Molecular characterization of new archaeal viruses from ...
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INSTITUTE OF BIOTECHNOLOGY AND DIVISION OF GENERAL MICROBIOLOGY DEPARTMENT OF BIOSCIENCES FACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES DOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF HELSINKI Molecular Characterization of New Archaeal Viruses from High Salinity Environments TATIANA DEMINA DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS, ALIMENTARIAE, BIOLOGICAE. UNIVERSITATIS HELSINKIENSIS 26/2016
Transcript of Molecular characterization of new archaeal viruses from ...
INSTITUTE OF BIOTECHNOLOGY AND DIVISION OF GENERAL MICROBIOLOGYDEPARTMENT OF BIOSCIENCESFACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCESDOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF HELSINKI
Molecular Characterization of New Archaeal Viruses from High Salinity Environments
TATIANA DEMINA
dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 26/2016
26/2016
Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2706-8
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Molecular characterization of new archaealviruses from high salinity environments
Tatiana A. Demina
Institute of Biotechnology andDivision of General Microbiology
Department of BiosciencesFaculty of Biological and Environmental Sciences and
Viikki Doctoral Programme in Molecular Biosciences andDoctoral Programme in Microbiology and Biotechnology
University of Helsinki
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Biological andEnvironmental Sciences of the University of Helsinki, for public examination
in lecture room 511 of Info Center Korona, Viikinkaari 11, Helsinki,on 16th of December 2016, at 12 noon.
Helsinki 2016
Supervisors
Docent Hanna M. OksanenInstitute of Biotechnology andDepartment of BiosciencesUniversity of HelsinkiFinland
Docent Maija K. PietiläDepartment of Food and EnvironmentalSciencesUniversity of HelsinkiFinland
PhD Nina S. AtanasovaInstitute of Biotechnology andDepartment of BiosciencesUniversity of HelsinkiFinland
Thesis advisory committee
Academy ProfessorDennis H. BamfordInstitute of Biotechnology andDepartment of BiosciencesUniversity of HelsinkiFinland
Docent Sari TimonenDepartment of Food and EnvironmentalSciencesUniversity of HelsinkiFinland
©Tatiana Demina
Cover image: Tatiana Demina
ISBN 978-951-51-2706-8 (paperback)ISBN 978-951-51-2707-5 (PDF)ISSN 2342-5423 (print)ISSN 2342-5431 (online)
Hansaprint
Helsinki 2016
Reviewers
Professor Kenneth StedmanDepartment of BiologyPortland State UniversityU.S.A.
Associate Professor Josefa AntónDepartment of Physiology, Genetics, andMicrobiologyUniversity of AlicanteSpain
Opponent
Professor Jarkko HantulaNatural Resources Institute (LUKE)Finland
Custos
Academy ProfessorDennis H. BamfordInstitute of Biotechnology andDepartment of BiosciencesUniversity of HelsinkiFinland
“Science is the poetry of reality”
Richard Dawkins
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List of original publicationsThis thesis is based on the following publications referred to by their Roman numerals:
I. Atanasova, N.S., Demina, T.A., Buivydas, A., Bamford, D.H., Oksanen, H.M.,2015. Archaeal viruses multiply: temporal screening in a solar saltern. Viruses 7,4, 1902-1926.
II. Demina, T.A., Pietilä, M.K., Svirskaite, J., Ravantti, J.J., Atanasova, N.S.,Bamford, D.H., Oksanen, H.M., 2016. Archaeal Haloarcula californiaeicosahedral virus 1 highlights conserved elements in icosahedral membrane-containing DNA viruses from extreme environments. mBio 7, 4.
III. Demina, T.A.*, Atanasova, N.S.*, Pietilä, M.K., Oksanen, H.M., Bamford, D.H.Vesicle-like virion of Haloarcula hispanica pleomorphic virus 3 preserves highinfectivity in saturated salt. Virology 499, 40-51. *These authors contributedequally.
Doctoral candidate’s contribution:
In I, TD participated in performing experiments, data analysis and handling, and writing themanuscript.
In II, TD contributed to the study design, acquisition of data, its analysis and interpretation,as well as drafring and editing the manuscript.
In III, TD was involved in project design, performing experiments, processing of obtaineddata, and writing the manuscript.
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AbbreviationsATP adenosine triphosphateATPase adenosine triphosphatase3-D three-dimensionalacc. no. accession numberBLAST Basic Local Alignment Search Toolbp base pairBREX bacteriophage exclusioncas clustered regularly interspaced short palindromic repeats-associated
proteinsCRISPR clustered regularly interspaced short palindromic repeatscryo-EM cryo-electron microscopyDNA deoxyribonucleic acidds double-strandedICTV International Committee on Taxonomy of VirusesITR inverted terminal repeatMCP major capsid proteinMEGA Molecular Evolutionary Genetics AnalysisMUSCLE Multiple Sequence Comparison by Log-Expectationn.r. non-redundantnt nucleotideORF open reading framep.i. post infectionPCR polymerase chain reactionPFU plaque forming unitRNA ribonucleic acidrRNA ribosomal RNASDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresisss single-strandedTEM transmission electron microscopyTLC thin layer chromatographytRNA transfer RNAVLP virus-like particleVP virion protein
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Abbreviated virus namesBTV Bluetongue virusEHP-1 environmental halophage 1EHP-12 environmental halophage 12EHP-6 environmental halophage 6HATV-1 Haloarcula head-tailed virus 1HATV-2 Haloarcula head-tailed virus 2HCIV-1 Haloarcula californiae icosahedral virus 1HCTV-1 Haloarcula californiae head-tailed virus 1HCTV-2 Haloarcula californiae head-tailed virus 2HCTV-5 Haloarcula californiae head-tailed virus 5HGPV-1 Halogeometricum pleomorphic virus 1HGTV-1 Halogeometricum head-tailed virus 1HHIV-2 Haloarcula hispanica icosahedral virus 2HHPV-1 Haloarcula hispanica pleomorphic virus 1HHPV-2 Haloarcula hispanica pleomorphic virus 2HHPV3 Haloarcula hispanica pleomorphic virus 3HHTV-1 Haloarcula hispanica head-tailed virus 1HHTV-2 Haloarcula hispanica head-tailed virus 2HJTV-1 Haloarcula japonica head-tailed virus 1HJTV-2 Haloarcula japonica head-tailed virus 2HRPV-1 Halorubrum pleomorphic virus 1HRPV-2 Halorubrum pleomorphic virus 2HRPV-3 Halorubrum pleomorphic virus 3HRPV-6 Halorubrum pleomorphic virus 6HRTV-1 Halorubrum head-tailed virus 1HRTV-10 Halorubrum head-tailed virus 10HRTV-11 Halorubrum head-tailed virus 11HRTV-12 Halorubrum head-tailed virus 12HRTV-2 Halorubrum head-tailed virus 2HRTV-3 Halorubrum head-tailed virus 3HRTV-4 Halorubrum head-tailed virus 4HRTV-5 Halorubrum head-tailed virus 5HRTV-6 Halorubrum head-tailed virus 6HRTV-7 Halorubrum head-tailed virus 7HRTV-8 Halorubrum head-tailed virus 8HRTV-9 Halorubrum head-tailed virus 9HSTV-1 Haloarcula sinaiiensis head-tailed virus 1HSTV-2 Halorubrum sodomense head-tailed virus 2HSTV-3 Halorubrum sodomense head-tailed virus 3HVTV-1 Haloarcula vallismortis head-tailed virus 1HVTV-2 Haloarcula vallismortis head-tailed virus 2NHV-1 nanohaloarchaeal virus 1OLPV Organic Lake phycodnavirus
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OLV Organic Lake virophagePBCV-1 Paramecium bursaria chlorella virus 1SCTP-1 Salicola head-tailed phage 1SCTP-2 Salicola head-tailed phage 2SCTP-3 Salicola head-tailed phage 3SIRV2 Sulfolobus islandicus rod-shaped virus 2SSIP-1 Salisaeta icosahedral phage 1STIV Sulfolobus turreted icosahedral virus
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SummaryViruses are important ecological and biological actors and one of the major evolutionaryforces that cells encounter. In contrast to their overwhelming genetic diversity, all knownvirus isolates display a limited number of morphotypes. It has been suggested that due tostructural constraints and limitations in protein fold space, virus morphological diversity isrestricted to certain possible architectures. Thus, the huge and complex virosphere can beordered by grouping viruses into a reasonably small number of structure-based lineages. Totest this hypothesis, more virus-host systems have to be isolated and characterized.Hypersaline environments, where archaea tend to dominate, are especially rich in viruses.Still very little is known about archaeal viruses in general, and halophilic archaeal viruses inparticular. This thesis is devoted to the isolation and characterization of new archaeal virusesfrom high salinity environments.
Thirty seven haloarchaeal viruses were obtained from a solar saltern in Samut Sakhon(Thailand), significantly increasing the total number of currently available haloarchaealvirus isolates. The majority of the isolated viruses had tailed icosahedral morphology, butpleomorphic and tailless icosahedral morphotypes were also found. When the infectivity ofthe isolated viruses was tested against haloarchaeal strains isolated from the same locationor distant hypersaline environments, numerous interactions were observed across samplesand host genera. Myoviruses and Halorubrum strains were the two components thatcontributed to ~75% of the observed interactions. From the isolated viruses, two werecharacterized in molecular detail: Haloarcula californiae icosahedral virus 1 (HCIV-1) andHaloarcula hispanica pleomorphic virus 3 (HHPV3),
HCIV-1 was isolated on Haloarcula californiae, but it could also infect three otherhaloarchaeal strains. The virus maintains its infectivity under high and low saltconcentrations, showing adaptability to environmental changes. HCIV-1 infection results incell lysis, which was demonstrated by traditional metrics, as well as by on-line monitoringof physiological changes in infected cells. The HCIV-1 virion is a tailless icosahedron of ~70nm in diameter, and it has an inner lipid membrane. The major virion components areproteins, lipids selectively acquired from the host, and a linear double-stranded (ds) DNAmolecule of 31,314 bp in length. The virus genome is predicted to contain 47 open readingframes (ORFs), the majority of which display similarities to the genes/ORFs ofalphasphaerolipoviruses SH1, PH1, and Haloarcula hispanica icosahedral virus 2 (HHIV-2). The genomes of these four viruses share gene synteny and demonstrate high conservationin the genes encoding core virion elements: major capsid proteins (MCPs) and putativeadenosine triphosphatases (ATPases). On the contrary, genes coding for (putative) vertexcomplex proteins share the lowest similarity among genes encoding structural proteins.HCIV-1 virion assembly model is predicted to mimic the one described for HHIV-2. TheInternational Committee on Taxonomy of Viruses (ICTV) has approved the proposal toassign HCIV-1 to the Alphasphaerolipovirus genus in Sphaerolipoviridae family, whichcomprises tailless icosahedral inner membrane-containing dsDNA viruses isolated fromextreme environments. In terms of structure-based virus classification, HCIV-1 conservedelements responsible for virion organization determine its place in the PRD1-adenovirus
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structural viral lineage, which unites archaeal, bacterial, and eukaryotic viruses that aredistributed world-wide.
HHPV3 was isolated on Har. hispanica, and its host range is limited to the isolationstrain. The HHPV3 life cycle is non-lytic, but virus infection results in significant host growthretardation. The virus withstands extremely high NaCl concentrations (3-5.5 M NaCl), butdemonstrates a peculiar, irreversible, pH-dependent loss of infectivity at 1.5 M NaCl. TheHHPV3 virion is a pleomorphic lipid vesicle of ~50 nm in diameter that contains only twomajor structural protein species: a membrane associated protein and a spike protein.HHPV3 acquires lipids non-selectively from the host membranes. The virus genome is acircular dsDNA molecule of 11,648 bp in length, which contains 17 predicted ORFs, includinga set of ORFs/genes characteristic to pleolipoviruses. HHPV3-related proviruses were foundin the chromosomes of many haloarchaeal strains, highlighting the natural abundance ofpleolipoviruses. We propose that HHPV3 is a new member in Betapleolipovirus genus ofthe Pleolipoviridae family. Whether pleolipoviruses form a distinct viral structural lineageremains to be seen.
The viruses isolated here provide a rich source for more detailed future studies, whichmay reveal valuable information on global virus diversity and evolutionary relationsbetween different viral groups. Accumulation of such information helps in resolving virusclassification, which is currently an extremely challenging task. Markedly, the 37 virusisolates obtained in this study display the previously known morphotypes. Moreover, HCIV-1 clearly resembles the other haloarchaeal tailless icosahedral viruses, while HHPV3 belongsto the group of pleolipoviruses. Notably, both viral groups are distributed world-wide. Thiswork supports the observation that highly similar virion architectures can be sampled allover the biosphere, highlighting limited morphological virus diversity. Further studies areneeded to portray true viral diversity and to verify whether all viruses can be efficientlyclassified into structure-based lineages.
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Table of contentsList of original publications ...................................................................................................... iAbbreviations ........................................................................................................................... iiAbbreviated virus names ....................................................................................................... iiiSummary .................................................................................................................................. vTable of contents .................................................................................................................... vii1. Introduction .......................................................................................................................... 1
1.1 Hypersaline environments and halophiles ..................................................................... 11.2 Viruses ............................................................................................................................. 31.3 Current virus classification ............................................................................................. 41.4 Viral structural lineages .................................................................................................. 51.5 Haloviruses ...................................................................................................................... 7
1.5.1 Eukaryotic and bacterial haloviruses ........................................................................ 71.5.2 Archaeal haloviruses ................................................................................................ 11
1.5.2.1 Tailed icosahedral viruses ................................................................................ 131.5.2.2 Spindle-shaped virus His1 ................................................................................ 141.5.2.3 Tailless icosahedral viruses .............................................................................. 151.5.2.4 Pleomorphic viruses ......................................................................................... 18
1.6 Virus-host interactions in hypersaline environments .................................................. 212. Aims of the Study ............................................................................................................... 243. Materials and Methods ...................................................................................................... 254. Results and Discussion ...................................................................................................... 27
4.1 New archaeal viruses isolated from high salinity ......................................................... 274.2 Virus-host interactions ................................................................................................ 284.3 Molecular characterization of HCIV-1 ......................................................................... 29
4.3.1 HCIV-1 infection cycle ............................................................................................ 294.3.2 HCIV-1 virion organization ................................................................................... 30
4.4 Molecular characterization of HHPV-3 ....................................................................... 324.4.1 HHPV3 infection cycle ........................................................................................... 324.4.2 HHPV3 infectivity at different salinities ............................................................... 334.4.3 HHPV3 virion organization ................................................................................... 33
5. Conclusions and Future Prospects .................................................................................... 376. Acknowledgements ............................................................................................................ 397. References .......................................................................................................................... 41
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1. Introduction1.1 Hypersaline environments and halophilesHypersaline environments, artificial and natural, are found all over our planet. Theexamples of such environments include saline lakes and soils, solar salterns, deep-seabrines, salt mines, halite deposits, and salty food products (Boetius and Joye, 2009).Hypersaline environments may be categorized into thalassohaline (also called thalassic) orathalassohaline (athalassic). Thalassohaline waters, such as solar salterns, haveapproximately the same ratio of ions as in seawater. On the contrary, athalassohalineenvironments have salts in proportions different from seawater, e.g. the Dead Sea is a low-Na+, high-Mg2+, and high-Ca2+ chloride brine (DasSarma and DasSarma, 2012, Oren, 2015).Some hypersaline environments are actually polyextreme, i.e. characterized by otherextreme conditions in addition to the extreme salinity. For example, halophiles thriving inthe Dead Sea are exposed not only to the saturated salt concentrations, but also to acidic pHand intensive UV radiation (Oren, 2010). Despite harsh conditions, the representatives ofall three domains of cellular life, Eukarya, Bacteria, and Archaea, as well as their virusesinhabit hypersaline environments (Boetius and Joye, 2009, Oren, 2015). The followingchapters will be devoted to halophilic viruses, while here, I will focus on halophilic cellularorganisms.
