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Recent Publications in this Series Molecular Characterization of New Archaeal from High Salinity Environments TATIANA DEMINA

9/2016 Satu Olkkola Antimicrobial Resistance and Its Mechanisms among Campylobacter coli and Campylobacter upsaliensis with a Special Focus on Streptomycin 10/2016 Windi Indra Muziasari Impact of Fish Farming on Antibiotic Resistome and Mobile Elements in Baltic Sea Sediment 11/2016 Kari Kylä-Nikkilä dissertationes schola doctoralis scientiae circumiectalis, Genetic Engineering of Lactic Acid to Produce Optically Pure Lactic Acid and to Develop alimentariae, biologicae. universitatis helsinkiensis 26/2016 a Novel Cell Immobilization Method Suitable for Industrial Fermentations 12/2016 Jane Etegeneng Besong epse Ndika Molecular Insights into a Putative RNA Encapsidation Pathway and Potyvirus Particles as Enzyme Nano-Carriers 13/2016 Lijuan Yan Bacterial Community Dynamics and Perennial Crop Growth in Motor Oil-Contaminated Soil in a TATIANA DEMINA Boreal Climate 14/2016 Pia Rasinkangas New Insights into the Biogenesis of Lactobacillus rhamnosus GG Pili and the in vivo Effects of Molecular Characterization of New Archaeal Pili 15/2016 Johanna Rytioja Viruses from High Salinity Environments Enzymatic Cell Wall Degradation by the White Rot Dichomitus squalens 16/2016 Elli Koskela Genetic and Environmental Control of Flowering in Wild and Cultivated Strawberries 17/2016 Riikka Kylväjä Staphylococcus aureus and Lactobacillus crispatus: Adhesive Characteristics of Two Gram- Positive Bacterial Species 18/2016 Hanna Aarnos Photochemical Transformation of Dissolved Organic Matter in Aquatic Environment – From a Boreal Lake and Baltic Sea to Global Coastal Ocean 19/2016 Riikka Puntila Trophic Interactions and Impacts of Non-Indigenous Species in the Baltic Sea Coastal Ecosystems 20/2016 Jaana Kuuskeri Genomics and Systematics of the White-Rot Fungus Phlebia radiata: Special Emphasis on Wood-Promoted Transcriptome and Proteome 21/2016 Qiao Shi Synthesis and Structural Characterization of Glucooligosaccharides and Dextran from Weissella confusa Dextransucrases 22/2016 Tapani Jokiniemi Energy Efficiency in Grain Preservation 23/2016 Outi Nyholm Virulence Variety and Hybrid Strains of Diarrheagenic Escherichia coli in Finland and Burkina Faso 24/2016 Anu Riikonen INSTITUTE OF BIOTECHNOLOGY AND Some Ecosystem Service Aspects of Young Street Tree Plantings DIVISION OF GENERAL MICROBIOLOGY 25/2016 Jing Cheng DEPARTMENT OF BIOSCIENCES The Development of Intestinal Microbiota in Childhood and Host-Microbe Interactions in Pediatric Celiac Diseases FACULTY OF BIOLOGICAL AND ENVIRONMENTAL SCIENCES DOCTORAL PROGRAMME IN MICROBIOLOGY AND BIOTECHNOLOGY UNIVERSITY OF HELSINKI 26/2016

Helsinki 2016 ISSN 2342-5423 ISBN 978-951-51-2706-8 Molecular characterization of new archaeal viruses from high salinity environments

Tatiana A. Demina

Institute of Biotechnology and Division of General Microbiology Department of Biosciences Faculty of Biological and Environmental Sciences and Viikki Doctoral Programme in Molecular Biosciences and Doctoral Programme in Microbiology and Biotechnology University of Helsinki

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Biological and Environmental 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 Reviewers

Docent Hanna M. Oksanen Professor Kenneth Stedman Institute of Biotechnology and Department of Biology Department of Biosciences Portland State University University of Helsinki U.S.A. Finland Associate Professor Josefa Antón Docent Maija K. Pietilä Department of Physiology, Genetics, and Department of Food and Environmental Microbiology Sciences University of Alicante University of Helsinki Spain Finland Opponent PhD Nina S. Atanasova Institute of Biotechnology and Professor Jarkko Hantula Department of Biosciences Natural Resources Institute (LUKE) University of Helsinki Finland Finland Custos Thesis advisory committee Academy Professor Academy Professor Dennis H. Bamford Dennis H. Bamford Institute of Biotechnology and Institute of Biotechnology and Department of Biosciences Department of Biosciences University of Helsinki University of Helsinki Finland Finland

Docent Sari Timonen Department of Food and Environmental Sciences University of Helsinki Finland

©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 “Science is the poetry of reality” Richard Dawkins

List of original publications This 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 californiae icosahedral 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 high infectivity in saturated salt. Virology 499, 40-51. *These authors contributed equally.

Doctoral candidate’s contribution: In I, TD participated in performing experiments, data analysis and handling, and writing the manuscript. 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 obtained data, and writing the manuscript.

i Abbreviations ATP adenosine triphosphate ATPase adenosine triphosphatase 3-D three-dimensional acc. no. accession number BLAST Basic Local Alignment Search Tool bp base pair BREX exclusion cas clustered regularly interspaced short palindromic repeats-associated CRISPR clustered regularly interspaced short palindromic repeats cryo-EM cryo-electron microscopy DNA deoxyribonucleic acid ds double-stranded ICTV International Committee on of Viruses ITR inverted terminal repeat MCP major capsid MEGA Molecular Evolutionary Genetics Analysis MUSCLE Multiple Sequence Comparison by Log-Expectation n.r. non-redundant nt nucleotide ORF open reading frame p.i. post infection PCR polymerase chain reaction PFU plaque forming unit RNA ribonucleic acid rRNA ribosomal RNA SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis ss single-stranded TEM transmission electron microscopy TLC thin layer chromatography tRNA transfer RNA VLP virus-like particle VP virion protein

ii Abbreviated virus names BTV Bluetongue virus EHP-1 environmental halophage 1 EHP-12 environmental halophage 12 EHP-6 environmental halophage 6 HATV-1 Haloarcula head-tailed virus 1 HATV-2 Haloarcula head-tailed virus 2 HCIV-1 Haloarcula californiae icosahedral virus 1 HCTV-1 Haloarcula californiae head-tailed virus 1 HCTV-2 Haloarcula californiae head-tailed virus 2 HCTV-5 Haloarcula californiae head-tailed virus 5 HGPV-1 Halogeometricum pleomorphic virus 1 HGTV-1 Halogeometricum head-tailed virus 1 HHIV-2 Haloarcula hispanica icosahedral virus 2 HHPV-1 Haloarcula hispanica pleomorphic virus 1 HHPV-2 Haloarcula hispanica pleomorphic virus 2 HHPV3 Haloarcula hispanica pleomorphic virus 3 HHTV-1 Haloarcula hispanica head-tailed virus 1 HHTV-2 Haloarcula hispanica head-tailed virus 2 HJTV-1 Haloarcula japonica head-tailed virus 1 HJTV-2 Haloarcula japonica head-tailed virus 2 HRPV-1 Halorubrum pleomorphic virus 1 HRPV-2 Halorubrum pleomorphic virus 2 HRPV-3 Halorubrum pleomorphic virus 3 HRPV-6 Halorubrum pleomorphic virus 6 HRTV-1 Halorubrum head-tailed virus 1 HRTV-10 Halorubrum head-tailed virus 10 HRTV-11 Halorubrum head-tailed virus 11 HRTV-12 Halorubrum head-tailed virus 12 HRTV-2 Halorubrum head-tailed virus 2 HRTV-3 Halorubrum head-tailed virus 3 HRTV-4 Halorubrum head-tailed virus 4 HRTV-5 Halorubrum head-tailed virus 5 HRTV-6 Halorubrum head-tailed virus 6 HRTV-7 Halorubrum head-tailed virus 7 HRTV-8 Halorubrum head-tailed virus 8 HRTV-9 Halorubrum head-tailed virus 9 HSTV-1 Haloarcula sinaiiensis head-tailed virus 1 HSTV-2 Halorubrum sodomense head-tailed virus 2 HSTV-3 Halorubrum sodomense head-tailed virus 3 HVTV-1 Haloarcula vallismortis head-tailed virus 1 HVTV-2 Haloarcula vallismortis head-tailed virus 2 NHV-1 nanohaloarchaeal virus 1 OLPV Organic Lake phycodnavirus

iii OLV Organic Lake PBCV-1 Paramecium bursaria chlorella virus 1 SCTP-1 Salicola head-tailed phage 1 SCTP-2 Salicola head-tailed phage 2 SCTP-3 Salicola head-tailed phage 3 SIRV2 Sulfolobus islandicus rod-shaped virus 2 SSIP-1 Salisaeta icosahedral phage 1 STIV Sulfolobus turreted icosahedral virus

iv Summary Viruses are important ecological and biological actors and one of the major evolutionary forces that cells encounter. In contrast to their overwhelming genetic diversity, all known virus isolates display a limited number of morphotypes. It has been suggested that due to structural constraints and limitations in protein fold space, virus morphological diversity is restricted to certain possible architectures. Thus, the huge and complex virosphere can be ordered by grouping viruses into a reasonably small number of structure-based lineages. To test this hypothesis, more virus-host systems have to be isolated and characterized. Hypersaline environments, where tend to dominate, are especially rich in viruses. Still very little is known about archaeal viruses in general, and halophilic archaeal viruses in particular. This thesis is devoted to the isolation and characterization of new archaeal viruses from 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 haloarchaeal virus isolates. The majority of the isolated viruses had tailed icosahedral morphology, but pleomorphic and tailless icosahedral morphotypes were also found. When the infectivity of the isolated viruses was tested against haloarchaeal strains isolated from the same location or distant hypersaline environments, numerous interactions were observed across samples and host genera. Myoviruses and Halorubrum strains were the two components that contributed to ~75% of the observed interactions. From the isolated viruses, two were characterized in molecular detail: Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica pleomorphic virus 3 (HHPV3), HCIV-1 was isolated on Haloarcula californiae, but it could also infect three other haloarchaeal strains. The virus maintains its infectivity under high and low salt concentrations, showing adaptability to environmental changes. HCIV-1 infection results in cell lysis, which was demonstrated by traditional metrics, as well as by on-line monitoring of physiological changes in infected cells. The HCIV-1 virion is a tailless icosahedron of ~70 nm in diameter, and it has an inner lipid membrane. The major virion components are proteins, lipids selectively acquired from the host, and a linear double-stranded (ds) DNA molecule of 31,314 bp in length. The virus is predicted to contain 47 open reading frames (ORFs), the majority of which display similarities to the genes/ORFs of alphasphaerolipoviruses SH1, PH1, and Haloarcula hispanica icosahedral virus 2 (HHIV- 2). The of these four viruses share gene synteny and demonstrate high conservation in the genes encoding core virion elements: major capsid proteins (MCPs) and putative adenosine triphosphatases (ATPases). On the contrary, genes coding for (putative) vertex complex 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. The International Committee on Taxonomy of Viruses (ICTV) has approved the proposal to assign HCIV-1 to the Alphasphaerolipovirus genus in Sphaerolipoviridae family, which comprises tailless icosahedral inner membrane-containing dsDNA viruses isolated from extreme environments. In terms of structure-based , HCIV-1 conserved elements responsible for virion organization determine its place in the PRD1-adenovirus

