This is the only highly conserved sequence within one variant genome exhibited a 270 bp duplication that the ITRs of all three viruses, therefore, it could be an generated two new open reading frames (ORFs) [15]. important signal for DNA replication [20,22]. The genome of the rudivirus SIRV1 was found to be extremely unstable [23]. The isolated virus invariably Transcription contained a population of variants with different but Although studies on transcription of the fusellovirus SSV1 closely related genomes. Upon propagation in a given host were crucial for our seminal understanding of mechan- strain, one or more genomes dominate in the viral isms of transcriptional regulation in archaea [27], these population. However, upon passage in a new host strain studies examined the induction of viral replication in the viral population undergoes changes and other variants lysogens, rather than the infection cycle. Recently, the are selected [23]. first detailed analysis of transcription over the complete replication cycle was performed on rudiviruses [28]. 12 bp genetic elements In vivo studies demonstrated a rather simple and ordered Genome sequencing of the SIRV1 variants demonstrated transcriptional pattern for both SIRV1 and SIRV2 that is that they contain a few highly variable genomic regions consistent with fairly unsophisticated virus–host relation- (Figure 4c; labeled A–F) in which deletions and/or insertions ships (Figure 4a,b). SIRV promoters, like those of their have occurred, in addition to gene transpositions [23]. hosts, carry a TATA-like box and a transcription factor B Comparison of the variant genomes also revealed the responsive element. However, many of the promoters presence of small genetic elements, invariably 12 bp in exhibit an additional virus-specific consensus element, the length or multiples thereof, which tend to be concentrated in trinucleotide GTC located immediately downstream from these variable regions (Figure 4c). Although their preva- the TATA-like box [28]. The same pattern is discernible for lence in the genome suggests that they are mobile, they show approximately one-third of the ARV1 promoter regions, no sequence conservation nor are they flanked by conserved although there was little evidence of the GTC motif being target sequences for integration or bordered by direct conserved for homologous genes in the three viral repeats indicative of transposition. However, some of their genomes [20] (Table 1). The results underline the probable transcripts could generate ‘bulge-helix-bulge’ splicing importance of both the host transcriptional machinery and motifs, which are characteristic of archaeal intron splicing virus-specific factors in generating and regulating viral junctions [23]. This suggested that they could have been transcripts. Many viral transcripts for single genes and mobilized at the RNA level by splicing, followed by reverse the first genes of operons are also leaderless, and lack the splicing and reverse transcription. consensus Shine-Dalgarno motif (GGTG) but this Many of the 12 bp elements occur within ORFs and, phenomenon also appears to prevail for transcription of given that they all contain multiples of three base pairs, viral host chromosomes [12,20,29]. they will extend the size of the gene product, possibly leading to modified or new protein functions. Therefore, Antisense RNAs the primary function of these elements is probably to One haloarchaeal virus øH1 produces an antisense RNA, facilitate viral gene and genome variation [23]. which generates a w150 bp RNA–RNA hybrid that is A search for such elements in other crenarchaeal processed by a ss/ds-specific RNase in vivo [30].Apart viruses revealed examples in the stable SIRV2 genome from an intron contained within a tRNALys encoded in the [23] and in the betalipothrixviruses (G. Vestergaard, AFV2 genome [13], no untranslated RNAs have been found R.A. Garrett and D. Prangishvili, unpublished data) but encoded in the crenarchaeal viral genomes. However, not in the circular fuselloviral genomes, suggesting that numerous putative antisense RNAs have been detected in they have a widespread presence at least within linear Sulfolobus cells some of which might be involved in viral genomes. www.sciencedirect.com Review TRENDS in Microbiology Vol.13 No.11 November 2005 541

(a) ITR ITR 2029 2029

SIRV1 ORFs 55a 76 399 131 81 179 134 64 486 74 562 268 90c 241 75 1 90a 105 56 440 59 158b 55c 77 154 114 417 209 95 252 90b 32312 102 306 119 207 91 101 335 110 121 1070 158a 356 112 98 55b

(b)

SIRV1 transcripts

(c)