Halophilic, or halophile, literally means “salt-loving”. Halophiles may be classified intoslight, moderate, and extreme, which require 1-3 % (0.2-0.5 M) NaCl, 3-15% (0.5-2.5 M)NaCl, or 15-30% (2.5-5.2 M) NaCl, respectively, for the optimal growth (Kushner andKamekura, 1988). This categorization is, however, nominal, and more widely, halophilerefers to an organism that inhabits an environment with salinity higher than in seawater(>3.5 % total salts). To survive in high salinity, halophiles use two main strategies, “high-salt-in” and “organic-solutes-in” (DasSarma and DasSarma, 2012, Edbeib et al., 2016). The“high-salt-in” strategy implies accumulation of ions inside the cells in concentration equalto that in the environment, while “organic-solutes-in” refers to the accumulation (de novosynthesis or uptake from the environment) of compatible solutes (osmolytes), such as aminoacids and sugars (Edbeib et al., 2016).
Hypersaline environments are dominated by prokaryotes, but eukaryotes also thrive atmoderate and even extreme salinities (Boetius and Joye, 2009, DasSarma and DasSarma,2012, Oren, 2014a, Ventosa et al., 2014). Dense populations of green algae, e.g. Dunaliellasalina, are main food sources for brine shrimp Artemia and larvae of brine flies. Otherinvertebrates are also known to survive in hypersaline environments: rotifers, flat worms,copepods, ostracods. In addition, halophytic plants, e.g. Laguncularia mangrove, can growin moderately saline soils. Even some vertebrates, e.g. Tilapia species, are observed tosurvive in moderately high salinity. It is also worth mentioning that some hypersaline pondsare important feeding sources for various birds, including flamingo. Among othereukaryotes inhabiting salty ponds are diatoms, protozoa (ciliate), and fungi.
Moderately or extremely halophilic bacteria are classified to at least eight differentphyla, among which Actinobacteria, Firmicutes, and Proteobacteria include most genera(Ventosa et al., 2012). The group is very heterogeneous, and contains Gram-positive/Gram-negative, aerobic/anaerobic, heterotrophic/phototrophic bacteria of different shapes (cocci,
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spirals, etc.) (Ventosa et al., 2012). Microbial mats formed by halophilic bacteria areobserved in hypersaline waters. Unicellular cyanobacteria constitute the upper level of themat, while filamentous cyanobacteria form the second layer. Anaerobic phototrophicbacteria (green and purple bacteria) grow beneath cyanobacterial layers, and strictlyanaerobic bacteria thrive in the bottom layers of the mat. In addition, methanogenic archaeaare also found in these bottom layers (Bolhuis et al., 2014, DasSarma and DasSarma, 2012).
In the saltiest ponds, halophilic archaea dominate, although some bacteria, e.g.Salinibacter ruber, may also be abundant (Ventosa et al., 2014, Zhaxybayeva et al., 2013).Halophilic archaea are taxonomically classified to the phylum Euryarchaeota, classesHalobacteria (extreme halophiles) and Methanomicrobia (halophilic or halotolerantmethanogens) (Oren, 2014b, Ventosa et al., 2012). In addition, a yet uncultivable lineage ofsmall-sized (~0.6 μm) archaea, called “Nanohaloarchaea”, seems to be globally distributedin hypersaline environments (Ghai et al., 2011, Grant et al., 1999, Narasingarao et al., 2012,Oh et al., 2010).
The majority of halophilic archaea belong to the class Halobacteria, which includes asingle order Halobacteriales with a single family Halobacteriaceae. By the year 2014, thefamily contained 47 genera and 165 species, with Halorubrum, Haloferax, and Haloarculabeing the largest genera (Oren, 2014b). Recently, it has been suggested to devide the classHalobacteria into three orders, Halobacteriales, Haloferacales, and Natrialbales (Gupta etal., 2015). The order Haloferacales has been proposed to include two families,Haloferacaceae and Halorubraceae, while the order Halobacteriales has been proposed toinclude three families, Halobacteriaceae, Haloarculaceae, and Halococcaceae (Gupta et al.,2016). The members of the Halobacteria are “the halophiles par excellence” (Oren, 2014b),meaning that many species may grow even in almost saturated brines. Haloarchaea displayvarious cell shapes: cocci, rods, disks, triangles (Haloarcula japonica), square(Haloquadratum walsbyi), and pleomorphic (DasSarma and DasSarma, 2012). Theproteins of haloarchaea are negatively charged, having an excess of acidic to basic aminoacids, which retains functionality at low water activity (DasSarma and DasSarma, 2015).Haloarchaeal lipids are ether-linked, as in other Archaea, and polar lipids profiles are usedfor chemotaxonomic differentiation (Ventosa et al., 2012). Some haloarchaea, e.g.Haloquadratum walsbyi and Halobacterium salinarum, may produce gas-filled rigidproteinaceous vesicles, which enables them to migrate vertically in water to the moreoxygenated surface layers (Pfeifer, 2015). Typically, the members of the Halobacteria areaerobic heterotrophs, but some species may have specific nutritional needs and have diversemetabolic pathways (Andrei et al., 2012). The species of the genera Haloferax andHaloarcula are usually easily grown in the laboratory conditions (Oren, 2002), while others,e.g. Halosimplex carlsbadense (Vreeland et al., 2002), have complex nutritional demands.Some haloarchaea may degrade aromic compounds, indicating a potential use inbioremediation of oil-polluted hypersaline environments (Al-Mailem et al., 2010). Somemembers of Halobacteria, e.g. Hlb. salinarum, are photoheterotrophs and use retinal-basedpigments to adsorb light energy (Andrei et al., 2012). These pink-pigmented archaea,together with Salinibacter ruber and Dunaliella salina, are responsible for unusual pink orred colors of highly saline ponds. Noticeably, a few halophilic archaea are polyextremophilic,as they grow at alkaline pH and high temperatures (Bowers and Wiegel, 2011). Interestingly,
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due to their remarkable ability to survive extreme desiccation, radiation, and starvation,halophilic archaea serve as model organisms in ground-based simulations, as well as in outerspace experiments, to explore whether microbial life could survive in space (Leuko et al.,2014, Stan-Lotter and Fendrihan, 2015).
1.2 VirusesSince the discovery of viruses in the 19th century, the concept of virus has changeddramatically from an infectious particle to the most abundant biological entity, importantecological actor, and a major factor in the evolution of cellular life (Dolja and Krupovič, 2013,Forterre and Prangishvili, 2013, Koonin and Dolja, 2013, Pennazio, 2003, Pradeu et al.,2016). Moreover, virus research has challenged our perception of life, i.e. its definition,boundaries, and origin (Kostyrka, 2016).
Many specific characteristics that were traditionally attributed to viruses now seem notto be universal. Historically, viruses were first determined as “filterable agents”, i.e. agentsthat bypass bacterial filters (Pennazio, 2003). However, this view is no longer applicablesince the discovery of giant amoebal viruses that are visible by light microscopy and possesslarge and complex genomes: e.g. Pandoravirus salinus genome is ~2.5 Mbp in length andcontains ~2,500 genes (Aherfi et al., 2016). Moreover, the perception of a virus particle, i.e.virion, as an inert infectious carrier of the virus genome has also been challenged with theobservation that the virions of hyperthermophilic spindle-shaped viruses, Acidianus two-tailed virus and Sulfolobus monocaudavirus 1, develop their tails outside (independently) ofthe host cells (Häring et al., 2005a, Prangishvili et al., 2006, Uldahl et al., 2016). The otherconceptual change lies in the definition of viruses as harmful obligate intracellular parasites:giant viruses may be called “quasi-autonomous” (Claverie and Abergel, 2010) and certainviruses may have relatively mutualistic relations with their hosts. For example,polydnaviruses are obligatory symbionts of parasitoid wasps and promote wasp larvaedevelopment in a body of another insect (Strand and Burke, 2012), and some cyanophagespromote photosynthesis in cyanobacteria during infection (Mann, 2003). In addition, giantviruses themselves may be preyed on by virophages (Aherfi et al., 2016).
Thus, recent discoveries suggest that “viruses are more about exceptions than therules” (Dolja and Krupovič, 2013). All this together with the accumulating data on capsid-less selfish elements has posed a reasonable need to revise the concept of virus. However,the way to define virus still raises hot debates. It has been suggested that virion may beconsidered as viral self, distinguishing viruses from all other replicative elements, such asplasmids, viroids, virusoids, and transposons (Bamford et al., 2002, Bamford, 2003,Krupovič and Bamford, 2010). Alternatively, virus can be defined as a type of replicatorwhich, together with other replicators, forms a continuum from the most selfish (virulentviruses) to the most cooperative (chromosomes, organelle DNA) replicators (Koonin andStarokadomskyy, 2016). Virus can also be viewed as a process, i.e. cyclic change of intra-and extracellular stages (Dupre and Guttinger, 2016). Raoult and Forterre (2008) haveproposed to define viruses and cells as capsid-encoding and ribosome-encoding organismsrespectively, while other replicating entities are grouped into “orphan replicons”.
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Viruses are extremely abundant in various environments (Rosario and Breitbart, 2011),imposing significant selective pressure on their hosts. It has been estimated that in marineenvironments virus particles are 5-100 times more abundant than prokaryotic cells (Suttle,2007). Moreover, in sea-surface waters, viruses appear to kill ~20-40% of microbial biomassdaily, thus substantially affecting composition of microbial communities and, indirectly,biogeochemical cycles (Suttle, 2007). Viruses are not only the most dominant biologicalentities, but also reservoirs of diverse genetic information. Many currently known viralsequences have no homologs in cellular or other viral sequences (Yin and Fischer, 2008). Ithas been suggested that gene flux from viruses to cells is common, and cells might beregarded as “giant pickpockets” of viral genes (Forterre and Prangishvili, 2013). Virus-derived sequences may constitute large portions of cellular genomes (Gogvadze and Buzdin,2009), and there are several examples of how originally, most probably, viral genes were“domesticated” by cells and recruited for crucial functions. For instance, expression ofendogenous retroviral (former) coat proteins, syncytins, is essential for the formation ofplacenta (Lokossou et al., 2014). Moreover, it has been hypothesized that viruses may havehad direct roles in such important evolutionary events as the origin of DNA and itsreplication mechanisms (Forterre, 2002), as well as development of cellular antiviraldefence systems, including “invention” of nucleus in eukaryotes and formation of complexcell wall in bacteria (Forterre and Prangishvili, 2009). All this, together with many moreexamples of possible virus impact on the evolution of life (Forterre and Prangishvili, 2013,Koonin and Dolja, 2013), suggests that viruses are one of the major forces in shaping of ourworld as we know it.
1.3 Current virus classificationApparently, an intrinsic feature of the human mind is a need for systematization of thesurrounding world in order to understand its ultimate complexity. The discovery of newviruses and accumulation of data regarding their shapes and properties resulted in thedevelopment of a concept of virus species. However, there is still no universal definition ofvirus species that would be satisfactory for all virologists, as different criteria have beenproposed to be used for assigning viruses into taxonomical categories.
Viruses display a variety of virion shapes and genomic structures. The majority of viruscapsids can be characterized by helical or icosahedral symmetry, although some morecomplex structures are also known. Notably, some viruses that infect thermophilic archaeadisplay quite unusual morphotypes, e.g. Acidianus bottle-shaped virus (Häring et al.,2005b, Pietilä et al., 2014, Prangishvili et al., 2016). In addition, a virion may harbour aninner or outer lipid envelope, derived from the host membranes. Virus genome may be aDNA or an RNA molecule, single-stranded (ss) or double-stranded (ds), circular or linear,continuous, nicked or segmented. The size of a genome and consequently, the size of a virionalso vary greatly. For example, porcine circoviruses have a circular ssDNA genome of ~2 kblong and an icosahedral capsid of only ~17 nm in diameter (Finsterbusch and Mankertz,2009), while Pithovirus sibericum has a circular dsDNA genome of ~610 kbp long and avirion of ~1500 nm in length and ~500 nm in diameter (Aherfi et al., 2016).
In the 1960s, Lwoff, Horne, and Tournier proposed a system of virus classificationbased on four essential virus features: a type of nucleic acid (DNA/RNA), a type of capsid
5
symmetry, presence/absence of envelope, and quantitative characteristics of capsid (e.g.number of capsomers) (Lwoff and Tournier, 1966). This system excluded some previouslyproposed criteria, such as symptoms that virus infection causes (Lwoff and Tournier, 1966).Another approach has been proposed by Baltimore, who suggested that all viruses can begrouped into seven classes based on how virus genetic information is expressed andreplicated (Baltimore, 1971).
In 1966, the International Union of Microbiological Societies delegated a task ofdeveloping and maintaining a universal, internationally agreed system of virus classificationto the newly established International Committee on Taxonomy of Viruses (ICTV). The maingoal of ICTV is “to categorize the multitude of known viruses into a single classificationscheme that reflects their evolutionary relationships” (http://www.ictvonline.org/). ICTVcurrently defines a virus species as “a monophyletic group of viruses whose properties canbe distinguished from those of other species by multiple criteria. […] These criteria mayinclude, but are not limited to, natural and experimental host range, cell and tissue tropism,pathogenicity, vector specificity, antigenicity, and the degree of relatedness of their genomesor genes” (http://www.ictvonline.org/codeOfVirusClassification.asp). The recognized taxa areorder, family, sub-family, genus, and species. The Virus Taxonomy Release 2015 reports 7orders, 111 families, 27 sub-families, 609 genera, and 3704 species. Of 111 families, 82 arenot assigned to any order.
Although some genes are widely present in virus genomes, e.g. genes encoding forcapsid proteins in icosahedral viruses, RNA and DNA polymerases, and other enzymesmediating genome replication or integration to the host genomes (Koonin and Dolja, 2013),there is, however, no single universal gene shared by all viruses. This makes it impossible toresolve virus phylogeny in a way similar to the usage of small subunit ribosomal RNAsequences in reconstructing the tree of cellular life. In addition, extensive horizontal genetransfer between different viruses and hosts (Hambly and Suttle, 2005) makes phylogenyresolution even more challenging. It has been proposed that instead of tree-like hierarchy, areticulated network topology might be more suitable for reflecting evolutionary pathways ofviruses (Morgan, 2016).
1.4 Viral structural lineagesWith the increasing number of resolved virion protein structures, striking similarities havebeen found in viruses previously thought to be unrelated (Benson et al., 1999). Inspired bythe observation that same structures are found all across the virosphere, Bamford and co-workers proposed to use similarities in virion architectural principles to group viruses intohigher-level taxa called viral structural lineages (Bamford et al., 2002, Bamford, 2003,Bamford et al., 2005a). The key point of this approach is that virion is what defines virusand distinguishes it from other replicators (Krupovič and Bamford, 2010). Virus geneticmodules responsible for forming a virion are considered to be vertically-inherited virus“self”, opposite to (often) horizontally exchanged modules involved in genome replicationand interactions with the host cell (Abrescia et al., 2010, Krupovič and Bamford, 2010). Thevirus “self” elements are conserved over evolutionary time, as only a limited number ofprotein folds are capable to constitute a functional virion (Bamford, 2003, Benson et al.,2004, Oksanen et al., 2012). Structural similarities may reflect deep evolutionary links, i.e.
