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. Author manuscript; available in PMC: 2024 May 27.
Published in final edited form as: Environ Microbiol. 2019 Jan 8;21(6):2002–2014. doi: 10.1111/1462-2920.14479

New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures

Ying Liu 1,†, David Brandt 2,†, Sonoko Ishino 3, Yoshizumi Ishino 3, Eugene V Koonin 4, Jörn Kalinowski 2,*, Mart Krupovic 1,*, David Prangishvili 1,*
PMCID: PMC11128462  NIHMSID: NIHMS1986679  PMID: 30451355

Summary

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.

Introduction

Viruses infecting archaea attract attention due to their diverse morphologies, distinct genome sequences and unusual properties. Around 100 isolated species of archaeal viruses are classified into 17 families, 5 of which are shared with the viruses of bacteria and 12 are specific for archaea. Viruses that genetically and structurally resemble bacteriophages are classified into families Myo-viridae, Siphoviridae, Podoviridae, Turriviridae and Sphaerolipoviridae and are referred to as ‘the cosmopolitan viruses’ (Prangishvili et al., 2017). With the exception of the members of the Turriviridae (Fulton et al., 2009), the cosmopolitan viruses infect hyperhalophiles and methanogens of the phylum Euryarchaeota (Pfister et al., 1998; Krupovic et al., 2010; Porter et al., 2013; Pawlowski et al., 2014; Pietilä et al., 2014)and, potentially, members of the phylum Thaumarchaeota residing in soil and marine environments (Krupovic et al., 2011; Chow et al., 2015; Labonté et al., 2015; Abby et al., 2018; Ahlgren et al., in press). By contrast, viruses specific to archaea are characterized by morphotypes that are not observed among viruses of bacteria and eukaryotes and, almost exclusively, infect hyperthermophilic members of the phylum Crenarchaeota (Prangishvili, 2013; Prangishvili et al., 2017). The uniqueness of the virion morphologies of crenarchaeal viruses is matched by their distinctive gene repertoires, with ~75% of the genes encoding unique proteins that so far have remained refractory to functional annotation based on sequence analyses (Krupovic et al., 2018).

Although, the details of the infection cycles for many creanarchaeal viruses remain to be fully understood, the available information has already illuminated the diversity of virus-host interaction strategies in archaea, some of which are similar to those of bacteriophages, others are shared with eukaryotic viruses, whereas yet others are unique to archaeal viruses (Prangishvili et al., 2017). For instance, many archaeal viruses with circular dsDNA genomes encode integrases and can lysogenize their hosts by site-specifically integrating into the host chromosome (Martin et al., 1984; Prangishvili et al., 2006; Mochizuki et al., 2011; Anderson et al., 2017; Wang et al., 2018). The entry process is poorly understood for archaeal viruses, but it has been shown that, similarly to certain bacteriophages, rod-shaped and filamentous viruses of the families Rudiviridae and Lipothrixviridae, specifically recognize and bind to pili-like appendages on the host cell surface (Bettstetter et al., 2003; Quemin et al., 2013). Notably, unlike bacterial viruses but similarly to eukaryotic viruses, many viruses of archaea contain lipid envelopes (Kristensen et al., 2015). The assembly of spindle-shaped fuselloviruses occurs concomitantly with the release from the host cell by a budding mechanism similar to that employed by enveloped eukaryotic viruses, such as influenza virus and HIV-1 (Quemin et al., 2016). An example of an archaeal virus-specific feature is provided the unique egress mechanism employed by lytic creanarchaeal viruses from three unrelated families that involves formation and subsequent opening of large pyramidal structures on the host cell surface (Brumfield et al., 2009; Daum et al., 2014; Wang et al., 2017).

During the past few years, following the emerging general trend in virus discovery (Simmonds et al., 2017), an increasing number of crenarchaeal viruses have been identified by culture-independent approaches (Schoenfeld et al., 2008; Bolduc et al., 2012; Gudbergsdóttir et al., 2016). Complete or near complete new viral genomes have been assembled for viruses that belong to the established families Rudiviridae (Inskeep et al., 2013; Gudbergsdóttir et al., 2016), Fuselloviridae (Servín-Garcidueñas et al., 2013), Bicaudaviridae and Ampullaviridae (Gudbergsdóttir et al., 2016), as well as the new proposed group of headtailed viruses infecting Marine Group II Euryarchaeota, the magroviruses (Nishimura et al., 2017; Philosof et al., 2017). However, the success of the metagenomic surveys depends on the feasibility of obtaining a sufficient concentration of virus genomes in the sample. In the case of extreme thermal environments, the natural habitats of crenarchaea, the concentration of extracellular virions is notoriously low (Inskeep et al., 2013; Bolduc et al., 2015; Munson-McGee et al., 2018a). Accordingly, direct metagenomic sequencing of environmental samples often yields incomplete viral genomes. To overcome this limitation, environmental samples can be enriched for viruses, for example, using specific growth media and conditions that favour virus replication. For instance, virus enrichment has been successfully applied for analysis of viruses in the sample from Obsidian pool at Yellowstone National Park, USA, resulting in the description of two new virus genomes which were tentatively attributed to two types of virions observed in the cell culture (Garrett et al., 2010). In the present study, we used virus enrichment for the survey of virus diversity in hot, acidic Umi Jigoku hot spring in Beppu, Japan, to describe seven new crenarchaeal viruses.