v structural viral lineage, which unites archaeal, bacterial, and eukaryotic viruses that are distributed world-wide. HHPV3 was isolated on Har. hispanica, and its host range is limited to the isolation strain. The HHPV3 cycle is non-lytic, but virus infection results in significant host growth retardation. The virus withstands extremely high NaCl concentrations (3-5.5 M NaCl), but demonstrates a peculiar, irreversible, pH-dependent loss of infectivity at 1.5 M NaCl. The HHPV3 virion is a pleomorphic lipid vesicle of ~50 nm in diameter that contains only two major structural protein species: a membrane associated protein and a spike protein. HHPV3 acquires lipids non-selectively from the host membranes. The virus genome is a circular dsDNA molecule of 11,648 bp in length, which contains 17 predicted ORFs, including a set of ORFs/genes characteristic to pleolipoviruses. HHPV3-related proviruses were found in the chromosomes of many haloarchaeal strains, highlighting the natural abundance of pleolipoviruses. We propose that HHPV3 is a new member in Betapleolipovirus genus of the family. Whether pleolipoviruses form a distinct viral structural lineage remains to be seen. The viruses isolated here provide a rich source for more detailed future studies, which may reveal valuable information on global virus diversity and evolutionary relations between different viral groups. Accumulation of such information helps in resolving virus classification, which is currently an extremely challenging task. Markedly, the 37 virus isolates obtained in this study display the previously known morphotypes. Moreover, HCIV- 1 clearly resembles the other haloarchaeal tailless icosahedral viruses, while HHPV3 belongs to the group of pleolipoviruses. Notably, both viral groups are distributed world-wide. This work supports the observation that highly similar virion architectures can be sampled all over the biosphere, highlighting limited morphological virus diversity. Further studies are needed to portray true viral diversity and to verify whether all viruses can be efficiently classified into structure-based lineages.

vi Table of contents List of original publications ...... i Abbreviations ...... ii Abbreviated virus names ...... iii Summary ...... v Table of contents ...... vii 1. Introduction ...... 1 1.1 Hypersaline environments and ...... 1 1.2 Viruses ...... 3 1.3 Current virus classification ...... 4 1.4 Viral structural lineages ...... 5 1.5 Haloviruses ...... 7 1.5.1 Eukaryotic and bacterial haloviruses ...... 7 1.5.2 Archaeal haloviruses ...... 11 1.5.2.1 Tailed icosahedral viruses ...... 13 1.5.2.2 Spindle-shaped virus His1 ...... 14 1.5.2.3 Tailless icosahedral viruses ...... 15 1.5.2.4 Pleomorphic viruses ...... 18 1.6 Virus-host interactions in hypersaline environments ...... 21 2. Aims of the Study ...... 24 3. Materials and Methods ...... 25 4. Results and Discussion ...... 27 4.1 New archaeal viruses isolated from high salinity ...... 27 4.2 Virus-host interactions ...... 28 4.3 Molecular characterization of HCIV-1 ...... 29 4.3.1 HCIV-1 infection cycle ...... 29 4.3.2 HCIV-1 virion organization ...... 30 4.4 Molecular characterization of HHPV-3 ...... 32 4.4.1 HHPV3 infection cycle ...... 32 4.4.2 HHPV3 infectivity at different salinities ...... 33 4.4.3 HHPV3 virion organization ...... 33 5. Conclusions and Future Prospects ...... 37 6. Acknowledgements ...... 39 7. References ...... 41

vii 1. Introduction 1.1 Hypersaline environments and halophiles Hypersaline environments, artificial and natural, are found all over our planet. The examples of such environments include saline lakes and soils, solar salterns, deep-sea brines, salt mines, halite deposits, and salty food products (Boetius and Joye, 2009). Hypersaline environments may be categorized into thalassohaline (also called thalassic) or athalassohaline (athalassic). Thalassohaline waters, such as solar salterns, have approximately the same ratio of ions as in seawater. On the contrary, athalassohaline environments 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 other extreme conditions in addition to the extreme salinity. For example, halophiles thriving in the Dead Sea are exposed not only to the saturated salt concentrations, but also to acidic pH and intensive UV radiation (Oren, 2010). Despite harsh conditions, the representatives of all three domains of cellular life, Eukarya, Bacteria, and Archaea, as well as their viruses inhabit hypersaline environments (Boetius and Joye, 2009, Oren, 2015). The following chapters will be devoted to halophilic viruses, while here, I will focus on halophilic cellular organisms. Halophilic, or , literally means “salt-loving”. Halophiles may be classified into slight, 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 and Kamekura, 1988). This categorization is, however, nominal, and more widely, halophile refers 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 equal to that in the environment, while “organic-solutes-in” refers to the accumulation (de novo synthesis or uptake from the environment) of compatible solutes (osmolytes), such as amino acids and sugars (Edbeib et al., 2016). Hypersaline environments are dominated by prokaryotes, but also thrive at moderate 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. Dunaliella salina, are main food sources for brine shrimp Artemia and larvae of brine flies. Other invertebrates are also known to survive in hypersaline environments: rotifers, flat worms, copepods, ostracods. In addition, halophytic , e.g. Laguncularia mangrove, can grow in moderately saline soils. Even some vertebrates, e.g. Tilapia species, are observed to survive in moderately high salinity. It is also worth mentioning that some hypersaline ponds are important feeding sources for various birds, including flamingo. Among other eukaryotes inhabiting salty ponds are diatoms, protozoa (ciliate), and fungi. Moderately or extremely halophilic bacteria are classified to at least eight different phyla, 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,

1 spirals, etc.) (Ventosa et al., 2012). Microbial mats formed by halophilic bacteria are observed in hypersaline waters. Unicellular cyanobacteria constitute the upper level of the mat, while filamentous cyanobacteria form the second layer. Anaerobic phototrophic bacteria (green and purple bacteria) grow beneath cyanobacterial layers, and strictly anaerobic bacteria thrive in the bottom layers of the mat. In addition, methanogenic archaea are 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, classes Halobacteria (extreme halophiles) and Methanomicrobia (halophilic or halotolerant methanogens) (Oren, 2014b, Ventosa et al., 2012). In addition, a yet uncultivable lineage of small-sized (~0.6 μm) archaea, called “Nanohaloarchaea”, seems to be globally distributed in 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 a single order Halobacteriales with a single family . By the year 2014, the family contained 47 genera and 165 species, with Halorubrum, Haloferax, and Haloarcula being the largest genera (Oren, 2014b). Recently, it has been suggested to devide the class Halobacteria into three orders, Halobacteriales, Haloferacales, and Natrialbales (Gupta et al., 2015). The order Haloferacales has been proposed to include two families, Haloferacaceae and Halorubraceae, while the order Halobacteriales has been proposed to include 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 display various cell shapes: cocci, rods, disks, triangles (Haloarcula japonica), square (Haloquadratum walsbyi), and pleomorphic (DasSarma and DasSarma, 2012). The proteins of haloarchaea are negatively charged, having an excess of acidic to basic amino acids, 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 used for chemotaxonomic differentiation (Ventosa et al., 2012). Some haloarchaea, e.g. Haloquadratum walsbyi and Halobacterium salinarum, may produce gas-filled rigid proteinaceous vesicles, which enables them to migrate vertically in water to the more oxygenated surface layers (Pfeifer, 2015). Typically, the members of the Halobacteria are aerobic heterotrophs, but some species may have specific nutritional needs and have diverse metabolic pathways (Andrei et al., 2012). The species of the genera Haloferax and Haloarcula 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 in bioremediation of oil-polluted hypersaline environments (Al-Mailem et al., 2010). Some members of Halobacteria, e.g. Hlb. salinarum, are photoheterotrophs and use retinal-based pigments to adsorb light energy (Andrei et al., 2012). These pink-pigmented archaea, together with Salinibacter ruber and Dunaliella salina, are responsible for unusual pink or red 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,

2 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 outer space experiments, to explore whether microbial life could survive in space (Leuko et al., 2014, Stan-Lotter and Fendrihan, 2015). 1.2 Viruses Since the discovery of viruses in the 19th century, the concept of virus has changed dramatically from an infectious particle to the most abundant biological entity, important ecological 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 not to be universal. Historically, viruses were first determined as “filterable agents”, i.e. agents that bypass bacterial filters (Pennazio, 2003). However, this view is no longer applicable since the discovery of giant amoebal viruses that are visible by light microscopy and possess large and complex genomes: e.g. salinus genome is ~2.5 Mbp in length and contains ~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 the observation that the virions of hyperthermophilic spindle-shaped viruses, two- tailed virus and Sulfolobus monocaudavirus 1, develop their tails outside (independently) of the host cells (Häring et al., 2005a, Prangishvili et al., 2006, Uldahl et al., 2016). The other conceptual change lies in the definition of viruses as harmful obligate intracellular parasites: giant viruses may be called “quasi-autonomous” (Claverie and Abergel, 2010) and certain viruses may have relatively mutualistic relations with their hosts. For example, are obligatory symbionts of parasitoid wasps and promote wasp larvae development in a body of another insect (Strand and Burke, 2012), and some cyanophages promote photosynthesis in cyanobacteria during infection (Mann, 2003). In addition, giant viruses themselves may be preyed on by (Aherfi et al., 2016). Thus, recent discoveries suggest that “viruses are more about exceptions than the rules” (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 be considered as viral self, distinguishing viruses from all other replicative elements, such as , , , and transposons (Bamford et al., 2002, Bamford, 2003, Krupovič and Bamford, 2010). Alternatively, virus can be defined as a type of replicator which, together with other replicators, forms a continuum from the most selfish (virulent viruses) to the most cooperative (chromosomes, organelle DNA) replicators (Koonin and Starokadomskyy, 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) have proposed to define viruses and cells as capsid-encoding and ribosome-encoding organisms respectively, while other replicating entities are grouped into “orphan replicons”.