6
common ancestry, even when there are no sequence similarities (Bamford, 2003).Moreover, one lineage may include viruses infecting hosts from all three domains of life,which suggests that viruses are ancient and that contemporary viruses are polyphyletic,originating from several types of viruses that infected the last universal common ancestor ofcells (Bamford, 2003, Krupovič and Bamford, 2010).
So far, four major lineages of icosahedral viruses have been established: HK97-like,Picorna-like, Bluetongue virus (BTV)-like, and PRD1-adenovirus-like (Abrescia et al., 2012,Bamford et al., 2005a, Oksanen et al., 2012). Each lineage unites viruses with a distinct foldof the major capsid protein (MCP) and its specific arrangement to form a capsid lattice.
(i) HK97-like viruses are dsDNA viruses with MCPs having a so-called canonical HK97fold, originally determined in bacteriophage HK97 (Wikoff et al., 2000). This lineage alsoincludes several tailed bacteriophages, e.g. T4 (Fokine et al., 2005), P22 (Jiang et al., 2003),and φ29 (Morais et al., 2005), eukaryotic herpesviruses (Baker et al., 2005), as well asarchaeal Haloarcula sinaiiensis tailed virus 1 (HSTV-1) (Pietilä et al., 2013c). Interestingly,HK97 fold was also identified in archaeal and bacterial encapsulins, proteins formingicosahedral nanocompartments inside cells, suggesting a common evolutionary origin ofencapsulins and capsid proteins of viruses belonging to the HK97-like structural lineage(Akita et al., 2007, McHugh et al., 2014).
(ii) Picorna-like viruses have MCPs of a single β-barrel fold, which are usuallypositioned tangentially to the capsid shell surface. Eukaryotic RNA viruses, such as foot-and-mouth disease virus (Acharya et al., 1989), rhinovirus (Rossmann et al., 1985), andpoliovirus (Hogle et al., 1985), as well as ssDNA tailless icosahedral bacteriophage ΦX174(Dokland et al., 1997) represent this lineage.
(iii) BTV-like viral lineage comprises dsRNA viruses with an α-helical MCP (Grimes etal., 1998). In addition to BTV, the members of this lineage are e.g. eukaryotic reovirus(Reinisch et al., 2000), rice dwarf virus (Nakagawa et al., 2003), yeast L-A virus (Naitow etal., 2002), and bacteriophage Φ6 (Huiskonen et al., 2006).
(iv) PRD1-adenovirus-like viruses are dsDNA tailless icosahedral viruses with aninternal lipid membrane (except adenovirus) and MCPs of a vertical double β-barrel fold,arranged into trimers forming pseudo-hexameric capsomers (Benson et al., 1999). Thislineage comprises e.g. bacteriophages PRD1 (Benson et al., 1999) and PM2 (Abrescia et al.,2008), Paramecium bursaria chlorella virus 1 (PBCV-1) (Nandhagopal et al., 2002), humanadenovirus (Athappilly et al., 1994, Rux et al., 2003), and archaeal virus Sulfolobus turretedicosahedral virus (STIV) (Rice et al., 2004). The same MCP fold was also found in virophageSputnik (Zhang et al., 2012a). In addition, vaccinia scaffolding protein D13, which forms atransitory lattice during virus morphogenesis, has also a double β-barrel fold (Bahar et al.,2011).
Recent findings suggest that PRD1-adenovirus lineage may be divided into “double β-barrel viruses” (described above) and “single β-barrel viruses”. Both groups of viruses shareoverall PRD1-type virion architecture, but the single β-barrel viruses have two MCPs of avertical single β-barrel fold, both forming the lattice. The suggested members of this groupare prokaryotic extremophilic viruses: thermophilic phage P23-77 (Rissanen et al., 2013)
7
and haloviruses Salisaeta icosahedral phage 1 (SSIP-1) (Aalto et al., 2012), SH1 (Jäälinojaet al., 2008) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) (Gil-Carton et al.,2015).
In PRD1, genome packaging into a pre-formed procapsid is aided by the packagingATPase P9, which is a virion protein located at the unique vertex (Gowen et al., 2003, Honget al., 2014, Strömsten et al., 2005). Comparison of P9 and other putative packagingadenosine triphosphatases (ATPases) of PRD1-type viruses revealed that in addition to theclassical Walker A and Walker B motifs (Walker et al., 1982), all these ATPases (exceptadenovirus ATPase) contain a region of conserved amino acid residues called P9/A32-specific motif (A32 for vaccinia virus ATPase A32) (Strömsten et al., 2005). A lack ofP9/A32-specific motif in adenovirus ATPase IVa2 may be related to the lack of an inner lipidmembrane in adenovirus virion (Strömsten et al., 2005).
Some other distinct viral structure-based lineages have been suggested in addition tothe four major established ones (Oksanen et al., 2012). Obviously, more information isneeded to identify all possible lineages, the number of which is, however, predicted to berelatively low (Bamford et al., 2005a). Current structure-based virus classification is limitedto the available high-resolution structures, but high-throughput methods for resolvingthree-dimensional (3-D) protein structures, as well as robust computational tools forstructure comparison, are under constant improvement (Abrescia et al., 2012, Ravantti etal., 2013). Recent advances in cryo-electron microscopy (cryo-EM) have already yieldedcomplex molecular structures of near-atomic resolution (Kuhlbrandt, 2014). Improvementsin the methodology within time might enable more comprehensive structure-basedreconstruction of viral phylogeny.
1.5 Haloviruses
1.5.1 Eukaryotic and bacterial halovirusesMany marine organisms may be called slight halophiles, but here, halophilic refers toorganisms inhabiting environments where salinity is higher than in seawater. To the best ofmy knowledge, and excluding this work, 74 haloviruses, i.e. viruses that infect halophilicorganisms, have been isolated and described to some extent (Atanasova et al., 2015b, Luk etal., 2014). The vast majority of these are archaeal viruses (54 viruses, see chapter 1.5.2).Very little is known about viruses that infect halophilic eukaryotes (5 viruses) or bacteria (15viruses) (Figures 1 A and B, Table 1). So far, eukaryotic haloviruses are represented by onlyfive amoebal icosahedral viruses, which were isolated from saline environments in Tunis(Boughalmi et al., 2013). Based on virion morphological features and comparisons of DNApolymerase or capsid protein sequences, four of these viruses were assigned to the familyMarseilleviridae and one to Mimiviridae family (Boughalmi et al., 2013) (Figure 1 A).
The known bacterial viruses isolated from hypersaline environments infect hosts fromgenera Halomonas, Pseudomonas, Tetragenococcus, Salinivibrio, Salicola, and Salisaeta,having either a lytic or lysogenic life cycle (Table 1). These viruses are tailed, morphologicallyresembling the members of the families Myoviridae (icosahedral head and contractile tail,further called myoviruses), Siphoviridae (icosahedral head and long non-contractile tail,further called siphoviruses), or Podoviridae (icosahedral head and short noncontractile tail,
8
further called podoviruses), except SSIP-1, which is a tailless icosahedral virus with an innerlipid membrane (Figure 1 B). Myovirus Salicola tailed phage 2 (SCTP-2) is a large virus witha head of ~125 nm in diameter and a tail of ~145 nm in length (Kukkaro and Bamford, 2009),while the other described bacterial haloviruses display smaller particles. The genomes ofbacterial haloviruses are either linear or circular DNA molecules of variable size up to 340kb, as in the temperate Halomonas phage ΦgspC (Seaman and Day, 2007) (Table 1). Cryo-EM-based reconstructions of virus particles are available for Salinivibrio phage CW02 (Shenet al., 2012) and for Salisaeta phage SSIP-1 (Aalto et al., 2012). CW02 is a member of theT7-like phage supergroup, and its virion reconstruction at 16-Å resolution permitted fittingof the HK97-fold (Shen et al., 2012), a conserved fold commonly found in dsDNAbacteriophages (see above, chapter 1.4). Salisaeta phage SSIP-1 shares common featureswith PRD1-like viruses, and the canonical double β-barrel MCP of bacteriophage PM2 wasfitted to SSIP-1 virion reconstruction at 12.5-Å resolution (Aalto et al., 2012). SSIP-1 has twoMCPs, thus it has been suggested to belong to the subgroup of single β-barrel viruses in thePRD1-adenovirus structural lineage (Aalto et al., 2012).
In addition, the analysis of a metavirome derived from Santa Pola solar saltern (Spain)allowed putative assignment of the identified halophages to the bacterium Salinibacterruber, one of the dominant players in the saltern microbiota (Garcia-Heredia et al., 2012,Santos et al., 2010). However, no viruses infecting S. ruber have yet been isolated. Similarly,metagenomic analysis of water surface samples from hypersaline Organic Lake in Antarcticaallowed reconstruction of a complete genome of virophage Organic Lake virophage (OLV),which is related to Sputnik, and a near-complete genome of its putative helper virus OrganicLake phycodnavirus (OLPV), which infects algae (Yau et al., 2011). Interestingly, thevirophage OLV MCP sequences were also found in metagenomes from other geographicallydistant hypersaline and freshwater reservoirs, suggesting its possibly global occurrence (Yauet al., 2011).
9
Figure 1. Morphotypes of isolated (A) eukaryotic, (B) bacterial, and (C) archaeal haloviruses.Representative viruses (virions) are drawn schematically. Scale bar, 100 nm. Tailed icosahedralviruses are coloured yellow, tailless icosahedral are green, and lipids are indicated by blue. Modifiedfrom (Atanasova et al., 2015b).
10
Tab
le 1
. Euk
aryo
tic
and
bact
eria
l hal
ovir
uses
.
Vir
us
Isol
atio
n h
ost
Mor
ph
otyp
e, s
izea
Lif
e cy
cle
Gen
ome,
size
Isol
atio
nso
urc
eR
efer
ence
Seb1
sol
Aca
ntha
moe
ba p
olyp
haga
Taill
ess
icos
ahed
ral (
Mrs
-lik
eb ), ~
220
nm
Ndc
DN
ASa
line
soil,
Tuni
sB
ough
alm
iet a
l.,20
13Se
b2so
lTa
illes
s ic
osah
edra
l (M
rs-l
ike)
, ~ 2
01 n
mN
dD
NA
Seb6
sol
Taill
ess
icos
ahed
ral (
Mrs
-lik
e), ~
206
nm
Nd
DN
ASe
b1ea
uTa
illes
s ic
osah
edra
l (M
rs-l
ike)
, ~ 2
26 n
mN
dD
NA
Salt
lake
wat
er, T
unis
Bou
g1Ta
illes
s ic
osah
edra
l (M
imi-
liked )
, ~ 5
19 n
mN
dD
NA
F9-1
1H
alom
onas
hal
ophi
laSi
phov
irus
,~ 6
0/20
0 nm
Lyso
geni
cN
dH
. hal
ophi
laF9
-11e
Cal
voet
al.,
198
8
G3
Pseu
dom
onas
sp. G
3M
yovi
rus
Lyti
cN
dSa
lt p
ond,
Can
ada
Kau
riet
al.,
199
1
φ71
16Te
trag
enoc
occu
s ha
loph
ilus
Myo
viru
s, 8
7-96
/200
nm
Lyti
cN
dFe
rmen
ting
soy
sauc
eU
chid
a an
d K
anbe
,19
93φ
D-8
6Te
trag
enoc
occu
s ha
loph
ilus
Siph
ovir
us 6
7/30
0 nm
Lyti
cN
dF5
-4H
alom
onas
hal
ophi
laSi
phov
irus
,~ 3
3/14
2 nm
Lyso
geni
cN
dH
. hal
ophi
lae
Cal
voet
al.,
199
4F1
2-9
Hal
omon
as h
alop
hila
Siph
ovir
us,~
42/
133
nmLy
soge
nic
Nd
UTA
KV
ibri
osp
. B1
Myo
viru
s, ~
86/
100
nmLy
tic
dsD
NA
,~
80 k
bpSo
lar
salt
ern,
Alic
ante
, Spa
inG
oele
t al.,
199
6
Φgs
pBH
alom
onas
sal
ina
Myo
viru
s,46
-51/
112
nmN
dds
DN
A,
~49
kb
Salin
e so
il,G
reat
Sal
tPl
ains
, USA
Seam
an a
nd D
ay,
2007
Φgs
pCH
alom
onas
sal
ina
Myo
viru
s, 5
3-86
/121
nm
Lyso
geni
cds
DN
A,
~34
0 kb
SCTP
-1Sa
licol
asp
. PV
3Si
phov
irus
, hea
d ~
95nm
Nd
Nd
Sola
r sa
lter
n,M
argh
erit
a di
Savo
ia, I
taly
Kuk
karo
and
Bam
ford
, 200
9SC
TP-2
Salic
ola
sp. P
V4
Myo
viru
s, ~
125
/145
nm
Nd
Nd
SCTP
-3Sa
licol
asp
. s3-
1M
yovi
rus
Nd
Nd
Ata
naso
vaet
al.,
2012
CW
02Sa
liniv
ibri
oSA
50Po
dovi
rus,
~60
nm
,T=
7la
evo
Nd
Line
ards
DN
A,
49,3
90 b
p
Gre
at S
alt
Lake
, USA
Shen
et a
l., 2
012
SSIP
-1Sa
lisae
tasp
. SP9
-1Ta
illes
s ic
osah
edra
l wit
h in
ner
lipid
mem
bran
e, ~
100
nm,T
= 4
9Ly
tic
Cir
cula
rds
DN
A,
43,7
88 b
p
Salt
wat
er,
Sedo
m P
onds
f ,Is
rael
Aal
toet
al.,
201
2
QH
HSV
-1H
alom
onas
ven
tosa
eSi
phov
irus
,~ 4
7/75
nm
Lyti
cds
DN
ASa
lt m
ine,
Qia
ohou
,C
hina
Fuet
al.,
201
6
a.C
apsi
d di
amet
er a
nd tr
iang
ulat
ion
num
ber
(T) f
or ta
illes
s ph
ages
and
pod
ovir
uses
; cap
sid
diam
eter
/tai
l len
gth
for
taile
d ph
ages
.b.
Mrs
-lik
e, M
arse
illev
irus
-lik
e.c.
Nd,
not
det
erm
ined
.d.
Mim
i-lik
e, M
imiv
irus
-lik
e.e.
Indu
ced
from
Hal
omon
as h
alop
hila
str
ains
isol
ated
from
hyp
ersa
line
soils
nea
r A
lican
te, S
pain
.f.
Sedo
m P
onds
are
exp
erim
enta
l pon
ds in
Isr
ael w
here
wat
er fr
om th
e D
ead
Sea
is d
ilute
d w
ith
wat
er fr
om th
e R
ed S
ea.
