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Spindle-shaped virus-like particles are abundant in extreme geothermal environments, from which five spindle-shaped viral species have been isolated to date. They infect members of the hyperthermophilic archaeal genus Sulfolobus, and constitute the Fuselloviridae, a family of double-stranded DNA viruses. Here we present four new members of this family, all from terrestrial acidic hot springs. Two of the new viruses exhibit a novel morphotype for their proposed attachment structures, and specific features of their genome sequences strongly suggest the identity of the host-attachment protein. All fuselloviral genomes are highly conserved at the nucleotide level, although the regions of conservation differ between virus-pairs, consistent with a high frequency of homologous recombination having occurred between them. We propose a fuselloviral specific mechanism for interviral recombination, and show that the spacers of the Sulfolobus CRISPR antiviral system are not biased to the highly similar regions of the fusellovirus genomes.
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In contrast to the rather uniform landscape of virion morphotypes in aquatic systems under moderate environmental conditions, mainly represented by tailed bacteriophages (reviewed by Prangishvili, 2003), virus-like particles observed in ecological niches at high temperatures, low pH or high salinity reveal a high diversity of complex morphotypes (Guixa-Boixareu et al., 1996; Oren et al., 1997; Rice et al., 2001; Rachel et al., 2002; Häring et al., 2005; Porter et al., 2007; Bize et al., 2008). About 40 virus species isolated from such environments, all carrying double-stranded (ds) DNA genomes, have been described, which infect members of the third domain of life, the Archaea (reviewed in Prangishvili et al., 2006a). Most common are viruses with an overall spindle-shaped morphology, either tail-less, tailed or even two-tailed, which taxonomically have been assigned to the viral families Fuselloviridae (SSV1, SSV2, SSV4, SSVrh and SSVk1, single-tailed), Bicaudaviridae (ATV, two-tailed) and the genus Salterprovirus (His 1 and His 2) while some still require classification (STSV1 and PAV1) (Schleper et al., 1992; Bath and Dyall-Smith, 1998; Arnold et al., 1999; Geslin et al., 2003; Wiedenheft et al., 2004; Xiang et al., 2005; Bath et al., 2006; Prangishvili et al., 2006b; Peng, 2008).
Five fuselloviruses have so far been isolated from acidic geothermal environments in different locations in Asia, Europe and North America, and they replicate in species of the hyperthermophilic archaeal genus Sulfolobus, which represents a significant percentage of the microbial population in most acidic terrestrial hot springs. Another major player in these environments is the genus Acidianus, from which several viruses have been isolated, including the linear filamentous and rod-shaped viruses AFV1 and ARV1, respectively, which have close viral relatives that also infect Sulfolobus (Prangishvili et al., 2006a; Snyder et al., 2007). Although the two genera coexist, no fusellovirus has yet been isolated from Acidianus, even though it appears to be the most predominant Sulfolobus viral type.
The circular dsDNA genomes of five known fuselloviruses are highly similar at both nucleotide and amino acid sequence levels, with the majority of gene products being of unknown function and lacking homologues in public sequence databases other than in other archaeal viruses (Wiedenheft et al., 2004). The viral DNA is protected against the harsh environment, at temperatures above 80°C and pH values below 2, within a spindle-shaped virion about 100 nm long and 60 nm wide, with a bunch of short, thin fibres at one of the pointed ends (Martin et al., 1984; Stedman et al., 2003; Wiedenheft et al., 2004; Peng, 2008). In the electron microscopy, the body is sometimes observed to be slightly elongated and more ‘cigar-shaped’, and the tail fibres appear to be quite sticky, readily attaching to cellular fragments, as well as linking virions to produce rosette-like aggregates (Fig. 1A– SSV7).