Results and discussion

Sample collection and establishment of enrichment cultures

Although, one of the first crenarchaeal viruses, the spindle-shaped virus SSV1 (family Fuselloviridae), has been isolated from a hot spring in Beppu, Japan (Yeats et al., 1982; Martin et al., 1984), the viral diversity in acidic hot springs of Japan has not been investigated in any detail. To remedy this situation, two environmental samples, J14 and J15, were collected at two locations in the acidic, hot spring Umi Jigoku in Beppu, Japan, with temperatures of 75 °C and 80 °C, respectively, and pH values around 3.5, in September, 2016. Both samples represented a mixture of translucent liquid with red sand. The samples were used to inoculate the medium favourable for the growth of members of the genus Sulfolobus, which are known to dominate thermal, acidic environments. After incubation at 75 °C for 10 days, the appearance in the culture of virus-like particles (VLPs) was observed by transmission electron microscopy (TEM), as described in Experimental Procedures. Five types of particles were observed in the two enrichment cultures (Fig. 1). Two of these types – the filamentous (Fig. 1A and B) and polyhedral (Fig. 1A and C) – were dominant in both cultures. In addition, in the J14 culture, we observed thin filamentous particles (Fig. 1D), rod-shaped particles (Fig. 1E) and pleomorphic, spherical particles (Fig. 1F). Viruses with similar virion morphologies have been previously observed in acidic hot springs from other geographical locations (Zillig et al., 1993; Rice et al., 2001; Rachel et al., 2002; Bize et al., 2008). Interestingly, although, spindle-shaped viruses are common in archaea-dominated environments (Krupovic et al., 2013), we did not observe any spindle-shaped VLP in our enrichment cultures. Conceivably, our culturing procedure counter-selected the hosts susceptible to infection with such viruses, or else, their propagation in the selected conditions was extremely low, with the amount insufficient for the detection by TEM. The latter possibility appears most likely, considering that spindle-shaped viruses of the family Fuselloviridae are temperate, integrate into the host chromosome (Pina et al., 2011) and specific conditions are required for the interruption of the lysogenic cycle. Genome sequencing of the isolated strains and/or induction experiments should provide further understanding on the life cycles of viruses in the established enrichment cultures.

Fig. 1.

Fig. 1.

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Electron micrographs of virions observed in the enrichment cultures J14 and J15.

A, B, C. Virion types observed in both cultures, shown on the example of virions from the J15 culture.

D, E, F. Virion types observed only in the J14 culture. Samples were negatively stained with 2% (wt/vol) uranyl acetate. The black arrows point to the pleomorphic, spherical particles and the white arrow points to the polyhedral particle. Bars, 200 nm in (A), and 100 nm in (B–F).

Metagenomic analysis of 16S rRNA sequences in the enrichment cultures

The microbial diversity in the two enrichment cultures was assessed by high-throughput sequencing of the PCR-amplified 16S rRNA genes, as described in Experimental Procedures. Analysis of the reads using the Ribosomal Database Project (RDP) classifier (Wang et al., 2007), with a 80% confidence threshold showed that all reads belonged to members of the order Sulfolobales. The reads representing the genus Sulfolobus were predominant in both enrichment cultures, with approximately 85% in J14 and 79% in J15 (Fig. 2). Most of the remaining reads were assigned to unclassified members of the family Sulfolobaceae (14% in J14 and 20% in J15; Fig. 2), whereas the genera Sulfurisphaera and Acidianus together were represented by 1% of reads in both samples. The microbial diversity in the enrichment cultures appears to be significantly less than in the natural habitats, as judged by the comparison with the reported community compositions of diverse hot, acidic terrestrial springs (Menzel et al., 2015; Munson-McGee et al., 2018b). The overall sequencing statistics are provided in Supporting Information Table S1.

Fig. 2.

Fig. 2.

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Microbial diversity at the genus level in the enrichment cultures J14 and J15.

The percentage of different genera was estimated by the abundance of the corresponding reads in the PCR-amplified 16S rRNA genes from the enrichment cultures.

General features of the assembled viral genomes

A survey of the viral diversity in the enrichment cultures was performed by metagenomic analysis (see Experimental Procedures). A total of 19 and 20 contigs, longer than 1.5 kb of double-stranded DNA, were assembled from J14 and J15 samples respectively (Supporting Information Table S2). A BLASTN analysis against Nucleotide collection database at NCBI showed that the contigs fell into three categories: (a) 10 contigs with sequence similarities to the genomes of known archaeal viruses; (b) 19 contigs without any similarities in the database; (c) 10 contigs with sequence similarities with Sulfolobus genomes (Supporting Information Table S2). The contigs from the latter category were not further analysed, whereas the sequences from the former two categories were aligned and assembled into longer contigs (Supporting Information Table S3). As a result, we obtained seven complete or near-complete viral genomes ranging from 20 222 to 38 091 bp in length, two of which were circular and five were linear. The ambiguous nucleotide positions in the merged regions of two viral genomes (Supporting Information Table S3) were verified by amplification and sequencing using custom designed oligo primers (Supporting Information Table S4). The general features of the seven new viral genomes are summarized in Table 1.

