The diversity of archaeal viruses is severely undersampled compared with that of viruses infecting bacteria and eukaryotes, limiting our understanding on their evolution and environmental impacts. Here, we describe the isolation and characterization of four new viruses infecting halophilic archaea from the saline Lake Retba, located close to Dakar on the coast of Senegal. Three of the viruses, HRPV10, HRPV11 and HRPV12, have enveloped pleomorphic virions and should belong to the family Pleolipoviridae, whereas the forth virus, HFTV1, has an icosahedral capsid and a long non-contractile tail, typical of bacterial and archaeal members of the order Caudovirales. Comparative genomic and phylogenomic analyses place HRPV10, HRPV11 and HRPV12 into the genus Betapleolipovirus, whereas HFTV1 appears to be most closely related to the unclassified Halorubrum virus HRTV-4. Differently from HRTV-4, HFTV1 encodes host-derived minichromosome maintenance helicase and PCNA homologues, which are likely to orchestrate its genome replication. HFTV1, the first archaeal virus isolated on a Haloferax strain, could also infect Halorubrum sp., albeit with an eightfold lower efficiency, whereas pleolipoviruses nearly exclusively infected autochthonous Halorubrum strains. Mapping of the metagenomic sequences from this environment to the genomes of isolated haloarchaeal viruses showed that these known viruses are underrepresented in the available viromes.
Conflict of Interest
The authors have no conflict of interest to declare.
|emi14604-sup-0001-supinfo.docxWord 2007 document , 12.6 KB||Appendix S1: Supporting Information|
|emi14604-sup-0002-TableS1.xlsxExcel 2007 spreadsheet , 27.3 KB||Supplementary Table 1. Annotation of pleolipoviral genomes|
|emi14604-sup-0003-TableS2.xlsxExcel 2007 spreadsheet , 20.5 KB||Supplementary Table 2. Blastp of pleolipoviruses|
|emi14604-sup-0004-TableS3.xlsxExcel 2007 spreadsheet , 39.3 KB||Supplementary Table 3. Annotation of HFTV1 genome|
|emi14604-sup-0005-TableS4.docxWord 2007 document , 25 KB||Supplementary Table 4. Haloferax and Halorubrum culture collection strains used in this study|
|emi14604-sup-0006-Figures.pdfPDF document, 1.1 MB||
Supplementary Figure 1. Plaque morphologies of the Lake Retba viruses. Four plaques for each virus have been circled. A. HRPV10 hazy plaques of 3-5 mm in diameter. B. HRPV11 hazy plaques of 5-10 in diameter (very difficult to document, but visible in optimal lighting conditions). C. HRPV12 hazy plaques of 5-8 mm in diameter. D. HFTV1 clear plaques of 2-4 mm in diameter.
Supplementary Figure 2. Comparison of genomic contigs obtained for HRPV11 using two different assembly algorithms (de novo assembly algorithm implemented in CLC Genomics Workbench v7 and SPAdes, respectively).
Supplementary Figure 3. A and B. Maximum likelihood phylogeny of ORF4 (spike protein) and ORF7 (NTPase) from pleolipoviruses. Members of the genera Alphapleolipovirus, Betapleolipovirus, and Gammapleolipovirus are colored blue, orange, and green, respectively, whereas HRPV10-12 are highlighted in grey. C. Maximum likelihood phylogenetic tree of the large subunit of the terminases from cultivated and uncultivated tailed haloviruses. Uncultivated and cultivated haloviruses are highlighted with blue and orange backgrounds, respectively, whereas HFTV1 is shown on a grey background. Sequences were aligned using MUSCLE (Edgar, 2004) and maximum likelihood trees were constructed using the program FastTree2 (Price et al., 2010). The numbers above the branches represent bootstrap support values from 100 replicates. The scale bars represent the number of substitutions per site.