Figure 1. A. Representative electron microscopy images of SSV6, SSV7 and ASV1. The end-filaments of SSV7 are very sticky, and the virus is almost always observed in ‘rosettes’ or attached to vesicles (white arrows). A rare single SSV7 is also shown (dotted white arrow). SSV6 and ASV1 do not have sticky ends and are always single, even when lying close together. Furthermore, SSV6 and ASV1 exhibit a wide range of morphotypes, varying from the standard spindle shape to an elongated sausage shape (indicated by dotted black arrows for SSV6). B. Magnification of the end-filaments of the three viruses. The filaments of SSV6 and ASV1 are thick, and seem to form a crown around the virus tips (black arrows) whereas SSV7 carries thinner filaments, that protrude directly from the virus tips. All samples were negatively stained with 2% Uranyl acetate and the scalebars are all 100 nm.
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SSV1 is the best studied fusellovirus, and the virion has been shown to contain proteins VP1, VP2, VP3 and small amounts of SSV1_D244 and SSV1_C792 (Reiter et al., 1987a; Menon et al., 2008). VP1 and VP3 are thought to be capsid proteins, whereas VP2 has been assigned a DNA-binding role, organizing DNA, but it is not encoded by other fuselloviruses (Stedman et al., 2003; Wiedenheft et al., 2004). Four non-structural SSV1 proteins have been characterized. SSV1_D63 is considered to link two different protein complexes, while SSV1_F93 and SSV1_F112 are DNA binding proteins implicated in transcriptional regulation (Kraft et al., 2004a,b; Menon et al., 2008). The fourth protein is an integrase of the tyrosine recombinase family, which catalyses site-specific integration of the viral genome into the host chromosome. As the viral recombination site (attP) is located within the integrase gene, integration leads to gene partition (Palm et al., 1991; Muskhelishvili et al., 1993). Despite this highly specialized adaptation, the integrase was recently shown to be non-essential for virus replication and basic viral functions (Clore and Stedman, 2007).
Replication of SSV1 and SSV2 can be induced by UV irradiation (Yeats et al., 1982; Stedman et al., 2003). The SSV1 transcription cycle, following UV induction, has also been elucidated by Northern analysis, physical mapping and DNA microarrays, and transcripts were classified as early (T5, T6 and T9), late (T3, Tx and T8) and UV inducible (Tind) (Fig. 2) (Reiter et al., 1987b; Fröls et al., 2007). The proteins encoded in the early transcripts of SSV1, and their homologues in other fuselloviruses, are often cysteine-rich compared with proteins encoded in the late transcripts (Palm et al., 1991; Stedman et al., 2003; Wiedenheft et al., 2004). This has recently been proposed to be due to intra- and extra-cellular localization of the early and late proteins respectively (Menon et al., 2008).
Figure 2. A. Graphical alignment of the nine circular fuselloviral genomes, linearized at the first nucleotide after the VP3 stop codon (following the convention of Wiedenheft et al., 2004). All ORFs larger than 50 amino acids indicated by arrows. Shades of blue and green: 13 ‘core’ genes. Dark grey: ORFs found in two or more fuselloviruses. Light grey: ORFs only found in one fusellovirus. Black: VP2. Yellow: SSV1_C792 homologues, both full length and partial. Red: SSV6_B1232 homologues. Orange: SSV1_B78 homologues. Light pink: SSV1_D244 homologues associated with the Integrase operon in all but ASV1 and SSVk1. Dark violet and light violet: Rad3-like helicase and Msed_2283 homologues substituting for a large part of the Integrase operon in ASV1, SSV7 and SSVk1. Dark pink: SSV1_F93 homologues. Brown: Highly conserved SSV1_C84 homologue overlapping with some of the other ‘core’ genes. Magenta: SSV1-C80 homologues and ASV1-A59. The transcripts identified by Fröls and colleagues (2007) are indicated below SSV1. B. The two different putative end-filament modules, exemplified by SSV1 and SSV6.