Table 1.

Genomic properties of the novel viruses from this study.

Virus Potential host Genome size (bp) Form TIR G + C% ORF Annotated ORF%a. Accession number
SBFV1 Sulfolobales 37 919 Linear + 35.6 66 18% MK064562
SBFV2 Sulfolobales 38 091 Linear + 39.5 68 18% MK064563
SBFV3 Sulfolobus tokadaii 31 324 Linear - 37.9 54 24% MK064564
SBRV1 Sulfolobus islandicus 34 025 Linear + 26.8 60 32% MK064565
SPV1 Sulfolobus 20 222 Circular - 38.3 45 20% KY780159
SPV2 Sulfolobus 20 424 Circular - 38.5 46 20% MK064567
SBV1 Sulfolobales 26 520 Linear + 34.2 50 12% MK064566
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a.

Virion structural proteins are not included in the annotation.

Analyses of viral genomes and their taxonomical assessment

New filamentous and rod-shaped viruses.

The linear genomes of two filamentous viruses, Sulfolobales Beppu filamentous virus 1 (SBFV1) and Sulfolobales Beppu filamentous virus 2 (SBFV2), show low similarity on the nucleotide sequence level to each other (about one third of their genomic sequences, mainly distributed in regions between ~12 kb and 36 kb, share ~50% identity). However, analysis of the protein sequences encoded in these two genomes showed that the ~75% of the putative proteins were homologous, with amino acid (aa) sequence identities ranging from 20% to 72% (Fig. 3A).

Fig. 3.

Fig. 3.

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Graphical alignment of the linear genomes of filamentous viruses SBFV1, SBFV2, SBFV3 and SBRV1 with each other, as well as with the genomes of known filamentous viruses and putative viral contigs from Ca. Korarchaeota isolate ARK-16.

The ORFs are represented with arrows that indicate the direction of transcription. The blue arrows denote ORFs homologous in viruses of the order Sulfolobales, and the yellow arrows denote ORFs with homologues in archaeal or bacterial cells. The predicted transmembrane proteins are indicted by asterisks. The terminal inverted repeats (TIRs) are depicted by black bars. The annotations of the putative ORFs are indicated above/below the corresponding ORFs. The homologous genes shared between viruses are connected by shading of different degrees of grey based on the aa-sequence identity represented in the bar in the right below.

The SBFV1 genome is 37 919 bp in length and carries 134 bp-long imperfect terminal inverted repeats (TIRs). The actual TIRs are likely to be longer, as indicated by the presence of 174 bp stretch only at one of the genomic ends. The genome contains 66 predicted open reading frames (ORFs) and shows, an overall 97% nucleotide sequence identity with the genome of Sulfolobus filamentous virus 1 (SFV1) isolated from the same enrichment culture (Liu et al., 2018). The major difference between the two genomes is the presence, in the SBFV1 genome, of an 840 bp insertion containing an ORF (SBFV1 ORF8) of unknown provenance, which is absent in the genome of SFV1 (Fig. 3A). The ORFs that are shared by the two viruses are 92.4% to 100% identical to each other at the aa sequence level. Only 11 of these predicted proteins showed significant similarity with sequences in the public databases (E-value cutoff of 1 × 10−3). In eight cases, the similarities were with ORFs of Sulfolobales viruses (Fig. 3A). Functions of 12 ORFs could be identified (Fig. 3A). The structural proteins were identified by comparisons with SFV1 (Fig. 3A).

The SBFV2 genome is 38 091 bp in length and carries 160 bp-long imperfect TIRs which actually could be longer, given the presence of a 61 bp extension at one end. SBFV2 encodes 68 putative ORFs, among which 49 are shared with SBFV1 and SFV1 (Fig. 3A, Supporting Information Table S5). Except for four ORFs (SBFV2 ORFs 12, 13, 14 and 26), the homologous ORFs are syntenic in all three genomes (Fig. 3A).

The linear genome of Sulfolobales Beppu filamentous virus 3 (SBFV3) is 31 324 bp in length, with no TIRs identified, which suggests that the sequence could be incomplete. It encodes 54 ORFs, among which 32 have homologues in archaeal viruses infecting members of the order Sulfolobales. The highest similarity was observed with the genome of Acidianus filamentous virus 2 (AFV2) (Häring et al., 2005), with which it shares 29 homologous ORFs (Fig. 3A). Based on the observed similarities, we assign the virus SBFV3 to the family Lipothrixviridae. Twelve SBFV3 ORFs could be functionally annotated, including four DNA-binding proteins (three ribbon-helix–helix domain-containing proteins and one zinc finger protein), two glycosyltransferases of the GT-B fold, an S-adenosylmethionine (SAM)-dependent methyltransferase (FkbM domain containing), an AAA+ ATPase domaincontaining superfamily 6 helicase, a protein phosphatase, a lectin, an ATPase subunit of an ABC transporter, and an ArclD1-like anti-CRISPR protein (Supporting Information Table S6).