ABCDEF

Figure 4. Genomic characteristics of the rudivirus SIRV1 and its variants. (a) Genome map of SIRV1 showing the locations of the ITRs and the direction and size of all the putative protein coding genes; the ORFs are labeled according to the number of codons they contain. The two ORFs denoted by red arrows are absent from the closely related virus SIRV2. (b) Transcriptome analysis of the SIRV1 variant VIII for which transcripts were detected after the following times, post infection: greenZ30 min, blueZ1h, purpleZ2 h and redZ3h [28]. (c) Map showing the main variable regions, labeled A–F, which were detected in the variants of SIRV1. The vertical lines indicate the approximate positions of the 12 bp elements, which were detected in multiple viral variants [23]. Variable low complexity sequence regions viruses differ from those of the host chromosome. Virus- Another exceptional mechanism of genome variation was encoded replication factors are probably required and observed in variants of the lipothrixvirus TTV1. The possibly include unknown types of DNA polymerase, genome sequence of TTV1 exhibits two regions of low which could have a high biotechnological potential. The complexity sequence extending more than w250 bp and viral strategies for transcriptional regulation also remain 475 bp, which consist predominantly of the repeated unknown; it is possible that they encode regulatory hexamer AC(T)CCX [32]. Viruses isolated from different proteins but these remain to be characterized. The purified Thermoproteus tenax colonies showed evidence of mechanism of genomic rearrangement based partly on 30–102 bp insertion and/or deletions located exclusively in putatively mobile 12 bp genetic elements is also intri- these two regions [33]. The insertions exhibit the same guing. These elements occur in several crenarchaeal viral repeated hexameric structure that occurs in both genomic genomes, some of which exist naturally as a mixture of regions, suggesting that they are produced by recombina- variants. tion. Moreover, at least for the larger region, both The extent to which the major difference between deletions and insertions seem to maintain an ORF that crenarchaeal and euryarchaeal viruses reflects a phyloge- produces a highly repetitive threonine-proline sequence netic division or an adaptation to high (or low) tempera- [33]: this protein appears to be expressed in large amounts ture remains unclear. However, the temperature inside the Thermoproteus tenax cell [33] and could have an adaptation explanation receives some support from the important role in modifying the defences of the cell. observation of a lower frequency of particles resembling crenarchaeal hyperthermophilic viruses in cooler aquatic Exciting challenges ecosystems where crenarchaea are also abundant. Clearly, Double-stranded DNAviruses that infect the Crenarchaeota further studies on viral diversity are necessary to resolve showno clear similarities in their morphologies and genomic this question and to understand its evolutionary impli- properties to either bacterial or eukaryal viruses, nor do cations. This and other fundamental questions that are they resemble viruses of the Euryarchaeota. Moreover, currently being addressed concerning crenarchaeal virus failure to detect homologues for most of their genes in public biology are listed in Box 1. databases suggests that they employ novel biochemical mechanisms for viral functions, which can now be studied in Acknowledgements detail (see Box 1). We are grateful to Gisle Vestergaard, Monika Ha¨ring, Kim Bru¨gger, Thus, the replication mechanisms for the crenarchaeal Xu Peng, Alexandra Kessler, Reinhard Rachel and Qunxin She for their help and discussions and to Guennadi Sezonov for help in preparing the viruses remain unknown and preliminary evidence figures. We dedicate this review to the memory of the late Wolfram Zillig suggests that strategies for genome replication of several who pioneered work on crenarchaeal viruses.

Box 1. Future crenarchaeal viral research References 1 Dyall-Smith, M. et al. (2003) Haloarchaeal viruses: how diverse are † Is the observed morphological diversity of the crenarchaeal they? Res. Microbiol. 154, 309–313 viruses only the tip of the iceberg? 2 Zillig, W. et al. (1996) Viruses, plasmids and other genetic elements of † Which mechanisms ensure the stable persistence and low copy thermophilic and hyperthermophilic archaea. FEMS Microbiol. Rev. number of many viral genomes within host cells? 18, 225–236 † How are the termini of the linear genomes of the lipothrixviruses, 3 Rice, G. et al. (2001) Viruses from extreme thermal environments. globuloviruses and ampullaviruses structured? Proc. Natl. Acad. Sci. U. S. A. 98, 13341–13345 † Which viral proteins facilitate replication and transcription of the 4 Rachel, R. et al. (2002) Remarkable morphological diversity of viruses viral genomes and what are their structural and biochemical and virus-like particles in hot terrestial environments. Arch. Virol. properties? 147, 2419–2429 www.sciencedirect.com 542 Review TRENDS in Microbiology Vol.13 No.11 November 2005