11
1.5.2 Archaeal halovirusesExcluding this work (see chaper 4.1), ~100 archaeal viruses have been isolated (Atanasovaet al., 2015b, Snyder et al., 2015). These are viruses that infect thermophilic crenarchaea,methanogenic euryarchaea, or halophilic euryarchaea (Dellas et al., 2014, Pietilä et al., 2014,Prangishvili, 2013). About 50 isolated viruses infect haloarchaea from eight different generaof the family Halobacteriaceae (Table 2) (Atanasova et al., 2015a). These virus isolatesoriginate from various hypersaline environments all over the world, and represent thefollowing morphotypes: tailed icosahedral, spindle-shaped (also called fusiform or lemon-shaped), tailless icosahedral, and pleomorphic (Figures 1 C and 2 A, Table 2) (Atanasova etal., 2015a). The Genbank non-redundant (n.r.) database (accessed 23.8.2016) also containsa genome sequence (dsDNA, ~40 kbp) of Halorubrum phage CGΦ46 (NC_021537.1), butno other information is available for this virus. In addition, one complete (environmentalhalophage 1 [EHP-1]) and 42 near-complete viral genomes were retrieved from metagenomicdata obtained from a solar saltern in Santa Pola (Alicante, Spain), and 25 of these weretentatively assigned to infect halophilic archaea Haloquadratum walsbyi (15 viruses),Halorubrum lacusprofundi (3), or nanohaloarchaea (7) (Garcia-Heredia et al., 2012, Santoset al., 2007). Recent methodological advances have also allowed unambiguous identificationof virus-host pairs in hypersaline environments, circumventing a need for cultivation, asdemonstrated by the identification of Nanohaloarchaeal virus 1 (NHV-1) most likelyinfecting a yet unculturable nanohaloarchaeon from Santa Pola saltern (Martínez-García etal., 2014).
12
Tab
le 2
. Arc
haea
l hal
ovir
uses
.
a.V
irus
es w
hose
gen
ome
sequ
ence
is a
vaila
ble
are
in b
old,
and
ast
eris
ks m
ark
viru
ses
who
se s
truc
ture
s ha
ve b
een
dete
rmin
edby
cry
o-E
M o
r cr
yo-e
lect
ron
tom
ogra
phy
and
3-D
imag
e re
cons
truc
tion
. For
full
viru
s na
mes
see
“A
bbre
viat
ed v
irus
nam
es”.
b.Fo
r Φ
H, o
nly
part
ial g
enom
e se
quen
ce is
ava
ilabl
e.c.
For
mor
e in
form
atio
n, s
ee T
able
3.
d.Fo
r m
ore
info
rmat
ion,
see
Tab
le 4
.
Mor
ph
otyp
eV
iru
sesa
Hos
t ge
ner
aR
efer
ence
s
Myo
viru
sH
s1,Φ
Hb ,
S510
0,H
F1,
HF
2, H
RTV
-1,
HR
TV-2
, HR
TV-3
,HR
TV
-5, H
RTV
-6,
HR
TV
-7,H
RT
V-8
, HR
TV-9
, HR
TV-1
0,H
RTV
-11,
HR
TV-1
2,H
ST
V-2
*,H
STV
-3,
HJT
V-1
, HJT
V-2
, HA
TV-1
, HA
TV-2
,HG
TV
-1,
ΦC
h1
Hal
obac
teri
um,
Hal
ofer
ax,
Hal
orub
rum
,H
aloa
rcul
a,H
alog
ranu
m,
Nat
rial
ba
Ata
naso
vaet
al.,
201
2, D
anie
ls a
nd W
ais,
1990
, Nut
tall
and
Dya
ll-Sm
ith,
199
3,Pi
etilä
et a
l., 2
013b
, Sch
nabe
l et a
l.,19
82b,
Senči
lo e
t al.,
201
3, T
ang
et a
l.,20
02, T
orsv
ik a
nd D
unda
s, 1
980,
Tor
svik
and
Dun
das,
197
4, W
itte
et a
l., 1
997
Siph
ovir
usH
h1, H
h3, J
a1, Φ
N, S
45, B
10,B
J1, H
RT
V-4
,H
HT
V-1
, HH
TV
-2,H
CT
V-1
, HC
TV
-2,
HC
TV
-5, H
VT
V-1
*,H
VT
V-2
Hal
obac
teri
um,
Hal
orub
rum
,H
aloa
rcul
a
Ata
naso
vaet
al.,
201
2, D
anie
ls a
ndW
ais,
1984
, Pag
alin
g et
al.,
200
7, P
aulin
g,19
82, P
ieti
lä e
t al.,
201
3b, S
enči
lo e
t al.,
2013
, Tor
svik
, 198
2, V
ogel
sang
-Wen
kean
d O
este
rhel
t, 19
88, W
ais
et a
l., 1
975
Podo
viru
sH
ST
V-1
*H
aloa
rcul
aA
tana
sova
et a
l., 2
012,
Pie
tilä
et a
l.,20
13c
Spin
dle
His
1*H
aloa
rcul
aB
ath
et a
l., 2
006,
Hon
get
al.,
201
5,Pi
etilä
et a
l., 2
013a
Taill
ess
icos
ahed
ralc
SH
1*, H
HIV
-2*,
PH
1,SN
J1H
aloa
rcul
a,H
alor
ubru
m,
Nat
rine
ma
Bam
ford
et a
l., 2
005b
, Gil-
Car
ton
et a
l.,20
15, J
aakk
ola
et a
l., 2
012,
Jää
linoj
a et
al.,
2008
, Por
ter
et a
l., 2
013,
Zha
ng e
tal
., 20
12b
Pleo
mor
phic
dH
HP
V-1
, HH
PV
-2, H
is2,
HR
PV
-1*,
HR
PV
-2, H
RP
V-3
, HR
PV
-6, H
GP
V-1
,S
NJ2
Hal
oarc
ula,
Hal
orub
rum
,H
alog
eom
etri
cum
,N
atri
nem
a
Ata
naso
vaet
al.,
201
2, B
ath
et a
l., 2
006,
Li e
t al.,
201
4, L
iu e
t al.,
201
5, P
ieti
lä e
tal
., 20
09, P
ieti
lä e
t al.,
201
0, P
ieti
lä e
tal
., 20
12, R
oine
et a
l., 2
010,
Senči
lo e
tal
., 20
12
13
1.5.2.1 Tailed icosahedral virusesThe majority of the isolated haloarchaeal viruses are tailed icosahedral resembling themembers of the families Myoviridae (24 viruses) or Siphoviridae (15 viruses) (Atanasova etal., 2015b). One podovirus that infects Haloarcula sinaiiensis (HSTV-1) has also beenisolated (Atanasova et al., 2012, Pietilä et al., 2013c). Although tailed haloarchaeal virusisolates are rather numerous, tailed virus-like particles (VLPs) are considered to constituteonly a minor part of natural viral communities in hypersaline environments (Santos et al.,2012).
Tailed viruses infect haloarchaea belonging to the genera Halobacterium, Haloarcula,Halorubrum, Haloferax, Halogranum, or Natrialba (Table 2), and these viruses (thosestudied for the life cycle) are virulent or temperate. However, no lysin or holin genes (Younget al., 2000) have been identified in the genomes of virulent haloarchaeal tailed viruses, andexact mechanisms of cell lysis are not known. Temperate haloarchaeal tailed viruses arerepresented by only two myoviruses: ΦH infecting Halobacterium salinarum and ΦCh1infecting Natrialba magadii. The ΦH partial genome (L segment) may reside in the hostcytoplasm as a plasmid (Schnabel, 1984), while ΦCh1 integrates into the host genome (Witteet al., 1997). In addition, a few other tailed haloarchaeal viruses may also be temperate, astheir genomes encode putative integrases (Senčilo et al., 2013).
ΦH and ΦCh1 are so far the best studied haloarchaeal tailed viruses in terms of genomefunctions. Molecular studies have showed that ΦH genome is highly unstable and duringinfection rounds, many genetically rearranged variants are produced (Schnabel et al.,1982a). The ΦH genome sequence, albeit not complete, is similar to that of ΦCh1.Noticeably, ΦCh1 is the only archaeal virus known to contain host-derived RNA along withvirus DNA in the virion (Witte et al., 1997). Similarly to ΦH, ΦCh1 also undergoes genomicrearrangements. The rearrangements occur in a certain region of ΦCh1 genome, allowingproduction of different types of tail fibres (Klein et al., 2012). Genomic rearrangements havealso been reported for a few other tailed haloarchaeal viruses (Daniels and Wais, 1998,Pagaling et al., 2007).
The genomes of 17 out of 40 tailed haloarchaeal viruses have been fully sequenced.These are dsDNA molecules, either circularly permuted and terminally redundant(suggesting headful DNA packaging mechanism) or non-permuted with direct terminalrepeats (suggesting replication through concatemeric intermediates) (Senčilo and Roine,2014). The genome sequences are diverse and mosaic, like those of tailed bacteriophages(Krupovič et al., 2010, Pietilä et al., 2013b, Senčilo et al., 2013). Some genes, e.g. onesencoding structural proteins and proteins involved in DNA and RNA metabolism, are alsosimilar to those in bacteriophages, but the majority of predicted genes have no assignedfunctions (Senčilo and Roine, 2014). In terms of sequence similarities observed within tailedhaloarchaeal viruses, nine viruses can be divided into three groups of closely related viruses:HF2-like, HRTV-7-like, and HCTV-1-like (Senčilo and Roine, 2014). Two other viruses(HCTV-2 and HHTV-2) are more distantly related, and six other viruses are singletons(Senčilo and Roine, 2014). Putative proviruses related to haloarchaeal tailed viruses havebeen identified in the genomes of halophilic and/or alkaliphilic, as well as in thermophilicand/or methanogenic euryarchaea (Krupovič et al., 2010, Senčilo et al., 2013).
14
Virion structures have been resolved for Hrr. sodomense myovirus HSTV-2, Har.vallismortis siphovirus HVTV-1, and Har. sinaiiensis podovirus HSTV-1 (Pietilä et al.,2013b, Pietilä et al., 2013c). All three viruses lose their infectivity at low salinity, but virusparticles display no major structural changes, and regain infectivity when high salinity isrestored (Pietilä et al., 2013b, Pietilä et al., 2013c). 3-D reconstruction revealed that HVTV-1 icosahedral capsid is built of MCP capsomers arranged in the lattice with the triangulationnumber (T) T = 13, 5-fold symmetric spikes project from the vertices, and trimeric decorativestructures are located in the center of each MCP hexamer (Pietilä et al., 2013b). In HSTV-2,MCP capsomers are arranged in T = 7 lattice and an additional minor structural protein isinserted between the MCPs, enlarging the capsid (Pietilä et al., 2013b). This was a newmechanism for capsid enlargement, that allows packaging of a larger DNA molecule thanexpected to fit into the icosahedron with T = 7 symmetry (Pietilä et al., 2013b). In HSTV-1,capsomers are arranged in T = 7 lattice, and cone-shaped towers decorate the virion at thecenter of each facet (Pietilä et al., 2013c). Notably, it was shown that HSTV-1 MCP has thecanonical HK97 fold (Pietilä et al., 2013c). Thus, structural analyses allowed placing thisarchaeal virus into the HK97-like structural viral lineage, together with tailedbacteriophages and eukaryotic herpesviruses (see chapter 1.4). Structure prediction andhomology modelling suggest that ΦCh1 may also adopt the HK97-fold, indicating that thelineage may be expanded to include more archaeal viruses (Krupovič et al., 2010).
1.5.2.2 Spindle-shaped virus His1Several archaeal spindle-shaped viruses have been isolated, and the majority of these virusesinfect hyperthermophilic crenarchaea (Krupovič et al., 2014, Pietilä et al., 2013a), and theonly one, His1, infects a halophilic euryarchaeon (Bath and Dyall-Smith, 1998). Notably,spindle-shaped morphotype is unique for archaeal viruses, as no such viruses have beenidentified for eukaryotes or bacteria. Currently, crenarchaeal spindle-shaped viruses areclassified into two families: Fuselloviridae, comprising viruses with one short tail, andBicaudaviridae, comprising viruses with one or two long tails. His1 is the only member ofSalterprovirus genus which is not assigned to any virus family. However, comparativeanalyses have showed that based on the similarities in MCP sequences, all spindle-shapedviruses may be assigned to the two above mentioned families, and His1 has been suggestedto form the Epsilonfusellovirus genus in the Fuselloviridae family (Krupovič et al., 2014).
His1 was isolated from a solar saltern in Victoria, Australia, on Haloarcula hispanica(Bath and Dyall-Smith, 1998). Originating from a highly saline environment, His1, however,tolerates a wide range of salinities (from 50 mM to 4 M NaCl) (Pietilä et al., 2013a). Theinfection is persistent, i.e. virus particles are released from the host cells continuously, whilethe host growth is retarded (Pietilä et al., 2013a, Svirskaitė et al., 2016). His1 genome, whichis a linear ~14.5 kbp long dsDNA molecule, encodes a putative type B DNA polymerase, hasinverted terminal repeats, and terminal proteins attached (Bath et al., 2006). This suggeststhat His1 replicates via protein priming. His1 MCP and a few minor structural proteins forma spindle-shaped virion, but pleomorphism in the virion shape has been observed (Pietilä etal., 2013a). No membrane has been detected in the virion, but the MCP exists in two forms,one of which is modified with a lipid moiety (Pietilä et al., 2013a). It has been suggested thatthe lipid modification enables hydrophobic interactions between the MCPs and thus may
15
account for the virion flexibility (Pietilä et al., 2013a). Cryo-electron tomography revealedthat His1 particles vary in size, but on average are ~92 nm long and ~40 nm wide, having~12 nm long tail-like structures with six tail spikes (Hong et al., 2015). The tail structure wasobserved to be more constant compared to the variable body shape (Hong et al., 2015). Ithas been speculated that His1 may recognize the host and attach to it with its tail spikestructures, and the tail hub may mediate the genome injection, after which spindle-shapedparticles could transform into empty tubes (Hong et al., 2015).
Curiously enough, while His1 is the only isolated spindle-shaped halovirus, culture-independent studies suggest that spindle-shaped viruses are abundant in hypersalineenvironments (Santos et al., 2012). Transmission electron microscopy (TEM) revealedspindle-shaped particles associated with square cells of Haloquadratum walsbyi, one of themost abundant extremely halophilic archaea (Guixa-Boixareu et al., 1996). Although Hqr.walsbyi was discovered already in 1980 by A. E. Walsby, it took more than 20 years to obtainit in pure culture (Burns, 2007, Walsby, 1980). Possibly, spindle-shaped viruses infectingHqr. walsbyi are yet awaiting to be isolated. In addition, in the recent metaviromic studiesof Namib Desert Salt pans, three obtained virus contigs showed sequence similarities to His1genes, including those coding for the MCP and minor structural proteins (Adriaenssens etal., 2016). It has been proposed that these contigs represent the genomes of virusesbelonging to the same virus lineage as His1 (Adriaenssens et al., 2016).
1.5.2.3 Tailless icosahedral virusesBefore this work, only six archaeal tailless icosahedral viruses were known, two of whichinfect thermophilic archaea (Sulfolobus viruses STIV and STIV2), and four infect halophilicarchaea (SH1, HHIV-2, PH1, and SNJ1) (Bamford et al., 2005b, Happonen et al., 2010,Jaakkola et al., 2012, Maaty et al., 2006, Porter et al., 2013, Zhang et al., 2012b). STIV andSTIV2 have been classified to the family Turriviridae, while SH1, HHIV-2, PH1, and SNJ1have been assigned to the family Sphaerolipoviridae together with Thermus phages P23-77and IN93 (Table 3). The Sphaerolipoviridae family includes three genera:Alphasphaerolipovirus (SH1, HHIV-2, PH1), Betasphaerolipovirus (SNJ1), andGammasphaerolipovirus (P23-77, IN93).
16
Tab
le 3
. Mem
bers
of t
he fa
mily
Spha
erol
ipov
irid
ae.