Supplementary Figure 4. Analysis of the HFTV1 genome by nuclease treatments. Lane 1, non-treated genome; Lane 2, genome treated with RQ1 DNase; Lane 3, genome treated with Exonuclease III; Lane 4, genome treated with mung bean nuclease (5 U / mg DNA). The positions of the molecular mass markers (bp) are indicated on left. The arrow indicates the position of the agarose gel wells.
Supplementary Figure 5. Classification of the HFTV1 open reading frames based on the best-blast hit analysis. Sequence similarity searches were performed using blastp algorithm against the NCBI non-redundant proteins database (for details, see Supplementary Table 3).
Supplementary Figure 6. In silico analysis of the HFTV1 genome using PhageTerm, a tool specifically designed for determination of genomic termini and packaging mechanism using next-generation sequencing data. A. Sequence coverage at the predicted genomic terminus of HFTV1. Exact terminal position is represented by a dotted red line. B. Evaluation of alternative start positions in the HFTV1 genome. The position 92 is determined as the most likely start site with the best p-value. C. Summary of the PhageTerm results indicating terminal redundancy of the HFTV1 genome; the likely start position; the lack of defined right end of the genome (i.e., distributed); the circular permutation; orientation; the class of packaging mechanism; and the typical representative (bacteriophage P1) with a similar genome packaging mechanism.
Supplementary Figure 7. A. Phylogenomic tree of cultivated and uncultivated tailed haloviruses was constructed using the Genome BLAST Distance Phylogeny (GBDP) strategy implemented in VICTOR (Meier-Kolthoff and Goker, 2017). The numbers above branches are GBDP pseudo-bootstrap support values from 100 replications. B. Recruitment plot for haloviruses against saltern viromes sequenced from the Lake Retba (Roux et al., 2016) and South Bay Salt Works (Rodriguez-Brito et al., 2010) was performed by BLASTN algorithm and expressed as Reads recruited Per Kb of genome per Gb of metagenome (RPKG) (see Experimental Procedures for details).
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
- 2012) Snapshot of virus evolution in hypersaline environments from the characterization of a membrane-containing Salisaeta icosahedral phage 1. Proc Natl Acad Sci U S A 109: 7079–7084.
- 2018) Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome. Front Microbiol 9: 28.
- 2012) Structure unifies the viral universe. Annu Rev Biochem 81: 795–822.
- 2019) Discovery of several novel, widespread, and ecologically distinct marine Thaumarchaeota viruses that encode amoC nitrification genes. ISME J: 13: 618–631.
- 2000) A novel lipothrixvirus, SIFV, of the extremely thermophilic crenarchaeon Sulfolobus. Virology 267: 252–266.
- 2015a) Haloarchaeal virus morphotypes. Biochimie 118: 333–343.
- 2015c) Archaeal viruses multiply: temporal screening in a solar saltern. Viruses 7: 1902–1926.
- 2018a) Extremely halophilic pleomorphic archaeal virus HRPV9 extends the diversity of pleolipoviruses with integrases. Res Microbiol 169: 500–504.
- 2018b) The unexplored diversity of pleolipoviruses: the surprising case of two viruses with identical major structural modules. Genes (Basel) 9: 131.
- 2015b) Haloviruses of archaea, bacteria, and eukaryotes. Curr Opin Microbiol 25: 40–48.
- 2012) Global network of specific virus-host interactions in hypersaline environments. Environ Microbiol 14: 426–440.
- ICTV Report Consortium. (2017) ICTV virus taxonomy profile: Pleolipoviridae. J Gen Virol 98: 2916–2917.
- 2005) Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica. J Virol 79: 9097–9107.
- 2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19: 455–477.
- 2013) The minichromosome maintenance replicative helicase. Cold Spring Harb Perspect Biol 5: a012807.
- 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
- 2016) Functional interactions of archaea, bacteria and viruses in a hypersaline endolithic community. Environ Microbiol 18: 2064–2077.
- 2007) Genomic plasticity in prokaryotes: the case of the square haloarchaeon. ISME J 1: 235–245.