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Here we report on the isolation and properties of four novel members of the Fuselloviridae, infecting species of the hyperthermophilic archaeal genera Sulfolobus and Acidianus, almost doubling the number of known fuselloviruses and extending their host-range to a new genus, Acidianus. This merited a revised comparative genomic analysis of fuselloviruses, which provided insights into functions of some viral proteins and addressed general questions concerning the evolution of the viruses and interactions with their hosts.
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In this paper we describe four new members of the family Fuselloviridae, SSV5, SSV6, SSV7 and ASV1, isolated from acidic hot springs of Iceland and USA, which infect members of the hyperthermophilic archaeal genera Sulfolobus and Acidianus.
Until now, fuselloviruses had only been found to replicate in Sulfolobus species. Our discovery of ASV1 in Acidianus brierleyi shows that fuselloviruses can propagate in both the major culturable genera from aerobic, acidic hot springs. Therefore, it is likely that fuselloviruses also infect other host species from these environments, such as Caldococcus, Vulcanisaeta and Stygiolobus (Snyder et al., 2007). Furthermore, the family Fuselloviridae presumably also extends its host range into the vast number of currently uncultured species found in other extreme environments, such as the acid mine drainage ecosystem, where a VP2 homologue, recently found by community genomics (Andersson and Banfield, 2008), indicate the presence of fuselloviruses.
By almost doubling the number of described fuselloviruses, we are refining the definion of ‘core’ genes of the family. The 18 conserved, or ‘core’ genes, that were defined for SSV1, SSV2, SSVk1 and SSVrh (Wiedenheft et al., 2004) can now be reduced to 13 (Table 1) and may have to be revised further as more fuselloviruses are sequenced, but our findings correlate well with a recent analysis of fuselloviral proviruses in S. islandicus strains (Held and Whitaker, 2009). We exclude the SSV1_C792 homologues from the list of ‘core’ genes, because we do not consider SSV6_C213 and ASV1_B208 to be able to fully complement the proteins found in other fuselloviruses, which are about four times larger (Table 1).
Six of the ‘core’ genes have no discernible function based on their primary sequence, except for some of them carrying predicted transmembrane segments (Table 1), and experimental data will be needed to determine their functional roles. Of the remaining seven, the integrase function was characterized experimentally (Muskhelishvili et al., 1993; Muskhelishvili, 1994; Serre et al., 2002; Letzelter et al., 2004; Clore and Stedman, 2007). Moreover, VP1 and VP3 are virion components in SSV1 virions, and VP1 is processed from the N-terminus in SSV1, to a length of 73 aa (Reiter et al., 1987a), which may explain the significant size difference we observe among the VP1 genes (Table 1). The remaining C-terminus of VP1 is similar in both length and sequence to the VP3 protein, and their roles might be partially interchangeable in the virion matrix. Bioinformatical analyses predict DnaA-like activity for SSV1_B251 homologues (Koonin, 1992) and transcriptional regulation activity for three other ‘core’ genes: SSV1_A79 and SSV1_B129, which are transcribed early, during infection and are probably involved in controlling the hosts transcriptional apparatus, and SSV1_B115, which is co-transcribed together with VP1, VP2, VP3 and SSV1_C792, later in infection, and may be involved in controlling the assembly and/or packaging of virions.
Genes that are highly conserved but present in a subset of the fuselloviruses could provide a possible way of classifying the fuselloviruses into subgroups, albeit subgroups that overlap.
Thus, ASV1, SSV6 and SSV1, encode a VP2 homologue, indicating that they all share a DNA packaging system. However, the difference to the SSV1 protein in the C-terminus may indicate an alternative mode of interaction of the protein and viral DNA with the major virion proteins, VP1 and VP3.
Another subgroup would be the SSVs, which all encode a highly conserved homologue of SSV1_C80, a protein containing the RHH 1 CopG domain. ASV1 does not encode any gene with obvious sequence similarity to SSV1_C80. However, ASV1_A59 also has an RHH 1 CopG domain, although it groups with other RHH1-containing genes, including a few Sulfolobus chromosomal genes (e.g. Saci_0942). Furthermore, ASV1_A59 occupies the same genomic position as the SSV1_C80 homologues do in the SSV genomes, and it very likely acts as a functional homologue of SSV1_C80.