5Ha¨ring, M. et al. (2005) Virusal diversity in hot springs of Pozzuoli, 20 Vestergaard, G. et al. (2005) ARV1, a novel rudivirus infecting the Italy, and characterisation of a unique archaeal virus Acidianus hyperthermophilic genus Acidianus. Virology 336, 83–92 bottle-shaped virus, from a new viral family, the Ampullaviridae. 21 Blum, H. et al. (2001) The linear genome of the archaeal virus SIRV1 J. Virol. 79, 9904–9911 has features in common with genomes of eukaryal viruses. Virology 6 Rice, G. et al. (2004) The structure of a thermophilic archaeal virus 281, 6–9 shows a double-stranded DNA viral capsid type that spans all domains 22 Peng, X. et al. (2001) Sequences and replication of genomes of the of life. Proc. Natl. Acad. Sci. U. S. A. 101, 7716–7720 archaeal rudiviruses SIRV1 and SIRV2: relationships to the archaeal 7 Xiang, X. et al. (2005) The Sulfolobus tengchongensis spindle-shaped lipothrixvirus SIFV and some eukaryal viruses. Virology 291, 226–234 virus STSV1: Virus-host interactions and genomic features. J. Virol. 23 Peng, X. et al. (2004) Multiple variants of the archaeal DNA rudivirus 79, 8677–8686 SIRV1 in a single host and a novel mechanism of genomic variation. 8 Arnold, H.P. et al. (2000) SNDV, a novel virus of the extremely Mol. Microbiol. 54, 366–375 thermophilic and acidophilic archaeon Sulfolobus. Virology 272, 24 Prangishvili, D. and Garrett, R.A. (2004) Exceptionally diverse 409–416 morphotypes and genomes of crenarchaeal hyperthermophilic 9 Prangishvili, D. et al. (1999) A novel virus family, the Rudiviridae: viruses. Biochem. Soc. Trans. 32, 204–208 Structure, virus-host interactions and genome variability of the 25 Birkenbihl, R.P. et al. (2001) Holliday junction resolving enzymes of Sulfolobus viruses SIRV1 and SIRV2. Genetics 152, 1387–1396 archaeal viruses SIRV1 and SIRV2. J. Mol. Biol. 309, 1067–1076 10 Janekovic, D. et al. (1983) TTV1, TTV2, and TTV3, a family of viruses 26 Baroudy, D. et al. (1982) Incompletely base-paired flip-flop terminal of the extremely thermophilic, anaerobic, sulfur-reducing archae- loops link the two DNA strands of the vaccinia virus genomes into one bacterium Thermoproteus tenax. Mol. Gen. Genet. 192, 39–45 uninterrupted polynucleotide chain. Cell 28, 315–324 11 Arnold, H.P. et al. (2000) A novel lipothrixvirus, SIFV, of the extremely 27 Zillig, W. et al. (1993) Transcription in archaea. In The Biochemistry of thermophilic crenarchaeon Sulfolobus. Virology 267, 252–266 Archaea (Kates, M. et al., eds), pp. 367–391, Elsevier 12 Bettstetter, M. et al. (2003) AFV1, a novel virus infecting hyperther- 28 Kessler, A. et al. (2004) Transcription of the rod-shaped viruses SIRV1 mophilic archaea of the genus Acidianus. Virology 315, 68–79 and SIRV2 of the hyperthermophilic archaeon Sulfolobus. 13 Ha¨ring, M. et al. (2005) Structure and genome organisation of AFV2, a J. Bacteriol. 186, 7745–7753 novel filamentous archaeal virus with unusual terminal structures. 29 Torarinsson, E. et al. (2005) Divergent transcriptional and transla- J. Bacteriol. 187, 3855–3858 tional signals in Archaea. Environ. Microbiol. 7, 47–54 14 Ha¨ring, M. et al. (2005) Independent virus development outside a host. 30 Stolt, P. and Zillig, W. (1993) Structure-specific ds/ss-RNase activity in Nature 436, 1101–1102 the extreme halophile Halobacterium salinarium. Nucleic Acids Res. 15 Ha¨ring, M. et al. (2004) Morphology and genome organization of the 21, 5595–5599 virus PSV of the hyperthermophilic archaea genera Pyrobaculum and 31 Tang, T-H. et al. (2005) Identification of novel non-coding RNAs as Thermoproteus: A novel virus family, the Globuloviridae. Virology potential antisense regulators in the archaeon Sulfolobus solfatar- 323, 233–242 icus. Mol. Microbiol. 55, 469–481 16 Reiter, W-D. et al. (1988) Archaebacterial viruses. Adv. Virus Res. 34, 32 Neumann, H. et al. (1989) Identification and characterisation of the 143–188 genes encoding three structural proteins of the Thermoproteus tenax 17 Schleper, C. et al. (1992) The particle from the extremely thermophilic virus TTV1. Mol. Gen. Genet. 217, 105–110 archaeon Sulfolobus is a virus. Proc. Natl. Acad. Sci. U. S. A. 89, 33 Neumann, H. and Zillig, W. (1990) Structural variability in the 7645–7649 genome of Thermoproteus tenax TTV1. Mol. Gen. Genet. 222, 435–437 18 Wiedenheft, B. et al. (2004) Comparative genomic analysis of 34 Palm, P. et al. (1991) Complete nucleotide sequence of the virus SSV1 hyperthermophilic archaeal Fuselloviridae viruses. J. Virol. 78, of the archaebacterium Sulfolobus shibatae. Virology 185, 242–250 1954–1961 35 Stedman, K.M. et al. (2003) Biological and genetic relationships 19 Snyder, J.C. et al. (2004) Effects of culturing on the population between fuselloviruses infecting the extremely thermophilic archaeon structure of a hyperthermophilic virus. Microb. Ecol. 48, 561–566 Sulfolobus: SSV1 and SSV2. Res. Microbiol. 154, 295–302

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