- 2016) Virus-mediated archaeal hecatomb in the deep seafloor. Sci Adv 2: e1600492.
- 2014) Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases. PLoS Genet 10: e1004181.
- 2013) High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake. Proc Natl Acad Sci U S A 110: 16939–16944.
- 2016a) Vesicle-like virion of Haloarcula hispanica pleomorphic virus 3 preserves high infectivity in saturated salt. Virology 499: 40–51.
- 2016b) Archaeal Haloarcula californiae icosahedral virus 1 highlights conserved elements in icosahedral membrane-containing DNA viruses from extreme environments. mBio 7: e00699-16.
- 2017) HCIV-1 and other tailless icosahedral internal membrane-containing viruses of the family Sphaerolipoviridae. Viruses 9: e32.
- 2016) Seasonal dynamics of extremely halophilic microbial communities in three Argentinian salterns. FEMS Microbiol Ecol 92: fiw184.
- 2009). Halohandbook. URL http://www.haloarchaea.com/resources/halohandbook/
- 2019) Halobacterium salinarum virus ChaoS9, a novel halovirus related to PhiH1 and PhiCh1. Genes (Basel) 10: e194.
- 2018) Complete genome sequence of the model halovirus phiH1 (phiH1). Genes (Basel) 9: e493.
- 2018) The PL6-family plasmids of Haloquadratum are virus-related. Front Microbiol 9: 1070.
- 2011) Haloquadratum walsbyi: limited diversity in a global pond. PLoS One 6: e20968.
- 1999) Novel 16S rRNA gene sequences retrieved from highly saline brine sediments of Kebrit Deep, Red Sea. Arch Microbiol 172: 213–218.
- 2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.
- 2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26: 2460–2461.
- 2019) The structure of a prokaryotic viral envelope protein expands the landscape of membrane fusion proteins. Nat Commun 10: 846.
- 2017) A plasmid from an Antarctic haloarchaeon uses specialized membrane vesicles to disseminate and infect plasmid-free cells. Nat Microbiol 2: 1446–1455.
- 1993) PHYLIP (phylogeny inference package), version 3.5 c: Joseph Felsenstein.
- 1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497–509.
- 2014) Plasmids from Euryarchaeota. Microbiol Spectr 2: PLAS-0027-2014.
- 2012) Reconstructing viral genomes from the environment using fosmid clones: the case of haloviruses. PLoS One 7: e33802.
- 2014) Protein-protein interactions leading to recruitment of the host DNA sliding clamp by the hyperthermophilic Sulfolobus islandicus rod-shaped virus 2. J Virol 88: 7105–7108.
- 2017) PhageTerm: a tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci Rep 7: 8292.
- 2018) Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations. FEMS Microbiol Rev 42: 353–375.
- 2001) TIGRFAMs: a protein family resource for the functional identification of proteins. Nucleic Acids Res 29: 41–43.
- 2010) Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11: 119.
- 2016b) Bipartite network analysis of the archaeal virosphere: evolutionary connections between viruses and capsidless mobile elements. J Virol 90: 11043–11055.
- 2016a) The double-stranded DNA virosphere as a modular hierarchical network of gene sharing. mBio 7: e00978–e00916.
- 1986) Haloarcula hispanica spec. Nov. and Haloferax gibbonsii spec, nov., two new species of extremely halophilic archaebacteria. Syst Appl Microbiol 8: 75–79.
- 2000) Genomic sequences of bacteriophages HK97 and HK022: pervasive genetic mosaicism in the lambdoid bacteriophages. J Mol Biol 299: 27–51.
- 1972) Techniques of Lipidology. In Techniques of Lipidology: Isolation, Analysis and Identification of Lipids. Amsterdam: North-Holland.
- 2016) The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes. Nucleic Acids Res 44: 4551–4564.
- 2011) Local biotic environment shapes the spatial scale of bacteriophage adaptation to bacteria. Am Nat 177: 440–451.