3 Viruses are extremely abundant in various environments (Rosario and Breitbart, 2011), imposing significant selective pressure on their hosts. It has been estimated that in marine environments 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 biomass daily, thus substantially affecting composition of microbial communities and, indirectly, biogeochemical cycles (Suttle, 2007). Viruses are not only the most dominant biological entities, but also reservoirs of diverse genetic information. Many currently known viral sequences have no homologs in cellular or other viral sequences (Yin and Fischer, 2008). It has been suggested that gene flux from viruses to cells is common, and cells might be regarded 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 of endogenous retroviral (former) coat proteins, syncytins, is essential for the formation of placenta (Lokossou et al., 2014). Moreover, it has been hypothesized that viruses may have had direct roles in such important evolutionary events as the origin of DNA and its replication mechanisms (Forterre, 2002), as well as development of cellular antiviral defence systems, including “invention” of nucleus in eukaryotes and formation of complex cell wall in bacteria (Forterre and Prangishvili, 2009). All this, together with many more examples 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 our world as we know it. 1.3 Current virus classification Apparently, an intrinsic feature of the human mind is a need for systematization of the surrounding world in order to understand its ultimate complexity. The discovery of new viruses and accumulation of data regarding their shapes and properties resulted in the development of a concept of virus species. However, there is still no universal definition of virus species that would be satisfactory for all virologists, as different criteria have been proposed to be used for assigning viruses into taxonomical categories. Viruses display a variety of virion shapes and genomic structures. The majority of virus capsids can be characterized by helical or icosahedral symmetry, although some more complex structures are also known. Notably, some viruses that infect thermophilic archaea display 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 an inner or outer lipid envelope, derived from the host membranes. Virus genome may be a DNA 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 virion also vary greatly. For example, porcine have a circular ssDNA genome of ~2 kb long and an icosahedral capsid of only ~17 nm in diameter (Finsterbusch and Mankertz, 2009), while sibericum has a circular dsDNA genome of ~610 kbp long and a virion 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 classification based on four essential virus features: a type of nucleic acid (DNA/RNA), a type of capsid

4 symmetry, presence/absence of envelope, and quantitative characteristics of capsid (e.g. number of capsomers) (Lwoff and Tournier, 1966). This system excluded some previously proposed 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 be grouped into seven classes based on how virus genetic information is expressed and replicated (Baltimore, 1971). In 1966, the International Union of Microbiological Societies delegated a task of developing and maintaining a universal, internationally agreed system of virus classification to the newly established International Committee on Taxonomy of Viruses (ICTV). The main goal of ICTV is “to categorize the multitude of known viruses into a single classification scheme that reflects their evolutionary relationships” (http://www.ictvonline.org/). ICTV currently defines a virus species as “a monophyletic group of viruses whose properties can be distinguished from those of other species by multiple criteria. […] These criteria may include, 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 genomes or genes” (http://www.ictvonline.org/codeOfVirusClassification.asp). The recognized taxa are order, family, sub-family, genus, and species. The Virus Taxonomy Release 2015 reports 7 orders, 111 families, 27 sub-families, 609 genera, and 3704 species. Of 111 families, 82 are not assigned to any order. Although some genes are widely present in virus genomes, e.g. genes encoding for capsid proteins in icosahedral viruses, RNA and DNA polymerases, and other enzymes mediating 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 to resolve virus phylogeny in a way similar to the usage of small subunit ribosomal RNA sequences in reconstructing the tree of cellular life. In addition, extensive between different viruses and hosts (Hambly and Suttle, 2005) makes phylogeny resolution even more challenging. It has been proposed that instead of tree-like hierarchy, a reticulated network topology might be more suitable for reflecting evolutionary pathways of viruses (Morgan, 2016). 1.4 Viral structural lineages With the increasing number of resolved virion protein structures, striking similarities have been found in viruses previously thought to be unrelated (Benson et al., 1999). Inspired by the 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 into higher-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 virus and distinguishes it from other replicators (Krupovič and Bamford, 2010). Virus genetic modules responsible for forming a virion are considered to be vertically-inherited virus “self”, opposite to (often) horizontally exchanged modules involved in genome replication and interactions with the host cell (Abrescia et al., 2010, Krupovič and Bamford, 2010). The virus “self” elements are conserved over evolutionary time, as only a limited number of protein 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. 5 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 of cells (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 fold of 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 HK97 fold, originally determined in bacteriophage HK97 (Wikoff et al., 2000). This lineage also includes several tailed , 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 as archaeal Haloarcula sinaiiensis tailed virus 1 (HSTV-1) (Pietilä et al., 2013c). Interestingly, HK97 fold was also identified in archaeal and bacterial encapsulins, proteins forming icosahedral nanocompartments inside cells, suggesting a common evolutionary origin of encapsulins 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 usually positioned tangentially to the capsid shell surface. Eukaryotic RNA viruses, such as foot- and-mouth disease virus (Acharya et al., 1989), (Rossmann et al., 1985), and poliovirus (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 et al., 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 et al., 2002), and bacteriophage Φ6 (Huiskonen et al., 2006). (iv) PRD1-adenovirus-like viruses are dsDNA tailless icosahedral viruses with an internal lipid membrane (except adenovirus) and MCPs of a vertical double β-barrel fold, arranged into trimers forming pseudo-hexameric capsomers (Benson et al., 1999). This lineage 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), human adenovirus (Athappilly et al., 1994, Rux et al., 2003), and archaeal virus Sulfolobus turreted icosahedral virus (STIV) (Rice et al., 2004). The same MCP fold was also found in virophage Sputnik (Zhang et al., 2012a). In addition, vaccinia scaffolding protein D13, which forms a transitory 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 share overall PRD1-type virion architecture, but the single β-barrel viruses have two MCPs of a vertical single β-barrel fold, both forming the lattice. The suggested members of this group are prokaryotic extremophilic viruses: thermophilic phage P23-77 (Rissanen et al., 2013)

6 and haloviruses Salisaeta icosahedral phage 1 (SSIP-1) (Aalto et al., 2012), SH1 (Jäälinoja et 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 packaging ATPase P9, which is a virion protein located at the unique vertex (Gowen et al., 2003, Hong et al., 2014, Strömsten et al., 2005). Comparison of P9 and other putative packaging adenosine triphosphatases (ATPases) of PRD1-type viruses revealed that in addition to the classical Walker A and Walker B motifs (Walker et al., 1982), all these ATPases (except adenovirus 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 of P9/A32-specific motif in adenovirus ATPase IVa2 may be related to the lack of an inner lipid membrane in adenovirus virion (Strömsten et al., 2005). Some other distinct viral structure-based lineages have been suggested in addition to the four major established ones (Oksanen et al., 2012). Obviously, more information is needed to identify all possible lineages, the number of which is, however, predicted to be relatively low (Bamford et al., 2005a). Current structure-based virus classification is limited to the available high-resolution structures, but high-throughput methods for resolving three-dimensional (3-D) protein structures, as well as robust computational tools for structure comparison, are under constant improvement (Abrescia et al., 2012, Ravantti et al., 2013). Recent advances in cryo-electron microscopy (cryo-EM) have already yielded complex molecular structures of near-atomic resolution (Kuhlbrandt, 2014). Improvements in the methodology within time might enable more comprehensive structure-based reconstruction of viral phylogeny. 1.5 Haloviruses 1.5.1 Eukaryotic and bacterial haloviruses Many marine organisms may be called slight halophiles, but here, halophilic refers to organisms inhabiting environments where salinity is higher than in seawater. To the best of my knowledge, and excluding this work, 74 haloviruses, i.e. viruses that infect halophilic organisms, have been isolated and described to some extent (Atanasova et al., 2015b, Luk et al., 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 (15 viruses) (Figures 1 A and B, Table 1). So far, eukaryotic haloviruses are represented by only five amoebal icosahedral viruses, which were isolated from saline environments in Tunis (Boughalmi et al., 2013). Based on virion morphological features and comparisons of DNA polymerase or capsid protein sequences, four of these viruses were assigned to the family and one to family (Boughalmi et al., 2013) (Figure 1 A). The known bacterial viruses isolated from hypersaline environments infect hosts from genera Halomonas, Pseudomonas, Tetragenococcus, Salinivibrio, Salicola, and Salisaeta, having either a lytic or lysogenic life cycle (Table 1). These viruses are tailed, morphologically resembling the members of the families (icosahedral head and contractile tail, further called myoviruses), (icosahedral head and long non-contractile tail, further called siphoviruses), or (icosahedral head and short noncontractile tail,

7 further called podoviruses), except SSIP-1, which is a tailless icosahedral virus with an inner lipid membrane (Figure 1 B). Myovirus Salicola tailed phage 2 (SCTP-2) is a large virus with a 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 of bacterial haloviruses are either linear or circular DNA molecules of variable size up to 340 kb, 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 (Shen et al., 2012) and for Salisaeta phage SSIP-1 (Aalto et al., 2012). CW02 is a member of the T7-like phage supergroup, and its virion reconstruction at 16-Å resolution permitted fitting of the HK97-fold (Shen et al., 2012), a conserved fold commonly found in dsDNA bacteriophages (see above, chapter 1.4). Salisaeta phage SSIP-1 shares common features with PRD1-like viruses, and the canonical double β-barrel MCP of bacteriophage PM2 was fitted to SSIP-1 virion reconstruction at 12.5-Å resolution (Aalto et al., 2012). SSIP-1 has two MCPs, thus it has been suggested to belong to the subgroup of single β-barrel viruses in the PRD1-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 Salinibacter ruber, 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 Antarctica allowed 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 Organic Lake phycodnavirus (OLPV), which infects algae (Yau et al., 2011). Interestingly, the virophage OLV MCP sequences were also found in metagenomes from other geographically distant hypersaline and freshwater reservoirs, suggesting its possibly global occurrence (Yau et al., 2011).

8 Figure 1. Morphotypes of isolated (A) eukaryotic, (B) bacterial, and (C) archaeal haloviruses. Representative viruses (virions) are drawn schematically. Scale bar, 100 nm. Tailed icosahedral viruses are coloured yellow, tailless icosahedral are green, and lipids are indicated by blue. Modified from (Atanasova et al., 2015b).