Gen
us
Vir
usa
Isol
atio
nh
ost
Lif
e cy
cle
Isol
atio
nso
urc
eS
tru
ctu
res
reso
lved
Cap
sid
dia
met
er,T
bM
CP
scG
enom
e, G
Bac
c. n
o.d
Ref
eren
ces
Alp
ha-
spha
ero-
lipov
irus
SH
1H
aloa
rcul
ahi
span
ica
Lyti
cSa
line
lake
(Ser
pent
ine
Lake
),A
ustr
alia
Vir
ion
(9.6
Å)
~80
nm
,T
= 2
8,de
xtro
VP7
, VP4
Line
ar d
sDN
A,
30,8
89 b
p,A
Y950
802
Bam
ford
et a
l.,20
05b,
Jääl
inoj
a et
al.,
2008
, Kiv
elä
etal
., 20
06,
Port
er e
t al.,
2005
HH
IV-2
Hal
oarc
ula
hisp
anic
aLy
tic
Sola
r sa
lter
n,(M
argh
erita
di S
avoi
a),
Ital
y
Vir
ion
(13
Å)
~80
nm
,T
= 2
8,de
xtro
VP7
, VP4
Line
ar d
sDN
A,
30,5
78 b
p,JN
9684
79
Gil-
Car
ton
etal
., 20
15,
Jaak
kola
et a
l.,20
12PH
1H
aloa
rcul
ahi
span
ica
Lyti
cSa
line
lake
(Pin
k La
kes)
,A
ustr
alia
~50
nm
VP7
, VP4
Line
ar d
sDN
A,
28,0
72 b
p,K
C25
2997
Port
e ret
al.,
2013
Bet
a-sp
haer
o-lip
ovir
us
SN
J1N
atri
nem
asp
. J7-
2Ly
soge
nic
Nat
rine
ma
sp. J
7-1
70-7
5 nm
PB6,
PB
2C
ircu
lar
dsD
NA
,16
,341
bp,
AY0
4885
0.1
Liu
et a
l., 2
015,
Zhan
g et
al.,
2012
b
Gam
ma-
spha
ero-
lipov
irus
P23
-77
Ther
mus
ther
mop
hilu
sLy
tic
Alk
alin
e ho
tsp
ring
,N
ew Z
eala
nd
Vir
ion
(14
Å),
MC
Ps (
3 Å
),~
78 n
m,
T =
28,
dext
roV
P16,
VP1
7C
ircu
lar
dsD
NA
,17
,036
bp,
GQ
4037
89
Jaat
inen
et a
l.,20
08,
Jala
svuo
ri e
tal
., 20
09IN
93Th
erm
usth
erm
ophi
lus
Lyso
geni
cTh
erm
usaq
uati
cus
TZ2
~13
0nm
VP1
3,V
P14
Cir
cula
rds
DN
A,
19,6
04 b
p,A
B06
3393
Mat
sush
ita
etal
., 19
95,
Mat
sush
ita
and
Yana
se, 2
009
a.Ty
pe s
peci
es n
ames
are
in b
old.
b.
T, tr
iang
ulat
ion
num
ber.
c.Sm
all a
nd la
rge
MC
Ps.
d.
Gen
Ban
k ac
cess
ion
num
bers
.
17
(i) Alphasphaerolipoviruses SH1 (Porter et al., 2005), HHIV-2 (Jaakkola et al., 2012),and PH1 (Porter et al., 2013) are virulent viruses isolated from distant hypersalineenvironments (saline lakes in Australia and a solar saltern in Spain) on Haloarculahispanica (Table 3). These viruses have narrow host ranges. In addition to Har. hispanica,SH1 is able to infect Haloarcula sp. PV7 and Halorubrum sp. CSW 2.09.4 which is closelyrelated to Hrr. sodomense (Jaakkola et al., 2012, Porter et al., 2005). HHIV-2 is also able toinfect Haloarcula sp. PV7 (Atanasova et al., 2012, Jaakkola et al., 2012), and PH1 infectsHalorubrum, sp. CSW 2.09.4 (Porter et al., 2013). It has been shown that in spite of thenarrow host ranges, SH1 and PH1 genomes can be transfected into several additionalarchaeal strains, suggesting that these virus genomes may replicate in a wide range of strains(Porter and Dyall-Smith, 2008, Porter et al., 2013). Although there have been somecontradictory proposals regarding SH1 exit strategy (Porter et al., 2007, Porter et al., 2005),recent studies, which included traditional metrics and biochemical measurements, haveshown that SH1 life cycle is lytic (Svirskaitė et al., 2016). Concurrently with virus release,cell culture density and viable cell counts drop, host cell membrane becomes compromised,cell respiration is declining, and adenosine triphosphate (ATP) leakage from the cells isdetected (Svirskaitė et al., 2016).
The genomes of alphasphaerolipoviruses are linear dsDNA molecules about 30 kbplong having inverted terminal repeats and terminal proteins attached (Bamford et al.,2005b, Jaakkola et al., 2012, Porter and Dyall-Smith, 2008, Porter et al., 2013). Whetherthese viruses replicate via protein-primed system remains to be seen, as no putative protein-primed DNA polymerase gene was found in their sequences. In these viruses, the mostdistinct genes encoding structural proteins are those for putative spikes (see below), whichmost probably serve as host recognition devices (Jaakkola et al., 2012, Jäälinoja et al.,2008). The genomes of alphasphaerolipoviruses have no predicted integrase genes.However, two related putative proviruses have been recognized in the chromosomes ofhalophilic archaea Halobiforma lacisalsi (provirus HaloLacP1) and Haladaptatuspaucihalophilus (provirus HalaPauP1) (Porter et al., 2013).
The virions of alphasphaerolipoviruses are icosahedrons of ~80 nm in diameter(except PH1), and they have an inner lipid membrane composed of lipids selectively acquiredfrom the host cell membrane (Bamford et al., 2005b, Gil-Carton et al., 2015, Jäälinoja et al.,2008). Cryo-EM structures of SH1 and HHIV-2 revealed that these viruses have capsidsarranged in an unusual T = 28 dextro lattice (same T number is also reported for Thermusphage P23-77, see below) (Gil-Carton et al., 2015, Jäälinoja et al., 2008). The capsids arebuilt of pseudo-hexameric capsomers having either two or three towers on the capsomersurface. The capsomers are formed by small and large MCPs, which are called virion proteins(VPs) VP7 (small MCP) and VP4 (large MCP) in both viruses. VP7 has one β-barrel domain,whereas VP4 has two single β-barrel domains, one on the top of the other (Gil-Carton et al.,2015, Jäälinoja et al., 2008). For HHIV-2, it has been proposed that VP4-VP4 homodimersand VP4-VP7 heterodimers make the lattice, which is stabilized by disulphide bridges acrossthe capsomers. The upper domain of VP4 (a β-barrel) forms the capsomer towers (Gil-Carton et al., 2015). The capsomer pseudo-hexameric footprint is conserved in SH1 andHHIV-2, while spike complexes located at 5-fold vertices have different architectures. SH1capsid is decorated with horn-like spikes (Jäälinoja et al., 2008), and HHIV-2 has propeller-
18
like spikes (Gil-Carton et al., 2015). This observation highlights conservation in the majorcapsid building blocks (virus “self” elements) and faster evolution in the host recognitionstructures.
(ii) Betasphaerolipovirus SNJ1 is a temperate virus, which resides as a provirus(plasmid) in Natrinema sp. J7-1, and produces plaques on Natrinema sp. J7-2 (Zhang et al.,2012b). SNJ1 life cycle mode depends on salinity: it is lytic at ≥ 3.4 M NaCl and lysogenic atlower salinity (3 M NaCl) (Mei et al., 2015). SNJ1 virions are 70-75 nm in diameter andcontain an inner lipid membrane (Liu et al., 2015). The genome is a circular DNA moleculeof about 16 kbp long (Zhang et al., 2012b). Little sequence similarity is observed betweenSNJ1 and alphasphaerolipoviruses.
(iii) Archaeal tailless icosahedral haloviruses share structural similarities with thebacterial tailless icosahedral viruses P23-77 and IN93 belonging to the genusGammasphaerolipovirus (Pawlowski et al., 2014). These viruses infect Thermusthermophilus strains (host range is narrow), undergo lytic (P23-77) or lysogenic (IN93) lifecycles, and have circular dsDNA genomes (Jaatinen et al., 2008, Jalasvuori et al., 2009,Matsushita and Yanase, 2009). P23-77 virion and MCPs structures have been resolved(Rissanen et al., 2013). Like in SH1 and HHIV-2, P23-77 icosahedral capsid of 78 nm indiameter is arranged in T = 28 dextro lattice. Small MCP VP16 and large MCP VP17 formpseudohexameric capsomers with two towers, which are located either on the same side orthe opposite corners of the capsomer (Rissanen et al., 2013). In all three viruses, P23-77,SH1, and HHIV-2, the lattice footprint is conserved, while P23-77 has yet another type ofspikes (elongated) which emerge from the 5-fold vertices (Gil-Carton et al., 2015, Rissanenet al., 2013).
Sphaerolipoviruses share virion architectural principles with the viruses belonging tothe PRD1-adenovirus structural lineage (see chapter 1.4). Together with SSIP-1 and severalrelated putative proviruses, sphaerolipoviruses constitute a distinct group of single β-barrelviruses within the lineage.
1.5.2.4 Pleomorphic virusesThe group of pleomorphic haloviruses includes eight viruses isolated from remotehypersaline locations on Haloarcula, Halorubrum, or Halogeometricum strains, and onetemperate virus induced from Natrinema sp. J7-1 (Table 4) (Liu et al., 2015, Pietilä et al.,2016). In addition, several related putative proviruses have been identified in thechromosomes or extrachromosomal elements of halophilic archaea belonging to severaldifferent genera (Liu et al., 2015). Pleomorphic archaeal viruses have been recently classifiedto the new family Pleolipoviridae (Pietilä et al., 2016). These viruses display virions ofspherical or pleomorphic shape, which are in fact vesicles built of lipids (which areunselectively derived from the host membrane) and typically only two structural proteinspecies: membrane-associated protein and spike protein (Pietilä et al., 2010, Pietilä et al.,2012). Cryo-EM reconstruction of Halorubrum pleomorphic virus 1 (HRPV-1) virions hasshown that the spike proteins are distributed irregularly on the virion surface (Pietilä et al.,2010). Pleolipoviruses undergo non-lytic life cycles, presumably entering the host cell byfusion of the virus lipid membrane with the host cell membrane (Pietilä et al., 2016).
19
Assembled virions are released continuously most probably via budding, retarding the hostgrowth, either slightly (as in case of HRPV-1 infection) or quite substantially (as in case ofHis2 infection) (Pietilä et al., 2009, Pietilä et al., 2012, Svirskaitė et al., 2016). The hostrange is typically narrow or limited to the isolation strain (Atanasova et al., 2012, Pietilä etal., 2009, Pietilä et al., 2012). The only temperate pleolipovirus, SNJ2, is able to integrateto and excise from the host chromosome (transfer RNA [tRNA] gene) (Liu et al., 2015).Peculiarly, SNJ2 production is dependent on the presence of SNJ1, the temperate virusresiding in Natrinema sp. J7-1 as a plasmid (Liu et al., 2015) (Table 3, see also chapter1.5.2.3). However, the nature of the interaction between SNJ1 and SNJ2 remains to beclarified. The genome type of pleolipoviruses is not conserved, it may be either circularssDNA or circular/linear dsDNA, depending on the virus (Pietilä et al., 2016). Interestingly,the genomes of HGPV-1, HRPV-3, and SNJ2 are circular dsDNA molecules with single-stranded interruptions, whose function is not yet clear (Liu et al., 2015, Senčilo et al., 2012).Although little sequence similarity is observed between pleolipovirus genomes, they allcontain a conserved set of genes, including those coding for structural proteins and aputative NTPase (Liu et al., 2015, Senčilo et al., 2012). Based on the gene content,pleolipoviruses are grouped into three genera: Alphapleolipovirus, Betapleolipovirus, andGammapleolipovirus (Table 4) (Pietilä et al., 2016).
20
Tab
le 4
. Arc
haea
l ple
omor
phic
vir
uses
in th
e fa
mily
Pleo
lipov
irid
ae.
Gen
us
Vir
usa
Isol
atio
n h
ost
Vir
ion
dia
me
ter
Lif
e cy
cle
Mem
bran
ep
rote
in,
spik
ep
rote
in
Gen
ome,
Gen
eBan
kac
cess
ion
nu
mbe
r
Isol
atio
n s
ourc
eR
efer
ence
s
Alp
ha-
pleo
lipo-
viru
s
HR
PV
-1H
alor
ubru
msp
.PV
6~
41 n
mPe
rsis
tent
VP3
, VP4
Cir
cula
r ss
DN
A,
7,04
8 nt
,FJ
6856
51
Sola
r sa
lter
n,Tr
apan
i, It
aly
Piet
iläet
al.,
200
9,Pi
etilä
et a
l., 2
010,
Piet
ilä e
t al.,
201
2H
RPV
-2H
alor
ubru
msp
.SS5
-4~
54 n
mPe
rsis
tent
VP4
,VP5
Cir
cula
r ss
DN
A,
10,6
56 n
t,JN
8822
64
Sola
r sa
lter
n, S
amut
Sakh
on, T
haila
ndA
tana
sova
et a
l., 2
012,
Piet
ilä e
t al.,
201
2,Se
nčilo
et a
l., 2
012
HR
PV-6
Hal
orub
rum
sp.S
S7-4
~49
nm
Pers
iste
ntV
P4, V
P5C
ircu
lar
ssD
NA
,8,
549
nt,
JN88
2266
Sola
r sa
lter
n,Sa
mut
Sakh
on, T
haila
ndPi
etil ä
et a
l., 2
012,
Senč
ilo e
t al.,
201
2
HH
PV-1
Hal
oarc
ula
hisp
anic
a~
52 n
mPe
rsis
tent
VP3
, VP4
Cir
cula
r ds
DN
A,
8,08
2 bp
,G
U32
1093
Sola
r sa
lter
n,M
argh
erit
a di
Sav
oia,
Ital
y
Roi
neet
al.,
201
0,Se
nčilo
et a
l., 2
012
HH
PV-2
Hal
oarc
ula
hisp
anic
a~
50nm
Pers
iste
ntO
RF3
pro
duct
,O
RF4
pro
duct
Cir
cula
r ss
DN
A,
8,17
6 nt
,K
F056
323
Sola
r sa
lter
n, H
ulu
Isla
nd, L
iaon
ing,
Chi
na
Liet
al.,
201
4
Bet
a-pl
eolip
o-vi
rus
HR
PV
-3H
alor
ubru
msp
.SP3
-3~
67 n
mPe
rsis
tent
VP1
, VP2
Cir
cula
rds
DN
Ab ,
8,77
0 bp
,JN
8822
65
Salt
wat
er,S
edom
Pond
s, I
srae
lA
tana
sova
et a
l., 2
012,
Piet
ilä e
t al.,
201
2,Se
nčilo
et a
l., 2
012
HG
PV-1
Hal
ogeo
met
ricu
msp
. CG
-9~
56 n
mPe
rsis
tent
VP2
and
VP3
,V
P4C
ircu
lar
dsD
NA
b ,9,
694
bp,
JN88
2267
Sola
r sa
lter
n, C
abo
deG
ata,
Spa
in
SNJ2
Nat
rine
ma
sp. J
7-1
70-8
0nm
Lyso
geni
cV
P12,
VP1
3C
ircu
lar
dsD
NA
b,c ,
16,9
92 b
p,A
JVG
0100
0023
Nat
rine
ma
sp. J
7-1
Liu
et a
l., 2
015
Gam
ma-
pleo
lipo-
viru
s
His
2H
aloa
rcul
ahi
span
ica
~71
nm
Pers
iste
ntV
P27,
VP2
8an
d V
P29
Line
ar d
sDN
A,
16,0
67 b
p,A
F191
797
Salin
e la
ke(P
ink
Lake
s), V
icto
ria,
Aus
tral
ia
Bat
het
al.,
200
6,Pi
etilä
et a
l., 2
012
a.Ty
pe s
peci
es a
re in
bol
d.b.