A third subgroup consists of ASV1, SSV7 and SSVk1, which all encode the Rad3-like helicase protein and the neighbouring Msed_2283 homologue (Fig. 2). The presence of the helicase strongly suggests that these two proteins are involved in DNA replication or recombination, and it is possible that the other fuselloviruses recruit host proteins to fulfill the same function.
A possible filament protein
The most striking genomic difference among the fuselloviruses is the ‘replacement’ of the SSV1_C792 module with the SSV6_B1232 module (Fig. 2B). It seems the C-terminal 170 aa from SSV1_C792 are essential, since they are retained as a small separate gene in both the ASV1 and SSV6 genomes; however, the remaining ∼620 aa of SSV1_C792 and the whole of SSV1_B78 are substituted by SSV6_B1232. The presence of the SSV6_B1232 module correlates with a difference in the number and structure of the sticky terminal filaments of the SSV6 and ASV1 virions, when compared with the SSV1_C792 module viruses (Fig. 1B). Possibly, there is a phenotype–genotype link, with the SSV1_C792 module being responsible for the multiple, thin, sticky filaments and the SSV6_B1232 module for the few, thick, less sticky filaments. In support of this hypothesis, small amounts of SSV1_C792 were recently found by mass-spectrometry in SSV1 virions (Menon et al., 2008). Moreover, the Phyre prediction tool suggested that both SSV1_C792 and SSV6_B1232 had a similar fold to the P2 receptor binding protein prd1, and it was recently shown that a large protein is responsible for the sticky end-fibres in the rudivirus SIRV2 (Steinmetz et al., 2008). Nevertheless, further studies will be needed to determine the exact functions of the SSV1_C792 and SSV6_B1232 modules in fuselloviruses.
Fuselloviral nucleotide similarity and a putative mechanism for interviral recombination
The multiple regions of high nucleotide similarity, or even identity, between the fuselloviral genomes do not represent a ‘core’ fusello-genome, since the regions of similarity differ between the various pairs of viruses, and often do not include the ‘core’ genes (Fig. 3). Instead, the pattern of similar and non-similar sections of DNA indicates frequent recombination events between fuselloviruses, similar to that observed for some bacteriophages (Hendrix et al., 1999). Possibly this occurs between pairs of fuselloviruses, present in the same host; however, we do not see a similar pattern of sequence similarity for the linear non-integrating archaeal viruses (Vestergaard et al., 2008a). Therefore, we suggest that a different mechanism is more likely.
Integrated fusellovirus genomes have been found in the Sulfolobus solfataricus P2 and in four S. islandicus chromosomes, where no trace of the covalently closed circular DNA (cccDNA) form was detected (Stedman et al., 2003; Held and Whitaker, 2009). Once a virus has been ‘caught’, a second, slightly different, fusellovirus might infect the same host, and insert itself into the same tRNA gene, resulting in a concatamer of the two fuselloviruses in the host chromosome (Fig. 5). This structure might be maintained for a couple of generations, but it would be inherently unstable if the two viral genomes are reasonably similar, as there would be a high chance of homologous recombination between the two integrated viruses. Such a recombination event would lead to the formation of one cccDNA virus and one inserted virus, both of which would consist of a part of each of the original two viruses (Fig. 5). Owing to the very short sequence similarity required for homologous recombination in Sulfolobus (Grogan, 2009), the cross-over point could potentially be in many different places, and each of these recombination events would form a unique mixture of the two viruses, similar to meiosis in eukaryotes. Thus, this offers a mechanism for rapidly generating a large number of diverse viral offspring. Our model does not exclude direct recombination between the cccDNA forms of fuselloviruses, but we propose that this type of ‘tandem insertion’ event happens frequently (on an evolutionary scale) in nature, and that repeated events, each involving a different pair of ‘parent’ fuselloviruses, would eventually produce the patchwork viral genomes we see today (Fig. 3).