9 Table 1. Eukaryotic and bacterial haloviruses.

Virus Isolation host Morphotype, sizea Life cycle Genome, Isolation Reference size source Seb1sol Acanthamoeba polyphaga Tailless icosahedral (Mrs-likeb), ~ 220 nm Ndc DNA Saline soil, Boughalmi et al., Seb2sol Tailless icosahedral (Mrs-like), ~ 201 nm Nd DNA Tunis 2013 Seb6sol Tailless icosahedral (Mrs-like), ~ 206 nm Nd DNA Seb1eau Tailless icosahedral (Mrs-like), ~ 226 nm Nd DNA Salt lake Boug1 Tailless icosahedral (Mimi-liked), ~ 519 nm Nd DNA water, Tunis F9-11 Halomonas halophila Siphovirus, ~ 60/200 nm Lysogenic Nd H. halophila Calvo et al., 1988 F9-11e G3 Pseudomonas sp. G3 Myovirus Lytic Nd Salt pond, Kauri et al., 1991 Canada φ7116 Tetragenococcus halophilus Myovirus, 87-96/200 nm Lytic Nd Fermenting Uchida and Kanbe, φD-86 Tetragenococcus halophilus Siphovirus 67/300 nm Lytic Nd soy sauce 1993 F5-4 Halomonas halophila Siphovirus, ~ 33/142 nm Lysogenic Nd H. halophilae Calvo et al., 1994 F12-9 Halomonas halophila Siphovirus, ~ 42/133 nm Lysogenic Nd UTAK Vibrio sp. B1 Myovirus, ~ 86/100 nm Lytic dsDNA, Solar saltern, Goel et al., 1996 ~80 kbp Alicante, Spain ΦgspB Halomonas salina Myovirus, 46-51/112 nm Nd dsDNA, Saline soil, Seaman and Day, ~49 kb Great Salt 2007 ΦgspC Halomonas salina Myovirus, 53-86/121 nm Lysogenic dsDNA, Plains, USA ~340 kb SCTP-1 Salicola sp. PV3 Siphovirus, head ~95 nm Nd Nd Solar saltern, Kukkaro and SCTP-2 Salicola sp. PV4 Myovirus, ~ 125/145 nm Nd Nd Margherita di Bamford, 2009 SCTP-3 Salicola sp. s3-1 Myovirus Nd NdSavoia, Italy Atanasova et al., 2012 CW02 Salinivibrio SA50 Podovirus, ~60 nm, T = 7 laevo Nd Linear Great Salt Shen et al., 2012 dsDNA, Lake, USA 49,390 bp SSIP-1 Salisaeta sp. SP9-1 Tailless icosahedral with inner lipid Lytic Circular Salt water, Aalto et al., 2012 membrane, ~100 nm, T = 49 dsDNA, Sedom Pondsf, 43,788 bp Israel QHHSV-1 Halomonas ventosae Siphovirus, ~ 47/75 nm Lytic dsDNA Salt mine, Fu et al., 2016 Qiaohou, China a. Capsid diameter and triangulation number (T) for tailless phages and podoviruses; capsid diameter/tail length for tailed phages. b. Mrs-like, -like. c. Nd, not determined. d. Mimi-like, -like. e. Induced from Halomonas halophila strains isolated from hypersaline soils near Alicante, Spain. f. Sedom Ponds are experimental ponds in Israel where water from the Dead Sea is diluted with water from the Red Sea.

10 1.5.2 Archaeal haloviruses Excluding this work (see chaper 4.1), ~100 archaeal viruses have been isolated (Atanasova et 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 genera of the family Halobacteriaceae (Table 2) (Atanasova et al., 2015a). These virus isolates originate from various hypersaline environments all over the world, and represent the following morphotypes: tailed icosahedral, spindle-shaped (also called fusiform or lemon- shaped), tailless icosahedral, and pleomorphic (Figures 1 C and 2 A, Table 2) (Atanasova et al., 2015a). The Genbank non-redundant (n.r.) database (accessed 23.8.2016) also contains a genome sequence (dsDNA, ~40 kbp) of Halorubrum phage CGΦ46 (NC_021537.1), but no other information is available for this virus. In addition, one complete (environmental halophage 1 [EHP-1]) and 42 near-complete viral genomes were retrieved from metagenomic data obtained from a solar saltern in Santa Pola (Alicante, Spain), and 25 of these were tentatively assigned to infect halophilic archaea Haloquadratum walsbyi (15 viruses), Halorubrum lacusprofundi (3), or nanohaloarchaea (7) (Garcia-Heredia et al., 2012, Santos et al., 2007). Recent methodological advances have also allowed unambiguous identification of virus-host pairs in hypersaline environments, circumventing a need for cultivation, as demonstrated by the identification of Nanohaloarchaeal virus 1 (NHV-1) most likely infecting a yet unculturable nanohaloarchaeon from Santa Pola saltern (Martínez-García et al., 2014).

11 Table 2. Archaeal haloviruses.

Morphotype Virusesa Host genera References

Myovirus Hs1, ΦHb, S5100, HF1, HF2, HRTV-1, Halobacterium, Atanasova et al., 2012, Daniels and Wais, HRTV-2, HRTV-3, HRTV-5, HRTV-6, Haloferax, 1990, Nuttall and Dyall-Smith, 1993, HRTV-7, HRTV-8, HRTV-9, HRTV-10, Halorubrum, Pietilä et al., 2013b, Schnabel et al., HRTV-11, HRTV-12, HSTV-2*, HSTV-3, Haloarcula, 1982b, Senčilo et al., 2013, Tang et al., HJTV-1, HJTV-2, HATV-1, HATV-2, HGTV-1, Halogranum, 2002, Torsvik and Dundas, 1980, Torsvik ΦCh1 Natrialba and Dundas, 1974, Witte et al., 1997 Siphovirus Hh1, Hh3, Ja1, ΦN, S45, B10, BJ1, HRTV-4, Halobacterium, Atanasova et al., 2012, Daniels and Wais, HHTV-1, HHTV-2, HCTV-1, HCTV-2, Halorubrum, 1984, Pagaling et al., 2007, Pauling, HCTV-5, HVTV-1*, HVTV-2 Haloarcula 1982, Pietilä et al., 2013b, Senčilo et al., 2013, Torsvik, 1982, Vogelsang-Wenke and Oesterhelt, 1988, Wais et al., 1975 Podovirus HSTV-1* Haloarcula Atanasova et al., 2012, Pietilä et al., 2013c Spindle His1* Haloarcula Bath et al., 2006, Hong et al., 2015, Pietilä et al., 2013a Tailless SH1*, HHIV-2*, PH1, SNJ1 Haloarcula, Bamford et al., 2005b, Gil-Carton et al., icosahedralc Halorubrum, 2015, Jaakkola et al., 2012, Jäälinoja et Natrinema al., 2008, Porter et al., 2013, Zhang et al., 2012b Pleomorphicd HHPV-1, HHPV-2, His2, HRPV-1*, Haloarcula, Atanasova et al., 2012, Bath et al., 2006, HRPV-2, HRPV-3, HRPV-6, HGPV-1, Halorubrum, Li et al., 2014, Liu et al., 2015, Pietilä et SNJ2 Halogeometricum, al., 2009, Pietilä et al., 2010, Pietilä et Natrinema al., 2012, Roine et al., 2010, Senčilo et al., 2012 a. Viruses whose genome sequence is available are in bold, and asterisks mark viruses whose structures have been determined by cryo-EM or cryo-electron tomography and 3-D image reconstruction. For full virus names see “Abbreviated virus names”. b. For ΦH, only partial genome sequence is available. c. For more information, see Table 3. d. For more information, see Table 4.

12 1.5.2.1 Tailed icosahedral viruses The majority of the isolated haloarchaeal viruses are tailed icosahedral resembling the members of the families Myoviridae (24 viruses) or Siphoviridae (15 viruses) (Atanasova et al., 2015b). One podovirus that infects Haloarcula sinaiiensis (HSTV-1) has also been isolated (Atanasova et al., 2012, Pietilä et al., 2013c). Although tailed haloarchaeal virus isolates are rather numerous, tailed virus-like particles (VLPs) are considered to constitute only 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 (those studied for the life cycle) are virulent or temperate. However, no lysin or holin genes (Young et al., 2000) have been identified in the genomes of virulent haloarchaeal tailed viruses, and exact mechanisms of cell lysis are not known. Temperate haloarchaeal tailed viruses are represented by only two myoviruses: ΦH infecting Halobacterium salinarum and ΦCh1 infecting Natrialba magadii. The ΦH partial genome (L segment) may reside in the host cytoplasm as a (Schnabel, 1984), while ΦCh1 integrates into the host genome (Witte et al., 1997). In addition, a few other tailed haloarchaeal viruses may also be temperate, as their genomes encode putative integrases (Senčilo et al., 2013). ΦH and ΦCh1 are so far the best studied haloarchaeal tailed viruses in terms of genome functions. Molecular studies have showed that ΦH genome is highly unstable and during infection 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 with virus DNA in the virion (Witte et al., 1997). Similarly to ΦH, ΦCh1 also undergoes genomic rearrangements. The rearrangements occur in a certain region of ΦCh1 genome, allowing production of different types of tail fibres (Klein et al., 2012). Genomic rearrangements have also 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 terminal repeats (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. ones encoding structural proteins and proteins involved in DNA and RNA metabolism, are also similar to those in bacteriophages, but the majority of predicted genes have no assigned functions (Senčilo and Roine, 2014). In terms of sequence similarities observed within tailed haloarchaeal 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 have been identified in the genomes of halophilic and/or alkaliphilic, as well as in thermophilic and/or methanogenic euryarchaea (Krupovič et al., 2010, Senčilo et al., 2013). 13 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 virus particles display no major structural changes, and regain infectivity when high salinity is restored (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 triangulation number (T) T = 13, 5-fold symmetric spikes project from the vertices, and trimeric decorative structures 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 is inserted between the MCPs, enlarging the capsid (Pietilä et al., 2013b). This was a new mechanism for capsid enlargement, that allows packaging of a larger DNA molecule than expected 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 the center of each facet (Pietilä et al., 2013c). Notably, it was shown that HSTV-1 MCP has the canonical HK97 fold (Pietilä et al., 2013c). Thus, structural analyses allowed placing this archaeal virus into the HK97-like structural viral lineage, together with tailed bacteriophages and eukaryotic herpesviruses (see chapter 1.4). Structure prediction and homology modelling suggest that ΦCh1 may also adopt the HK97-fold, indicating that the lineage may be expanded to include more archaeal viruses (Krupovič et al., 2010).

1.5.2.2 Spindle-shaped virus His1 Several archaeal spindle-shaped viruses have been isolated, and the majority of these viruses infect hyperthermophilic crenarchaea (Krupovič et al., 2014, Pietilä et al., 2013a), and the only 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 been identified for eukaryotes or bacteria. Currently, crenarchaeal spindle-shaped viruses are classified into two families: , comprising viruses with one short tail, and , comprising viruses with one or two long tails. His1 is the only member of genus which is not assigned to any virus family. However, comparative analyses have showed that based on the similarities in MCP sequences, all spindle-shaped viruses may be assigned to the two above mentioned families, and His1 has been suggested to 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). The infection is persistent, i.e. virus particles are released from the host cells continuously, while the host growth is retarded (Pietilä et al., 2013a, Svirskaitė et al., 2016). His1 genome, which is a linear ~14.5 kbp long dsDNA molecule, encodes a putative type B DNA polymerase, has inverted terminal repeats, and terminal proteins attached (Bath et al., 2006). This suggests that His1 replicates via protein priming. His1 MCP and a few minor structural proteins form a spindle-shaped virion, but pleomorphism in the virion shape has been observed (Pietilä et al., 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 that the lipid modification enables hydrophobic interactions between the MCPs and thus may

14 account for the virion flexibility (Pietilä et al., 2013a). Cryo-electron tomography revealed that 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 was observed to be more constant compared to the variable body shape (Hong et al., 2015). It has been speculated that His1 may recognize the host and attach to it with its tail spike structures, and the tail hub may mediate the genome injection, after which spindle-shaped particles 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 hypersaline environments (Santos et al., 2012). Transmission electron microscopy (TEM) revealed spindle-shaped particles associated with square cells of Haloquadratum walsbyi, one of the most 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 obtain it in pure culture (Burns, 2007, Walsby, 1980). Possibly, spindle-shaped viruses infecting Hqr. walsbyi are yet awaiting to be isolated. In addition, in the recent metaviromic studies of Namib Desert Salt pans, three obtained virus contigs showed sequence similarities to His1 genes, including those coding for the MCP and minor structural proteins (Adriaenssens et al., 2016). It has been proposed that these contigs represent the genomes of viruses belonging to the same virus lineage as His1 (Adriaenssens et al., 2016).