Bet
aple
olip
ovir
us g
enom
es a
re d
sDN
A m
olec
ules
with
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21
1.6 Virus-host interactions in hypersaline environmentsThe number of VLPs in hypersaline environments is high (up to 109-101o VLP/ml), increasingalong the salinity gradient (Bettarel et al., 2011, Boujelben et al., 2012, Guixa-Boixareu etal., 1996, Santos et al., 2012). Moreover, in many hypersaline reservoirs, free virusessignificantly outnumber cells (up to 100 VLP/cell) (Santos et al., 2012). In the saltiest ponds(above 25 % total salinity), viruses are usually the only predators controlling prokaryoticgrowth rates and mortality (Guixa-Boixareu et al., 1996, Pedros-Alio et al., 2000). Asrevealed by electron microscopy, the major virus morphotypes in hypersaline environmentsare spindle-shaped, spherical, tailed, and filamentous (Boujelben et al., 2012, Guixa-Boixareu et al., 1996, Sime-Ngando et al., 2011). Spindle-shaped VLPs are often reported asthe most abundant ones, and their abundance is increasing along the salinity gradient,correlating with the abundance of square cells of Hqr. walsbyi (Bettarel et al., 2011, Guixa-Boixareu et al., 1996, Sime-Ngando et al., 2011). Surprisingly, VLPs of very unusual shapeswere observed in hypersaline Lake Retba (Senegal): stars, rods, hooks, and hairpins amongothers (Sime-Ngando et al., 2011). It is, however, questionable whether these unusual shapesrepresent real virions or TEM-related artefacts. In addition, it is important to remember thatmicroscopy examinations, especially those of environmental samples, may not be accurate,because along with virus particles, similar sized extracellular vesicles (Forterre et al., 2013,Soler et al., 2015) or recently discovered nanohaloarchaeal cells may be counted as sphericalVLPs.
Culture-independent approaches, such as pulsed-field gel electrophoresis of total viralDNA, as well as metagenomics, metatranscriptomics, and metaproteomics, have beenapplied to assess the diversity and activity of viral communities in hypersalineenvironments. In general, these studies have shown that halovirus communities are diverseand specific for hypersaline environments (Boujelben et al., 2012, Díez et al., 2000,Emerson et al., 2013b, Roux et al., 2016, Sandaa et al., 2003). However, the literaturecontains contradictory reports about (i) temporal and (ii) spatial variation in haloviruscommunities:
(i) Temporal: Metagenomic analyses of water samples from San Diego salterns takenat different time points have shown that the most abundant viruses, as well as the mostabundant microbial species, were stable, while the variation in microdiversity (changing ofgenotypes) was observed (Rodriguez-Brito et al., 2010). Emerson et al. (2013a, 2012)reanalysed the same dataset and suggested that virus community was actually dynamic atthe taxon level and that variation observed at the genotype level in fact reflected shifts at thepopulation level. In a set of studies, Emerson and colleagues analysed metaviromes fromLake Tyrell samples collected over different timescales, and concluded that haloviruspopulations were stable over days, relatively equally stable and dynamic over weeks, anddynamic over months to years (Emerson et al., 2013a, Emerson et al., 2012, Emerson et al.,2013b).
(ii) Spatial: Several studies have suggested that common traits can be found in themetaviromes reconstructed from distant hypersaline environments (Garcia-Heredia et al.,2012, Roux et al., 2016, Santos et al., 2010, Sime-Ngando et al., 2011). For example, commonfeatures were identified in the metaviromes from hypersaline ponds in Senegal, Australia,
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and Spain, suggesting that virus communities are stable across continents (Roux et al.,2016). However, the usage of different methods for the preparation of environmental DNA,different sequencing techniques, and sequence analyses tools impose challenges for directdata comparisons. The lack of universal virus genes also makes it difficult to makecomparisons across samples. In addition, different conclusions regarding similarity may bedrawn depending on whether nucleotide or protein sequences are compared. For example,in the above mentioned study of Lake Tyrell virus community dynamics, the viromes werecompared at the nucleotide sequence level, with the threshold of 90% identity, andconsidered to be highly dynamic over months to years (Emerson et al., 2012). When thesame datasets were used in a large comparison of ~40 viromes and the similarity wasassessed at the protein sequence level, the Lake Tyrell viromes were closely relatedcompared to the other datasets (Roux et al., 2016). In addition, the stringency of comparisonhas to be taken into account, as demonstrated in the same study of the Lake Tyrell viruscommunity: when the fragment recruitment detection threshold was reduced from 90% to75% nucleotide identity, some previously unseen similarities to the sequenced halovirusesand San Diego metavirome sequences were detected (Emerson et al., 2012).
Typically, a high percentage of environmental virus sequences do not match to thedatabases (Boujelben et al., 2012, Santos et al., 2010, Sime-Ngando et al., 2011). However,some studies have reported sequence similarities between metaviromes and the isolatedhaloviruses. Metagenomic analyses of the samples from hypersaline ponds in Senegalrevealed sequence matches to the isolated haloarchaeal tailed viruses, pleolipoviruses, andHis1, as well as environmental halophages assembled from Santa Pola salterns (Roux et al.,2016). In metaproteomics studies of the virus community in Deep Lake, Antarctica, eightdistinct MCPs were detected, five matching to the known isolated haloarchaeal siphoviruses,two to the putative tailed environmental halophages, EHP-12 and EHP-6, and one to Vibriophage VBM1 (Tschitschko et al., 2015). In addition to the MCPs, a prohead protease wasdetected in the metaproteomic dataset, indicating active virus life cycles in Deep Lake(Tschitschko et al., 2015). The activity of viruses in the environment may also be assessedby metatranscriptomics. For instance, total environmental RNA from Santa Pola salternswas analysed using microarrays constructed based on the previously obtained metaviromefrom the same environment (Santos et al., 2011). This work has shown that halovirus geneswere actively expressed at the sampling time and that the most active viruses were those thattentatively infect haloarchaea and Salinibacter strains (Santos et al., 2011).
Low number of matches between metagenomic sequences and databases indicates thatonly a small part of haloviruses have been characterized. A global sampling of distanthypersaline environments performed in 2012 doubled the number of isolated haloarchaealviruses, and revealed numerous virus-host pairs within and across samples (Atanasova etal., 2012). Although the analysis of environmental sequences allows reconstruction ofcomplete or almost complete virus genomes and tentative identification of virus-host pairs(Garcia-Heredia et al., 2012, Martínez-García et al., 2014, Santos et al., 2007), moleculardetails of virus-host interactions may be studied in depth only in isolated virus-host systems.Obviously, rapidly accumulating sequence data needs functional annotation, and in thisrespect, cultivation-based studies cannot be neglected.
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Although hypersaline environments, such as solar salterns, may be seen as simplifiedsystems for ecological studies due to low species diversity, high cell abundance, and shortfood chains, there is still little understanding of virus-host interactions in hypersalineeconiches. Multiple factors may affect virus-host interplay. It has been shown that salinityaffects the adsorption of haloviruses to the host cells (Kukkaro and Bamford, 2009, Mei etal., 2015), as well as regulates switches between lytic and lysogenic virus life cycles (Bettarelet al., 2011, Daniels and Wais, 1990, Mei et al., 2015). Another important aspect of virus-host interplay is the ability of host cells to resist virus attacks. Virus defense systems arecommonly found in archaea and include clustered regularly interspaced short palindromicrepeats (CRISPR) and CRISPR-associated proteins (Cas) immune system (Garrett et al.,2011, Sorek et al., 2008), toxin-antitoxin system (Yamaguchi et al., 2011), superinfectionexclusion mechanism (Stolt and Zillig, 1992), and bacteriophage exclusion (BREX)mechanism (Goldfarb et al., 2015). Finally, more spatial and temporal studies in hypersalineenvironments are needed to test the current models of virus-host dynamics, such as Lotka-Volterra “kill the winner” (Thingstad, 2000), constant-diversity dynamics (Rodriguez-Valera et al., 2009), or recently proposed “piggyback the winner” (Knowles et al., 2016). Toconclude, only coupling of various approaches to study virus-host interplay would provide acomplete view on the true virus diversity, as well as temporal and spatial dynamics inhypersaline environments.
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2. Aims of the StudyIt has been estimated that viruses are present virtually in all types of environments that onecan imagine, being a significant component of the biosphere (Breitbart and Rohwer, 2005,Dolja and Krupovič, 2013, Forterre and Prangishvili, 2013, Rohwer et al., 2009). Virusesdisplay huge genetic diversity, but more modest variety of virion shapes (Krupovič andBamford, 2011). One way to comprehend the diversity in the virosphere is to percept virionas a “vertically-inherited” viral “self” (Abrescia et al., 2010, Krupovič and Bamford, 2010).It has been suggested that only a limited number of virion architectures exist, and deepevolutionary links have been found between viruses that infect hosts from all three domainsof cellular life (Abrescia et al., 2012, Bamford et al., 2002, Bamford, 2003, Oksanen et al.,2012). The key to understand ultimate viral diversity, virus functions and complexevolutionary relations of different virus groups is to isolate new virus-host systems and tocharacterize them in detail. Hypersaline environments, where archaea dominate, are a richsource of new viruses, and only very little is known about archaeal viruses. This thesis isdevoted to the isolation of new haloarchaeal viruses in order to probe their morphologicaldiversity. Virus-host interactions in a hypersaline environment are also addressed. Inaddition, comprehensive descriptions of two new isolates are provided.
The specific aims of this work were:
- To isolate new archaeal viruses from high salinity environments and to determine theirmorphotypes;
- To reveal virus-host interactions in high salinity environments using culture-basedapproach;
- To describe certain isolated viruses in molecular detail, including characterization ofvirus infection cycle and virion components.
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3. Materials and MethodsSalt samples used in this study included saltwater and salt crystals collected from a solarsaltern in Samut Sakhon (Thailand) in 2009 and 2010 (Table S1 in I). Fifty one halophilicprokaryotic strains isolated from the salt samples (Tables 1 and S2 in I), as well as elevenculture collection strains (Table 1 in I) were used here. The studied viruses included 45isolates listed in publication I (Tables 2 and S3 in I) and Haloarcula hispanica pleomorphicvirus 3 (HHPV3) described in publication III. Methods and bioinformatics tools used in thiswork are listed in Table 5 and Table 6, respectively. Virus genome sequences were depositedin the GenBank database. For more detailed information about the methods, please see thepublications I-III and references therein.
Table 5. Laboratory methods used in this study.
Method Used inCultivation of halophilic prokaryotes and their viruses I, II, IIIPlaque assay I, II, IIIIsolation of halophilic prokaryotes from salt samples IIsolation of archaeal viruses from salt samples I, IIISpot-on-lawn test for virus infectivity I, IIIVirus adsorption assay II, IIIVirus life cycle analysis II, IIIElectrochemical and ATP measurements during virus infection IINegative staining and transmission electron microscopy I, II, IIIThin-section TEM IIPurification of virus particles by NaCl-polyethylene glycol 6000precipitation, rate zonal and equilibrium centrifugation
I, II, III
Bradford assay for measuring protein concentration I, IISodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) I, II, IIICoomassie blue staining of gels I, II, IIIPr0-Q Emerald, SYPRO-Ruby, and Sudan Black B staining of gels II, IIIIdentification of proteins by N-terminal sequencing IIIdentification of proteins by mass spectrometry II, IIIExtraction of lipids and thin-layer chromatography (TLC) II, IIIDNA extraction and enzymatic digestion II, IIIAgarose gel electrophoresis and ethidium bromide staining II, IIIPolymerase chain reaction (PCR) I, IIISanger sequencing I, II, IIIIllumina sequencing III
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Table 6. Bioinformatic tools used in this study.
Type of analysis Tool(s) Used inPhylogenetic analysis Molecular Evolutionary Genetics
Analysis (MEGA)I, II, III
Sequence alignment Multiple Sequence Comparison byLog-Expectation (MUSCLE)
I, II, III
EMBOSS Stretcher, EMBOSS Needle II, IIIVisualization, transfer, andalignment of sequences
Geneious v. 6.1.6 by Biomatters I, II, III
Prediction of open reading frames(ORFs)
GenMarkS, Glimmer II, III
Prediction of replication origin Ori-Finder 2 IIICalculating of guanine-cytosinecontent
Science Buddies “Genomics %G~CContent Calculator”
II, III
Calculating of isoelectric point andmolecular mass
Compute pI/MW tool of ExPASy tools II, III
Prediction of tRNA sequences ARAGORN v1.2.36 IIDetection of repeats Tandem Repeats Finder, EMBOSS
EinvertedII, III
Homology detection Basic local alignment search tool(BLAST)
II, III
Detection of conserved domains NCBI Conserved Domain Search II, IIIInterProScan at EMBL-EBI III
Prediction of transmembranehelices
TMHMM v 2.0 II, III
Prediction of coiled-coil regions COILS tool II, IIIGraphical visualization Circos IIPrediction of signal peptides Signal P v 4.1 III
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4. Results and Discussion4.1 New archaeal viruses isolated from high salinityIn order to isolate new archaeal viruses, salt samples (Table SI in I) were collected from asolar saltern in Samut Sakhon, Thailand in 2009 and 2010. From these samples, halophilicprokaryotes were isolated: 36 archaeal and 15 bacterial strains (Table 1 in I). Based on theirpartial 16S rRNA gene sequences, the strains were classified at genus level. Bacterial strainsbelonged to four genera of two phyla: Proteobacteria (Rhodovibrio) and Firmicutes(Halobacillus, Pontibacillus, and Sedimibacillus). The isolated archaea were assigned to tengenera of Halobacteriaceae family, with Halorubrum and Haloarcula strains being themost numerous (10 and 6, respectively). The strains of these genera are commonly reportedas prevalent in culture-based samplings of hypersaline environments (Birbir et al., 2007,Boujelben et al., 2014, Burns et al., 2004). Using the isolated archaeal strains, as well as ourculture collection strains (Table 1 in I), we isolated 46 archaeal viruses from the abovementioned salt samples (Tables 2 and S3 in I; publication III, see chapter 4.4), for 37 ofwhich purification protocols were developed and virion morphotype was determined byTEM. The 37 purified viruses differed one from another by plaque morphology (Figures S1in I and S1 in III), virion morphotype (Figures 2 in I, 5 in II, and 3 in III), sensitivity of virusinfectivity to chloroform (Table 2 in I, and chapter 4.4), host ranges (Tables S4 and S5 in I,and chapter 4.4), and structural protein patterns of purified virus particles (Figures S2 in I,2 in II, and 4 in III) and thus were considered unique. These isolates were named accordingto their isolation host and morphology (Table 2 in I, and chapter 4.4). The isolated virusesdisplayed four different morphotypes: myovirus (27 isolates), siphovirus (4), pleomorphic(5) and tailless icosahedral (1) (Figures 2 in I, 5 in II, and 3 in III).
Among 37 isolated viruses with known morphotypes (I, III), one pleomorphic virus(HRPV-6) had been described in detail elsewhere (see chapter 1.5.2.4) (Pietilä et al., 2012,Senčilo et al., 2012). Thus, in this thesis, only 36 isolated viruses are counted as new oneswhen compared to the number of all haloarchaeal viruses reported in the currently availableliterature (see chapter 1.5.2.4). However, HRPV-6, which had been isolated from the samelocation during the same sampling, was included in virus-host interactions tests, so I willdiscuss the interactions of all 37 isolates in chapter 4.2.