- 2010) Order to the viral universe. J Virol 84: 12476–12479.
- 2018) Viruses of archaea: structural, functional, environmental and evolutionary genomics. Virus Res 244: 181–193.
- 2010a) Comparative analysis of the mosaic genomes of tailed archaeal viruses and proviruses suggests common themes for virion architecture and assembly with tailed viruses of bacteria. J Mol Biol 397: 144–160.
- 2010b) The evolutionary history of archaeal MCM helicases: a case study of vertical evolution combined with hitchhiking of mobile genetic elements. Mol Biol Evol 27: 2716–2732.
- 2011a) Genomics of bacterial and archaeal viruses: dynamics within the prokaryotic virosphere. Microbiol Mol Biol Rev 75: 610–635.
- 2011b) A thaumarchaeal provirus testifies for an ancient association of tailed viruses with archaea. Biochem Soc Trans 39: 82–88.
- 2015) FastME 2.0: a comprehensive, accurate, and fast distance-based phylogeny inference program. Mol Biol Evol 32: 2798–2800.
- 2013) Characterization of CRISPR RNA biogenesis and Cas6 cleavage-mediated inhibition of a provirus in the haloarchaeon Haloferax mediterranei. J Bacteriol 195: 867–875.
- 2015) Identification and characterization of SNJ2, the first temperate pleolipovirus integrating into the genome of the SNJ1-lysogenic archaeal strain. Mol Microbiol 98: 1002–1020.
- 2010) Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs. Nucleic Acids Res 38: 3952–3962.
- 2019) Novel Caudovirales associated with marine group I Thaumarchaeota assembled from metagenomes. Environ Microbiol. https://doi.org/10.1111/1462-2920.14462. [Epub ahead of print].
- 1997) tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25: 955–964.
- 2015) The adaptive immune system of Haloferax volcanii. Life (Basel) 5: 521–537.
- 2014) Dark matter in archaeal genomes: a rich source of novel mobile elements, defense systems and secretory complexes. Extremophiles 18: 877–893.
- 2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 14: 60.
- 2017) VICTOR: genome-based phylogeny and classification of prokaryotic viruses. Bioinformatics 33: 3396–3404.
- 1993) HF1 and HF2: novel bacteriophages of halophilic archaea. Virology 197: 678–684.
- 2010) Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds. Extremophiles 14: 161–169.
- 2002) Molecular ecology of extremely halophilic archaea and bacteria. FEMS Microbiol Ecol 39: 1–7.
- 1997) Occurrence of virus-like particles in the Dead Sea. Extremophiles 1: 143–149.
- 2007) Sequence analysis of an archaeal virus isolated from a hypersaline lake in Inner Mongolia, China. BMC Genomics 8: 410.
- 2011) The archaeal PCNA proteins. Biochem Soc Trans 39: 20–24.
- 2003) Origins of highly mosaic mycobacteriophage genomes. Cell 113: 171–182.
- 2017) Novel abundant oceanic viruses of uncultured marine group II Euryarchaeota. Curr Biol 27: 1362–1368.
- 2012) Virion architecture unifies globally distributed pleolipoviruses infecting halophilic archaea. J Virol 86: 5067–5079.
- 2013a) Modified coat protein forms the flexible spindle-shaped virion of haloarchaeal virus His1. Environ Microbiol 15: 1674–1686.
- 2014) Archaeal viruses and bacteriophages: comparisons and contrasts. Trends Microbiol 22: 334–344.
- 2010) The single-stranded DNA genome of novel archaeal virus halorubrum pleomorphic virus 1 is enclosed in the envelope decorated with glycoprotein spikes. J Virol 84: 788–798.
- 2013c) Insights into head-tailed viruses infecting extremely halophilic archaea. J Virol 87: 3248–3260.
- 2013b) Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story. Proc Natl Acad Sci U S A 110: 10604–10609.
- 2009) An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope. Mol Microbiol 72: 307–319.