The genome of the Sulfolobales Beppu rod-shaped virus 1 (SBRV1) is 34 025 bp in length, carries 1322 bp long perfect TIRs, suggesting that it represents a complete viral genome. The genome contains 60 ORFs, of which 31 ORFs have homologues in archaeal viruses of the order Sulfolobales (Fig. 3B). The highest similarity, with 25 homologous genes, is with SIRV2 (Peng et al., 2001). Moreover, SBRV1 ORF20 is highly similar (82% identity) to the major capsid protein (MCP) of SIRV2, suggesting that SBRV1 is a bona fide member of the family Rudiviridae. Nineteen ORFs of SBRV1 could be functionally annotated (Supporting Information Table S6), including five DNA-binding proteins of three types (these proteins contain zinc finger, helix-turn-helix and ribbon-helix–helix domains), two GT-B fold glycosyltransferases, two SAM-dependent methyltransferases, a GNAT domain-containing acetyltransferase, a phosphoadenosine phosphosulfate reductase, a queuine tRNA-ribosyltransferase, a dCTP deaminase (dUTPase superfamily), a rolling-circle replication initiation protein of the HUH superfamily, a single-stranded DNA-binding protein, an AAA+ ATPase domain-containing protein, an amino acid transporter, a Cas4-like nuclease and a Holliday junction resolvase. With the exception of some genes encoding DNA-binding proteins, all of the shared genes are conserved in other rudiviruses (Prangishvili et al., 2013). Interestingly, SBRV1 does not encode a single identifiable homologue of the ArclD1 anti-CRISPR protein, which has undergone a substantial diversification and expansion in the genome of SIRV2 and, to a lesser extent, in several other rudiviruses (He et al., 2018). In some of the rudiviruses, ArclD1 homologues are clustered at both termini of the linear genome (He et al., 2018). In SBRV1, the corresponding loci are occupied by short ORFs with no apparent homologues in public sequence databases, except for ORF55 which encodes PCNA-interacting helixturn-helix DNA-binding protein (Gardner et al., 2014) with homologues in SIRV1, SIRV2 and SIRV3. In those three rudiviruses, the homologous helix-turn-helix proteins are adjacent to the genes encoding AcrID1 proteins and, accordingly, are thought to regulate the expression of acr genes (He et al., 2018). Thus, SBRV1 might encode novel anti-CRISPR proteins, which are located at the genome termini, probably, downstream of ORF55.

A new group of filamentous archaeal viruses.

Several predicted genes of SBFV1, SBFV2 and SFV1 show pronounced similarities to the ORFs from two metagenomic contigs present in public databases. Both these contigs, NODE_60 (PNIS01000177) and NODE_193 (PNIS01000064), are annotated as representing Candidatus Korarchaeota, an archaeal isolate ARK-16 originating from a thermal pool in the Uzon Caldera on Kamchatka peninsula, Russia (Wilkins et al., 2018) (Fig. 3A). The contig NODE_60 (42 819 bp in length) shares 15 homologues with the three viral genomes, including two helicases, three glycosyltransferases, the Cas4-like nuclease and the GDP-mannose 4,6-dehydratase (Fig. 3A, Supporting Information Table S6). Notably, HHpred analysis suggests that NODE_60 C0182_01370 (P = 94.3) and SBFV2 ORF33 (P = 94.6), but not their homologues in SBFV1 (ORF24) and SFV1 (ORF23, MCP), are homologous to the MCP of SIRV2 (DiMaio et al., 2015) (Fig. 3A, Supporting Information Table S6). The protein sequences of these ORFs, however, can be predicted to encode MCPs, and this assignment indeed has been confirmed by detailed biochemical and structural analyses in the case of SFV1, where ORF23 encodes one of the two paralogous MCPs structurally related to the single MCP of SIRV2 (Liu et al., 2018). Comparison of the contig NODE_60 with known lipothrixviruses revealed the highest similarity to Sulfolobus islandicus filamentous virus (SIFV) (Arnold et al., 2000), with 13 homologues (Fig. 3A). This analysis indicates that, most likely, NODE_60 is a virus contig. Although, no TIR was detected, the contig resembles a near complete viral genome according to the alignment with other filamentous virus genomes (Fig. 3A). In contrast, the 25 339 bp long contig NODE_193 carries 55 bp TIRs which are probably partial, as indicated by the presence of a 171 bp extension at one end. Although, the contig shares only five homologues with SBFV1, SBFV2 and SFV1, more than half of its predicted ORFs have homologues in known lipothrixviruses. One terminal region of about 11 kb in the contig NODE_193 is shared with SIFV, whereas the other terminal region of about 6 kb is shared with AFV2 (Häring et al., 2005) (Fig. 3A). These observations suggest that contig NODE_193 also is a near complete viral genome. Notably, NODE_60 and NODE_193 might represent the first identified virus infecting hyperthermophilic archaea of the putative phylum Korarchaeota. However, some ambiguities exist in the taxonomical assignment of the isolate ARK-16: although, it was considered to be a member of the Korarchaeota in the taxonomical assignment, in the phylogenetic tree the isolate clustered with Crenarchaeota (Wilkins et al., 2018). Regardless of the taxonomy of the putative hosts, the two contigs expand our understanding of the diversity of filamentous archaeal viruses.