1.5.2.3 Tailless icosahedral viruses Before this work, only six archaeal tailless icosahedral viruses were known, two of which infect thermophilic archaea (Sulfolobus viruses STIV and STIV2), and four infect halophilic archaea (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 and STIV2 have been classified to the family , while SH1, HHIV-2, PH1, and SNJ1 have been assigned to the family Sphaerolipoviridae together with Thermus phages P23-77 and IN93 (Table 3). The Sphaerolipoviridae family includes three genera: Alphasphaerolipovirus (SH1, HHIV-2, PH1), Betasphaerolipovirus (SNJ1), and Gammasphaerolipovirus (P23-77, IN93).

15 Table 3. Members of the family Sphaerolipoviridae. Genus Virusa Isolation Life cycle Isolation Structures Capsid MCPsc Genome, GB References host source resolved diameter, Tb acc. no.d Alpha- SH1 Haloarcula Lytic Saline lake Virion (9.6 Å)~80 nm, VP7, VP4 Linear dsDNA, Bamford et al., sphaero- hispanica (Serpentine T = 28, dextro 30,889 bp, 2005b, lipovirus Lake), AY950802 Jäälinoja et al., Australia 2008, Kivelä et al., 2006, Porter et al., 2005 HHIV-2 Haloarcula Lytic Solar saltern, Virion (13 Å)~80 nm, VP7, VP4 Linear dsDNA, Gil-Carton et hispanica (Margherita T = 28, dextro 30,578 bp, al., 2015, di Savoia), JN968479 Jaakkola et al., Italy 2012 PH1 Haloarcula Lytic Saline lake ~50 nm VP7, VP4 Linear dsDNA, Porter et al., hispanica (Pink Lakes), 28,072 bp, 2013 Australia KC252997 Beta- SNJ1 Natrinema Lysogenic Natrinema 70-75 nm PB6, PB2 Circular Liu et al., 2015, sphaero- sp. J7-2 sp. J7-1 dsDNA, Zhang et al., lipovirus 16,341 bp, 2012b AY048850.1 Gamma- P23-77 Thermus Lytic Alkaline hot Virion (14 Å), ~78 nm, VP16, Circular Jaatinen et al., sphaero- thermophilus spring, MCPs (ޒ3 Å), T = 28, dextro VP17 dsDNA, 2008, lipovirus New Zealand 17,036 bp, Jalasvuori et GQ403789 al., 2009 IN93 Thermus Lysogenic Thermus ~130 nm VP13, Circular Matsushita et thermophilus aquaticus VP14 dsDNA, al., 1995, TZ2 19,604 bp, Matsushita and AB063393 Yanase, 2009 a. Type species names are in bold. b. T, triangulation number. c. Small and large MCPs. d. GenBank accession numbers.

16 (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 hypersaline environments (saline lakes in Australia and a solar saltern in Spain) on Haloarcula hispanica (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 closely related to Hrr. sodomense (Jaakkola et al., 2012, Porter et al., 2005). HHIV-2 is also able to infect Haloarcula sp. PV7 (Atanasova et al., 2012, Jaakkola et al., 2012), and PH1 infects Halorubrum, sp. CSW 2.09.4 (Porter et al., 2013). It has been shown that in spite of the narrow host ranges, SH1 and PH1 genomes can be transfected into several additional archaeal 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 some contradictory proposals regarding SH1 exit strategy (Porter et al., 2007, Porter et al., 2005), recent studies, which included traditional metrics and biochemical measurements, have shown 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 is detected (Svirskaitė et al., 2016). The genomes of alphasphaerolipoviruses are linear dsDNA molecules about 30 kbp long 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). Whether these 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 most distinct genes encoding structural proteins are those for putative spikes (see below), which most 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 of halophilic archaea Halobiforma lacisalsi (provirus HaloLacP1) and Haladaptatus paucihalophilus (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 acquired from 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 capsids arranged in an unusual T = 28 dextro lattice (same T number is also reported for Thermus phage P23-77, see below) (Gil-Carton et al., 2015, Jäälinoja et al., 2008). The capsids are built of pseudo-hexameric capsomers having either two or three towers on the capsomer surface. 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 homodimers and VP4-VP7 heterodimers make the lattice, which is stabilized by disulphide bridges across the 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 and HHIV-2, while spike complexes located at 5-fold vertices have different architectures. SH1 capsid is decorated with horn-like spikes (Jäälinoja et al., 2008), and HHIV-2 has propeller- 17 like spikes (Gil-Carton et al., 2015). This observation highlights conservation in the major capsid building blocks (virus “self” elements) and faster evolution in the host recognition structures. (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 at lower salinity (3 M NaCl) (Mei et al., 2015). SNJ1 virions are 70-75 nm in diameter and contain an inner lipid membrane (Liu et al., 2015). The genome is a circular DNA molecule of about 16 kbp long (Zhang et al., 2012b). Little sequence similarity is observed between SNJ1 and alphasphaerolipoviruses. (iii) Archaeal tailless icosahedral haloviruses share structural similarities with the bacterial tailless icosahedral viruses P23-77 and IN93 belonging to the genus Gammasphaerolipovirus (Pawlowski et al., 2014). These viruses infect Thermus thermophilus strains (host range is narrow), undergo lytic (P23-77) or lysogenic (IN93) life cycles, 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 in diameter is arranged in T = 28 dextro lattice. Small MCP VP16 and large MCP VP17 form pseudohexameric capsomers with two towers, which are located either on the same side or the 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 of spikes (elongated) which emerge from the 5-fold vertices (Gil-Carton et al., 2015, Rissanen et al., 2013). Sphaerolipoviruses share virion architectural principles with the viruses belonging to the PRD1-adenovirus structural lineage (see chapter 1.4). Together with SSIP-1 and several related putative proviruses, sphaerolipoviruses constitute a distinct group of single β-barrel viruses within the lineage.

1.5.2.4 Pleomorphic viruses The group of pleomorphic haloviruses includes eight viruses isolated from remote hypersaline locations on Haloarcula, Halorubrum, or Halogeometricum strains, and one temperate 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 the chromosomes or extrachromosomal elements of halophilic archaea belonging to several different genera (Liu et al., 2015). Pleomorphic archaeal viruses have been recently classified to the new family Pleolipoviridae (Pietilä et al., 2016). These viruses display virions of spherical or pleomorphic shape, which are in fact vesicles built of lipids (which are unselectively derived from the host membrane) and typically only two structural protein species: 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 has shown 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 by fusion of the virus lipid membrane with the host cell membrane (Pietilä et al., 2016).

18 Assembled virions are released continuously most probably via budding, retarding the host growth, either slightly (as in case of HRPV-1 infection) or quite substantially (as in case of His2 infection) (Pietilä et al., 2009, Pietilä et al., 2012, Svirskaitė et al., 2016). The host range is typically narrow or limited to the isolation strain (Atanasova et al., 2012, Pietilä et al., 2009, Pietilä et al., 2012). The only temperate pleolipovirus, SNJ2, is able to integrate to 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 virus residing in Natrinema sp. J7-1 as a plasmid (Liu et al., 2015) (Table 3, see also chapter 1.5.2.3). However, the nature of the interaction between SNJ1 and SNJ2 remains to be clarified. The genome type of pleolipoviruses is not conserved, it may be either circular ssDNA 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 all contain a conserved set of genes, including those coding for structural proteins and a putative NTPase (Liu et al., 2015, Senčilo et al., 2012). Based on the gene content, pleolipoviruses are grouped into three genera: Alphapleolipovirus, Betapleolipovirus, and Gammapleolipovirus (Table 4) (Pietilä et al., 2016).

19 Table 4. Archaeal pleomorphic viruses in the family Pleolipoviridae. Genus Virusa Isolation host Virion Life cycle Membrane Genome, Isolation source References diame protein, GeneBank ter spike accession protein number Alpha- HRPV-1 Halorubrum sp. PV6 ~41 nm Persistent VP3, VP4 Circular ssDNA, Solar saltern, Pietilä et al., 2009, pleolipo- 7,048 nt, Trapani, Italy Pietilä et al., 2010, virus FJ685651 Pietilä et al., 2012 HRPV-2 Halorubrum sp. SS5-4 ~54 nm Persistent VP4, VP5 Circular ssDNA, Solar saltern, Samut Atanasova et al., 2012, 10,656 nt, Sakhon, Thailand Pietilä et al., 2012, JN882264 Senčilo et al., 2012 HRPV-6 Halorubrum sp. SS7-4 ~49 nm Persistent VP4, VP5 Circular ssDNA, Solar saltern, Samut Pietilä et al., 2012, 8,549 nt, Sakhon, Thailand Senčilo et al., 2012 JN882266 HHPV-1 Haloarcula hispanica ~52 nm Persistent VP3, VP4 Circular dsDNA, Solar saltern, Roine et al., 2010, 8,082 bp, Margherita di Savoia, Senčilo et al., 2012 GU321093 Italy HHPV-2 Haloarcula hispanica ~50 nm Persistent ORF3 product, Circular ssDNA, Solar saltern, Hulu Li et al., 2014 ORF4 product 8,176 nt, Island, Liaoning, KF056323 China Beta- HRPV-3 Halorubrum sp. SP3-3 ~67 nm Persistent VP1, VP2 Circular Salt water, Sedom Atanasova et al., 2012, pleolipo- dsDNAb, Ponds, Israel Pietilä et al., 2012, virus 8,770 bp, Senčilo et al., 2012 JN882265 HGPV-1 Halogeometricum ~56 nm Persistent VP2 and VP3, Circular Solar saltern, Cabo de sp. CG-9 VP4 dsDNAb, Gata, Spain 9,694 bp, JN882267 SNJ2 Natrinema sp. J7-1 70-80 Lysogenic VP12, VP13 Circular Natrinema sp. J7-1 Liu et al., 2015 nm dsDNAb,c, 16,992 bp, AJVG01000023 Gamma- His2 Haloarcula hispanica ~71 nm Persistent VP27, VP28 Linear dsDNA, Saline lake (Pink Bath et al., 2006, pleolipo- and VP29 16,067 bp, Lakes), Victoria, Pietilä et al., 2012 virus AF191797 Australia a. Type species are in bold. b. Betapleolipovirus genomes are dsDNA molecules with single-stranded interruptions. c. SNJ2 genome corresponds to the region in Natrinema sp. J7-1 chromosome (WGS contig04, nt 19,792-36,797).