The isolation of new viruses has increased the total number of isolated archaealhaloviruses from 54 to 90 (Figures 2 A and B). Noticeably, the morphotype distributionobserved in this study is highly similar to that in the previous global sampling of distanthypersaline environments (Atanasova et al., 2012), with tailed icosahedral virus isolatesbeing the most numerous. Obviously, the isolated viruses represented only a part of naturalviral diversity in the studied environment, as a culture-based approach is limited to thestrains that can be cultivated on artificial growth media and viruses that form detectableplaques on host lawns. However, this does not diminish a possible role of the isolated virusesin the dynamics of halophilic microbial communities (see chapter 4.2 for virus-hostinteractions). So far, culture-dependent samplings of viral diversity in hypersalineenvironments yielded a relatively low number of different virus morphotypes (Atanasova etal., 2015a), supporting the idea that all viruses can be grouped into a limited number of
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structural lineages (Bamford et al., 2002, Oksanen et al., 2012). Future isolations of virus-host pairs from hypersaline environments will show if haloarchaeal viruses with virions ofunusual shape, e.g. possibly resembling those of thermophilic crenarchaeal viruses(Prangishvili et al., 2016) or the particles observed in TEM examinations of environmentalsamples (Sime-Ngando et al., 2011), also exist.
Figure 2. Distribution of morphotypes of isolated haloarchaeal viruses (A) excluding and (B)including current study. Total numbers of isolates of a given morphotype are indicated in brackets.
4.2 Virus-host interactionsTo reveal virus-host interactions, all isolated viruses were tested against all isolatedhalophilic prokaryotes (except Halorubrum sp. SS13-13 and Halogranum sp. SS13-5 whichdid not form a dense lawn) and additional archaeal culture collection strains. For 37 viruseswith known morphotypes, we detected 269 specific interactions across genera and samples,all archaea-specific (Tables S4 and S5 in I; publication III, see chapter 4.4). In addition, 73interactions were observed for nine virus isolates of unknown morphotype (Table S3 in I).Further, I discuss the interactions of the 37 unique virus isolates (Table 2 in I and publicationIII).
Originally, all the viruses were isolated on Halorubrum or Haloarcula strains, butcross-testing showed that strains from other genera (Halobacterium, Halobellus, andHaloterrigena) were also susceptible to the isolated viruses. However, no viruses wereisolated for Halolamina, Halogranum, Haloferax, Halogeometricum, and Natrinemastrains. Myoviruses had the broadest host ranges (from 3 to 14 hosts, 9 on average), andsome Halorubrum strains were susceptible to many viruses (up to 26 different viruses).These two components accounted for the majority (~75%) of the interactions that wereobserved. Viruses of the other morphotypes had narrower host ranges.
Viruses isolated from Samut Sakhon saltern were able to infect the strains isolatedfrom the same location (from both 2009 and 2010 samples), as well as culture collectionstrains originating from geographically distant hypersaline environments (Tables S4 and S5in I and publication III). For example, Halorubrum sodomense isolated from the Dead Seawaters (Israel) (Oren, 1983) could be infected by 18 myoviruses, and “Haloarcula
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californiaea” isolated from coastal salt deposits in Baja California (Mexico) (Javor et al.,1982) was a host for 12 viruses (11 myoviruses and one icosahedral virus). These diverseinteractions indicate a constant interplay between haloarchaea and their viruses (especiallymyoviruses) extending over time, genus, and location “barriers” and are in line with theprevious observations of global distribution of microbes and viruses in hypersalineenvironments (Atanasova et al., 2012, Dyall-Smith et al., 2011, Oh et al., 2010, Roux et al.,2016).
To gain more insights into viruses inhabiting hypersaline environments, here, Iprovide a detailed molecular characterization of two isolates obtained from Samut Sakhonsaltern: Haloarcula californiae icosahedral virus 1 (HCIV-1, see chapter 4.3) andHaloarcula hispanica pleomorphic virus 3 (HHPV3, see chapter 4.4).
4.3 Molecular characterization of HCIV-1
4.3.1 HCIV-1 infection cycleAlthough HCIV-1 had been isolated from salt crystals (I), it tolerated a wide range ofsalinities. No major changes in infectivity were detected in a range of 3 to 23 % total salinityfor a 24-h period tested (Figure S1 in II). The ability to tolerate high and low ionic strengthmay be advantageous in such environments as a solar saltern, where salinity is changing dueto natural rainfalls and water evaporation.
HCIV-1 was isolated on Har. californiae, but could also infect Har. japonica withapproximately same efficiency of plating, and with much lower efficiency, Har. hispanicaand Halorubrum sp. SS7-4 (I). It seems that a narrow host range is typical for all currentlyknown haloarchaeal tailless icosahedral viruses (see chapter 1.5.2.3). The adsorption ofHCIV-1 particles to Har. californiae cells was relatively slow, but efficient: at ~1.5 h postinfection (p.i.), ~45 % of virus particles adsorbed to the cells, and by ~5 h p.i, the maximumof 80% adsorption was reached (Figure S2A in II). The adsorption rate constant was 5.7 ×10-11 ml/min (calculated for the first h p.i.), which is in a range typical for halophilic archaealviruses (Jaakkola et al., 2012, Kukkaro and Bamford, 2009, Svirskaitė et al., 2016). At 2.5 hp.i., tail-like structures between Har. californiae cells and icosahedral virus particles werevisualized by TEM (Figures 1 B and C in II). To my knowledge, similar structures have notbeen reported for other tailless archaeal viruses, but were described for tailless icosahedralbacteriophages, e.g. PRD1 (Peralta et al., 2013) and ΦX174 (Sun et al., 2014), as the “devices”for genome delivery.
The single-step growth curve of HCIV-1 infection showed that at 10-12 h p.i., theturbidity of the infected culture started to decrease (Figures 1 A and G in II), while thenumber of free progeny viruses was rapidly increasing (Figure 1G in II). At the same time,cell debris were accumulating, as revealed by light (Figure S2 B in II) and electronmicroscopy (Figure 1 E in II). In addition, the leakage of ATP from the cells and binding ofa lipophilic indicator anion (phenyldicarbaundecaborane) to the cells occurred also at ~10 hp.i., indicating that cell integrity was compromised (Figures 1 F and H in II). Moreover, thelevel of oxygen consumption was significantly decreasing compared to the uninfectedculture (Figure 1 I in II). All this suggested that assembled HCIV-1 particles exit cells via cell
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lysis. The same type of study approach has been applied to SH1, showing that the virus isvirulent (Svirskaitė et al., 2016). However, in SH1 infection, cell lysis occurs earlier (at ~6 hp.i.), which probably reflects specificity of virus-host interactions. We could not identify anylysis-related genes in HCIV-1 genome (see below). However, this is not surprising takinginto account that no lysis-related genes similar to those of bacteriophages are found in thegenomes of archaeal viruses. Moreover, the only currently described archaeal virus exitstrategy is that of viruses STIV and Sulfolobus islandicus rod-shaped virus 2 (SIRV2)(Snyder et al., 2013). These viruses exit Sulfolobus cells through pyramide-like openings,not observed for any other virus-host systems (Snyder et al., 2013).
4.3.2 HCIV-1 virion organizationAn optimized multistep purification protocol was used to obtain highly purified HCIV-1particles (Table S1 and Figure 2 in II). The major structural components of HCIV-1 virionwere determined (Figures 2-5 in II). TEM of highly purified particles revealed that theicosahedral HCIV-1 virion was ~70 nm in diameter and contained an inner layer(membrane) (Figure 4). The virion consisted of at least 12 protein species (Figures 2 and 4A in II), which were designated according to their amino acid sequence similarities (seebelow) to the corresponding proteins of SH1, PH1, and HHIV-2. Notably, the overallstructural protein profiles were similar, but not identical in HCIV-1, SH1, and HHIV-2(Figure 4 A in II). HCIV-1 lipid profile consisted of three major phospholipid species:phosphatidylglycerol, phosphatidylglycerophosphate methyl ester, andphosphatidylglycerosulfate (Figure 3 in II). HCIV-1 and Har. californiae lipid compositionsdiffered (Figure 3 in II), suggesting that HCIV-1 acquires lipids selectively from the host cellmembrane. A selective acquisition of the host phospholipids has been previously reportedfor other tailless inner membrane-containing icosahedral dsDNA viruses, e.g. SH1 (Bamfordet al., 2005b), HHIV-2 (Gil-Carton et al., 2015), PRD1 (Laurinavičius et al., 2004b), Bam35(Laurinavičius et al., 2004b), as well as for the enveloped tailless icosahedral dsRNAbacteriophage Φ6 (Laurinavičius et al., 2004a).
Based on the sequence similarities with SH1 and HHIV-2, for which virion structureshave been resolved (Gil-Carton et al., 2015, Jäälinoja et al., 2008), we propose the followingvirion model for HCIV-1. HCIV-1 proteins VP4 and VP7 are the MCPs, while proteins VP2,VP3 and VP4 form the vertex complex, and proteins VP10 and VP12 are associated with theinner membrane (Figure 4). Based on the similar sizes of SH1, HHIV-2, and HCIV-1 virions,HCIV-1 capsid lattice could also be arranged in T = 28 (Gil-Carton et al., 2015, Jäälinoja etal., 2008). Moreover, HCIV-1 virion assembly model might mimic that described for HHIV-2 (Gil-Carton et al., 2015) (see chapter 1.5.2.3). We suggest that the small MCP VP7, whichhas one single β-barrel domain, and the large MCP VP4, which has two single β-barreldomains, form pseudo-hexameric capsomers in HCIV-1 capsid. It has been proposed that inHHIV-2, disulphide bridges formed between cysteine residues in the MCPs stabilize thecapsid lattice (Gil-Carton et al., 2015). However, in HCIV-1, only VP7 has the conservedcysteine residue. It remains to be elucidated how the MCPs are linked in HCIV-1 capsid andwhat mechanisms are used to stabilize the lattice.
31
The HCIV-1 genome is a linear dsDNA molecule of 31,314 bp long (Genbank acc. no.KT809302) (II). The genome was predicted to contain 47 open reading frames (ORFs), tenof which were confirmed to be genes encoding structural proteins (Figure 4 C and Table S2in II). A BLASTx search against the NCBI n.r. database found that 43 out of the 47 ORFs hadmatches to haloarchaeal viral sequences, and the majority of these hits were toalphasphaerolipoviruses SH1, PH1, and HHIV-2 (Table S2 in II). The overall gene syntenyand sequence similarities suggested that these viruses are closely related (Figure 3, FigureS5 in III). In addition, a BLAST search yielded hits to haloarchaeal sequences (Table S5 inII), including previously known alphasphaerolipovirus-related proviruses HaloLakP1 andHalaPauP1 (Pawlowski et al., 2014, Porter et al., 2013). We also identified a new putativeHCIV-1-related provirus in Haladaptatus cibarius (NZ_JDTH01000002.1, nt 488,665-496,650) and designated it as HalaCibP1 (Table S5 in II).
Figure 3. The genomes of HCIV-1 and alphasphaerolipoviruses SH1, PH1, and HHIV-2. ORFs/genesare shown as grey arrows, and homologous ones encoding (putative) structural proteins are in thesame colours. Inverted terminal repeats (ITRs) are shown as light blue arrows. ORFs/genes productsare indicated next to the coding regions. The structural protein names (VPs) are the same for all fourviruses, except spike proteins (asterisk): VP3 and VP6 for SH1, PH1, and HCIV-1, but VP16 andVP17 for HHIV-2. Amino acid similarities (%) between proteins VP12, MCPs and putative ATPasesare shown in between the genomes.
HCIV-1 putative protein 13 is predicted to be an ATPase, as it contains canonicalWalker A and B motifs (Walker et al., 1982), as well as the P9/A32-specific motif found inATPases of the viruses belonging to the PRD1-adenovirus structural lineage (Strömsten etal., 2005). The core capsid proteins, MCPs and VP12, as well as putative ATPases, are themost conserved proteins in HCIV-1 and alphasphaerolipoviruses (Figure 3), which is in linewith the idea of conserved viral “self” elements. On the contrary, vertex complex proteins,most likely involved in host cell recognition, have the lowest level of similarity (II) (Jaakkolaet al., 2012). HCIV-1 putative spike proteins, VP3 and VP6, are similar to those of SH1, notHHIV-2, and thus expected to have a horn-like shape, as in SH1 (Jaakkola et al., 2012,Jäälinoja et al., 2008).
Based on all of these characteristics, HCIV-1 belongs to Alphasphaerolipovirus genusof the Sphaerolipoviridae family, which comprises icosahedral dsDNA viruses infectingextremophilic archaea or bacteria (Pawlowski et al., 2014). The phylogenetic analyses of
32
MCPs and ATPase sequences of sphaerolipoviruses, as well as related putative proviruses,indicated that HCIV-1 clusters with the group of alphasphaerolipoviruses (Figure 6 in II).Our proposal to classify HCIV-1 as a new species (Haloarcula virus HCIV1) in the genusAlphasphaerolipovirus has been recently approved by ICTV. In respect to the structure-based virus classification, the conserved structural elements responsible for virionorganization in HCIV-1 indicate that this virus belongs to the “single β-barrel” subgroup ofPRD1-adenovirus structural lineage (Gil-Carton et al., 2015, Oksanen et al., 2012).Amazingly, four of the five currently known haloarchaeal tailless icosahedral viruses appearto be closely related, despite being isolated from geographically distant locations (Figure 4).In terms of taxonomy, eukaryotic, bacterial, or archaeal viruses that are distributed world-wide in moderate and extreme environments and which are currently assigned to a numberof different families may be related by the principles of virion architecture and thus mayconstitute just one structural lineage (Abrescia et al., 2012, Oksanen et al., 2012). Forexample, PRD1-adenovirus lineage unites the viruses currently classified to the familiesTectiviridae (PRD1, Bam35), Adenoviridae (adenovirus), Corticoviridae (PM2),Turriviridae (STIV and STIV2), and Sphaerolipoviridae (HCIV-1, SH1, PH1, HHIV-2, SNJ1,P23-77) (Abrescia et al., 2012).
Figure 4. HCIV-1 virion schematic model and global distribution of alphasphaerolipoviruses(geographic locations from which viruses were isolated are indicated). Map source: Wikimediacommons.
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4.4 Molecular characterization of HHPV3
4.4.1 HHPV3 infection cycleHHPV3 was isolated from salt crystals on Har. hispanica and could not infect any otherhaloarchaeal strains of the 47 tested ones (III). The adsorption of HHPV3 particles to Har.hispanica cells reached ~75 % in 2 h (Figure 1 A in III), and the adsorption rate constant was2.4×10-12 ml/min (calculated for the first 30 min p.i.). Starting at ~6 h p.i., the titer of freeprogeny viruses was increasing and reached the maximum of ~8×1010 plaque forming units(PFU)/ml by ~13 h p.i. (Figure 1 B in III). Approximately at the same time (~12 h p.i.), theturbidity of the infected culture stopped increasing and remained at the same level,indicating growth retardation, while the uninfected culture continued growing. Starting at~6 h p.i., the number of viable cells was decreasing, and by ~12 h p.i. the difference in viablecell counts between the infected and uninfected cultures was about two orders of magnitude(Figure 1 C in III). Thus, although HHPV3 life cycle seems to be non-lytic, the infectionresults in significant growth retardation. Very narrow host ranges, and similar non-lyticinfection cycles with various degrees of host growth retardation have been reported forpleolipoviruses (see chapter 1.5.2.4), which presumably enter the cell via fusion of the virionand the cell membrane and exit the cell via budding (Pietilä et al., 2016, Svirskaitė et al.,2016).