Figure 4 shows positions of the newly isolated viruses SFV1, SBFV1, SBFV2, SBRV1 and the two newly identified putative viruses, ‘Korarchaeon isolate ARK-16’ contig NODE_60 and contig NODE_193, in a phylogenetic tree of the whole proteome sequences of all known members of the order Ligamenvirales of filamentous dsDNA viruses. The tree supports the assignment of the SBRV1 to the family Rudiviridae whereas SBFV3 and contig NODE_193 are assigned to the genus Deltalipothrixvirus of the family Lipothrixviridae (Fig. 4). Notably, SFV1, SBFV1, SBFV2 and the contig NODE_60 form a clade that is distinct from the currently recognized genera in the families Rudiviridae and Lipothrixviridae (Fig. 4).

Fig. 4.

Fig. 4.

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Inferred phylogenetic tree of archaeal filamentous and rodshaped viruses based on whole genome VICTOR (Meier-Kolthoff and Göker, 2017) analysis at the amino acid level.

The tree is rooted at mid-point, and the branch length is scaled in terms of the Genome BLAST Distance Phylogeny (GBDP) distance formula D6. The branch which support value >70% is shown. For each genome, the virus name and the GenBank accession number are indicated. The new viruses from this study are marked by filled stars, and the viruses newly identified from public metagenomic databases are marked by open stars.

New species in the family Portogloboviridae.

The two circular viral genomes, 20 222 bp and 20 424 bp long, were recovered from different enrichment cultures (Supporting Information Table S3). They share an overall 91.7% nucleotide sequence identity to each other. The former is identical to the genome of Sulfolobus polyhedral virus 1 (SPV1), which was previously isolated from the same enrichment culture and represents the first member of the proposed virus family Portogloboviridae (Liu et al., 2017). The viral contig of 20 424 bp represents a member of the same family and is designated Sulfolobus polyhedral virus 2 (SPV2).

SPV2 encodes 46 ORFs, among which 43 have homologues in SPV1, with 37 homologues showing aa-sequence identities from 69.9% to 99.7% (Fig. 5, Supporting Information Table S7). In addition, there are three insertions, two deletions and one duplication in the SPV2 genome, when compared to SPV1 (Fig. 5). Insertion 1 encompasses a region of 60 bp, which shows no similarity to any sequences in the public databases (Fig. 5). A 141 bp region from the 5′ distal terminus of SPV2 ORF26 to its upstream intergenic region was duplicated, followed by an insertion of 130 bp sequence that showed high similarity (81% identity) to the intergenic region between SPV1 ORF31 and ORF32 (Fig. 5). These rearrangements resulted in the loss of SPV1 ORF28 in the SPV2 genome due to a frameshift (Fig. 5). Insertion 3 resulted in the gain of a 286 bp region with a high similarity (77% identity) to the genome of S. solfataricus strain P1 that encompasses two new ORFs, ORF40 and ORF41. ORF40 shows no similarity to sequence in the databases, whereas ORF41 has an uncharacterized homologue in the genome of an archaeon Metallosphaera yellowstonensis (Fig. 5). Deletion 1, of a 274 bp region, resulted in the removal of SPV1 ORF21 from the SPV2 genome, and Deletion 2, of 120 bp, affected the intergenic region between SPV2 ORF30 and ORF31 (Fig. 5).

Fig. 5.

Fig. 5.

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Graphical comparison of SPV1 and SPV2 genomes.

The deletions (DEL), insertions (INS) and duplication (DUP) of sequences in SPV2 compared to SPV1 are marked. Annotations are shown above the corresponding ORFs.

A putative member of a new virus family.

The 26 520 bplong, linear genome of the Sulfolobales Beppu virus 1 (SBV1), has a GC content of 34.2% and 230 bp imperfect TIRs that are likely to be incomplete, as indicated by the presence of a 73 bp extension at one genome terminus. SBV1 encompasses 50 ORFs, among which 19 encode predicted transmembrane domains (Fig. 6, Supporting Information Table S8). Only one-fifth (10 ORFs) of the ORFs show significant similarities to sequences in the database (Fig. 6, Supporting Information Table S8). Nine ORFs have homologues in members of the virus families Rudiviridae, Lipothrixviridae and Portogloboviridae as well as the Sulfolobus ellipsoid virus 1 (SEV1), which has been recently classified into a new family Ovaliviridae (Wang et al., 2017), and Metallosphaera turreted icosahedral virus (MTIV) (Wagner et al., 2017) (Supporting Information Table S8). Four of these predicted proteins (ORFs 6, 11, 27 and 28) share homologues exclusively with SEV1, with aa sequence identities from 23% to 38% (Fig. 6). Notably, ORF6 encodes a 109 aa-long, α-helical protein, homologous to the SEV1 MCP VP4 (Wang et al., 2017). ORF27 containing three transmembrane domains is a homologue of SEV1 ORF641 and its C-terminal part is homologous to the SEV1 capsid protein VP2. Besides, ORF7 is homologous to a newly identified ORF of SEV1, sharing with it 52% aa sequence identity (Fig. 6, Supporting Information Table S8).

Fig. 6.

Fig. 6.

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Graphical comparison of SBV1 and SEV1 genomes.