20 1.6 Virus-host interactions in hypersaline environments The number of VLPs in hypersaline environments is high (up to 109-101o VLP/ml), increasing along the salinity gradient (Bettarel et al., 2011, Boujelben et al., 2012, Guixa-Boixareu et al., 1996, Santos et al., 2012). Moreover, in many hypersaline reservoirs, free viruses significantly 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 prokaryotic growth rates and mortality (Guixa-Boixareu et al., 1996, Pedros-Alio et al., 2000). As revealed by electron microscopy, the major virus morphotypes in hypersaline environments are 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 as the 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 shapes were observed in hypersaline Lake Retba (Senegal): stars, rods, hooks, and hairpins among others (Sime-Ngando et al., 2011). It is, however, questionable whether these unusual shapes represent real virions or TEM-related artefacts. In addition, it is important to remember that microscopy 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 spherical VLPs. Culture-independent approaches, such as pulsed-field gel electrophoresis of total viral DNA, as well as metagenomics, metatranscriptomics, and metaproteomics, have been applied to assess the diversity and activity of viral communities in hypersaline environments. In general, these studies have shown that halovirus communities are diverse and 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 literature contains contradictory reports about (i) temporal and (ii) spatial variation in halovirus communities: (i) Temporal: Metagenomic analyses of water samples from San Diego salterns taken at different time points have shown that the most abundant viruses, as well as the most abundant microbial species, were stable, while the variation in microdiversity (changing of genotypes) was observed (Rodriguez-Brito et al., 2010). Emerson et al. (2013a, 2012) reanalysed the same dataset and suggested that virus community was actually dynamic at the taxon level and that variation observed at the genotype level in fact reflected shifts at the population level. In a set of studies, Emerson and colleagues analysed metaviromes from Lake Tyrell samples collected over different timescales, and concluded that halovirus populations were stable over days, relatively equally stable and dynamic over weeks, and dynamic 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 the metaviromes 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, common features were identified in the metaviromes from hypersaline ponds in Senegal, Australia,

21 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 direct data comparisons. The lack of universal virus genes also makes it difficult to make comparisons across samples. In addition, different conclusions regarding similarity may be drawn depending on whether nucleotide or protein sequences are compared. For example, in the above mentioned study of Lake Tyrell virus community dynamics, the viromes were compared at the nucleotide sequence level, with the threshold of 90% identity, and considered to be highly dynamic over months to years (Emerson et al., 2012). When the same datasets were used in a large comparison of ~40 viromes and the similarity was assessed at the protein sequence level, the Lake Tyrell viromes were closely related compared to the other datasets (Roux et al., 2016). In addition, the stringency of comparison has to be taken into account, as demonstrated in the same study of the Lake Tyrell virus community: when the fragment recruitment detection threshold was reduced from 90% to 75% nucleotide identity, some previously unseen similarities to the sequenced haloviruses and San Diego metavirome sequences were detected (Emerson et al., 2012). Typically, a high percentage of environmental virus sequences do not match to the databases (Boujelben et al., 2012, Santos et al., 2010, Sime-Ngando et al., 2011). However, some studies have reported sequence similarities between metaviromes and the isolated haloviruses. Metagenomic analyses of the samples from hypersaline ponds in Senegal revealed sequence matches to the isolated haloarchaeal tailed viruses, pleolipoviruses, and His1, 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, eight distinct 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 Vibrio phage VBM1 (Tschitschko et al., 2015). In addition to the MCPs, a prohead protease was detected 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 assessed by metatranscriptomics. For instance, total environmental RNA from Santa Pola salterns was analysed using microarrays constructed based on the previously obtained metavirome from the same environment (Santos et al., 2011). This work has shown that halovirus genes were actively expressed at the sampling time and that the most active viruses were those that tentatively infect haloarchaea and Salinibacter strains (Santos et al., 2011). Low number of matches between metagenomic sequences and databases indicates that only a small part of haloviruses have been characterized. A global sampling of distant hypersaline environments performed in 2012 doubled the number of isolated haloarchaeal viruses, and revealed numerous virus-host pairs within and across samples (Atanasova et al., 2012). Although the analysis of environmental sequences allows reconstruction of complete 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), molecular details 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 this respect, cultivation-based studies cannot be neglected.

22 Although hypersaline environments, such as solar salterns, may be seen as simplified systems for ecological studies due to low species diversity, high cell abundance, and short food chains, there is still little understanding of virus-host interactions in hypersaline econiches. Multiple factors may affect virus-host interplay. It has been shown that salinity affects the adsorption of haloviruses to the host cells (Kukkaro and Bamford, 2009, Mei et al., 2015), as well as regulates switches between lytic and lysogenic virus life cycles (Bettarel et 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 are commonly found in archaea and include clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) immune system (Garrett et al., 2011, Sorek et al., 2008), toxin-antitoxin system (Yamaguchi et al., 2011), superinfection exclusion mechanism (Stolt and Zillig, 1992), and bacteriophage exclusion (BREX) mechanism (Goldfarb et al., 2015). Finally, more spatial and temporal studies in hypersaline environments 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). To conclude, only coupling of various approaches to study virus-host interplay would provide a complete view on the true virus diversity, as well as temporal and spatial dynamics in hypersaline environments.

23 2. Aims of the Study It has been estimated that viruses are present virtually in all types of environments that one can imagine, being a significant component of the biosphere (Breitbart and Rohwer, 2005, Dolja and Krupovič, 2013, Forterre and Prangishvili, 2013, Rohwer et al., 2009). Viruses display huge genetic diversity, but more modest variety of virion shapes (Krupovič and Bamford, 2011). One way to comprehend the diversity in the virosphere is to percept virion as 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 deep evolutionary links have been found between viruses that infect hosts from all three domains of 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 complex evolutionary relations of different virus groups is to isolate new virus-host systems and to characterize them in detail. Hypersaline environments, where archaea dominate, are a rich source of new viruses, and only very little is known about archaeal viruses. This thesis is devoted to the isolation of new haloarchaeal viruses in order to probe their morphological diversity. Virus-host interactions in a hypersaline environment are also addressed. In addition, 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 their morphotypes; - To reveal virus-host interactions in high salinity environments using culture-based approach; - To describe certain isolated viruses in molecular detail, including characterization of virus infection cycle and virion components.

24 3. Materials and Methods Salt samples used in this study included saltwater and salt crystals collected from a solar saltern in Samut Sakhon (Thailand) in 2009 and 2010 (Table S1 in I). Fifty one halophilic prokaryotic strains isolated from the salt samples (Tables 1 and S2 in I), as well as eleven culture collection strains (Table 1 in I) were used here. The studied viruses included 45 isolates listed in publication I (Tables 2 and S3 in I) and Haloarcula hispanica pleomorphic virus 3 (HHPV3) described in publication III. Methods and bioinformatics tools used in this work are listed in Table 5 and Table 6, respectively. Virus genome sequences were deposited in the GenBank database. For more detailed information about the methods, please see the publications I-III and references therein. Table 5. Laboratory methods used in this study. Method Used in Cultivation of halophilic prokaryotes and their viruses I, II, III Plaque assay I, II, III Isolation of halophilic prokaryotes from salt samples I Isolation of archaeal viruses from salt samples I, III Spot-on-lawn test for virus infectivity I, III Virus adsorption assay II, III Virus life cycle analysis II, III Electrochemical and ATP measurements during virus infection II Negative staining and transmission electron microscopy I, II, III Thin-section TEM II Purification of virus particles by NaCl-polyethylene glycol 6000 I, II, III precipitation, rate zonal and equilibrium centrifugation Bradford assay for measuring protein concentration I, II Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) I, II, III Coomassie blue staining of gels I, II, III Pr0-Q Emerald, SYPRO-Ruby, and Sudan Black B staining of gels II, III Identification of proteins by N-terminal sequencing II Identification of proteins by mass spectrometry II, III Extraction of lipids and thin-layer chromatography (TLC) II, III DNA extraction and enzymatic digestion II, III Agarose gel electrophoresis and ethidium bromide staining II, III Polymerase chain reaction (PCR) I, III Sanger sequencing I, II, III Illumina sequencing III

25 Table 6. Bioinformatic tools used in this study. Type of analysis Tool(s) Used in Phylogenetic analysis Molecular Evolutionary Genetics I, II, III Analysis (MEGA) Sequence alignment Multiple Sequence Comparison by I, II, III Log-Expectation (MUSCLE) EMBOSS Stretcher, EMBOSS Needle II, III Visualization, transfer, and Geneious v. 6.1.6 by Biomatters I, II, III alignment of sequences Prediction of open reading frames GenMarkS, Glimmer II, III (ORFs) Prediction of replication origin Ori-Finder 2 III Calculating of guanine-cytosine Science Buddies “Genomics %G~C II, III content Content Calculator” Calculating of isoelectric point and Compute pI/MW tool of ExPASy tools II, III molecular mass Prediction of tRNA sequences ARAGORN v1.2.36 II Detection of repeats Tandem Repeats Finder, EMBOSS II, III Einverted Homology detection Basic local alignment search tool II, III (BLAST) Detection of conserved domains NCBI Conserved Domain Search II, III InterProScan at EMBL-EBI III Prediction of transmembrane TMHMM v 2.0 II, III helices Prediction of coiled-coil regions COILS tool II, III Graphical visualization Circos II Prediction of signal peptides Signal P v 4.1 III