- 2016) Pleolipoviridae, a newly proposed family comprising archaeal pleomorphic viruses with single-stranded or double-stranded DNA genomes. Arch Virol 161: 249–256.
- 2015) Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. Elife 4: e06416.
- 2017) The enigmatic archaeal virosphere. Nat Rev Microbiol 15: 724–739.
- 2010) FastTree 2–;approximately maximum-likelihood trees for large alignments. PLoS One 5: e9490.
- 2012) SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes. Bioinformatics 28: 1823–1829.
- 2006) FigTree 1.4.3 - a graphical viewer of phylogenetic trees and a program for producing publication-ready figures. URL http://treebioedacuk/software/figtree/
- 2014) Global phylogenomic analysis disentangles the complex evolutionary history of DNA replication in archaea. Genome Biol Evol 6: 192–212.
- 2010) Viral and microbial community dynamics in four aquatic environments. ISME J 4: 739–751.
- 2004) Inversion within the haloalkaliphilic virus phiCh1 DNA results in differential expression of structural proteins. Mol Microbiol 52: 413–426.
- 2015) VirSorter: mining viral signal from microbial genomic data. PeerJ 3: e985.
- 2016) Analysis of metagenomic data reveals common features of halophilic viral communities across continents. Environ Microbiol 18: 889–903.
- 2014) Metavir 2: new tools for viral metagenome comparison and assembled virome analysis. BMC Bioinformatics 15: 76.
- 2019) Structural basis for assembly of vertical single beta-barrel viruses. Nat Commun 10: 1184.
- 1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368–379.
- 2013) Snapshot of haloarchaeal tailed virus genomes. RNA Biol 10: 803–816.
- 2012) Related haloarchaeal pleomorphic viruses contain different genome types. Nucleic Acids Res 40: 5523–5534.
- 2011) Diversity of virus-host systems in hypersaline Lake Retba, Senegal. Environ Microbiol 13: 1956–1972.
- 2016) Monitoring physiological changes in haloarchaeal cell during virus release. Viruses 8: 59.
- 2004) Haloviruses HF1 and HF2: evidence for a recent and large recombination event. J Bacteriol 186: 2810–2817.
- 2003) The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4: 41.
- 1998) Review of the biogeography of the genus Artemia (Crustacea, Anostraca). J Biogeogr 25: 213–226.
- 2018) Genomic variation and biogeography of Antarctic haloarchaea. Microbiome 6: 113.
- 2015) Microbial diversity of hypersaline environments: a metagenomic approach. Curr Opin Microbiol 25: 80–87.
- 2017) Putative archaeal viruses from the mesopelagic ocean. PeerJ 5: e3428.
- 2018) Characterization of ecologically diverse viruses infecting co-occurring strains of cosmopolitan hyperhalophilic Bacteroidetes. ISME J 12: 424–437.
- 2009) Local adaptation of bacteriophages to their bacterial hosts in soil. Science 325: 833.
- 2018a) A novel family of tyrosine integrases encoded by the temperate pleolipovirus SNJ2. Nucleic Acids Res 46: 2521–2536.
- 2018b) Rolling-circle replication initiation protein of haloarchaeal sphaerolipovirus SNJ1 is homologous to bacterial transposases of the IS91 family insertion sequences. J Gen Virol 99: 416–421.
- 2013) Phage-bacteria infection networks. Trends Microbiol 21: 82–91.
- 1998) Bacteriophage diversity in the North Sea. Appl Environ Microbiol 64: 4128–4133.
- 1970) Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40: 734–744.
- 2018) Vast diversity of prokaryotic virus genomes encoding double jelly-roll major capsid proteins uncovered by genomic and metagenomic sequence analysis. Virol J 15: 67.
- 2012) Temperate membrane-containing halophilic archaeal virus SNJ1 has a circular dsDNA genome identical to that of plasmid pHH205. Virology 434: 233–241.
- 2018) A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J Mol Biol 430: 2237–2243.