The ORFs are represented with arrows that indicate the direction of transcription. The blue arrows denote ORFs with homologues in viruses of the Sulfolobales, the yellow arrows denote ORFs with archaeal and/or bacterial homologues. The ORFs containing predicted transmembrane domains are indicted by asterisks. The newly predicted SEV1 ORF is marked by a star. The terminal inverted repeats (TIRs) are depicted by black bars. Annotations are shown above/below the corresponding ORFs. The genes shared by SBV1 and SEV1 are connected by shading of different degrees of grey based on the aa-sequence identity represented in the bar in the lower right corner.

The putative functions of six SBV1 ORFs could be predicted. Two ORFs encode DNA-binding proteins with helix-turn-helix (ORF1) and ribbon-helix–helix (ORF35) domains respectively (Fig. 6, Supporting Information Table S8). ORF17 encodes a superfamily 2 helicase, which has homologues in several lipothrixviruses. ORF10, ORF32 and ORF39 are predicted to encode a glycosyltransferase (GT-B fold), a nucleotidyltransferase (Rossmann-like, alpha/beta fold) and a SAM-dependent methyltransferase (Rossmann-like fold) respectively. Homologues of ORFs 10 and 39 were found in archaeal viruses, whereas the closest homologues of ORF32 are encoded in archaeal/bacterial chromosomes (Fig. 6, Supporting Information Table S8).

Although, SBV1 shares with SEV1 the morphogenetic module, including homologous MCPs unique to these two viruses, the genome replication modules of these viruses are unrelated. In contrast to SEV1, SBV1 does not encode a DNA polymerase, but instead encodes other putative components of the replication machinery, including the helicase and nucleotidyltransferase (Fig. 6). This modular evolution, whereby functional modules are shuffled by recombination among distinct viruses infecting the same host, is common across the virosphere (Krupovic and Bamford, 2010; Iranzo et al., 2016a). Based on the results of the genome analysis, we suggest that SBV1 might become the founding member of a new virus family.

CRISPR spacer matches to the viral genomes

The CRISPR arrays in archaeal and bacterial genomes contain short spacer sequences, which record the past invasions by extrachromosomal mobile genetic elements, including viruses and plasmids (Makarova and Koonin, 2015). According to the CRISPR Finder database (CRISPRdb, the last update 05/2017) (Grissa et al., 2007), a total of 5342 unique spacers are present in the 34 complete genomes of the members of the order Sulfolobales, including Sulfolobus, Acidianus and Metallosphaera. We attempted to find the potential hosts of the new viruses identified in this work by comparing their genomes with the CRISPR spacers in the CRISPRdb using blastn. By using a setting of an E-value cutoff of 0.1 and over 90% identity between the viral genome fragment (i.e., the proto-spacer) and the full-length spacer, significant spacer matches were only found in two of the newly identified viruses, SBFV3 and SBRV1. For the former, two spacer matches were in the S. tokodaii strain 7, and for the latter, two spacers matches were in S. islandicus strains HVE10/4 and REY15A (Supporting Information Table S9). The results suggest that S. tokodaii and S. islandicus are potential hosts for SBFV3 and SBRV1, correspondingly. No spacer matches were found in the database for the other five viruses, and the newly identified filamentous virus contigs Ca. Korarchaeota NODE_60 and NODE_193, suggesting that the CRISPR spacer information in the dozens of sequenced Sulfolobales strains is still sparse compared with the divergent viral genomes in the environments.

Concluding remarks

The explosive increase of viral genome sequences in the age of metagenomics has greatly increased our appreciation of the global virus diversity (Labonté and Suttle, 2013; Dayaram et al., 2015; Rosario et al., 2015). Driven by the growing importance of metagenomics for virus discovery, the International Committee on Taxonomy of Viruses (ICTV) has recently modified the criteria for recognition of new virus species, for which now the availability of the genome sequence can serve as the sole basis (Simmonds et al., 2017). However, difficulties remain for classification of novel viruses which lack significant genomic similarities to known viruses. This problem is especially pertinent in the case of crenarchaeal viruses which show an astounding diversity of unusual morphologies and genome contents (Iranzo et al., 2016a,b).

Following the new ICTV regulations, dsDNA viral genomes that have been assembled in the studies described here and elsewhere (Rosario et al., 2015; Gudbergsdóttir et al., 2016; Yutin et al., 2018) can be officially classified as bona fide new viruses. Comparative genomics analysis of the viruses from the Umi Jigoku hot springs suggests that 4 of the 7 identified viruses should be assigned to known virus families: SBRV1 to the Rudiviridae (Prangishvili and Krupovic, 2012), SBFV3 to the Lipothrixviridae (Prangishvili and Krupovic, 2012) and SPV1 and SPV2 to the Portogloboviridae (Liu et al., 2017). These taxonomic assignments are consistent with the presence, in the enrichment cultures, of virions typical in morphology and size for members of these families. The ~910 nm long, rigid, rod-shaped virions (Fig. 1E) apparently belong to the rudivirus SBRV1. The ~950 nm long filamentous virions (Fig. 1D), most likely, represent the lipothrixvirus SBFV3. The polyhedral virions with the diameter of about 85 nm (Fig. 1A and F) appear to represent virions of SPV1 and SPV2.