4.4.2 HHPV3 infectivity at different salinitiesTesting the stability of HHPV3 infectivity in different salt solutions (Figures 2 and S3 in III)revealed that CaCl2 and NaCl are critical for the maintenance of HHPV3 infectivity.Noticeably, no major changes in HHPV3 infectivity were observed when incubated in abuffer containing 3 to 5.5 M NaCl over a 24-h period (Figure 2 A in III). The solution of 5.5M NaCl was saturated and contained salt crystals. The fact that HHPV3 was isolated fromsalt crystals attests to its ability to survive in saturated salt. However, at 1.5 M NaCl, HHPV3demonstrated a peculiar, irreversible, pH-dependent loss of infectivity (Figure 2 A in III).NaCl may affect the virus isoelectric point, as shown for some bacteriophages (Langlet et al.,2008). Possibly, changes in HHPV3 virion net charge resulted in clustering of virus particlesat certain pH.
Here, we also tested the effect of NaCl concentration on the infectivity of thepleolipoviruses HRPV-1 (Pietilä et al., 2009, Pietilä et al., 2010, Pietilä et al., 2012),Halogeometricum pleomorphic virus 1 (HGPV-1), and Halorubrum pleomorphic virus 3(HRPV-3) (Atanasova et al., 2012, Pietilä et al., 2012, Senčilo et al., 2012). For HRPV-1, theconcentration of 2-3 M NaCl was optimal, while HGPV-1 and HRPV-3 demonstrated nosignificant infectivity changes when incubated in their optimal buffers containing from 0 to5.5 M (saturated) NaCl. Such maintenance of high infectivity over a full range of NaClconcentrations observed in betapleolipoviruses HGPV-1 and HRPV-3 indicates their highresistance to natural salinity changes that occur in hypersaline environments.Unfortunately, very little is known about virus survival strategies in extreme conditions. Itwould be intriguing to see how virus particles can cope with osmotic stress, lacking anymeans to actively regulate their interactions with the surrounding environment. Theproperties of a pleolipoviral virion, a flexible membrane vesicle decorated by spike proteins
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(Pietilä et al., 2010, Pietilä et al., 2012), may be a key to survival. However, more insightsinto virion structural organization are needed to understand what features underlie thedifferences observed in the adaptability of different pleolipoviruses to salinity changes.
4.4.3 HHPV3 virion organizationHighly purified HHPV3 particles were obtained with optimized purification protocols(Figure 3 A and Table S3 in III) and further used for the analyses of virion components.HHPV3 particles are spherical, but slightly pleomorphic, ~50 nm in diameter (Figure 3 B inIII). This type of pleomorphic morphology is characteristic to a group of haloarchaealpleolipoviruses (Pietilä et al., 2016). HHPV3 has two major viral structural protein species,which are not glycosylated, and a lipid fraction (Figure 4 in III). TLC of the lipids extractedfrom HHPV3 particles or Har. hispanica cells (Bamford et al., 2005b) showed that the virusand the host had the same lipid species in approximately same proportions (Figure 5 in III),indicating a non-selective mode of lipid acquisition by HHPV3 from the host membrane.Pleolipoviruses are considered to acquire lipids non-selectively, although only His2 and itshost, Har. hispanica, display identical lipid profiles, while in lipid compositions of the othervirus-host pairs minor differences are observed, with virus lipids lacking one or more hostlipid species (Liu et al., 2015, Pietilä et al., 2012). Thus, some degree of selectivity might becharacteristic to certain pleolipoviruses.
The HHPV3 genome is a circular 11,648 bp-long dsDNA molecule (Genbank acc. no.KX344510) (Figures S4 A and B in III). Fragmentation patterns observed after HHPV3 DNAwas treated with Mung Bean endonuclease and Bal-31 exonuclease (Figures S4 C and D inIII) suggest that the dsDNA molecule might have nicks or ssDNA regions. This type ofgenome organization (discontinuous dsDNA) has been previously reported forbetapleolipoviruses HGPV-1, HRPV-3, and SNJ2 (Liu et al., 2015, Senčilo et al., 2012). InHRPV-3 and HGPV-1, the discontinuities are ssDNA regions (Senčilo et al., 2012), while inSNJ2, both nicks and ssDNA regions are found (Liu et al., 2015). In HRPV-3, theinterruptions are located mainly on the coding strand, while in SNJ2, the gaps aredistributed on both strands. Both in HRPV-3 and SNJ2, the discontinuities are present incoding and in intergenic regions, and a specific motif GCCCA often precedes them (Liu etal., 2015, Senčilo et al., 2012). This motif can also be found in HHPV3 genome on bothstrands (Figure 6 A in III). Site-specific nicks in genomic DNA have also been reported forsome bacteriophages, e.g. T5 (Abelson and Thomas, 1966) and T7 (Khan et al., 1995), buttheir biological function is unknown. The question of how the nicks/gaps are introduced andwhat role they may have in virus replication remains to be answered. Liu and colleagues(2015) have speculated that the nicks and ssDNA regions in SNJ2 and related viruses mightinduce DNA damage responses from the host, which could promote virus replication.
The HHPV3 genome was predicted to contain 17 ORFs, two of which were confirmedto be genes (gene 1 and gene 3) encoding two major structural protein species, VP1 and VP3(Figures 4 and 6, Table S6 in III). A set of HHPV3 ORFs/genes has sequence similarity,albeit not high, to the conserved cluster of genes observed in pleolipoviruses (Table S6 in III,Figure 5). Moreover, HHPV3 and other pleolipovirus genomes are collinear (Figure 5).Based on the sequence similarities with HRPV-1, the type species of the family
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Pleolipoviridae (Pietilä et al., 2016), HHPV3 VP1 is predicted to be a membrane-associatedprotein and VP3 is most probably a spike protein. Putative protein 6 contains a domaincharacteristic to NTPases (Table S7 in III) and displays similarity to the previously detectedputative NTPases of pleolipoviruses (Figure 6 C in III, Figure 5). Based on the observed genesynteny, HHPV3 can be assigned to the group of betapleolipoviruses (Figure 5).
Figure 5. The genomes of HHPV3 and pleolipoviruses: (A) alphapleolipoviruses, (B)betapleolipoviruses, (C) gammapleolipovirus. ORFs/genes are shown as grey arrows, andhomologous ones are in the same colours. In HHPV3, the products of genes 1 and 3, i.e. VP1 andVP3, are indicated. (D) Schematic model of HHPV3 virion.
HHPV3-related sequence elements are present in many haloarchaea (Figures 6 D andS5). Some of these elements have been previously reported as betapleolipovirus-relatedputative proviruses, all flanked by an integrase and tRNA genes in haloarchaealchromosomes (Liu et al., 2015, Pietilä et al., 2016). The highest sequence similarities wereobserved between HHPV3 and putative proviruses in Har. hispanica and Har. marismortui(Figure 6 D in III). In addition to the known ones (Bath et al., 2006, Chen et al., 2014, Dyall-Smith et al., 2011, Liu et al., 2015, Pietilä et al., 2009, Roine et al., 2010, Senčilo et al., 2012),eight more putative pleolipovirus-related proviruses were identified in this work (Figure S5
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in III). The fact that the known pleolipoviruses were isolated from distant locations and thatrelated proviruses are commonly found in the genomes of archaea originating from varioushypersaline environments suggests the extreme abundance of pleolipoviruses in nature. Aninteresting example of pleolipovirus-related proviruses is the provirus that was identified inthe genome of ‘Halanaeroarchaeum sulfurireducens’ M27-SA2, isolated from deep-sea (~3km depth) salt-saturated anoxic lake Medee (Messina et al., 2016). In this prophage, thepleolipovirus-like ORFs are interrupted by a single CRISPR array and eight associated cas-genes. It seems that the system might have a role in abolishing cellular anti-phagemechanisms, but additional data is needed to understand its functions (Messina et al.,2016).
Based on the cumulative data, HHPV3 virion is suggested to be a lipid vesicle, whichcontains one membrane-associated protein (VP1), and the vesicle surface is decorated by aspike protein (VP3) (Figure 5 D). HHPV3 is proposed to be a member of Betapleolipovirusgenus of the family Pleolipoviridae, which comprises haloarchaeal pleomorphic virusesdistributed worldwide. The discovery of pleolipoviruses further highlights the observationthat similar virion types can be found all across the globe (Liu et al., 2015, Pietilä et al., 2016,Roine et al., 2010, Senčilo et al., 2012).
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5. Conclusions and Future ProspectsIn this work, 37 viruses were obtained from the samples collected from Samut Sakhonsaltern in 2009 and 2010. These isolates have significantly increased the total number ofisolated archaeal viruses. All new viruses displayed the previously known morphotypes.Moreover, the overall morphotype distribution observed in the known haloarchaeal virusesremains approximately the same when the viruses isolated here are added. Thus far, culture-dependent surveys of the hypersaline environments have revealed only a handful of differentvirus morphotypes (Atanasova et al., 2015a, Atanasova et al., 2015b). Whether this reflectsthe natural diversity in these extreme environments remains to be revealed by future studies,coupling culture-dependent and -independent methods. However, only the culture-basedapproach provides an opportunity for a comprehensive characterization of viruses inmolecular detail. The isolates obtained here are a valuable source for further more detailedresearch, including genome sequencing and annotation, as well as structural studies.
Multiple virus-host interactions were observed between the isolated viruses andhalophilic archaeal strains derived from the same location or from distant hypersalineenvironments. The interactions occurred across time and genus barriers, suggesting that thestudied viruses and haloarchaeal strains constitute a dynamic part of the natural microbiotaof hypersaline environments. Although tailed viruses are considered to be rare in highsalinity environments (Santos et al., 2012), numerous interactions observed betweenmyoviruses and a group of Halorubrum strains indicate that these tailed viruses may havea significant input in natural virus-host interplay.
A more in-depth characterization was performed for two new viruses with differentmorphotypes, tailless icosahedral virus HCIV-1 and pleomorphic virus HHPV3. Based onmany lines of evidence, the closest relatives for HCIV-1 are the other haloarchaeal taillessicosahedral viruses, SH1, PH1, and HHIV-2. Thus, HCIV-1 is classified as a new species inAlphasphaerolipovirus genus of the family Sphaerolipoviridae, which comprises taillessicosahedral dsDNA viruses that infect extremophilic archaea or bacteria. The conservedelements responsible for HCIV-1 virion architecture suggest that the virus belongs to thePRD1-adenovirus structural lineage together with other sphaerolipoviruses. HHPV3 issuggested to belong to the group of pleolipoviruses, and is proposed to be classified as a newspecies in Betapleolipovirus genus of the family Pleolipoviridae.
Due to the astronomical number of viruses in the biosphere, the probability to isolatesame viruses all over the globe seems to be low (Hendrix, 2002, Saren et al., 2005, Suttle,2007). However, the cases of HCIV-1 and HHPV3, together with the examples from othervirus groups (Saren et al., 2005), suggest the global distribution and resemblance of distinctvirion architectures. The results obtained in this work are in line with the idea that theenormous virus diversity resides at sequence level, while the number of different virionarchitectures is limited (Krupovič and Bamford, 2011). So far, striking structural similaritieshave been observed between various viruses classified into numerous families which are notassigned to any higher taxa (Abrescia et al., 2012). In this respect, structure-basedclassification is potentially a highly efficient approach to resolve current challenges of virustaxonomy. More sampling and detailed studies on the currently available virus isolates will
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reveal how many ways there are to build an infectious virus particle and what evolutionarypathways underlie current virus diversity.
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6. AcknowledgementsThis work was carried out during the years 2012-2016 at the University of Helsinki, Instituteof Biotechnology and Department of Biosciences, Programme on Molecular Virology headedby Academy Professor Dennis Bamford and supervised by Docent Hanna Oksanen, DocentMaija Pietilä, and PhD Nina Atanasova.
I owe my deepest gratitude to my supervisor, Hanna, for expert advice, constantsupport, and guidance in everything related to our projects, especially in project planningand academic writing. You have always had time for my questions, and I learned so muchthrough our discussions. Thank you for your trust, patience, and kind attitude.
With great pleasure, I thank Dennis for welcoming me for PhD studies in his researchgroup. Thank you for taking care of all of us, for your wisdom, kindness, and sense ofhumour. Your immense knowledge and experience have been of great support for this thesiswork. I also deeply appreciate your work as a member of my thesis advisory committee.
I am deeply thankful to my co-supervisors, Maija and Nina. Maija, thank you forguiding me in learning laboratory techniques and in data analyses. Your excellentorganizational skills and clarity of scientific thinking have been inspiring for me. Nina, thankyou for your passion in hunting for new viruses and for outstanding courage and persistencyin taming the “odd” ones. Thank you also for our lively discussions over a wide range oftopics!
I am grateful to Docent Sari Timonen for being a member of my thesis advisorycommittee. Thank you for sincere interest, enthusiasm, and helpful advice in my researchand studies. Thank you for such inspiring and fun meetings!
I express special gratitude to the pre-examiners of this thesis, Professor KennethStedman and Associate Professor Josefa Antón. Thank you for reading my thesis, its criticalassessment, and valuable comments.
I sincerely appreciate the financial support, which made this work possible, providedby the Centre for International Mobility CIMO, the Viikki Doctoral Programme in MolecularBiosciences (VGSB), the Doctoral Programme in Microbiology and Biotechnology (MBDP),the Ella and Georg Ehrnrooth Foundation, and the Alfred Kordelin Foundation. I alsoacknowledge the support of employees and the use of experimental resources of EU ESFRIInstruct Centre for Virus and Macromolecular Complex Production (ICVIR) at theUniversity of Helsinki, Finland.
I share the credit of my work with all my co-authors. I thank PhD Andrius Buivydas forhis contribution to the discovery of new archaeal viruses. I am grateful to Docent JanneRavantti for guiding me in the (exciting) world of Bioinformatics, for general computersupport, and for challenging me with “suomi-perjantai”. I am thankful to Julija Svirskaitefor the enthusiasm in fighting against electrodes (and winning them!) and being mycompanion in long virus life cycle experiments. Thank you also for your friendship!
My gratitude is extended to all of our excellent technicians, who have contributed somuch to the efficiency and productivity of our research laboratory. I express specialgratitude to Helin Veskiväli for being a highly skilled assistant for the work presented in this
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thesis. I also thank Sari Korhonen and Riitta Tarkiainen for their helpful advice in laboratorypractices.
I would like to acknowledge all members of the Programme on Molecular Virology forcreating friendly and relaxed atmosphere. I especially thank Irma Orientaite, AlesiaRomanovskaya, and Daria Starkova for interesting conversations over lunch/tea. I alsoexpress my appreciation to Outi Lyytinen for being a great companion in travelling and for(patiently) teaching me Finnish.
I would like to give special thanks to my friends, Luiza, Könul, and Konstantin. Despitebusy schedules and geographical distances, I have felt your presence and moral support.Thank you for all the bright and joyful moments and our conversations full of laugh!
Finally, I express heartfelt gratitude to my beloved husband Sergey and my dearparents, Alexey and Galina, for their endless love and absolute support. Your presence in mylife fills it with happiness and joy. Without you, it would be hardly feasible to accomplishthis work.
Tatiana
October 2016
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INSTITUTE OF BIOTECHNOLOGY AND DIVISION OF GENERAL MICROBIOLOGYDEPARTMENT OF BIOSCIENCESFACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCESDOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF HELSINKI
Molecular Characterization of New Archaeal Viruses from High Salinity Environments
TATIANA DEMINA
dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 26/2016
26/2016
Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2706-8
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