26 4. Results and Discussion 4.1 New archaeal viruses isolated from high salinity In order to isolate new archaeal viruses, salt samples (Table SI in I) were collected from a solar saltern in Samut Sakhon, Thailand in 2009 and 2010. From these samples, halophilic prokaryotes were isolated: 36 archaeal and 15 bacterial strains (Table 1 in I). Based on their partial 16S rRNA gene sequences, the strains were classified at genus level. Bacterial strains belonged to four genera of two phyla: Proteobacteria (Rhodovibrio) and Firmicutes (Halobacillus, Pontibacillus, and Sedimibacillus). The isolated archaea were assigned to ten genera of Halobacteriaceae family, with Halorubrum and Haloarcula strains being the most numerous (10 and 6, respectively). The strains of these genera are commonly reported as 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 our culture collection strains (Table 1 in I), we isolated 46 archaeal viruses from the above mentioned salt samples (Tables 2 and S3 in I; publication III, see chapter 4.4), for 37 of which purification protocols were developed and virion morphotype was determined by TEM. The 37 purified viruses differed one from another by plaque morphology (Figures S1 in I and S1 in III), virion morphotype (Figures 2 in I, 5 in II, and 3 in III), sensitivity of virus infectivity 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 according to their isolation host and morphology (Table 2 in I, and chapter 4.4). The isolated viruses displayed 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 ones when compared to the number of all haloarchaeal viruses reported in the currently available literature (see chapter 1.5.2.4). However, HRPV-6, which had been isolated from the same location during the same sampling, was included in virus-host interactions tests, so I will discuss the interactions of all 37 isolates in chapter 4.2. The isolation of new viruses has increased the total number of isolated archaeal haloviruses from 54 to 90 (Figures 2 A and B). Noticeably, the morphotype distribution observed in this study is highly similar to that in the previous global sampling of distant hypersaline environments (Atanasova et al., 2012), with tailed icosahedral virus isolates being the most numerous. Obviously, the isolated viruses represented only a part of natural viral diversity in the studied environment, as a culture-based approach is limited to the strains that can be cultivated on artificial growth media and viruses that form detectable plaques on host lawns. However, this does not diminish a possible role of the isolated viruses in the dynamics of halophilic microbial communities (see chapter 4.2 for virus-host interactions). So far, culture-dependent samplings of viral diversity in hypersaline environments yielded a relatively low number of different virus morphotypes (Atanasova et al., 2015a), supporting the idea that all viruses can be grouped into a limited number of

27 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 of unusual shape, e.g. possibly resembling those of thermophilic crenarchaeal viruses (Prangishvili et al., 2016) or the particles observed in TEM examinations of environmental samples (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 interactions To reveal virus-host interactions, all isolated viruses were tested against all isolated halophilic prokaryotes (except Halorubrum sp. SS13-13 and Halogranum sp. SS13-5 which did not form a dense lawn) and additional archaeal culture collection strains. For 37 viruses with 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, 73 interactions 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 publication III). Originally, all the viruses were isolated on Halorubrum or Haloarcula strains, but cross-testing showed that strains from other genera (Halobacterium, Halobellus, and Haloterrigena) were also susceptible to the isolated viruses. However, no viruses were isolated for Halolamina, Halogranum, Haloferax, Halogeometricum, and Natrinema strains. Myoviruses had the broadest host ranges (from 3 to 14 hosts, 9 on average), and some Halorubrum strains were susceptible to many viruses (up to 26 different viruses). These two components accounted for the majority (~75%) of the interactions that were observed. Viruses of the other morphotypes had narrower host ranges. Viruses isolated from Samut Sakhon saltern were able to infect the strains isolated from the same location (from both 2009 and 2010 samples), as well as culture collection strains originating from geographically distant hypersaline environments (Tables S4 and S5 in I and publication III). For example, Halorubrum sodomense isolated from the Dead Sea waters (Israel) (Oren, 1983) could be infected by 18 myoviruses, and “Haloarcula

28 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 diverse interactions indicate a constant interplay between haloarchaea and their viruses (especially myoviruses) extending over time, genus, and location “barriers” and are in line with the previous observations of global distribution of microbes and viruses in hypersaline environments (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, I provide a detailed molecular characterization of two isolates obtained from Samut Sakhon saltern: Haloarcula californiae icosahedral virus 1 (HCIV-1, see chapter 4.3) and Haloarcula hispanica pleomorphic virus 3 (HHPV3, see chapter 4.4). 4.3 Molecular characterization of HCIV-1 4.3.1 HCIV-1 infection cycle Although HCIV-1 had been isolated from salt crystals (I), it tolerated a wide range of salinities. No major changes in infectivity were detected in a range of 3 to 23 % total salinity for a 24-h period tested (Figure S1 in II). The ability to tolerate high and low ionic strength may be advantageous in such environments as a solar saltern, where salinity is changing due to natural rainfalls and water evaporation. HCIV-1 was isolated on Har. californiae, but could also infect Har. japonica with approximately same efficiency of plating, and with much lower efficiency, Har. hispanica and Halorubrum sp. SS7-4 (I). It seems that a narrow host range is typical for all currently known haloarchaeal tailless icosahedral viruses (see chapter 1.5.2.3). The adsorption of HCIV-1 particles to Har. californiae cells was relatively slow, but efficient: at ~1.5 h post infection (p.i.), ~45 % of virus particles adsorbed to the cells, and by ~5 h p.i, the maximum of 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 archaeal viruses (Jaakkola et al., 2012, Kukkaro and Bamford, 2009, Svirskaitė et al., 2016). At 2.5 h p.i., tail-like structures between Har. californiae cells and icosahedral virus particles were visualized by TEM (Figures 1 B and C in II). To my knowledge, similar structures have not been reported for other tailless archaeal viruses, but were described for tailless icosahedral bacteriophages, 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., the turbidity of the infected culture started to decrease (Figures 1 A and G in II), while the number 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 electron microscopy (Figure 1 E in II). In addition, the leakage of ATP from the cells and binding of a lipophilic indicator anion (phenyldicarbaundecaborane) to the cells occurred also at ~10 h p.i., indicating that cell integrity was compromised (Figures 1 F and H in II). Moreover, the level of oxygen consumption was significantly decreasing compared to the uninfected culture (Figure 1 I in II). All this suggested that assembled HCIV-1 particles exit cells via cell

29 lysis. The same type of study approach has been applied to SH1, showing that the virus is virulent (Svirskaitė et al., 2016). However, in SH1 infection, cell lysis occurs earlier (at ~6 h p.i.), which probably reflects specificity of virus-host interactions. We could not identify any lysis-related genes in HCIV-1 genome (see below). However, this is not surprising taking into account that no lysis-related genes similar to those of bacteriophages are found in the genomes of archaeal viruses. Moreover, the only currently described archaeal virus exit strategy 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 organization An optimized multistep purification protocol was used to obtain highly purified HCIV-1 particles (Table S1 and Figure 2 in II). The major structural components of HCIV-1 virion were determined (Figures 2-5 in II). TEM of highly purified particles revealed that the icosahedral 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 4 A in II), which were designated according to their amino acid sequence similarities (see below) to the corresponding proteins of SH1, PH1, and HHIV-2. Notably, the overall structural 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, and phosphatidylglycerosulfate (Figure 3 in II). HCIV-1 and Har. californiae lipid compositions differed (Figure 3 in II), suggesting that HCIV-1 acquires lipids selectively from the host cell membrane. A selective acquisition of the host phospholipids has been previously reported for other tailless inner membrane-containing icosahedral dsDNA viruses, e.g. SH1 (Bamford et 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 dsRNA bacteriophage Φ6 (Laurinavičius et al., 2004a). Based on the sequence similarities with SH1 and HHIV-2, for which virion structures have been resolved (Gil-Carton et al., 2015, Jäälinoja et al., 2008), we propose the following virion 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 the inner 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 et al., 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, which has one single β-barrel domain, and the large MCP VP4, which has two single β-barrel domains, form pseudo-hexameric capsomers in HCIV-1 capsid. It has been proposed that in HHIV-2, disulphide bridges formed between cysteine residues in the MCPs stabilize the capsid lattice (Gil-Carton et al., 2015). However, in HCIV-1, only VP7 has the conserved cysteine residue. It remains to be elucidated how the MCPs are linked in HCIV-1 capsid and what mechanisms are used to stabilize the lattice.

30 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), ten of which were confirmed to be genes encoding structural proteins (Figure 4 C and Table S2 in II). A BLASTx search against the NCBI n.r. database found that 43 out of the 47 ORFs had matches to haloarchaeal viral sequences, and the majority of these hits were to alphasphaerolipoviruses SH1, PH1, and HHIV-2 (Table S2 in II). The overall gene synteny and sequence similarities suggested that these viruses are closely related (Figure 3, Figure S5 in III). In addition, a BLAST search yielded hits to haloarchaeal sequences (Table S5 in II), including previously known alphasphaerolipovirus-related proviruses HaloLakP1 and HalaPauP1 (Pawlowski et al., 2014, Porter et al., 2013). We also identified a new putative HCIV-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/genes are shown as grey arrows, and homologous ones encoding (putative) structural proteins are in the same colours. Inverted terminal repeats (ITRs) are shown as light blue arrows. ORFs/genes products are indicated next to the coding regions. The structural protein names (VPs) are the same for all four viruses, except spike proteins (asterisk): VP3 and VP6 for SH1, PH1, and HCIV-1, but VP16 and VP17 for HHIV-2. Amino acid similarities (%) between proteins VP12, MCPs and putative ATPases are shown in between the genomes.

HCIV-1 putative protein 13 is predicted to be an ATPase, as it contains canonical Walker A and B motifs (Walker et al., 1982), as well as the P9/A32-specific motif found in ATPases of the viruses belonging to the PRD1-adenovirus structural lineage (Strömsten et al., 2005). The core capsid proteins, MCPs and VP12, as well as putative ATPases, are the most conserved proteins in HCIV-1 and alphasphaerolipoviruses (Figure 3), which is in line with 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) (Jaakkola et al., 2012). HCIV-1 putative spike proteins, VP3 and VP6, are similar to those of SH1, not HHIV-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 genus of the Sphaerolipoviridae family, which comprises icosahedral dsDNA viruses infecting extremophilic archaea or bacteria (Pawlowski et al., 2014). The phylogenetic analyses of

31 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 genus Alphasphaerolipovirus has been recently approved by ICTV. In respect to the structure- based virus classification, the conserved structural elements responsible for virion organization in HCIV-1 indicate that this virus belongs to the “single β-barrel” subgroup of PRD1-adenovirus structural lineage (Gil-Carton et al., 2015, Oksanen et al., 2012). Amazingly, four of the five currently known haloarchaeal tailless icosahedral viruses appear to 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 number of different families may be related by the principles of virion architecture and thus may constitute just one structural lineage (Abrescia et al., 2012, Oksanen et al., 2012). For example, PRD1-adenovirus lineage unites the viruses currently classified to the families Tectiviridae (PRD1, Bam35), (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: Wikimedia commons.