Two of the new viruses, SBFV1 and SBFV2, are highly similar at the nucleotide and/or protein sequence levels to the recently described filamentous virus SFV1 (Liu et al., 2018). Together with the previously described metagenomic contig NODE_60, which we identify here as a viral genome, SFV1, SBFV1 and SBFV2 form a clade that is distinct from the known genera in the families of filamentous and rodshaped dsDNA viruses (Fig. 4). The abundance of the SBFV1 and SBFV2 genome sequences in the metagenomic data corresponds with the abundance of SFV1-like filamentous virions (Fig. 1A and B) in the enrichment cultures, suggesting that these virions belong to SBFV1 and SBFV2.

The genome of the new virus SBV1 shares limited sequence similarity with the recently described Sulfolobus ellipsoid virus SEV1, the sole representative of the recently created family Ovaliviridae (Wang et al., 2017) (Fig. 6). Although, the two viruses share the morphogenetic module, their genome replication modules are unrelated (Fig. 6). Thus far, we have been unable to isolate a pure strain of SBV1 for detailed structural and biochemical characterization. No particles similar to the virions of SEV1 were observed in the enrichment cultures. The only virion type that potentially could represent SBV1 consists slightly pleomorphic, spherical particles, measuring about 115 × 100 nm (Fig. 1F, black arrows).

The examination of microbial diversity in the enrichment cultures shows that potential hosts of the 7 new dsDNA viruses are members of the order Sulfolobales of the phylum Crenarchaeaota. CRISPR spacer analysis suggested that the most likely hosts of SBFV3 and SBRV1 are S. tokodaii and S. islandicus respectively (Supporting Information Table S9).

The approach for assessing viral diversity in environmental samples used in the present study, based on enriching environmental samples for viruses of specific hosts, has certain advantages over the classical metagenomics approach. In addition to providing sufficient amounts of genetic material for analysis, it allows the establishment of links between genome sequences and virion morphotypes, as well as identification of host strains. This is especially important for the discovery and description of novel virus types, as exemplified by SBV1.

Experimental procedures

Sampling sites and enrichment cultures

Two environmental samples J14 and J15 were collected from acidic, hot springs Umi Jigoku in Beppu, Japan in September, 2016. The samples were stored at 4 °C before enrichment culture for one month. Each sample of 10 ml was inoculated to 40 ml Sulfolobus growth medium (Zillig et al., 1993) and incubated at 75 °C for 10 days under aerobic conditions without shaking.

Cellular DNA extraction, amplification of 16S rRNA genes and sequencing

Cell pellets were collected from 2 ml enrichment cultures of sample J14 and J15 by centrifugation (12 000 r.p.m., 2 min, Eppendorf desktop centrifuge). DNA from cell fractions was isolated as described previously (Rensen et al., 2016). The 16S rRNA gene libraries were prepared according to the 16S Metagenomic Sequencing Library Preparation protocol for the Illumina Miseq system (15044223 B). The 16S rRNA gene fragments of V3-V4 region were amplified with universal primer sets ARC344F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGACGGGGYGCAGCAGGCGCGA-3′) and Arch806R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACVSGGGTATCTAAT-3′) (Takahashi et al., 2014) with Illumina adapters as underlined in the primer sequences. The libraries were sequenced by Illumina Miseq (Illumina San Diego, CA) with pair-end 250 bp read lengths (Genomics Platform, Institut Pasteur, France).

Taxonomic analysis of cell fractions based on 16S rRNA gene

The reads from sequencing of 16S rRNA amplicons were subjected to basic trimming on Galaxy (Afgan et al., 2016). Briefly, after demultiplexing, the sequences were trimmed by quality with Phred score ≥ 20 and reads longer than 180 bp were subjected to further analysis. The paired-end reads were merged by Pear (Afgan et al., 2016), and the redundant sequences were clustered. The chimeric sequences were removed using Ribosomal Database Project (RDP) chimera check pipeline (Edgar et al., 2011), whereas the remaining sequences were subjected to RDP classifier (Wang et al., 2007) with 80% confidence threshold for taxonomic analysis.

VLP concentration and transmission electron microscopy

The VLPs in the culture were separated from the cell fractions by centrifugation (9000 r.p.m., 15 min, Sorvall 1500 rotor), and concentrated by ultracentrifugation (35 000 r.p.m., 3 h, 4 °C, Beckman rotor type 60 Ti). The pellets were suspended in the sample buffer containing 20 mM Tris acidic buffer (pH 6) and 3% (wt/vol) NaCl. For TEM observations, samples were applied to carboncoated copper grids, negatively stained with 2% uranyl acetate and observed under the transmission electron microscope FEI Tecnai Biotwin. The images were digitally recorded, using a CCD camera connected to a computer.