Figure 5. Proposed model for recombination between integrated fuselloviruses. A. The first fusellovirus (SSVa) infects the host, and integrates into the chromosome. B. The second fusellovirus (SSVb) infects the host, and integrates into the same tRNA as SSVa. C. The ‘tandem integration’ of SSVa and SSVb. The dashed arrows indicate examples of homologous recombination sites. D. Examples of ‘offspring’ cccDNA fuselloviruses from the recombination of SSVa and SSVb.
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Our model also serves to explain why fuselloviruses have developed an integrase that is inactivated upon integration. The integrase is not essential for viral propagation (Clore and Stedman, 2007) but if the proposed recombination mechanism is correct, then the unique SSV-type integrase will help the virus in the long term, by promoting recombination with closely related viruses, since the inactivation provides a high chance of the viral genome being ‘caught’ in an integrated form in the chromosome. Nevertheless, inactivation of the integrase is not required for recombination between tandem insertions. Studies of the Sulfolobus plasmids pARN3 and pARN4 reveal stretches of nucleotide identity, which might have been generated by tandem insertions, even though these plasmids carry non-inactivatable integrases (Greve et al., 2004).
The inherent instability of a tandem insertion makes it difficult, if not impossible, to detect in nature. However, a concatamer of inserted viral genomes, similar to the one proposed in our model, was recently discovered in the chromosome of Methanococcus voltae A3. There, the two viral genomes integrated into the same tRNA gene are very different, preventing homologous recombination, thus ‘trapping’ the viral concatamer in the host chromosome (Krupovic and Bamford, 2008). The attP sites of SSV2 and SSV7 as well as SSV5 and SSV6 match the same tRNA in S. solfataricus P2 (Table 1), making it likely that fuselloviruses are also able to integrate into the same tRNA, forming concatamers, which are unstable due to the similarity between the fuselloviruses. Moreover, it was shown that SSVk1 is able to integrate into several different sites in the host genome (Wiedenheft et al., 2004), increasing the likelihood of finding a ‘partner’ for recombination. Finally, examples of related viruses infecting the same host at the same time are known for Sulfolobales, such as AFV6, AFV7 and AFV8 in Acidianus convivator (Vestergaard et al., 2008b).
If the ‘tandem insertion’ model is correct, then an evolutionary tree of an entire viral genome has no meaning, nor would that from individual ‘core’ genes (since two halves of the same gene might originate from different ‘parent’ viruses). One might instead analyse genes, described in the previous section, that are not shared by all fuselloviruses, since these genes cannot serve as cross-over points for homologous recombination. Although for the moment, the data set is too small for a phylogenetic analysis based on these genes, the presence or absence of certain genes in a subset of the viruses, has provided important clues to understanding protein functions in the fuselloviruses, including the putative filament proteins SSV1_C792 and SSV6_B1232.
With the current understanding of the CRISPR antiviral system, high nucleotide similarity between viruses should be disadvantageous, since a single spacer, matching a conserved region, will provide a host with immunity to several virus strains (Lillestøl et al., 2009). Nevertheless, the puzzling fact remains that fuselloviruses do possess highly similar, sometimes identical, nucleotide regions, and it is possible that the integration and/or the frequent recombination somehow provide the fuselloviruses with the means to evade the CRISPR system in their hosts.
It has been proposed that thermoacidophilic archaeal viruses are highly mobile, even between distant hot springs in the same geothermal area, and that different fuselloviruses continuously infect a more-or-less stable population of host species (Snyder et al., 2007). The high nucleotide similarity we have found, even between fuselloviruses isolated on different continents, seems to confirm that they do manage to exchange genetic material over the intercontinental distances that separate some of the geothermal ‘islands’ in the cold ‘ocean’.