32 4.4 Molecular characterization of HHPV3 4.4.1 HHPV3 infection cycle HHPV3 was isolated from salt crystals on Har. hispanica and could not infect any other haloarchaeal 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 was 2.4×10-12 ml/min (calculated for the first 30 min p.i.). Starting at ~6 h p.i., the titer of free progeny 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.), the turbidity 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 viable cell 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 infection results in significant growth retardation. Very narrow host ranges, and similar non-lytic infection cycles with various degrees of host growth retardation have been reported for pleolipoviruses (see chapter 1.5.2.4), which presumably enter the cell via fusion of the virion and the cell membrane and exit the cell via budding (Pietilä et al., 2016, Svirskaitė et al., 2016). 4.4.2 HHPV3 infectivity at different salinities Testing 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 a buffer containing 3 to 5.5 M NaCl over a 24-h period (Figure 2 A in III). The solution of 5.5 M NaCl was saturated and contained salt crystals. The fact that HHPV3 was isolated from salt crystals attests to its ability to survive in saturated salt. However, at 1.5 M NaCl, HHPV3 demonstrated 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 particles at certain pH. Here, we also tested the effect of NaCl concentration on the infectivity of the pleolipoviruses 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, the concentration of 2-3 M NaCl was optimal, while HGPV-1 and HRPV-3 demonstrated no significant infectivity changes when incubated in their optimal buffers containing from 0 to 5.5 M (saturated) NaCl. Such maintenance of high infectivity over a full range of NaCl concentrations observed in betapleolipoviruses HGPV-1 and HRPV-3 indicates their high resistance to natural salinity changes that occur in hypersaline environments. Unfortunately, very little is known about virus survival strategies in extreme conditions. It would be intriguing to see how virus particles can cope with osmotic stress, lacking any means to actively regulate their interactions with the surrounding environment. The properties of a pleolipoviral virion, a flexible membrane vesicle decorated by spike proteins

33 (Pietilä et al., 2010, Pietilä et al., 2012), may be a key to survival. However, more insights into virion structural organization are needed to understand what features underlie the differences observed in the adaptability of different pleolipoviruses to salinity changes. 4.4.3 HHPV3 virion organization Highly 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 in III). This type of pleomorphic morphology is characteristic to a group of haloarchaeal pleolipoviruses (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 extracted from HHPV3 particles or Har. hispanica cells (Bamford et al., 2005b) showed that the virus and 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 its host, Har. hispanica, display identical lipid profiles, while in lipid compositions of the other virus-host pairs minor differences are observed, with virus lipids lacking one or more host lipid species (Liu et al., 2015, Pietilä et al., 2012). Thus, some degree of selectivity might be characteristic 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 DNA was treated with Mung Bean endonuclease and Bal-31 exonuclease (Figures S4 C and D in III) suggest that the dsDNA molecule might have nicks or ssDNA regions. This type of genome organization (discontinuous dsDNA) has been previously reported for betapleolipoviruses HGPV-1, HRPV-3, and SNJ2 (Liu et al., 2015, Senčilo et al., 2012). In HRPV-3 and HGPV-1, the discontinuities are ssDNA regions (Senčilo et al., 2012), while in SNJ2, both nicks and ssDNA regions are found (Liu et al., 2015). In HRPV-3, the interruptions are located mainly on the coding strand, while in SNJ2, the gaps are distributed on both strands. Both in HRPV-3 and SNJ2, the discontinuities are present in coding and in intergenic regions, and a specific motif GCCCA often precedes them (Liu et al., 2015, Senčilo et al., 2012). This motif can also be found in HHPV3 genome on both strands (Figure 6 A in III). Site-specific nicks in genomic DNA have also been reported for some bacteriophages, e.g. T5 (Abelson and Thomas, 1966) and T7 (Khan et al., 1995), but their biological function is unknown. The question of how the nicks/gaps are introduced and what 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 might induce DNA damage responses from the host, which could promote virus replication. The HHPV3 genome was predicted to contain 17 ORFs, two of which were confirmed to 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

34 Pleolipoviridae (Pietilä et al., 2016), HHPV3 VP1 is predicted to be a membrane-associated protein and VP3 is most probably a spike protein. Putative protein 6 contains a domain characteristic to NTPases (Table S7 in III) and displays similarity to the previously detected putative NTPases of pleolipoviruses (Figure 6 C in III, Figure 5). Based on the observed gene synteny, 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, and homologous ones are in the same colours. In HHPV3, the products of genes 1 and 3, i.e. VP1 and VP3, are indicated. (D) Schematic model of HHPV3 virion.

HHPV3-related sequence elements are present in many haloarchaea (Figures 6 D and S5). Some of these elements have been previously reported as betapleolipovirus-related putative proviruses, all flanked by an integrase and tRNA genes in haloarchaeal chromosomes (Liu et al., 2015, Pietilä et al., 2016). The highest sequence similarities were observed 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

35 in III). The fact that the known pleolipoviruses were isolated from distant locations and that related proviruses are commonly found in the genomes of archaea originating from various hypersaline environments suggests the extreme abundance of pleolipoviruses in nature. An interesting example of pleolipovirus-related proviruses is the provirus that was identified in the genome of ‘Halanaeroarchaeum sulfurireducens’ M27-SA2, isolated from deep-sea (~3 km depth) salt-saturated anoxic lake Medee (Messina et al., 2016). In this prophage, the pleolipovirus-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-phage mechanisms, 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, which contains one membrane-associated protein (VP1), and the vesicle surface is decorated by a spike protein (VP3) (Figure 5 D). HHPV3 is proposed to be a member of Betapleolipovirus genus of the family Pleolipoviridae, which comprises haloarchaeal pleomorphic viruses distributed worldwide. The discovery of pleolipoviruses further highlights the observation that 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).

36 5. Conclusions and Future Prospects In this work, 37 viruses were obtained from the samples collected from Samut Sakhon saltern in 2009 and 2010. These isolates have significantly increased the total number of isolated archaeal viruses. All new viruses displayed the previously known morphotypes. Moreover, the overall morphotype distribution observed in the known haloarchaeal viruses remains 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 different virus morphotypes (Atanasova et al., 2015a, Atanasova et al., 2015b). Whether this reflects the natural diversity in these extreme environments remains to be revealed by future studies, coupling culture-dependent and -independent methods. However, only the culture-based approach provides an opportunity for a comprehensive characterization of viruses in molecular detail. The isolates obtained here are a valuable source for further more detailed research, including genome sequencing and annotation, as well as structural studies. Multiple virus-host interactions were observed between the isolated viruses and halophilic archaeal strains derived from the same location or from distant hypersaline environments. The interactions occurred across time and genus barriers, suggesting that the studied viruses and haloarchaeal strains constitute a dynamic part of the natural microbiota of hypersaline environments. Although tailed viruses are considered to be rare in high salinity environments (Santos et al., 2012), numerous interactions observed between myoviruses and a group of Halorubrum strains indicate that these tailed viruses may have a significant input in natural virus-host interplay. A more in-depth characterization was performed for two new viruses with different morphotypes, tailless icosahedral virus HCIV-1 and pleomorphic virus HHPV3. Based on many lines of evidence, the closest relatives for HCIV-1 are the other haloarchaeal tailless icosahedral viruses, SH1, PH1, and HHIV-2. Thus, HCIV-1 is classified as a new species in Alphasphaerolipovirus genus of the family Sphaerolipoviridae, which comprises tailless icosahedral dsDNA viruses that infect extremophilic archaea or bacteria. The conserved elements responsible for HCIV-1 virion architecture suggest that the virus belongs to the PRD1-adenovirus structural lineage together with other sphaerolipoviruses. HHPV3 is suggested to belong to the group of pleolipoviruses, and is proposed to be classified as a new species in Betapleolipovirus genus of the family Pleolipoviridae. Due to the astronomical number of viruses in the biosphere, the probability to isolate same 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 other virus groups (Saren et al., 2005), suggest the global distribution and resemblance of distinct virion architectures. The results obtained in this work are in line with the idea that the enormous virus diversity resides at sequence level, while the number of different virion architectures is limited (Krupovič and Bamford, 2011). So far, striking structural similarities have been observed between various viruses classified into numerous families which are not assigned to any higher taxa (Abrescia et al., 2012). In this respect, structure-based classification is potentially a highly efficient approach to resolve current challenges of virus taxonomy. More sampling and detailed studies on the currently available virus isolates will

37 reveal how many ways there are to build an infectious virus particle and what evolutionary pathways underlie current virus diversity.

38 6. Acknowledgements This work was carried out during the years 2012-2016 at the University of Helsinki, Institute of Biotechnology and Department of Biosciences, Programme on Molecular Virology headed by Academy Professor Dennis Bamford and supervised by Docent Hanna Oksanen, Docent Maija Pietilä, and PhD Nina Atanasova. I owe my deepest gratitude to my supervisor, Hanna, for expert advice, constant support, and guidance in everything related to our projects, especially in project planning and academic writing. You have always had time for my questions, and I learned so much through 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 research group. Thank you for taking care of all of us, for your wisdom, kindness, and sense of humour. Your immense knowledge and experience have been of great support for this thesis work. 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 for guiding me in learning laboratory techniques and in data analyses. Your excellent organizational skills and clarity of scientific thinking have been inspiring for me. Nina, thank you for your passion in hunting for new viruses and for outstanding courage and persistency in taming the “odd” ones. Thank you also for our lively discussions over a wide range of topics! I am grateful to Docent Sari Timonen for being a member of my thesis advisory committee. Thank you for sincere interest, enthusiasm, and helpful advice in my research and studies. Thank you for such inspiring and fun meetings! I express special gratitude to the pre-examiners of this thesis, Professor Kenneth Stedman and Associate Professor Josefa Antón. Thank you for reading my thesis, its critical assessment, and valuable comments. I sincerely appreciate the financial support, which made this work possible, provided by the Centre for International Mobility CIMO, the Viikki Doctoral Programme in Molecular Biosciences (VGSB), the Doctoral Programme in Microbiology and Biotechnology (MBDP), the Ella and Georg Ehrnrooth Foundation, and the Alfred Kordelin Foundation. I also acknowledge the support of employees and the use of experimental resources of EU ESFRI Instruct Centre for Virus and Macromolecular Complex Production (ICVIR) at the University of Helsinki, Finland. I share the credit of my work with all my co-authors. I thank PhD Andrius Buivydas for his contribution to the discovery of new archaeal viruses. I am grateful to Docent Janne Ravantti for guiding me in the (exciting) world of Bioinformatics, for general computer support, and for challenging me with “suomi-perjantai”. I am thankful to Julija Svirskaite for the enthusiasm in fighting against electrodes (and winning them!) and being my companion 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 so much to the efficiency and productivity of our research laboratory. I express special gratitude to Helin Veskiväli for being a highly skilled assistant for the work presented in this

39 thesis. I also thank Sari Korhonen and Riitta Tarkiainen for their helpful advice in laboratory practices. I would like to acknowledge all members of the Programme on Molecular Virology for creating friendly and relaxed atmosphere. I especially thank Irma Orientaite, Alesia Romanovskaya, and Daria Starkova for interesting conversations over lunch/tea. I also express 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. Despite busy 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 dear parents, Alexey and Galina, for their endless love and absolute support. Your presence in my life fills it with happiness and joy. Without you, it would be hardly feasible to accomplish this work.

Tatiana October 2016

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51 YEB

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