VLP purification, viral DNA isolation and sequencing

For purification, the concentrated VLPs were subjected to isopycnic gradient centrifugation in 0.45 g/ml CsCl (in the sample buffer, Beckman SW60 Ti rotor, 40 000 r.p.m., 20 h, 15 °C). The fractions containing VLPs were taken from the CsCl gradients based on the light scattering bands, yielding four fractions from J14 sample and five fractions from J15 sample (Supporting Information Fig. S1). Nucleic acids were extracted from each CsCl fraction as described previously (Rensen et al., 2016). Sequencing libraries were prepared from 50 ng input DNA of each fraction using the Illumina Nextera library preparation kit and sequenced on Illumina HiSeq 1500 (Illumina, San Diego, CA). Sequenced reads (2 × 250 bp, paired-end mode) were quality-trimmed using Trimmomatic v0.3.5 with a sliding-window approach (window size: 4; quality threshold: 30) (Bolger et al., 2014) and assembled using MEGAHIT v1.1.1 in meta-sensitive mode (Li et al., 2015). Remaining ambiguous regions in assembled contigs were amplified using specifically designed oligonucleotide primers (Supporting Information Table S4). PCR product sequences were determined on an Applied Biosystems Prism 3730xl (Weiterstadt, Germany) Sanger sequencer using BigDye-terminator chemistry.

Analysis of viral genomes

ORFs were predicted that are larger than 35 codons by RAST v2.0 (Overbeek et al., 2013), and confirmed manually by searching for a putative ribosome-binding site upstream of the start codon. The in silico-translated protein sequences were used as queries to search for sequence homologues in the nonredundant protein database using PSI-BLAST (Altschul et al., 1990) with an upper threshold E-value of 1 × 10−3 at National Center for Biotechnology Information (NCBI). The global alignment of amino acid sequences were carried out by EMBOSS Needle tool (McWilliam et al., 2013). Searches for distant homologues were performed using HHpred (Söding et al., 2005) against different protein databases, including PFAM (Database of Protein Families), PDB (Protein Data Bank), CDD (Conserved Domains Database) and COG (Clusters of Orthologous Groups), which are accessible via the HHpred website. Transmembrane domains were predicted using TMHMM (Krogh et al., 2001), whereas the secondary structure was predicted using Jpred (Drozdetskiy et al., 2015) and PsiPred (Jones, 1999). Pair-wise genomic comparison was visualized by EasyFig (Sullivan et al., 2011).

Phylogenetic analysis of filamentous dsDNA viruses

The phylogenetic analysis of viral whole proteome sequences was carried out by the VICTOR online resource, using the distance formula D6 (Meier-Kolthoff and Göker, 2017).

Analysis of CRISPR spacer matches

The virus genomes were searched against the CRISPR database (CRISPRdb) (Grissa et al., 2007) using BLAST with an upper threshold E-value of 0.1. Matches that had over 90% of sequence identity between the putative proto-spacer and the full-length spacer were considered significant.

Accession numbers

The viral genome sequences have been deposited in the GenBank database as listed in Table 1.

Supplementary Material

Supplementary Information

Fig. S1. Purification of VLPs from J14 and J15 enrichment cultures by CsCl isopycnic gradient centrifugation. The four (F1-F4) and five (F1-F5) light scattering bands were collected for viral genome isolation, as indicated.

Table S1. Statistics of reads of 16S rRNA gene from Illumina sequencing.

Table S2. Viral contigs assembled from each CsCl fraction.

Table S3. Complete viral genomes assembled from individual or different CsCl fractions.

Table S4. Primer sequences for amplification and sequencing of ambiguous sequence regions (Oligonucleotides were purchased from Metabion (Planegg, Germany)).

Table S5. Amino acid sequence identity between homologues of SFV1 and SBFV1, as well as SBFV1 and SBFV2.

Table S6 Annotations of SBFV3, SBRV1, Ca. Korarchaeota isolate ARK-16 NODE_60 and NODE_193.

Table S7. The similarities between homologues of SPV1 and SPV2.

Table S8 Annotation of SBV1 genome.

Table S9 Significant CRISPR spacer matches to the new archaeal viruses in this study.

Acknowledgements

This work was supported by the European Union’s Horizon 2020 research and innovation program under grant agreement 685778 (project VIRUS-X), and l’Agence Nationale de la Recherche (projects EXAVIR and ENVIRA). We acknowledge Prisca Viehöver (Genetics and Genomics of Plants, Bielefeld University, for Sanger sequencing as well as Anika Winkler and Katharina Hanuschka (Center for Biotechnology, Bielefeld University) for technical assistance during next-generation Illumina sequencing.

Footnotes

Supporting Information

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

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Associated Data

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Supplementary Materials

Supplementary Information

Fig. S1. Purification of VLPs from J14 and J15 enrichment cultures by CsCl isopycnic gradient centrifugation. The four (F1-F4) and five (F1-F5) light scattering bands were collected for viral genome isolation, as indicated.

Table S1. Statistics of reads of 16S rRNA gene from Illumina sequencing.

Table S2. Viral contigs assembled from each CsCl fraction.

Table S3. Complete viral genomes assembled from individual or different CsCl fractions.

Table S4. Primer sequences for amplification and sequencing of ambiguous sequence regions (Oligonucleotides were purchased from Metabion (Planegg, Germany)).

Table S5. Amino acid sequence identity between homologues of SFV1 and SBFV1, as well as SBFV1 and SBFV2.

Table S6 Annotations of SBFV3, SBRV1, Ca. Korarchaeota isolate ARK-16 NODE_60 and NODE_193.

Table S7. The similarities between homologues of SPV1 and SPV2.

Table S8 Annotation of SBV1 genome.

Table S9 Significant CRISPR spacer matches to the new archaeal viruses in this study.

NIHMS1986679-supplement-Supplementary_Information.docx (487KB, docx)

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